專(zhuān)利名稱(chēng)：具有降低吸附作用提高水解作用的蛋白酶k變異體的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及用于各種洗滌劑的新的酶變異體及編碼這些酶變異體的DNA序列。
背景技術(shù)：
在天然存在的蛋白質(zhì)中，酶組成了其中最大的一類(lèi)。每類(lèi)酶通常催化(加速一個(gè)反應(yīng)而本身不被消耗)不同類(lèi)型的化學(xué)反應(yīng)。其中一類(lèi)酶如蛋白酶，已知它們具有水解(將一個(gè)化合物斷裂成兩個(gè)或更多的簡(jiǎn)單化合物，并在斷裂的化學(xué)鍵的任何一端吸收水分子的H和OH部分)其它蛋白質(zhì)的能力。這種水解蛋白酶的能力已通過(guò)在洗衣用洗滌劑制品中加入天然存在的或蛋白工程改造的蛋白酶作為添加劑而得到利用。衣服上許多污跡都是蛋白質(zhì)性的，而特異性寬的蛋白酶能基本上增強(qiáng)去除這些污跡的能力。
遺憾的是，這些蛋白質(zhì)在它們天然的細(xì)菌環(huán)境中的效力水平常常不能轉(zhuǎn)移到相對(duì)非天然的洗滌環(huán)境中去。尤其是，當(dāng)不在這些酶的天然環(huán)境中使用時(shí)，蛋白酶的一些特征，如熱穩(wěn)定性、pH穩(wěn)定性、氧化穩(wěn)定性和底物特異性不是所需的最佳狀態(tài)。
蛋白酶的氨基酸序列決定了蛋白酶的性質(zhì)。蛋白酶中氨基酸序列的改變，根據(jù)改變氨基酸序列的位置、性質(zhì)和/或數(shù)量，可不同程度地改變酶的性質(zhì)，甚至可使酶滅活。已采用了幾種方法改變野生型蛋白酶的氨基酸序列以試圖改良它們的性質(zhì)，達(dá)到在洗滌環(huán)境中提高蛋白酶效力的目的。這些方法包括改變氨基酸序列以增強(qiáng)熱穩(wěn)定性并提高在不同環(huán)境下的氧化穩(wěn)定性。
盡管本領(lǐng)域中已描述了多種方法，但仍需繼續(xù)尋找新的有效的蛋白酶的變異體，用于清潔多種表面。
發(fā)明目的本發(fā)明的一個(gè)目的是提供相對(duì)于野生型蛋白酶K具有更強(qiáng)的水解作用的蛋白酶K酶變異體。
本發(fā)明的另一目的是提供含有這些枯草桿菌蛋白酶變異體的洗滌劑。
發(fā)明概述本發(fā)明涉及蛋白酶K的變異體，它們對(duì)野生型蛋白酶K的氨基酸序列中某些序列進(jìn)行改良，野生型氨基酸序列包括第一環(huán)區(qū)、第二環(huán)區(qū)、第三環(huán)區(qū)、第四環(huán)區(qū)和第五環(huán)區(qū)和第六環(huán)區(qū)；其中改良的氨基酸序列是在一個(gè)或多個(gè)環(huán)區(qū)的特定位置上含有與野生型蛋白酶K不同的氨基酸(即替換)，因此，與野生型蛋白酶K相比，蛋白酶K變異體降低了對(duì)不溶底物的吸附作用，并提高了對(duì)不溶底物的水解作用。本發(fā)明也涉及編碼這些蛋白酶K變異體的DNA序列。本發(fā)明還涉及包含有這些蛋白酶K變異體的用于清潔多種表面的組合物。
發(fā)明描述I.蛋白酶K變異體本發(fā)明涉及枯草桿菌蛋白酶，尤其是蛋白酶K，通過(guò)使編碼這些酶的各種核苷酸序列發(fā)生突變從而改變?cè)撁傅陌被嵝蛄衼?lái)改良這些酶。本發(fā)明改良的枯草桿菌蛋白酶(下文稱(chēng)為“蛋白酶K變異體”)與野生型枯草桿菌蛋白酶相比，降低了對(duì)不溶底物的吸附作用并提高了對(duì)不溶底物的水解作用。本發(fā)明還涉及編碼這些蛋白酶K變異體的DNA序列。
本發(fā)明中的枯草桿菌蛋白酶屬于已知為蛋白酶的一類(lèi)酶。蛋白酶是一種斷裂肽鍵的催化劑。蛋白酶中的一類(lèi)是絲氨酸蛋白酶，絲氨酸蛋白酶的特征是在其活性中心有一個(gè)必需的絲氨酸殘基。
有資料證實(shí)了酶對(duì)可溶底物的水解速率隨酶濃度的增大而提高這種觀測(cè)結(jié)果。對(duì)表面結(jié)合的底物也似乎是這樣，例如在許多洗滌應(yīng)用中遇到的，水解速率隨表面濃度的增大而提高，這已為事實(shí)所證實(shí)。(Brode，P.F.III和D.S.Rauch，LANGMUIR，“枯草桿菌蛋白酶BPN對(duì)固定化底物的活性(Subtilisin BPNActivity on an ImmobilizedSubstrate)”，1992年第8卷1325-1329頁(yè))。事實(shí)上，已發(fā)現(xiàn)當(dāng)酶的表面濃度發(fā)生變化時(shí)，對(duì)不溶底物的水解速率與表面濃度呈線(xiàn)性依賴(lài)關(guān)系。(Rubingh，D.N.和M D.Bauer，“蛋白酶在蛋白-溶液界面的水解催化作用(Catalysis of Hydrolysis by Proteases at theProtein-Solution Interface)”見(jiàn)I.Noda和D.N.Rubingh編，Elsevier1992年出版“聚合物溶液，混合物和界面(POLYMER SOLUTIONS，BLENDS AND INTERFACES)”第464頁(yè))。令人吃驚的是，當(dāng)我們?cè)噲D利用這一原則尋找具有更好的洗滌性能的變異的蛋白酶時(shí)，我們找不到吸附越好性能越好的酶。實(shí)際上，我們意外地測(cè)定到了相反的情況降低酶對(duì)底物的吸附作用將提高底物的水解作用(即更好的洗滌性能)。
雖然不希望拘泥于理論，但可以認(rèn)為當(dāng)變異體相互比較時(shí)，性能的提高是源于這樣一個(gè)事實(shí)，即吸附作用小的酶結(jié)合力也小，從而在表面上有更高的移動(dòng)性，將不溶的蛋白質(zhì)底物從表面上除去。在可比較的酶溶液濃度下，這種提高的移動(dòng)性足以勝過(guò)在表面施加更高濃度的酶所帶來(lái)的好處。
本文所述的突變被指定為改變(即降低)酶對(duì)表面結(jié)合的污垢的吸附作用。在蛋白酶K中，某些氨基酸在酶分子上形成外環(huán)。為了討論的方便，這些環(huán)被稱(chēng)作第一、第二、第三、第四和第五環(huán)區(qū)。具體地說(shuō)，64-71位形成第一環(huán)區(qū)；95-107位形成第二環(huán)區(qū)；133-140位形成第三環(huán)區(qū)；160-170位形成第四環(huán)區(qū)；190-194位形成第五環(huán)區(qū)；203-223位形成第六環(huán)區(qū)(位置序號(hào)類(lèi)似于野生型枯草桿菌蛋白酶K中氨基酸序列的位置(SEQ ID NO.1)。
據(jù)信這些環(huán)區(qū)在酶分子對(duì)表面結(jié)合的肽的吸附作用中起著重要作用，一個(gè)或更多的環(huán)區(qū)上特定的突變將顯著影響這種吸附作用。雖然不希望受理論的限制，但至少有兩個(gè)原因可使人相信這些環(huán)區(qū)對(duì)蛋白酶K分子的吸附作用是非常重要的。第一，組成環(huán)區(qū)的氨基酸可與酶分子作用的表面緊密接觸。第二，環(huán)區(qū)與蛋白酶K分子上活性中心和結(jié)合口袋的鄰近使其在酶對(duì)表面結(jié)合的底物(肽/蛋白質(zhì)污垢)的催化產(chǎn)生的吸附作用中起著作用。
本文中，“變異體”指氨基酸序列與野生型酶不同的酶。
本文中，“突變的蛋白酶K DNA”指編碼蛋白酶K變異體的DNA序列。
本文中，“野生型蛋白酶K”指SEQ ID NO.1所代表的酶。Gunkel，F(xiàn).A.和Gassen H.G.對(duì)蛋白酶K的氨基酸序列有更多的描述，見(jiàn)“來(lái)自Tritirachium album Limber的蛋白酶K染色體基因的鑒定及其cDNA在大腸桿菌中的表達(dá)(Proteinase K from Tritirachium albumLimberCharacterization of the Chromosomal Gene andExpressim of the cDNA in Escherichia coli)”歐洲生物化學(xué)雜志(EUR.J.BIOCHEM.)1989年179卷185-194頁(yè)，在此引入作為參考。
本文中，術(shù)語(yǔ)“蛋白酶K野生型氨基酸序列”包括SEQ ID NO1以及除64-71，95-107，133-140，160-170，190-194和203-223位外有修飾氨基酸序列的SEQ ID NO1。
本文中，“更親水的氨基酸”指比參照下面的親水性表的對(duì)照氨基酸具有更高親水性的其它任何氨基酸。下面的親水性表(表1)按親水性升高由上向下順序排列出了氨基酸(見(jiàn)Hopp，T.P.，和Woods，K.R.，“由氨基酸序列推測(cè)蛋白質(zhì)的抗原決定簇(Prediction of ProteinAntigenic Determinants from Amino Acid Sequences)”，美國(guó)國(guó)家科學(xué)院學(xué)報(bào)(PROCEEDINGS OF THENATIONALACADEMY OFSCIENCEUSA)1981年78卷3824-3828頁(yè)，在此引入作為參考)。
表1氨基酸 親水性數(shù)值Trp -3.4Phe -2.5Tyr -2.3Leu，Ile -1.8Val -1.5Met -1.3Cys -1.0Ala，His -0.5Thr -0.4Pro，Gly -0.0Gln，Asn 0.2Ser0.3Arg+，Lys+，Glu-，Asp-3.0
表1也指明了哪些氨基酸帶有電荷(該特征是基于pH為大約8-9)。帶正電荷的氨基酸是Arg和Lys，帶負(fù)電荷的氨基酸是Glu和Asp，剩下的氨基酸是中性的。在本發(fā)明的-個(gè)優(yōu)選實(shí)施方案中，替代的氨基酸為中性或帶負(fù)電荷的，更優(yōu)選帶負(fù)電荷的(即Glu或Asp)。
因此，例如，“用中性或帶負(fù)電荷的親水性相同或更強(qiáng)的氨基酸替換Gln”是指Gln將被Asn(與Gln有相同親水性)或Ser、Glu或Asp(它們均比Gln親水性強(qiáng))替換；與Gln相比，它們均是中性的或帶負(fù)電荷的并都具有更高的親水性值。同樣，“用中性或帶負(fù)電荷的親水性更強(qiáng)的氨基酸替換Pro”是指Pro將被Gln、Asn、Ser、Glu或Asp替換。
在本發(fā)明的一個(gè)實(shí)施方案中，蛋白酶K變異體是對(duì)蛋白酶K野生型氨基酸序列進(jìn)行改良，其中在野生型氨基酸序列的第一環(huán)區(qū)、第二環(huán)區(qū)、第三環(huán)區(qū)、第四環(huán)區(qū)、第五環(huán)區(qū)或第六環(huán)區(qū)中的一個(gè)或多個(gè)環(huán)區(qū)中的一個(gè)或多個(gè)位置包含替換；因此蛋白酶K變異體與野生型蛋白酶K相比，降低了對(duì)不溶底物的吸附作用，而提高了對(duì)不溶底物的水解作用。
在本發(fā)明的一個(gè)優(yōu)選實(shí)施方案中，一個(gè)或多個(gè)環(huán)區(qū)中一個(gè)或多個(gè)位置的替換氨基酸(參照表1)是中性或帶負(fù)電荷的，并且與野生型氨基酸序列中被替換位置的氨基酸相比，具有相同或更強(qiáng)的親水性，優(yōu)選具有更強(qiáng)親水性的氨基酸。A.在第一環(huán)區(qū)中的替換當(dāng)替換發(fā)生在第一環(huán)區(qū)時(shí)，替換發(fā)生在64、65、66、67、68、70或71位中的一個(gè)或多個(gè)位置。
當(dāng)替換發(fā)生在64位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在65位時(shí)，替代氨基酸是Glu。
當(dāng)替換發(fā)生在66位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在67位時(shí)，替代氨基酸是Asp、Gln、Glu或Ser。
當(dāng)替換發(fā)生在68位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在70位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在71位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。B.在第二環(huán)區(qū)中的替換當(dāng)替換發(fā)生在第二環(huán)區(qū)時(shí)，替換發(fā)生在95、96、97、98、99、100、101、102、103、104、105、106或107位中的一個(gè)或多個(gè)位置。
當(dāng)替換發(fā)生在95位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
當(dāng)替換發(fā)生在96位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
當(dāng)替換發(fā)生在97位時(shí)，替代氨基酸是Glu。
當(dāng)替換發(fā)生在98位時(shí)，替代氨基酸是Glu。
當(dāng)替換發(fā)生在99位時(shí)，替代氨基酸是Asp、Gln、Glu或Ser。
當(dāng)替換發(fā)生在100位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在101位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在102位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在103位時(shí)，替代氨基酸是Asn、Asp、Glu或Ser。
當(dāng)替換發(fā)生在104位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
當(dāng)替換發(fā)生在105位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在106位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)替換發(fā)生在107位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。C.在第三環(huán)區(qū)中的替換當(dāng)替換發(fā)生在第三環(huán)區(qū)時(shí)，替代發(fā)生在133、134、135、136、137、138、139或140位中的一個(gè)或多個(gè)位置。其中當(dāng)替換發(fā)生在133位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
當(dāng)替換發(fā)生在134位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在135位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在136位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在137位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
當(dāng)替換發(fā)生在138位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在139位時(shí)，替代氨基酸是Asp或Glu。和當(dāng)替換發(fā)生在140位時(shí)，替代氨基酸是Asp或Glu。D.在第四環(huán)區(qū)中的替換當(dāng)替換發(fā)生在第四環(huán)區(qū)時(shí)，替換發(fā)生在160、161、162、163、164、165、166、167、168、169或170位中的一個(gè)或多個(gè)位置。其中當(dāng)替換發(fā)生在160位時(shí)，替換氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在161位時(shí)，替換氨基酸是Asp、Gln、Glu或Ser。
當(dāng)替換發(fā)生在162位時(shí)，替換氨基酸是Asp、Gln、Glu或Ser。
當(dāng)替換發(fā)生在163位時(shí)，替換氨基酸是Asp、Gln、Glu或Ser。
當(dāng)替換發(fā)生在164位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
當(dāng)替換發(fā)生在165位時(shí)，替代氨基酸是Glu。
當(dāng)替換發(fā)生在166位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
當(dāng)替換發(fā)生在167位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在168位時(shí)，替代氨基酸是Asp、Gln、Glu或Ser。
當(dāng)替換發(fā)生在169位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。和當(dāng)替換發(fā)生在170位時(shí)，替代氨基酸是Asp或Glu。E.在第五環(huán)區(qū)中的替換當(dāng)替換發(fā)生在第五環(huán)區(qū)時(shí)，替換發(fā)生在190、191、192、193或194位中的一個(gè)或多個(gè)位置。其中當(dāng)替換發(fā)生在190位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在191位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在192位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr、Tyr或Val。
當(dāng)替換發(fā)生在193位時(shí)，替代氨基酸是Asp或Glu。和當(dāng)替換發(fā)生在194位時(shí)，替代氨基酸是Asp、Gln、Glu或Ser。F.在第六環(huán)區(qū)中的替換當(dāng)替換發(fā)生在第六環(huán)區(qū)時(shí)，替換發(fā)生在203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222或223位中的一個(gè)或多個(gè)位置。其中當(dāng)替換發(fā)生在203位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在204位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
當(dāng)替換發(fā)生在205位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在206位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)替換發(fā)生在207位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在208位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
當(dāng)替換發(fā)生在209位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
當(dāng)替換發(fā)生在210位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在211位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)替換發(fā)生在212位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Phe、Pro、Ser、Thr、Tyr或Val。
當(dāng)替換發(fā)生在213位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
當(dāng)替換發(fā)生在214位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在215位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在216位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在217位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)替換發(fā)生在218位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在219位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在220位時(shí)，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
當(dāng)替換發(fā)生在221位時(shí)，替代氨基酸是Asp或Glu。
當(dāng)替換發(fā)生在222位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)替換發(fā)生在223位時(shí)，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。G.酶變異體的制備實(shí)施例1突變型蛋白酶K DNA構(gòu)建一個(gè)含野生型蛋白酶K基因的噬菌粒(“PKP”)。質(zhì)粒pUC119(Vieira.J和Messing，J.“單鏈質(zhì)粒DNA的制備(Production ofSingle-Stranded Plasmid DNA)”，酶學(xué)方法(METHODSINENZYMOLOGY)1989年153卷3-11頁(yè))中2.8Kbp PvuII限制酶片段克隆到質(zhì)粒pUB110(Bacillus Genetic Stock Center，Columbus，OH 1E9)的PvuII位點(diǎn)上。pUC119-pUB110雜交質(zhì)粒被命名為pJMA 601。將解淀粉芽孢桿菌枯草桿菌蛋白酶基因克隆到質(zhì)粒pJMA 601中?？莶輻U菌蛋白酶基因被修飾后含有兩個(gè)BamHI位點(diǎn)。一個(gè)BamHI位點(diǎn)在編碼Gly和Lys的DNA之間，即前區(qū)中第二和第三個(gè)氨基酸殘基的DNA之間。另一個(gè)BamHI位點(diǎn)位于TAA終止序列之后。利用反轉(zhuǎn)錄和聚合酶鏈反應(yīng)從RNA中擴(kuò)增出蛋白酶K的cDNA，其中寡核苷酸引物上除含與蛋白酶K cDNA相同的序列外，還各含一個(gè)BamHI位點(diǎn)。被擴(kuò)增的片段包含從蛋白酶K前區(qū)起始處Ala到成熟蛋白酶C末端Ala間的DNA序列。擴(kuò)增的片段用于替換解淀粉芽孢桿菌枯草桿菌蛋白酶基因中兩個(gè)BamHI位點(diǎn)間的DNA序列。將噬菌粒PKP轉(zhuǎn)化到大腸桿菌Ung-菌株CJ 236中并用VCSM 13輔助噬菌體產(chǎn)生含尿嘧啶的單鏈DNA模板(Kunkel，T.A.，J.D.Roberts和R.A.Zakour，“快速有效無(wú)需表型選擇的位點(diǎn)專(zhuān)一誘變(Rapid and efficient site-specificmutagenesis without phenotypic selection)”，酶學(xué)方法(METHODSINENZYMOLOGY)，1987年154卷367-382頁(yè)；改良的方法見(jiàn)Yuckenberg，P.D.，F(xiàn).Witney，J.Geisselsoder和J.McClary“利用含尿嘧啶的DNA和噬菌粒載體的體外定點(diǎn)誘變(Site-directed in vitro mutagenesisusing urail-containing DNA and phagemid vectors)”，M.J.McPherson編1991年版“定向誘變實(shí)用方法(DIRECTEDMUTAGENESIS-A PRACTICALAPPROACH)”27-48頁(yè)，兩者均引于此以作參考)。用Zoller和Smith的單引物定點(diǎn)誘變修飾方法(Zoller，M.J.和M.Smith，“用M13衍生載體進(jìn)行寡核苷酸指導(dǎo)的誘變一種在任何DNA片段上產(chǎn)生點(diǎn)突變的有效而常用的方法(0ligonucleotide-directedmutagenesis using M13-derived vectorsan efficient andgeneral procedure for the production of point mutations inany fragment of DNA)”，核酸研究(NUCLEICACIDSRESEARCH)，1982年10卷6487-6500頁(yè)，在此引入作為參考)產(chǎn)生所有的突變體(基本上如以上Yuckenberg等(1991)介紹的)。寡核苷酸用AppliedBiosystem Inc.公司380B DNA合成儀合成。誘變反應(yīng)產(chǎn)物轉(zhuǎn)化到大腸桿菌MM 294菌株(American Type Culture Collection E.coli.33625)中。所有突變體均由DNA測(cè)序所證實(shí)且分離的DNA被轉(zhuǎn)化到枯草芽胞桿菌表達(dá)型菌株BG 2036中(Yang，M.Y.，E.Ferrari和D.J.Henner，“枯草芽胞桿菌中性蛋白酶基因的克隆以及用克隆基因產(chǎn)生體外誘導(dǎo)的缺失突變(Cloning of the Neutral Protease Gene ofBacillus subtilis and the Use of the Cloned Gene to Createan In Vitro-derived Deletion Mutation)”，細(xì)菌學(xué)雜志(JOURNAL OFBACTERIOLOGY)1984年160卷15-21頁(yè))。對(duì)于一些環(huán)區(qū)突變體，使用在相應(yīng)環(huán)區(qū)含終止密碼子移碼突變的修飾PKP產(chǎn)生尿嘧啶模板。寡核苷酸被設(shè)計(jì)成可恢復(fù)正確的閱讀框并編碼64，65，66，67，68，70，71，95，96，97，98，99，100，101，102，103，104，105，106，107，133，134，135，136，137，138，139，140，160，161，162，163，164，165，166，167，168，169，170，190，191，192，193，194，203，204，205，206，207，208，209，210，211，212，213，214，215，216，217，218，219，220，221，222或223位的隨機(jī)替換(這些密碼子中所有三種堿基用等摩爾和/或比例不同的所有四種核苷酸的混合物)。修正了移碼終止并產(chǎn)生有功能酶的突變通過(guò)它們消化酪蛋白的能力得到鑒別。隨機(jī)替換由DNA測(cè)序確定。
實(shí)施例2發(fā)酵含有研究中的枯草桿菌蛋白酶突變體的枯草芽胞桿菌細(xì)胞(BG2036)在1升LB-葡萄糖肉湯培養(yǎng)基中長(zhǎng)至對(duì)數(shù)中期后，接種至BiostatED發(fā)酵罐(B.Braun Biotech，Inc.，Allentown，Pennsylvania)中，體積為10升。發(fā)酵培養(yǎng)基中含酵母抽提物、淀粉、抗泡沫劑、緩沖劑和痕量的礦質(zhì)(見(jiàn)“發(fā)酵實(shí)用方法(FERMENTATIONA PRACTICALAPPROACH)”，B.McNeil和L.M.Harvey編，1990年版)。在發(fā)酵過(guò)程中肉湯培養(yǎng)基pH保持在7.0。加入氯霉素以對(duì)誘變質(zhì)粒進(jìn)行抗生素選擇。細(xì)胞在37℃生長(zhǎng)過(guò)夜至A600為約60，然后收獲。
實(shí)施例3純化通過(guò)下面的步驟取用發(fā)酵肉湯獲得純酶。通過(guò)離心從肉湯培養(yǎng)基中除去枯草桿菌細(xì)胞，然后用100K的截止濾膜除去細(xì)小的顆粒使肉湯澄清。接著用10K的截止濾膜濃縮并通過(guò)流動(dòng)透析降低離子強(qiáng)度，并用0.025M MES緩沖劑(2-(N-嗎啉代)乙磺酸)調(diào)pH至5.5。酶上樣于陽(yáng)離子交換色譜柱或親和吸附色譜柱進(jìn)一步純化，用NaCl或丙二醇梯度液從柱中洗脫該酶(見(jiàn)Scopes.R.K.“蛋白純化原理與方法(PROTEINPURIFICATIONPRINCIPLESAND PRACTICE)”，在此引入作為參考)。
pNA實(shí)驗(yàn)(DelMar，E.G.，C.Largman，J.W.Brodrick和M.C.Geokas，分析生物化學(xué)(ANAL.BIOCHEM.)1979年99卷316-320頁(yè)，在此引入作為參考)用來(lái)檢測(cè)梯度洗脫時(shí)分部收集液中的活性酶濃度。該實(shí)驗(yàn)測(cè)量了當(dāng)酶水解可溶性合成底物琥珀酰-丙氨酸-丙氨酸-脯氨酸-苯丙氨酸-對(duì)硝基苯胺(sAAPF-pNA)時(shí)，對(duì)硝基苯胺的釋放速率。用分光光度計(jì)于410nm處測(cè)量水解反應(yīng)中黃色的生成速率，其與活性酶的濃度成比例。另外，280nm處測(cè)定的吸光值用來(lái)確定總蛋白濃度?；钚悦?總蛋白的比值表示酶純度，并用來(lái)確定將合并為貯存液的各分部收集液。
為了避免貯存中的酶自水解，向合并的從色譜柱中得到的分部收集液中加入等重的丙二醇。純化完成后，貯存酶溶液的純度用SDS-PAGE(十二烷基硫酸鈉聚丙烯酰胺凝膠電泳)檢測(cè)，絕對(duì)酶濃度用購(gòu)自Sigma化學(xué)公司(St.Louis，Missouri)的胰蛋白酶抑制劑II-T型火雞卵清，通過(guò)活性位點(diǎn)滴定法確定。測(cè)量所得的轉(zhuǎn)換因數(shù)將告訴我們?cè)诿阜肿又胁煌恢玫哪男└淖儗a(chǎn)生比野生型酶對(duì)可溶底物pNA具有更高活性的酶變異體。
使用之前，酶貯存液用Sephadex G 25(Pharmacia，Piscataway，NewJersey)分子排阻過(guò)濾柱洗脫除去丙二醇并更換緩沖劑。酶貯存液中MES緩沖劑被換成含0.01M CaCl2并用HCl調(diào)pH至8.6的0.1M Tris緩沖劑(Tris(羥甲基氨基甲烷))。所有實(shí)驗(yàn)在25℃，pH8.6 Tris緩沖劑中進(jìn)行。H.酶變異體的鑒定實(shí)施例4模型表面的制備購(gòu)自CPG公司的氨丙基人工微孔玻璃(CPG)用作支持物，共價(jià)附著購(gòu)自Bachem公司(Torrence，California)的sAAPF-pNA底物。反應(yīng)在二甲亞砜中進(jìn)行，(1-乙基-3-[3-(二甲基氨基)丙基]碳二亞胺鹽酸鹽)(EDC)用作偶合劑。反應(yīng)完后(用pNA實(shí)驗(yàn)檢測(cè))，除去過(guò)量的溶劑，CPGsAAPF-pNA用二甲亞砜(DMSO)和雙蒸水淋洗，隨后在干燥箱中約70℃用N2吹干。反應(yīng)方案和固定化底物的制備按Brode，P.F.III，和D.S.Rauch所述進(jìn)行，見(jiàn)“枯草桿菌蛋白酶BPN對(duì)固定化底物的活性(Subtilisin BPNActivity on anImmobilized Substrate)”，LANGMUIR，1992年8卷1325-1329頁(yè)，在此引入作為參考。
CPG表面具有62,000±7,000 pNA分子/μm2。其表面積與CPG公司為我們所購(gòu)的CPG提供的數(shù)據(jù)50.0m2/g相比沒(méi)有改變。這表明往CPG上加sAAPF-pNA的方法沒(méi)有損壞其多孔的結(jié)構(gòu)(平均直徑486)。
實(shí)施例5表面水解實(shí)驗(yàn)利用CPGsAAPF-pNA，酶變異體的吸附作用和對(duì)CPG結(jié)合肽的水解作用可通過(guò)一個(gè)簡(jiǎn)單的實(shí)驗(yàn)來(lái)測(cè)量。將一小體積的酶變異體貯存液加入含Tris緩沖劑和已抽氣的CPGsAAPF-pNA的燒瓶中。將燒瓶放入作曲形運(yùn)動(dòng)的搖床中搖90分鐘，其中在不同的時(shí)間間隔停機(jī)幾次(例如，在吸附水解的早期，如頭20分鐘，每2分鐘停一次，而在實(shí)驗(yàn)的后期則每10分鐘停一次)。CPGsAAPF-pNA允許沉淀并在溶液中取樣。實(shí)驗(yàn)方法及吸附作用和水解作用的計(jì)算均按以上Brode等人1992年描述的方法進(jìn)行。
所有酶都檢測(cè)了其抗自水解的穩(wěn)定性而且在整個(gè)實(shí)驗(yàn)過(guò)程中不應(yīng)有明顯的自水解損失。因而酶的吸附作用可通過(guò)測(cè)溶液的消耗來(lái)確定。酶變異體初始濃度和在不同時(shí)刻所測(cè)得的酶濃度的差異體現(xiàn)了被吸附的酶變異體的量。從表面水解下來(lái)的pNA的量可通過(guò)測(cè)等份試樣在410nm處的吸收得到。將試樣量與燒瓶中剩余的量相加計(jì)算出水解下來(lái)的pNA的總量。這個(gè)值通過(guò)減去當(dāng)沒(méi)有酶時(shí)被pH8.6的Tris緩沖劑水解下來(lái)的pNA的量得到校正。依據(jù)酶的效率，這個(gè)本底水解作用占總水解作用的7-29％。
實(shí)施例6可溶底物動(dòng)力學(xué)分析可溶底物sAAPF-pNA的水解速率通過(guò)在DU-70分光光度計(jì)上于410nm處測(cè)吸收升高與時(shí)間的函數(shù)得到檢測(cè)。對(duì)每個(gè)動(dòng)力學(xué)測(cè)量，酶濃度保持恒定，它被制成6-10納摩爾，而底物濃度為90-700μMsAAPF-pNA。在900秒內(nèi)每秒取一個(gè)吸收值且將數(shù)據(jù)轉(zhuǎn)到LOTUSTM記錄紙(Lotus Development Corporation，Cambridge，Massachusetts)上。動(dòng)力學(xué)參數(shù)的分析可按標(biāo)準(zhǔn)的Lineweaver Burk分析進(jìn)行，在LineweaverBurk分析中，曲線(xiàn)起始部分(通常第一分鐘)的數(shù)據(jù)符合線(xiàn)性回歸曲線(xiàn)，可得出vo。將vo與so數(shù)值以標(biāo)準(zhǔn)的倒數(shù)形式作圖可得出KM和kcatI.蛋白酶K變異體實(shí)例本發(fā)明中降低了對(duì)表面結(jié)合底物的吸附作用并提高了對(duì)表面結(jié)合底物的水解作用的蛋白酶K變異體在下面的表2-36中列出。描述特定突變時(shí)，首先給出的是野生型酶的原始氨基酸，其后為位置序號(hào)，最后是置換氨基酸。
表2環(huán)區(qū)1-單突變變異體Arg64AspArg64GluAsp65GluGly66AsnGly66AspGly66GlnGly66GluGly66ProGly66SerAsn67AspAsn67GlnAsn67GluAsn67SerGly68AsnGly68AspGly68GlnGly68GluGly68ProGly68SerGly70AsnGly70AspGly70GlnGly70GluGly70ProGly70SerThr71AsnThr71AspThr71GlnThr71GluThr71GlyThr71ProThr71Ser
表3環(huán)區(qū)1-雙突變變異體Asn67Ser + Thr71AspGly66Ser + Thr71SerArg64Asp + Thr71AsnArg64Asp + Gly68SerGly66Glu + Gly70SerArg64Glu + Gly68SerArg64Glu + Gly66GlnAsn67Glu + Gly70AsnAsn67Gln + Thr71AspGly66Ser + Gly70AsnAsp65Glu + Gly70SerArg64Glu + Gly70AsnGly66Pro + Thr71AsnGly70Gln + Thr71GlnGly68Glu + Gly70AsnAsp65Glu + Thr71SerGly68Asp + Gly70GlnAsp65Glu + Gly68GlnGly70Gln + Thr71AspGly68Gln + Gly70ProAsn67Asp + Gly70GlnGly66Ser + Asn67GlnAsp65Glu + Gly68AsnGly68Asp + Gly70ProGly66Gln + Thr71ProAsp65Glu + Thr7lProArg64Asp + Gly68AsnGly68Asn + Thr71GluGly66Asn + Thr71GluGly68Ser + Thr71AspGly68Ser + Thr71GlyGly66Ser + Gly68GlnGly66Gln + Gly70SerGly66Glu + Gly70ProArg64Glu + Gly70ProAsp65Glu + Thr71GlyArg64Glu + Thr71GlyAsp65Glu + Gly70ProArg64Asp + Gly68GlnArg64Glu + Gly68ProGly66Asn + Gly70Ser表4環(huán)區(qū)1-三突變變異體Asn67Ser + Gly68Asp + Gly70SerAsn67Asp + Gly70Asn + Thr71GlyArg64Glu + Gly66Asn + Gly70ProGly66Asp + Gly68Ser + Gly70SerAsp65Glu + Gly70Gln + Thr71GlnGly66Glu + Gly68Pro + Gly70AsnArg64Asp + Gly66Ser + Gly70GlnGly66Gln + Asn67Gln + Gly68GluGly66Glu + Asn67Ser + Thr7lProGly66Asn + Gly68Pro + Thr71GlnArg64Glu + Gly66Gln + Gly68GlnGly66Asn + Gly68Ser + Gly70ProArg64Asp + Gly70Gln + Thr71GlyAsp65Glu + Gly68Gln + Gly70SerArg64Glu + Gly66Pro + Gly68SerGly66Gln + Asn67Gln + Thr71GlyArg64Glu + Gly68Pro + Thr71SerAsn67Asp + Gly70Ser + Thr71GlyArg64Glu + Gly70Pro + Thr71GlnAsp65Glu + Gly68Gln + Gly70GlnGly66Pro + Gly68Gln + Thr71ProGly66Glu + Asn67Gln + Gly70ProGly66Gln + Gly70Ser + Thr71GlnAsp65Glu + Gly68Gln + Gly70AsnArg64Glu + Gly66Ser + Thr71ProAsp65Glu + Gly68Ser + Gly70AsnAsp65Glu + Asn67Ser + Gly70SerArg64Glu + Gly66Ser + Gly70ProAsp65Glu + Gly70Gln + Thr71GlyAsp65Glu + Gly68Asn + Gly70AsnGly68Asn + Gly70Pro + Thr71AspArg64Asp + Gly68Ser + Gly70ProGly66Asn + Gly68Glu + Gly70GlnAsn67Glu + Gly68Gln + Gly70AsnGly66Glu + Gly68Pro + Gly70GlnAsn67Asp + Gly68Glu + Gly70SerAsn67Asp + Gly68Asp + Gly70AsnArg64Asp + Asp65Glu + Asn67SerArg64Asp + Asp65Glu + Gly70ProArg64Glu + Asp65Glu + Asn67Gln表5環(huán)區(qū)1-四突變變異體Asp65Glu + Gly68Pro + Gly70Asn + Thr71AsnAsp65Glu + Asn67Gln + Gly68Gln + Gly70SerGly66Glu + Asn67Gln + Gly68Asn + Gly70GlnAsp65Glu + Gly66Asn + Gly70Ser + Thr71GlnAsn67Gln + Gly68Glu + Gly70Gln + Thr71GlyArg64Glu + Gly66Gln + Gly70Asn + Thr71GlnGly66Pro + Gly68Ser + Gly70Asn + Thr71GlnArg64Glu + Gly68Pro + Gly70Gln + Thr71ProGly66Glu + Gly68Pro + Gly70Asn + Thr71ProAsp65Glu + Gly66Glu + Asn67Ser + Gly70ProAsp65Glu + Gly66Asp + Gly68Asn + Thr71SerAsp65Glu + Gly66Glu + Gly68Pro + Gly70SerAsp65Glu + Gly66Glu + Gly70Asn + Thr71GlnArg64Asp + Asp65Glu + Asn67Gln + Gly70AsnArg64Asp + Asp65Glu + Gly70Pro + Thr71GlnGly66Glu + Asn67Asp + Gly68Ser + Gly70SerGly66Glu + Asn67Glu + Gly70Asn + Thr71SerAsp65Glu + Gly66Asp + Asn67Glu + Gly68SerArg64Glu + Asp65Glu + Gly66Asp + Gly68ProGly66Glu + Asn67Asp + Gly68Asp + Gly70AsnAsp65Glu + Asn67Glu + Gly68Ser + Gly70GlnAsp65Glu + Asn67Asp + Gly68Gln + Gly70SerAsp65Glu + Asn67Glu + Gly68Gln + Gly70AsnAsp65Glu + Gly66Asn + Asn67Glu + Gly70ProAsp65Glu + Gly66Asp + Gly68Asp + Thr71AsnAsp65Glu + Gly66Glu + Asn67Gln + Gly68Asp表6環(huán)區(qū)2-單突變變異體Val 95AlaVal 95AsnVal 95AspVal 95CysVal 95GlnVal 95GluVal 95GlyVal 95HisVal 95MetVal 95ProVal 95SerVal 95ThrLeu 96AlaLeu 96AsnLeu 96AspLeu 96CysLeu 96GlnLeu 96GluLeu 96GlyLeu 96HisLeu 96IleLeu 96MetLeu 96ProLeu 96SerLeu 96ThrLeu 96ValAsp 97GluAsp 98GluAsn 99AspAsn 99GlnAsn 99GluAsn 99SerGly100AsnGly100AspGlyl00GlnGly100GluGly100ProGly100SerSer101AspSer101GluGly102AsnGly102AspGly102GlnGly102GluGly102ProGly102SerGln103AsnGln103AspGln103GluGln103SerTyr104AlaTyr104AsnTyr104AspTyr104CysTyr104GlnTyr104GluTyr104GlyTyr104HisTyr104IleTyr104LeuTyr104MetTyr104ProTyr104SerTyr104ThrTyr104ValSer105AspSer105GluThr106AsnThr106AspThr106GlnThr106GluThr106GlyThr106ProThr106SerIle107AlaIle107AsnIle107AspIle107CysIle107GlnIle107GluIle107GlyIle107HisIle107LeuIle107MetIle107ProIle107Ser
20Ile107ThrIle107Val表7環(huán)區(qū)2-雙突變變異體Val 95Gln + Ser101GluTyr104His + Ile107AspVal 95Glu + Ile107AsnAsn 99Gln + Gln103AspLeu 96Gly + Ser105GluVal 95Met + Gln103AspSer101Asp + Ile107ThrVal 95His + Asp 97GluAsp 98Glu + Tyr104LeuLeu 96Cys + Asp 97GluGln103Glu + Tyr104GlnGly102Ser + Tyr104GlyLeu 96Ala + Thr106ProThr106Gln + Ile107AsnAsn 99Gln + Ile107AspAsn 99Glu + Thr106GlyGly102Pro + Gln103AspAsn 99Asp + Tyr104ThrLeu 96Ile + Ser101GluVal 95Gly + Gln103SerTyr104Leu + Ser105GluGly102Asn + Tyr104ProLeu 96Asp + Ile107LeuAsp 98Glu + Gly102AsnLeu 96Pro + Gly100GluSer101Asp + Thr106SerGly100Ser + Gln103AsnGly102Pro + Tyr104ThrLeu 96Glu + Ile107LeuLeu 96Ile + Gty100AspGly100Pro + Ile107SerAsp 97Glu + Asn 99GlnAsp 97Glu + Ile107CysGly102Ser + Gln103GluGly100Asn + Tyr104AspGly100Pro + Thr106AspVal 95Pro + Asp 97GluVal 95Thr + Gly100GluThr106Pro + Ile107GluSer101Asp + Gly102AsnSer105Glu + Ile107ThrAsn 99Gln + Ser105AspGln103Ser + Ile107MetLeu 96Asp + Thr106GlyVal 95Thr + Gly102AspVal 95Ala + Ser105Asp
21Gly100Asn + Gln103SerGly102Asp + Thr106GlnLeu 96Asn + Ser105AspGly100Glu + Gly102ProGln103Asp + Ile107HisTyr104Ala + Ile107ProAsp 97Glu + Ile107ProAsn 99Glu + Tyr104AsnVal 95Thr + Asp 98GluGly100Gln + Thr106GlyAsn 99Glu + Gln103SerVal 95His + Ser105AspGly102Pro + Ile107AlaAsp 97Glu + Gly100Gln表8環(huán)區(qū)2-三突變變異體Val 95Gln + Leu 96Thr + Ser101GluSer101Asp + Thr106Ser + Ile107LeuVal 95Asn + Leu 96Asn + Asp 98GluGln103Glu + Tyr104Ser + Ile107SerVal 95Cys + Leu 96Glu + Gly102SerVal 95His + Tyr104Thr + Ser105GluLeu 96Pro + Asn 99Asp + Gln103SerVal 95Ser + Leu 96Asp + Gln103SerLeu 96Cys + Gly102Gln + Ile107LeuVal 95Ser + Leu 96His + Thr106ProLeu 96Gln + Gly102Pro + Ser105AspLeu 96Met + Asn 99Ser + Ser105AspLeu 96His + Ser101Asp + Tyr104ValAsn 99Asp + Gly100Asn + Gly102SerVal 95Gln + Gly100Asp + Thr106ProLeu 96Asn + Tyr104Glu + Ile107GlnAsn 99Asp + Gly102Ser + Tyr104HisVal 95Met + Leu 96Gly + Gly100ProVal 95Ala + Asp 98Glu + Asn 99SerAsp 97Glu + Tyr104Thr + Ile107HisLeu 96Thr + Gly102Gln + Gln103AspTyr104Met + Thr106Glu + Ile107LeuGly100Ser + Gln103Asn + Tyr104IleGln103Asn + Tyr104Pro + Thr106GluVal 95Met + Asp 98Glu + Asn 99GlnLeu 96His + Asp 97Glu + Ile107MetGly100Asn + Gln103Ser + Ser105AspGly102Asn + Gln103Glu + Thr106ProVal 95Met + Gly100Ser + Gly102AspVal 95Asp + Leu 96Met + Tyr104AsnGln103Glu + Tyr104Cys + Ile107AsnVal 95Met + Leu 96Ile + Tyr104MetVal 95Ser + Ser101Glu + Thr106GlyVal 95Pro + Asn 99Asp + Gly100SerGly100Ser + Gln103Glu + Ile107ValGln103Asp + Tyr104Val + Ile107HisGly100Gln + Tyr104Met + Thr106AspAsn 99Asp + Gly100Ser + Tyr104HisSer101Asp + Gly102Asp + Gln103SerAsp 98Glu + Asn 99Asp + Tyr104AlaAsp 98Glu + Asn 99Glu + Gly100GlnGly102Asp + Gln103Asp + Thr106GlnTyr104Glu + Ser105Asp + Thr106ProAsp 97Glu + Asp 98Glu + Tyr104ThrAsp 97Glu + Asp 98Glu + Asn 99AspAsn 99Asp + Gly100Asp + Ser101GluGln103Glu + Tyr104Asp + Ile107GluLeu 96Glu + Asp 97Glu + Gly102AspLeu 96Glu + Ser101Asp + Gln103AsnAsp 97Glu + Gly100Asp + Ile107HisAsp 97Glu + Asn 99Ser + Gly100AspGln103Asp + Ser105Glu + Thr106AsnLeu 96Ser + Ser105Asp + Ile107AspAsp 97Glu + Ser101Glu + Gln103SerAsp 97Glu + Ser101Glu + Tyr104MetAsn 99Asp + Gly100Ser + Ser101AspGln103Asp + Tyr104His + Ile107GluAsp 97Glu + Gly102Glu + Tyr104ProVal 95Asp + Gly102Glu + Tyr104GlyVal 95Asp + Asp 97Glu + Ile107Asn表9環(huán)區(qū)2-四突變變異體Val 95Gln + Asp 97Glu + Gly100Ser + Ile107GlnVal 95Ser + Leu 96Pro + Asp 97Glu + Gln103AsnLeu 96Gln + Gly100Ser + Ser101Asp + Thr106AsnVal 95Cys + Asp 97Glu + Gly100Gln + Gly102SerLeu 96Pro + Gly102Asn + Ser105Asp + Ile107GlnVal 95Ser + Gly100Gln + Ser101Glu + Ile107AsnLeu 96Met + Ser101Glu + Gln103Asn + Thr106ProAsn 99Asp + Gly100Pro + Gly102Ser + Tyr104AlaGly100Pro + Gly102Pro + Thr106Gly + Ile107GluVal 95Ser + Gly100Gln + Ser101Asp + Gly102ProVal 95Gly + Leu 96Val + Thr106Pro + Ile107AspVal 95Gln + Leu 96Val + Asn 99Asp + Ile107ThrLeu 96Ser + Asp 97Glu + Thr106Gln + Ile107MetLeu 96Thr + Asn 99Gln + Tyr104Val + Ile107CysAsn 99Glu + Gly100Gln + Gly102Asn + Gln103SerVal 95Gly + Ser101Glu + Gln103Ser + Thr106GlyVal 95Asn + Leu 96Gln + Asp 98Glu + Gly100AsnVal 95Cys + Gly102Pro + Tyr104Val + Ile107AspVal 95Met + Leu 96Met + Gly102Glu + Tyr104ThrVal 95Asp + Leu 96Cys + Thr106Ser + Ile107GlyLeu 96Met + Gly100Glu + Ser101Glu + Tyr104CysAsp 98Glu + Asn 99Glu + Gly102Asn + Ile107SerVal 95Ser + Asp 97Glu + Asp 98Glu + Asn 99SerVal 95Gly + Asp 97Glu + Asp 98Glu + Tyr104IleLeu 96Gly + Asp 97Glu + Asp 98Glu + Asn 99AspAsp 98Glu + Asn 99Glu + Gly100Glu + Thr106GlyLeu 96Glu + Asp 97Glu + Ser101Asp + Tyr104IleAsp 97Glu + Gly100Asp + Ser101Glu + Tyr104LeuAsp 97Glu + Asp 98Glu + Gly100Glu + Tyr104MetGly100Glu + Ser101Asp + Gly102Asp + Ile107GlyVal 95Met + Leu 96Glu + Asp 97Glu + Gly102AspAsp 97Glu + Gly100Glu + Tyr104Ala + Thr106GlnAsp 97Glu + Asn 99Glu + Tyr104His + Thr106GlyGly102Ser + Gln103Asp + Ser105Asp + Thr106SerAsp 97Glu + Ser101Asp + Gly102Pro + Tyr104IleAsn 99Asp + Gly100Gln + Ser101Asp + Thr106ProAsn 99Asp + Gly100Ser + Ser101Asp + Ile107CysGly102Ser + Gln103Glu + Ser105Asp + Ile107AspAsp 98Glu + Gly100Glu + Ser101Glu + Gly102GlnVal 95Gly + Asp 98Glu + Asn 99Glu + Ser101AspAsp 97Glu + Gly102Asp + Gln103Ser + Ile107MetLeu 96Ser + Asp 97Glu + Gly100Glu + Gly102GluVal 95Thr + Leu 96Glu + Asp 98Glu + Ser101AspGly102Glu + Tyr104Ala + Ser105Asp + Thr106AspLeu 96Met + Ser101Asp + Gln103Glu + Ile107GlnSer101Asp + Gln103Asp + Tyr104Ser + Ile107ThrAsp 98Glu + Gly100Asn + Ser101Glu + Tyr104ValVal 95Glu + Gly102Glu + Gln103Asn + Tyr104GluVal 95Thr + Ser101Glu + Gln103Asp + Ile107GluSer101Glu + Gln103Asp + Tyr104Met + Ile107GluVal 95Glu + Ser101Asp + Tyr104His + Ile107GluVal 95Gly + Asp 97Glu + Gly102Glu + Thr106GluVal 95Ser + Leu 96Asp + Asn 99Gln + Gln103GluLeu 96Glu + Gly102Asn + Tyr104Gln + Thr106GluVal 95Asp + Asp 98Glu + Gly100Asp + Thr106GlyLeu 96Glu + Gly102Asp + Gln103Ser + Ser105GluGly100Ser + Ser101Glu + Gln103Asp + Ser105AspVal 95Met + Gly100Glu + Gly102Asp + Ile107AspLeu 96Ala + Asp 97Glu + Ser101Asp + Ile107AspLeu 96Gln + Asp 97Glu + Gln103Asp + Ile107Glu表10環(huán)區(qū)3-單突變變異體Leu133AlaLeu133AsnLeu133AspLeu133CysLeu133GlnLeu133GluLeu133GlyLeu133HisLeu133IleLeu133MetLeu133ProLeu133SerLeu133ThrLeu133ValGly134AsnGly134AspGly134GlnGly134GluGly134ProGly134SerGly135AsnGly135AspGly135GlnGly135GluGly135ProGly135SerGly136AsnGly136AspGly136GlnGly136GluGly136ProGly136SerTyr137AlaTyr137AsnTyr137AspTyr137CysTyr137GlnTyr137GluTyr137GlyTyr137HisTyr137IleTyr137LeuTyr137MetTyr137ProTyr137SerTyr137ThrTyr137ValSer138AspSer138GluSer139AspSer139GluSer140AspSer140Glu表11環(huán)區(qū)3-雙突變變異體Gly134Asn + Ser140AspLeu133Ala + Gly135GluLeu133Thr + Ser139AspLeu133Gln + Ser140AspGly136Gln + Ser138GluGly134Ser + Ser138AspGly136Pro + Ser139AspGly135Asn + Ser138AspGly135Glu + Gly136ProLeu133Ile + Ser140AspLeu133Cys + Ser140AspGly134Asn + Gly135ProLeu133Cys + Gly136AspTyr137Asn + Ser139AspGly136Asn + Ser140GluGly134Glu + Gly135ProTyr137Met + Ser140AspGly135Gln + Ser139GluTyr137Thr + Ser138GluLeu133Asn + Ser139GluGly135Ser + Gly136GlnLeu133Gly + Gly136SerLeu133Ala + Tyr137HisLeu133Val + Gly136GluTyr137Ile + Ser139AspLeu133Ile + Tyr137GlnGly136Ser + Ser140AspGly134Asn + Ser138GluGly134Ser + Ser138GluGly135Ser + Ser138AspGly136Ser + Ser140GluGly136Ser + Ser138GluLeu133Glu + Gly134AsnLeu133Glu + Gly135GlnGly135Asn + Tyr137GluTyr137Thr + Ser139GluTyr137Ala + Ser139GluGly134Gln + Tyr137GlyGly135Pro + Tyr137HisLeu133Pro + Ser138GluLeu133Thr + Gly135SerGly136Pro + Ser138GluGly134Ser + Ser140AspLeu133Met + Tyr137AlaTyr137Val + Ser138GluGly134Pro + Ser140GluLeu133Thr + Tyr137SerGly135Glu + Gly136AsnLeu133Ala + Ser140GluGly134Gln + Ser139GluLeu133Pro + Tyr137GlyLeu133Val + Gly136SerGly136Asp + Tyr137ProGly136Asn + Tyr137ValLeu133Ala + Ser138AspGly135Gln + Gly136ProTyr137Met + Ser138GluLeu133Gln + Tyr137AspTyr137Ser+Ser138GluG1y134Asp+Gly135Pro表12環(huán)區(qū)3-三突變變異體Leu133Cys + Gly135Gln + Tyr137GluLeu133Ile + Gly134Ser + Gly136GluLeu133Gly + Gly135Glu + Tyr137ValLeu133Gln + Gly135Gln + Ser139AspLeu133Asp + Gly135Pro + Tyr137AsnLeu133Val + Gly135Gln + Gly136SerGly135Asn + Tyr137Ile + Ser139GluLeu133Met + Gly136Pro + Ser140AspGly134Pro + Gly136Pro + Ser138GluGly135Asn + Tyr137Met + Ser138AspLeu133Ser + Gly136Asn + Tyr137ValGly134Glu + Gly135Ser + Gly136ProGly136Asn + Tyr137Met + Ser140GluLeu133Ile + Gly136Pro + Tyr137GluLeu133Met + Gly136Gln + Tyr137GlnLeu133Val + Gly135Pro + Ser139AspLeu133Asp + Gly135Gln + Tyr137CysGly135Pro + Tyr137Met + Ser138GluGly136Gln + Tyr137Gly + Ser140AspLeu133Ser + Gly134Pro + Ser140AspLeu133Thr + Gly134Asp + Gly135ProLeu133Ile + Gly135Asn + Ser139GluGly135Glu + Gly136Pro + Tyr137HisGly135Asn + Gly136Pro + Ser140GluLeu133Met + Gly134Ser + Gly136GlnGly134Pro + Gly135Asn + Ser140GluGly135Gln + Gly136Gln + Ser138AspGly136Ser + Tyr137Met + Ser138GluGly134Pro + Gly136Ser + Ser139AspGly134Ser + Tyr137Thr + Ser138AspLeu133Thr + Gly134Pro + Ser140AspGly134Gln + Gly135Pro + Ser140GluGly134Pro + Tyr137His + Ser138AspLeu133Met + Gly134Gln + Ser139AspLeu133Ser + Gly134Asn + Ser138GluGly136Gln + Tyr137Ala + Ser139GluLeu133Ile + Gly136Asn + Ser139AspLeu133Ala + Gly135Asp + Gly136ProLeu133Asn + Ser138Asp + Ser139GluGly136Asn + Ser138Asp + Ser139AspGly135Asn + Ser138Asp + Ser139GluLeu133Asp + Gly134Asp + Tyr137HisLeu133Asp + Gly134Glu + Gly135ProLeu133Gly + Gly136Glu + Tyr137AspTyr137Ser + Ser139Glu + Ser140AspGly134Gln + Ser139Asp + Ser140AspGly136Asn + Ser139Asp + Ser140GluTyr137Pro + Ser139Asp + Ser140AspGly135Ser + Tyr137Glu + Ser138GluSer138Asp + Ser139Asp + Ser140AspTyr137Glu + Ser138Asp + Ser139GluTyr137Ala + Ser138Asp + Ser140AspTyr137Thr + Ser138Asp + Ser140GluTyr137Gln + Ser138Asp + Ser140AspLeu133Thr + Ser138Asp + Ser140GluGly135Ser + Ser138Asp + Ser140GluGly136Gln + Ser138Asp + Ser140AspGly135Ser + Ser138Glu + Ser140AspGly136Gln + Ser138Glu + Ser140AspLeu133Val + Ser138Glu + Ser140Glu表13環(huán)區(qū)3-四突變變異體Leu133Ala + Gly134Asn + Tyr137Ser + Ser138AspGly134Glu + Gly135Asn + Gly136Asn + Tyr137CysLeu133Cys + Gly135Asn + Gly136Gln + Ser138AspLeu133Pro + Gly134Asn + Gly135Ser + Ser139AspLeu133Val + Gly134Gln + Gly136Gln + Ser140GluLeu133Asp + Gly134Gln + Gly136Gln + Tyr137ThrLeu133His + Gly135Gln + Gly136Pro + Tyr137SerLeu133Gly + Gly134Ser + Tyr137Ala + Ser138AspLeu133Gln + Gly134Gln + Gly135Asn + Ser140GluLeu133Ser + Gly135Ser + Tyr137Pro + Ser139GluLeu133Asn + Gly134Asn + Gly136Glu + Tyr137CysLeu133Met + Gly134Asn + Gly135Gln + Ser138GluLeu133Asn + Gly134Ser + Tyr137Val + Ser138GluGly135Asn + Gly136Pro + Tyr137Cys + Ser139AspLeu133Ile + Gly135Asp + Gly136Asn + Tyr137IleGly134Asp + Gly135Glu + Gly136Gln + Tyr137ThrLeu133Gln + Gly134Glu + Gly135Asp + Tyr137ThrGly134Ser + Tyr137Val + Ser138Glu + Ser139GluGly134Ser + Tyr137Val + Ser138Asp + Ser139GluLeu133His + Gly135Gln + Ser138Asp + Ser139AspLeu133Gly + Gly135Pro + Ser138Glu + Ser139AspLeu133Val + Gly135Pro + Ser138Asp + Ser139GluLeu133Ser + Gly134Gln + Gly135Asp + Gly136GluLeu133Ile + Gly134Gln + Ser139Asp + Ser140AspLeu133Cys + Tyr137Asn + Ser139Asp + Ser140GluLeu133Ser + Tyr137His + Ser139Glu + Ser140GluGly134Pro + Gly136Gln + Ser139Asp + Ser140GluGly134Gln + Gly135Ser + Ser139Asp + Ser140AspLeu133Cys + Gly134Pro + Ser139Glu + Ser140AspLeu133Ala + Gly136Asn + Tyr137Glu + Ser138AspGly136Pro + Ser138Asp + Ser139Glu + Ser140AspTyr137Leu + Ser138Asp + Ser139Glu + 5er140GluGly135Asn + Ser138Asp + Ser139Asp + Ser140GluLeu133Gly + Ser138Asp + Ser139Glu + Ser140GluLeu133Ser + Tyr137Asp + Ser138Asp + Ser139GluGly136Gln + Tyr137Asp + Ser138Asp + Ser139AspGly136Asn + Tyr137Asp + Ser138Asp + Ser139GluGly134Glu + Gly135Asp + Gly136Asp + Tyr137IleLeu133Gln + Tyr137Val + Ser138Glu + Ser140AspGly134Pro + Tyr137Gly + Ser138Asp + Ser140AspLeu133His + Tyr137Leu + Ser138Glu + Ser140GluGly134Gln + Gly135Ser + Ser138Glu + Ser140GluLeu133Cys + Gly136Ser + Ser138Glu + Ser140GluGly135Gln + Gly136Gln + Ser138Asp + Ser140AspGly134Pro + Tyr137His + Ser138Asp + Ser140GluLeu133Gln + Gly134Pro + Gly136Glu + Ser138AspLeu133His + Gly134Pro + Gly136Asp + Ser138GluLeu133Ile + Gly135Glu + Tyr137Glu + Ser138GluGly134Asn + Gly135Asp + Tyr137Asp + Ser138GluGly135Gln + Tyr137Glu + Ser138Asp + Ser140GluGly136Asn + Tyr137Glu + Ser138Asp + Ser140GluLeu133Asn + Tyr137Asp + Ser138Glu + Ser140AspLeu133Cys + Gly136Pro + Tyr137Glu + Ser139AspGly134Glu + Gly135Pro + Gly136Asp + Tyr137GluGly136Pro + Tyr137Glu + Ser139Asp + Ser140AspGly135Ser + Tyr137Glu + Ser139Glu + Ser140GluLeu133Pro + Gly134Glu + Gly136Asp + Tyr137CysGly134Gln + Gly136Asp + Ser138Glu + Ser139AspGly135Ser + Gly136Glu + Ser138Glu + Ser139AspGly134Asp + Gly135Glu + Gly136Ser + Ser138Asp表14環(huán)區(qū)4-單突變變異體Gly160AsnGly160AspGly160GlnGly160GluGly160ProGly160SerAsn161AspAsn161GlnAsn161GluAsn161SerAsn162AspAsn162GlnAsn162GluAsn162SerAsn163AspAsn163GlnAsn163GluAsn163SerAla164AsnAla164AspAla164GlnAla164GluAla164GlyAla164HisAla164ProAla164SerAla164ThrAsp165GluAla166AsnAla166AspAla166GlnAla166GluAla166GlyAla166HisAla166ProAla166SerAla166ThrArg167AspArg167GluAsn168AspAsn168GlnAsn168GluAsn168SerTyr169AlaTyr169AsnTyr169AspTyr169CysTyr169GlnTyr169GluTyr169GlyTyr169HisTyr169IleTyr169LeuTyr169MetTyr169ProTyr169SerTyr169ThrTyr169ValSer170AspSer170Glu表15環(huán)區(qū)4-雙突變變異體Gly160Gln + Asp165GluAsn161Ser + Asn162SerAla164Thr + Asn168AspGly160Asn + Ser170GluAsn161Gln + Asn163GluAsn162Ser + Ser170AspGly160Gln + Asn168GluAla166Gly + Ser170AspAsn161Glu + Asn162SerAsn162Gln + Ser170AspAla166Thr + Ser170GluAsn162Ser + Ala164ProGly160Asn + Asn161GlnGly160Glu + Ala166SerAla166Gly + Ser170GluAla164His + Ala166GlyAla164Asn + Asp165GluAsn162Asp + Asn168SerAla164Pro + Asn168AspAsn162Asp + Ala164AsnTyr169Thr + Ser170AspAla164Glu + Tyr169HisAsp165Glu + Tyr169HisAsn162Glu + Asn168SerGly160Asp + Tyr169MetAsn162Glu + Ala166AsnAsn161Glu + Ala166ThrAsp165Glu + Ala166AsnAsn161Gln + Tyr169ProAsn162Asp + Ala164ThrAsn162Ser + Arg167GluAla166Pro + Arg167AspAla164Thr + Ser170AspAsn163Asp + Ala164AsnAsp165Glu + Asn168GlnAsn163Asp + Tyr169ThrAsn168Asp + Tyr169GlyArg167Asp + Asn168SerAsn161Ser + Arg167GluAsn161Ser + Ser170GluGly160Asp + Ala166GlyAla164Asn + Ala166GlnGly160Asn + Asn161GluAsn162Glu + Ala166SerAla166His + Asn168AspGly160Pro + Ala164GlyGly160Asn + Ala164GlyAla164Gln + Ser170AspAsn161Glu + Ala164ThrGly160Glu + Ala166GlyAla166Pro + Ser170AspAla166Pro + Tyr169AlaGly160Ser + Arg167GluGly160Asp + Ala164SerAsn161Gln + Ala164ProAsn163Glu + Ala166GlnAla164Ser + Asn168GlnGly160Glu + Ala164SerGly160Asp + Ala166GlnAla164Thr + Asp165Glu
表16環(huán)區(qū)4-三突變變異體Gly160Gln + Asn161Ser + Asp165GluAla166Gly + Arg167Glu + Tyr169CysAsn162Ser + Ala166Asn + Ser170AspGly160Gln + Asn162Asp + Asn168SerAla164Asn + Asn168Gln + Ser170GluAla164Asp + Ala166Thr + Asn168SerAsn163Gln + Ala166Gly + Arg167GluAsn162Ser + Ala166His + Asn168GlnAsp165Glu + Ala166Thr + Asn168GlnAsn163Ser + Ala164Pro + Asn168AspGly160Glu + Ala164His + Tyr169GlnAsn161Gln + Asn163Ser + Ser170GluAla166Thr + Tyr169Leu + Ser170AspGly160Ser + Ala164Asn + Asn168GluAsn161Gln + Asn162Glu + Asn163SerAsn163Ser + Ala166Ser + Arg167GluAla164Thr + Ala166Gly + Arg167GluAla164Ser + Ala166Gly + Ser170GluAsn162Gln + Ala164Gln + Ala166ProAsn162Glu + Asn163Gln + Ala164GlyAsn161Asp + Ala164Asn + Tyr169ValGly160Asn + Ala164Glu + Ala166ProAsn162Gln + Ala164Pro + Arg167AspAsn163Ser + Ala164Ser + Tyr169GlyAla166Asn + Arg167Glu + Asn168SerAsn163Gln + Asp165Glu + Ala166SerAsn162Ser + Ala164His + Asn168AspAla164Ser + Arg167Asp + Asn168AspAsn162Ser + Arg167Asp + Asn168AspAsn161Asp + Asn162Glu + Asn163GlnAsn161Asp + Asn162Glu + Asn168GlnGly160Glu + Asn161Glu + Ala166AsnGly160Glu + Asn161Glu + Ala164SerGly160Pro + Asn162Asp + Asn163AspAsn162Asp + Asn163Glu + Ala166AsnAsn161Glu + Asn162Asp + Asn163GluAsn162Glu + Asn163Ser + Ala164AspAsp165Glu + Arg167Asp + Tyr169ProAsp165Glu + Arg167Asp + Asn168AspAla164Gln + Arg167Asp + Tyr169AspAsn161Asp + Asn162Asp + Ala164AspAsn161Asp + Asn163Asp + Tyr169ThrAsn161Asp + Asn163Asp + Ala164AsnGly160Glu + Asn162Glu + Asn163GlnAla164Asp + Asp165Glu + Arg167GluAla164Pro + Asp165Glu + Asn168AspGly160Asp + Asn162Glu + Ala164GluGly160Pro + Asn168Glu + Ser170GluAla164Glu + Arg167Glu + Asn168GluAsn161Glu + Asn163Glu + Asp165GluGly160Asp + Asn162Glu + Asp165GluAsp165Glu + Ala166Gly + Tyr169GluGly160Glu + Asn161Asp + Asp165GluArg167Asp + Asn168Gln + Ser170AspAsn162Gln + Arg167Asp + Ser170GluAsn163Gln + Arg167Glu + Ser170GluAsn162Asp + Asp165Glu + Asn168GluAsn162Asp + Asp165Glu + Arg167GluGly160Asp + Asn168Gln + Ser170GluGly160Asp + Asp165Glu + Tyr169Gly表17環(huán)區(qū)4-四突變變異體Gly160Gln + Asn161Ser + Asn162Ser + Asp165GluGly160Glu + Asn161Ser + Asn162Gln + Asn168SerAsn161Ser + Asn163Asp + Ala164Asn + Tyr169ThrGly160Ser + Asp165Glu + Ala166Asn + Tyr169LeuGly160Gln + Ala166Ser + Arg167Asp + Tyr169HisGly160Ser + Asn163Ser + Ala166Gly + Ser170AspAsn161Gln + Asn163Gln + Ala164Glu + Ala166GlyAsn161Glu + Asn163Gln + Ala166His + Tyr169AsnAsn163Gln + Ala166His + Arg167Asp + Asn168GlnGly160Gln + Asn162Ser + Asn163Asp + Tyr169GlnAsn162Glu + Ala166Gln + Asn168Ser + Tyr169GlyAsn163Glu + Ala166His + Asn168Gln + Tyr169GlnGly160Asn + Asn161Ser + Ala164Thr + Ala166SerAsn161Gln + Asn162Ser + Asn168Glu + Tyr169SerAsn161Gln + Ala166His + Arg167Glu + Tyr169AlaAsn161Gln + Ala164Gly + Ala166Gln + Tyr169HisAla164Gln + Asp165Glu + Asn168Ser + Tyr169MetAla164Gln + Ala166His + Arg167Glu + Asn168GluGly160Asn + Asn162Ser + Ala164Glu + Asp165GluAsnl61Asp + Asn162Asp + Ala164Asn + Ala166SerAsn161Asp + Asn162Asp + Ala164Asn + Ala166ThrAla164Gln + Ala166Asn + Asn168Asp + Tyr169AspGly160Pro + Asn163Asp + Ala164Asp + Ala166ThrAsn161Ser + Asn163Asp + Ala164Asp + Asn168GlnAsn162Asp + Asn163Asp + Ala166Pro + Tyr169MetAsn161Ser + Asn163Gln + Tyr169Asp + Ser170AspAsn161Ser + Ala166His + Tyr169Asp + Ser170GluAsn161Glu + Asn162Asp + Asn163Glu + Tyr169CysAsn161Glu + Asn162Glu + Asn163Asp + Tyr169AlaAsp165Glu + Ala166Gly + Arg167Asp + Asn168GlnAsp165Glu + Arg167Asp + Asn168Gln + Tyr169ValAsn161Asp + Asn163Glu + Ala164Asn + Ala166GlyAsn163Gln + Ala164Asp + Asp165Glu + Arg167AspAsp165Glu + Ala166Asn + Asn168Asp + Tyr169GluAsn163Gln + Asp165Glu + Arg167Glu + Tyr169AspAsp165Glu + Arg167Asp + Asn168Ser + Tyr169AspAsn163Ser + Asp165Glu + Arg167Asp + Tyr169GluAsn163Gln + Ala166Thr + Asn168Glu + Ser170GluAsn161Gln + Ala166Gly + Asn168Asp + Ser170GluAsn163Glu + Asp165Glu + Ala166Gly + Tyr169LeuGly160Pro + Asn162Gln + Asn163Asp + Asp165GluGly160Glu + Asn163Gln + Asn168Asp + Tyr169GluGly160Asp + Ala164Glu + Asp165Glu + Tyr169SerAsn161Glu + Ala164Asp + Asp165Glu + Tyr169GlnGly160Asp + Asn162Asp + Ala166Gly + Tyr169AspAsn161Glu + Asn162Glu + Asp165Glu + Ala166AsnAsn161Asp + Asn162Asp + Asp165Glu + Ala166GlnAsn161Asp + Asn163Asp + Asp165Glu + Asn168GlnGly160Asp + Asn161Ser + Asn163Glu + Tyr169GlyGly160Asp + Asn162Asp + Asp165Glu + Asn168GlnAsn161Ser + Arg167Glu + Tyr169Thr + Ser170AspAsn161Gln + Arg167Glu + Asn168Gln + Ser170AspGly160Asp + Asn163Asp + Asp165Glu + Tyr169AlaGly160Ser + Asn162Glu + Ala164Pro + Asp165GluGly160Glu + Arg167Glu + Asn168Glu + Tyr169GlnAsn161Ser + Ala164Asp + Arg167Glu + Tyr169AsnAsn162Ser + Asn163Asp + Ala164Asp + Asn168AspAsn163Glu + Asp165Glu + Ala166Asn + Arg167GluAsn163Asp + Asp165Glu + Arg167Asp + Asn168SerGly160Glu + Ala164Gly + Asn168Asp + Ser170Asp表18環(huán)區(qū)5-單突變變異體Ser190AspSer190GluSer191AspSer191GluPhe192AlaPhe192AsnPhe192AspPhe192CysPhe192GlnPhe192GluPhe192GlyPhe192HisPhe192IlePhe192LeuPhe192MetPhe192ProPhe192SerPhe192ThrPhe192TyrPhe192ValSer193AspSer193GluAsn194AspAsn194GlnAsn194GluAsn194Ser表19環(huán)區(qū)5-雙突變變異體Ser190Asp + Phe192LeuSer190Glu + Phe192GlnPhe192Glu + Asn194GlnPhe192Tyr + Asn194GluSer190Asp + Phe192IleSer191Glu + Phe192SerSer191Asp + Phe192GlySer191Asp + Asn194SerSer191Glu + Pne192ThrSer191Glu + Asn194GlnSer191Glu + Phe192HisSer191Asp + Phe192AsnSer190Asp + Phe192GlySer191Asp + Asn194GlnSer191Asp + Phe192AlaSer190Glu + Phe192CysSer190Glu + Phe192IleSer191Asp + Phe192SerSer190Asp + Asn194GlnSer191Glu + Phe192CysPhe192Ser + Asn194GluSer191Glu + Phe192GlyPhe192Gly + Asn194GlnPhe192Cys + Asn194GlnPhe192Asn + Asn194AspSer190Glu + Phe192TyrSer191Glu + Phe192AlaSer190Glu + Asn194SerPhe192Ile + Asn194SerSer191Glu + Phe192MetSer190Asp + Phe192MetSer190Asp + Asn194SerPhe192Gly + Asn194AspPhe192His + Asn194SerSer190Glu + Asn194GlnPhe192Ala + Asn194GluPhe192Thr + Asn194GluSer190Glu + Phe192SerSer191Glu + Phe192IlePhe192Val + Asn194GluPhe192Thr + Asn194AspSer191Glu + Phe192ProSer191Asp + Phe192LeuPhe192Ile + Asn194GluSer190Asp + Phe192ThrSer191Asp + Phe192IlePhe192Thr + Asn194GlnPhe192Ala + Asn194GlnSer191Asp + Phe192TyrSer190Glu + Phe192ThrPhe192Leu + Asn194GluPhe192His + Asn194GlnSer190Asp + Phe192TyrPhe192Val + Asn194SerPhe192Asp + Asn194GlnPhe192Gly + Asn194SerSer191Asp + Phe192GlnPhe192Asp + Asn194SerPhe192Thr + Asn194SerSer190Glu + Phe192Ala表20環(huán)區(qū)5-三突變變異體Ser191Asp + Phe192Met + Asn194GlnSer191Glu + Phe192Ala + Asn194GlnSer191Asp + Phe192Leu + Asn194SerSer191Glu + Phe192Asn + Asn194GlnSer190Asp + Phe192Gln + Asn194GlnSer191Asp + Phe192Ile + Asn194GlnSer190Asp + Phe192Asn + Asn194SerSer191Glu + Phe192Ile + Asn194SerSer190Asp + Phe192Leu + Asn194GlnSer190Asp + Phe192Val + Asn194SerSer190Glu + Phe192Gln + Asn194SerSer191Asp + Phe192Val + Asn194SerSer191Glu + Phe192Ala + Asn194SerSer191Glu + Phe192Cys + Asn194GlnSer190Glu + Phe192Val + Asn194SerSer190Glu + Phe192Ile + Asn194GlnSer190Glu + Phe192Ser + Asn194SerSer190Glu + Phe192Tyr + Asn194GlnSer191Asp + Phe192Ile + Asn194SerSer190Asp + Ser191Glu + Asn194SerSer190Asp + Ser191Asp + Phe192MetSer190Glu + Ser191Glu + Phe192ValSer190Asp + Ser191Glu + Phe192SerSer190Asp + Ser191Asp + Phe192ThrSer190Glu + Ser191Asp + Asn194SerSer190Glu + Ser191Asp + Phe192AsnSer190Asp + Ser191Asp + Phe192AsnSer190Asp + Ser191Asp + Phe192ValSer190Glu + Ser191Glu + Phe192CysSer190Asp + Ser191Glu + Phe192AsnSer190Glu + Ser191Glu + Phe192LeuSer190Glu + Ser191Asp + Phe192GlySer190Glu + Ser191Glu + Phe192SerSer190Glu + Ser191Glu + Asn194SerSer190Asp + Ser191Asp + Asn194GlnSer190Asp + Ser191Asp + Phe192TyrSer190Asp + Ser191Glu + Phe192MetSer190Asp + Ser191Asp + Phe192GlySer190Glu + Ser191Asp + Asn194GlnSer190Asp + Ser191Glu + Phe192LeuSer190Asp + Ser191Glu + Phe192GlySer190Glu + Ser191Asp + Phe192TyrSer190Asp + Ser191Glu + Phe192ThrSer190Glu + Serl91Asp + Phe192HisSer190Asp + Ser191Asp + Phe192GlnSer190Glu + Ser191Asp + Phe192IleSer190Asp + Ser191Glu + Asn194GlnSer190Asp + Ser191Asp + Phe192ProSer190Glu + Ser191Asp + Phe192CysSer190Asp + Ser191Asp + Asn194SerSer190Asp + Ser191Glu + Phe192G1nSer190Glu + Ser191Asp + Phe192ProSer191Asp + Phe192Asp + Asn194GlnSer191Asp + Phe192Glu + Asn194SerSer191Asp + Phe192Glu + Asn194GlnSer190Asp + Ser191Glu + Phe192AspSer190Asp + Ser191Asp + Phe192GluSer190Glu + Ser191Glu + Phe192AspSer190Glu + Ser191Asp + Phe192AspSer190Glu + Ser191Asp + Phe192Glu表21環(huán)區(qū)5-四突變變異體Ser190Glu + Ser191Asp + Phe192Ile + Asn194SerSer190Asp + Ser191Asp + Phe192Gly + Asn194GlnSer190Glu + Ser191Asp + Phe192His + Asn194SerSerl90G1u + Ser191Asp + Phe192Gln + Asn194GlnSer190Asp + Ser191Asp + Phe192Met + Asn194SerSer190Asp + Ser191Glu + Phe192Leu + Asn194GlnSer190Glu + Ser191Glu + Phe192Ala + Asn194SerSer190Asp + Ser191Asp + Phe192Pro + Asn194GlnSer190Glu + Ser191Glu + Phe192Leu + Asn194SerSer190Glu + Ser191Asp + Phe192His + Asn194GlnSer190Glu + Ser191Asp + Phe192Cys + Asn194GlnSer190Glu + Ser191Asp + Phe192Thr + Asn194SerSer190Glu + Ser191Asp + Phe192Ser + Asn194GlnSer190Glu + Ser191Asp + Phe192Thr + Asn194GlnSer190Asp + Ser191Glu + Phe192Ile + Asn194SerSer190Glu + Ser191Asp + Phe192Gln + Asn194SerSer190Asp + Ser191Asp + Phe192Cys + Asn194GlnSer190Asp + Ser191Asp + Phe192Pro + Asn194SerSer190Glu + Ser191Glu + Phe192Ala + Asn194GlnSer190Asp + Ser191Glu + Phe192Thr + Asn194SerSer190Glu + Ser191Asp + Phe192Leu + Asn194GlnSer190Glu + Ser191Asp + Phe192Gly + Asn194GlnSer190Asp + Ser191Asp + Phe192Cys + Asn194SerSer190Glu + Ser191Glu + Pne192His + Asn194GlnSer190Glu + Ser191Asp + Phe192Asn + Asn194SerSer190Asp + Ser191Asp + Phe192His + Asn194GlnSer190Glu + Ser191Glu + Phe192Ile + Asn194GlnSer190Asp + Ser191Asp + Phe192Tyr + Asn194SerSer190Glu + Ser191Asp + Phe192Tyr + Asn194GlnSer190Glu + Ser191Asp + Phe192Ala + Asn194SerSer190Asp + Ser191Glu + Phe192Ala + Asn194SerSer190Asp + Ser191Glu + Phe192Asn + Asn194SerSer190Glu + Ser191Asp + Phe192Val + Asn194SerSer190Asp + Ser191Asp + Phe192Gln + Asn194SerSer190Asp + Ser191Asp + Phe192Gln + Asn194GlnSer190Asp + Ser191Asp + Phe192Leu + Asn194GlnSer190Asp + Ser191Asp + Phe192Ser + Asn194GlnSer190Glu + Ser191Asp + Phe192Glu + Asn194SerSer190Asp + Ser191Asp + Phe192Asp + Asn194SerSer190Glu + Ser191Asp + Phe192Glu + Asn194GlnSer190Glu + Ser191Glu + Phe192Glu + Asn194SerSer190Asp + Ser191Glu + Phe192Glu + Asn194GlnSer190Asp + Ser191Asp + Phe192Glu + Asn194SerSer190Glu + Ser191Glu + Phe192Asp + Asn194SerSer190Glu + Ser191Asp + Phe192Asp + Asn194SerSer190Glu + Ser191Glu + Phe192Asp + Asn194GlnSer190Asp + Ser191Asp + Phe192Asp + Asn194GlnSer190Asp + Ser191Glu + Phe192Glu + Asn194SerSer190Glu + Ser191Glu + Phe192Tyr + Asn194GluSer190Glu + Ser191Glu + Phe192Met + Asn194GluSer190Asp + Ser191Glu + Phe192Gln + Asn194AspSer190Asp + Ser191Asp + Phe192Ala + Asn194AspSer190Glu + Ser191Glu + Phe192Leu + Asn194AspSer190Glu + Ser191Glu + Phe192Ser + Asn194AspSer190Glu + Ser191Glu + Phe192Met + Asn194AspSer190Asp + Ser191Glu + Phe192Tyr + Asn194GluSer190Asp + Ser191Asp + Phe192His + Asn194AspSer190Glu + Ser191Asp + Phe192Gln + Asn194AspSer190Asp + Ser191Asp + Phe192Asn + Asn194GluSer190Asp + Ser191Glu + Phe192Ile + Asn194Asp表22環(huán)區(qū)5-單突變變異體Gly203AsnGly203AspGly203GlnGly203GluGly203ProGly203SerPro204AsnPro204AspPro204GlnPro204GluPro204GlyPro204SerGly205AsnGly205AspGly205GlnGly205GluGly205ProGly205SerThr206AsnThr206AspThr206GlnThr206GluThr206GlyThr206ProThr206SerSer207AspSer207GluIle208AlaIle208AsnIle208AspIle208CysIle208GlnIle208GluIle208GlyIle208HisIle208LeuIle208MetIle208ProIle208SerIle208ThrIle206ValLeu209AlaLeu209AsnLeu209AspLeu209CysLeu209GlnLeu209GluLeu209GlyLeu209HisLeu209IleLeu209MetLeu209ProLeu209SerLeu209ThrLeu209ValSer210AspSer210GluThr211AsnThr211AspThr211GlnThr211GluThr211GlyThr211ProThr211SerTrp212AlaTrp212AsnTrp212AspTrp212CysTrp212GlnTrp212GluTrp212GlyTrp212HisTrp212IleTrp212LeuTrp212MetTrp212PheTrp212ProTrp212SerTrp212ThrTrp212TyrTrp212ValIle213AlaIle213AsnIle213AspIle213CysIle213GlnIle213GluIle213GlyIle213HisIle213LeuIle213MetIle213ProIle213SerIle213ThrIle213ValGly214AsnGly214AspGly214GlnGly214GluGly214ProGly214SerGly215AsnGly215AspGly215GlnGly215GluGly215ProGly215SerSer216AspSer216GluThr217AsnThr217AspThr217GlnThr217GluThr217GlyThr217ProThr217SerArg218AspArg218GluSer219AspSer219GluIle220AlaIle220AsnIle220AspIle220CysIle220GlnIle220GluIle220GlyIle220HisIle220LeuIle220MetIle220ProIle220SerIle220ThrIle220ValSer221AspSer221GluGly222AsnGly222AspGly222GlnGly222GluGly222ProGly222SerThr223AsnThr223AspThr223GlnThr223GluThr223GlyThr223ProThr223Ser表23環(huán)區(qū)6-雙突變變異體Gly203Gln + Ser219GluThr206Gly + Leu209SerTrp212Phe + Ser221GluArg218Glu + Gly222ProGly214Asp + Ile220SerGly215Asp + Thr217GlyGly203Asn + Ser219AspTrp212Ile + Ser221GluTrp212Gln + Ser219AspTrp212Cys + Ser219AspThr217Glu + Thr223ProGly214Pro + Ile220ValIle208Pro + Thr223SerLeu209Gln + Ile220GluThr206Asn + Ile220HisIle208Ala + Thr217SerPro204Gln + Ser221AspTrp212Thr + Thr223AsnGly203Asn + Ser219GluGly203Gln + Gly214ProGly214Asp + Gly215GlnGly203Ser + Arg218GluThr206Gln + Trp212AsnPro204Ser + Ile213GluIle208Leu + Gly222GluGly215Gln + Thr217GlnIle208Thr + Ser219AspGly205Ser + Gly214AspIle208Ser + Arg218GluIle208Ser + Ile220ValThr211Gly + Arg218AspIle213Asp + Ile220SerPro204Gln + Ser219GluThr211Gly + Ile220MetIle208His + Leu209AlaGly205Asn + Ser207GluThr206Ser + Gly215AsnGly205Asn + Gly222SerGly203Pro + Ile213CysThr206Gln + Gly215SerIle208Ser + Gly214GluThr217Gln + Ser219GluPro204Ser + Ser216AspGly214Asn + Ser216AspIle208Leu + Thr211AsnPro204Gly + Ser207GluSer219Asp + Gly222AsnTrp212Tyr + Ile213ThrThr206Ser + Trp212ValIle220Asp + Thr223ProIle208Pro + Trp212ProIle208Leu + Thr217AspPro204Ser + Gly214SerGly203Ser + Gly222ProGly205Ser + Arg218GluGly203Pro + Ser207AspIle208Val + Ser219GluSer216Asp + Gly222GlnSer207Asp + Trp212AlaIle220Cys + Gly222GluThr206Asn + Ile213CysThr206Asp + Gly215GlnSer207Asp + Thr211SerIle208Leu + Ser216GluLeu209Val + Gly214AspThr206Glu + Ile213AsnPro204Ser + Thr211SerIle213Asp + Gly215ProGly214Asp + Thr223SerGly205Asn + Leu209CysGly205Gln + Ile208AlaIle208Leu + Ser219AspLeu209Glu + Thr211ProIle208Cys + Thr217GlnPro204Asn + Leu209AsnGly214Pro + Ile220ProGly203Gln + Thr211GlyPro204Gly + Thr223AspGly215Ser + Thr217ProThr206Gln + Thr211AsnGly205Pro + Gly215ProGly203Pro + Ser207GluSer207Glu + Ile208HisPro204Ser + Thr223GlyGly205Asn + Gly215AspLeu209Gln + Ser219GluGly215Pro + Thr223SerIle208Gln + Ser221AspGly203Gln + Arg218AspGly203Asn + Ile220AspGly205Pro + Arg218AspThr211Asn + Arg218GluGly205Asn + Thr223GluThr206Glu + Leu209HisThr206Pro + Gly215AsPTrp212Gln + Ser221AspSer216Glu + Ile220MetGly215Asn + Gly222AspIle213Cys + Ser219AspPro204Ser + Thr211AsnPro204Ser + Thr223ProPro204Gln + Ile213GluGly214Ser + Ser221AspIle208Leu + Thr223ProSer207Asp + Ile208GlyIle208Leu + Arg218GluLeu209Ser + Ile220HisIle213Leu + Arg218AspPro204Ser + Ser219AspGly203Ser + Gly222GluGly205Pro + Thr223AsnTrp212Thr + Gly214SerThr206Ser + Ser216AspGly203Pro + Thr206GlyThr211Pro + Gly222SerIle213Thr + Thr223GluThr206Pro + Ile213GluSer207Asp + Gly222AsnIle213Val + Gly214AsnThr211Asn + Thr223AsnIle213Cys + Thr223ProPro204Ser + Gly222GlnGly205Gln + Gly215GluLeu209Pro + Gly222GlnGly205Ser + Gly222AspPro204Ser + Ile208LeuPro204Gln + Thr211GlyLeu209Ser + Ser219AspThr217Glu + Thr223AsnGly203Gln + Thr217SerGly205Pro + Ile213GlnLeu209Glu + Thr217SerSer216Asp + Thr217SerTrp212Gln + Ile213AlaArg218Glu + Ile220GlnLeu209Ile + Thr223AsnIle208Met + Gly215GlnArg218Asp + Ile220AlaGly203Asn + Pro204AsnIle208His + Ser221AspIle213Asn + Ser219AspTrp212Met + Ser219GluThr206Pro + Ser216GluPro204Gln + Thr206AspThr206Gly + Gly215ProPro204Asn + Gly222GlnSer219Glu + Gly222SerIle220Asn + Gly222AsnGly205Asn + Ile213AsnGly203Ser + Ile213LeuSer207Asp + Thr211AsnLeu209Pro + Ile213GluIle208Pro + Thr223AspThr211Ser + Gly222AspThr206Ser + Thr217AspGly203Gln + Gly214GluGly205Ser + Gly215SerGly203Gln + Leu209AlaGly203Gln + Gly222AsnSer207Asp + Thr211GlyGly205Gln + Ser219GluTrp212Cys + Ser216AspGly203Gln + Thr217GluArg218Glu + Ile220CysThr211Gly + Ser221GluPro204Asn + Ser216AspArg218Asp + Gly222ProGly205Pro + Ser221GluArg218Glu + Thr223SerLeu209Asn + Ser216GluGly205Pro + Ile220MetThr206Asn + Thr217AspGly203Ser + Ile208ThrThr217Ser + Arg218GluPro204Asn + Thr211AsnGly205Asn + Thr217GluSer207Asp + Thr223GlyTrp212Tyr + Ile213ProGly214Gln + Ser216AspThr206Asp + Ile208Thr表24環(huán)區(qū)6-三突變變異體Gly203Gln + Thr206Gly + Ser219GluIle213Ala + Gly215Asp + Thr217GlyGly203Asn + Ser219Asp + Thr223SerGly215Pro + Ser216Glu + Thr223SerGly203Gln + Trp212Pro + Gly222AspPro204Ser + Gly205Ser + Ser207GluGly205Pro + Ile208Met + Ser219AspThr211Gly + Gly215Gln + Thr217AsnThr211Ser + Thr217Gln + Thr223AspGly205Gln + Thr211Asn + Gly222AsnThr211Pro + Gly214Asn + Thr217AsnGly205Pro + Leu209Asn + Thr223GlnGly203Gln + Trp212Leu + Ile220AlaTrp212Thr + Gly215Glu + Ile220ThrGly205Gln + Ser207Asp + Trp212AlaGly205Asn + Trp212Phe + Ser216GluSer207Glu + Thr211Gly + Gly222AsnPro204Gln + Gly205Ser + Ser221GluThr206Pro + Leu209Gly + Thr223AsnIle208Met + Thr217Gly + Thr223GlnThr211Ser + Gly215Pro + Gly222GluIle208Pro + Trp212Pro + Ser216GluGly203Ser + Thr211Gln + Gly222ProGly203Pro + Ile208Val + Ser219GluThr206Pro + Gly215Ser + Ser216GluThr206Pro + Ile220Asn + Thr223GlyPro204Gln + Gly205Pro + Arg218GluTrp212His + Ile213Met + Gly215AspIle208Ser + Gly222Gln + Thr223GlnGly205Gln + Trp212Leu + Arg218AspGly214Gln + Ser216Glu + Gly222SerGly203Asn + Ser216Glu + Gly222GlnLeu209Asn + Gly222Glu + Thr223SerThr206Asp + Thr211Asn + Gly214SerPro204Asn + Thr211Gly + Trp212ThrGly203Ser + Ile208Ala + Ser216GluThr211Ser + Gly214Pro + Gly222GluGly215Gln + Arg218Asp + Ile220ValGly215Ser + Ser216Asp + Thr223SerThr206Asn + Thr211Asn + Arg218GluPro204Asn + Thr217Glu + Thr223SerThr206Ser + Ile213Met + Gly215SerPro204Gly + Gly205Ser + Trp212GlnThr211Pro + Trp212Ala + Thr223AspGly215Glu + Ile220Ala + Thr223GlnThr206Gln + Ile208Ser + Leu209AspThr211Ser + Trp212Val + Ser219GluGly205Pro + Thr211Pro + Ser216AspGly205Pro + Trp212His + Ser219GluGly205Pro + Trp212Asn + Ser219AspLeu209Ser + Thr211Gln + Arg218AspPro204Gly + Thr211Asn + Gly215GluGly203Pro + Pro204Ser + Ser216AspGly203Gln + Ile213Val + Arg218GluIle208Gly + Thr217Asn + Gly222SerPro204Ser + Gly205Gln + Trp212IlePro204Gln + Gly214Glu + Gly215GlnTrp212Cys + Gly215Gln + Ser221GluGly203Asn + Leu209Val + Thr217GluGly205Asn + Ser207Asp + Thr217GlyPro204Gln + Thr206Asp + Gly215SerIle208Gln + Thr211Pro + Ser219AspGly205Ser + Ile213His + Ser219GluGly203Ser + Ile208Ser + Trp212MetThr211Pro + Trp212Tyr + Ser219AspGly205Gln + Ile208His + Gly222AsnGly203Ser + Thr211Ser + Ile220GluPro204Gly + Leu209Ile + Arg218GluTrp212Tyr + Ile213Ser + Thr217ProIle208Asn + Ile213Pro + Gly214SerGly203Gln + Ile213His + Ser219AspGly205Asn + Thr211Gln + Ser221GluGly205Asn + Ser207Glu + Gly214GlnIle208Val + Gly214Glu + Gly222GlnGly203Ser + Thr206Gly + Ile213LeuPro204Gly + Ile208Pro + Gly215AspThr206Glu + Thr217Gly + Ile220ProIle213Ser + Thr217Asp + Gly222GlnIle208Val + Gly215Asn + Thr223SerGly205Pro + Ser207Glu + Thr217SerGly203Pro + Thr206Glu + Ile208HisGly205Ser + Trp212Cys + Ser216AspPro204Ser + Thr206Asp + Leu209IleThr206Glu + Ile208Thr + Ile220GlyIle213Gln + Ile220Glu + Gly222GlnIle208Val + Gly214Gln + Gly215ProLeu209Pro + Ser216Glu + Thr217AsnPro204Gln + Trp212Met + Gly222GluIle208Met + Thr211Asn + Thr223GlnPro204Gln + Leu209Ile + Arg218AspIle208Leu + Ser216Glu + Ile220ProPro204Gly + Gly205Gln + Thr206GluIle208Ser + Leu209His + Gly214ProGly203Pro + Ile208Gln + Trp212SerGly205Asn + Gly215Glu + Gly222ProGly203Ser + Thr217Gln + Ile220LeuLeu209Ser + Gly214Ser + Gly222GlnIle208Gly + Leu209Pro + Ser221AspGly205Asn + Thr206Ser + Ile220GlySer207Glu + Leu209Met + Gly222AsnGly203Ser + Trp212Gln + Ser216AspGly205Asn + Ile208Cys + Thr211GlnThr211Gly + Trp212Gly + Thr223GluGly205Asn + Gly214Pro + Ser221GluGly205Ser + Trp212Ala + Ser216AspGly203Gln + Arg218Glu + Ile220LeuSer207Asp + Thr211Asn + Thr223GlyTrp212Cys + Gly215Asn + Thr223ProGly205Asn + Gly214Ser + Arg218GluGly214Pro + Thr217Glu + Gly222AsnThr206Gln + Ile208Asn + Thr211SerGly203Gln + Gly214Pro + Ser219GluTrp212His + Ile213Gln + Arg218GluIle208Ala + Ile213Ala + Ser221GluPro204Asn + Ile208Leu + Trp212ProGly205Asn + Ser219Asp + Ile220HisGly205Gln + Ile208Leu + Thr211GlnTrp212Tyr + Ile213Gly + Gly215GlnTrp212Met + Gly215Glu + Gly222AsnThr206Ser + Thr211Ser + Thr217AsnIle208Leu + Ile213Ser + Ile220ProIle208Ser + Gly214Pro + Gly222AsnThr211Ser + Ser216Glu + Thr223ProGly203Ser + Thr217Pro + Thr223GlyGly203Ser + Trp212Ala + Gly214AspGly205Asn + Ile208Val + Ser219AspGly203Ser + Pro204Gln + Ser221AspGly203Pro + Trp212Gly + Gly222ProIle208Gln + Thr217Gly + Arg218GluThr206Gln + Ser207Glu + Thr223GlySer207Asp + Leu209His + Thr211ProIle208Thr + Trp212Val + Ile220MetLeu209Ala + Ser221Asp + Thr223GlnGly203Asn + Thr211Gly + Gly214GluGly214Pro + Gly215Glu + Ile220ProGly205Ser + Gly214Asn + Ser216GluThr206Gln + Ile213Thr + Ser219GluPro204Gly + Thr211Gly + Ser221AspGly203Pro + Gly205Asn + Gly215AspGly203Pro + Thr211Asn + Thr223AsnPro204Gly + Leu209Pro + Gly215GluGly203Pro + Ile213Glu + Ile220GlyPro204Gln + Ile213Met + Ile220ProGly214Pro + Thr217Gln + Gly222GluIle208His + Ser221Glu + Thr223AsnGly203Pro + Gly214Asn + Arg218AspIle213Ala + Ser216Asp + Gly222SerGly203Asn + Thr211Gly + Thr217AsnLeu209Met + Thr211Gln + Ser219AspThr206Ser + Leu209Ile + Thr211GlyIle213Met + Gly215Gln + Ser221AspPro204Asn + Ser207Asp + Ile220HisGly203Gln + Ile208Val + Ser221AspThr217Gln + Ile220Val + Thr223GluGly205Asn + Ile208Val + Ile213HisThr206Asn + Thr211Gly + Thr217AspSer207Glu + Ile208Cys + Gly215ProPro204Asn + Gly205Pro + Trp212IlePro204Gly + Ile208Val + Thr223AspPro204Gly + Gly215Asn + Ile220HisGly205Asn + Thr211Asn + Trp212ProPro204Ser + Thr206Glu + Ile213ValGly203Pro + Ser207Glu + Gly214AsnPro204Gln + Gly214Glu + Gly215GluGly205Ser + Thr217Asp + Arg218AspLeu209Met + Thr217Asp + Arg218AspThr217Asp + Arg218Asp + Ile220CysThr211Gln + Ser221Glu + Gly222AspGly205Pro + Thr206Asp + Ser207GluPro204Gly + Thr206Asp + Ser207GluThr211Gln + Ile220Glu + Ser221AspTrp212Tyr + Arg218Asp + Ser219GluIle208Val + Arg218Glu + Ser219GluGly205Gln + Gly215Asp + Ser216AspLeu209Glu + Trp212Phe + Ser219AspThr206Glu + Ile208Ala + Ser221AspGly205Asn + Ser207Glu + Ser221GluSer207Glu + Thr211Asn + Ile220AspGly203Pro + Ser207Glu + Ile220GluSer207Asp + Trp212His + Ser219Asp表25環(huán)區(qū)6-四突變變異體Gly203Gln + Thr206Gly + Leu209Ser + Ser219GluIle208Val + Thr217Ser + Ser219Asp + Gly222ProGly203Asn + Ile213Thr + Gly214Pro + Ser219GluGly203Gln + Trp212Phe + Gly214Asn + Gly215AspSer207Asp + Leu209Thr + Thr211Pro + Trp212GlyGly205Pro + Trp212Asn + Ile213His + Ile220AspGly203Gln + Pro204Ser + Trp212Ile + Ile220SerPro204Gln + Thr211Gly + Ser219Glu + Ile220MetGly203Gln + Trp212Gly + Gly215Glu + Ile220AlaThr206Gln + Thr211Pro + Gly215Asn + Ile220ThrLeu209Val + Thr211Ser + Arg218Asp + Ile220SerGly203Gln + Ile208Cys + Leu209Ser + Gly214GluGly205Gln + Ser207Glu + Gly215Gln + Ile220AlaIle208Ala + Leu209Val + Ser216Glu + Thr223ProPro204Gly + Ile208His + Ser219Glu + Ile220SerThr206Asp + Ile208Gly + Thr217Asn + Thr223SerGly203Asn + Leu209Ser + Gly214Ser + Ser221AspIle213Asp + Gly214Ser + Ile220Gln + Gly222AsnTrp212Thr + Ile213Gly + Gly214Ser + Ile220AlaGly203Asn + Leu209Asp + Trp212Phe + Gly222GlnThr206Asn + Thr211Ser + Ile213Gly + Ser219AspPro204Asn + Gly205Gln + Thr206Asn + Ser207GluGly203Gln + Gly205Asn + Ser207Asp + Leu209ThrGly205Gln + Ile213Val + Gly214Pro + Arg218GluThr206Pro + Ile213Pro + Gly222Pro + Thr223AspGly203Asn + Gly214Asn + Ser221Asp + Thr223GlyIle213Asn + Ser216Asp + Ile220Asn + Gly222AsnPro204Ser + Leu209Pro + Ile213Glu + Ile220ProGly203Gln + Gly205Ser + Leu209Ala + Gly215SerGly203Gln + Ser207Asp + Thr211Gly + Gly222AsnPro204Gln + Leu209His + Thr217Gly + Ser219GluThr206Asp + Ile208Thr + Leu209Ala + Thr217AsnIle208Thr + Leu209Gly + Arg218Asp + Ile220LeuPro204Asn + Ile208Val + Leu209His + Arg218GluIle208Asn + Thr211Gln + Ser219Glu + Gly222GlnThr206Gly + Ser207Glu + Ile208Thr + Ile213GlyLeu209Glu + Gly214Gln + Ile220Val + Gly222GlnGly205Gln + Gly214Asn + Thr217Gln + Thr223GluGly203Gln + Leu209Val + Gly215Pro + Ile220GluThr211Gln + Trp212Gln + Gly215Asn + Ser219GluPro204Ser + Ile208Ser + Gly222Glu + Thr223GlnPro204Gly + Ile208Ser + Gly215Gln + Ser221AspGly203Gln + Thr211Gly + Ile213Leu + Gly214AsnPro204Asn + Thr211Ser + Gly214Asp + Thr217AsnPro204Gly + Trp212Met + Gly215Glu + Gly222AsnThr206Glu + Ile208Leu + Ile213Ser + Ile220ProThr206Gly + Trp212Leu + Gly214Asp + Gly222SerGly203Ser + Gly214Asp + Thr217Pro + Thr223GlyGly203Ser + Pro204Asn + Ile208Ala + Gly215GluGly203Ser + Thr211Ser + Ser219Glu + Gly222AsnSer207Glu + Ile208Gly + Thr211Pro + Ile220ProGly203Asn + Gly205Pro + Leu209Ala + Gly222GluLeu209Asn + Thr211Gly + Ile213Thr + Thr223GlnGly203Pro + Pro204Asn + Ile213His + Thr217GlnIle208Asn + Thr217Pro + Ile220Leu + Ser221GluThr206Ser + Thr211Gln + Ile213Gly + Ser221AspGly205Pro + Gly215Asp + Ile220His + Thr223GlyPro204Gln + Ile208Thr + Ser219Asp + Gly222AsnGly203Pro + Leu209Met + Thr211Pro + Ile220CysThr206Gly + Leu209Cys + Ile213His + Ser216AspIle208Gly + Leu209Met + Thr217Gly + Ile220ProGly203Asn + Thr206Ser + Gly214Gln + Ser221GluIle208His + Thr211Ser + Trp212Pro + Arg218AspThr206Ser + Ile208Pro + Leu209Ile + Gly214AspGly205Pro + Thr206Glu + Ile213His + Gly222AsnThr211Asn + Ile213Gln + Thr217Asp + Thr223SerThr206Asn + Trp212Val + Arg218Glu + Ile220ProPro204Asn + Ser207Asp + Thr211Gln + Gly214SerPro204Ser + Ile213Met + Gly214Asp + Thr217GlyGly203Pro + Ile208Ala + Ser216Glu + Thr217ProSer207Asp + Trp212Cys + Gly215Pro + Thr217GlyPro204Asn + Gly214Gln + Ser216Glu + Ile220HisPro204Ser + Trp212Ala + Gly214Asp + Gly215ProPro204Asn + Leu209Gly + Thr211Gly + Ile213ThrPro204Ser + Gly205Gln + Ile208His + Ser219AspGly203Asn + Gly205Ser + Leu209Ser + Arg218GluThr206Pro + Thr211Gln + Ile213Glu + Gly214AsnSer207Asp + Gly214Asn + Ile220Val + Gly222AsnTrp212Tyr + Ile213Val + Gly214Asn + Gly222SerGly205Pro + Ile208Gly + Ser216Asp + Ile220HisGly203Asn + Leu209Gly + Gly214Pro + Arg218AspPro204Gln + Ile208Cys + Ile213Gly + Gly215GluPro204Asn + Gly205Pro + Leu209Ala + Thr211ProGly203Pro + Ile213Ala + Thr217Gly + Arg218GluGly203Gln + Pro204Gly + Ile220Gly + Ser221GluGly205Asn + Ile213Gly + Gly214Pro + Gly222SerGly203Asn + Thr211Gln + Gly214Glu + Gly222GlnIle213Thr + Gly215Gln + Ser219Asp + Ile220ProGly203Asn + Pro204Ser + Arg218Asp + Thr223ProGly203Ser + Thr211Gln + Arg218Glu + Ile220ValGly203Ser + Pro204Ser + Ile208Ser + Thr211GlnPro204Gly + Gly214Asn + Gly215Glu + Thr223AsnIle208Cys + Thr211Pro + Trp212Ser + Gly215ProPro204Asn + Ile213Met + Thr217Glu + Ile220GlnPro204Gly + Gly205Pro + Ile208Gly + Thr211ProGly203Ser + Thr206Gly + Ile208Asn + Ser219AspPro204Asn + Ile213Glu + Gly215Pro + Thr217AsnGly203Asn + Thr211Gln + Arg218Glu + Ile220GlyGly203Gln + Ile208Cys + Gly215Ser + Arg218AspPro204Gln + Gly205Gln + Gly214Ser + Thr223GlyPro204Gln + Leu209Met + Trp212Phe + Ser219GluGly205Asn + Ile208Cys + Leu209Ile + Ile220AsnGly203Pro + Leu209Cys + Thr217Gly + Ile220GlyGly205Asn + Thr206Gly + Ile208Asn + Ser216GluPro204Gln + Thr206Pro + Ser207Glu + Gly214AsnGly203Gln + Ile208Met + Trp212His + Thr223ProPro204Asn + Leu209Val + Thr211Asn + Gly215SerGly203Asn + Ile208His + Ser221Asp + Gly222SerPro204Asn + Leu209Pro + Ser219Glu + Ile220GluThr211Pro + Ile213Asn + Gly214Glu + Gly215AspThr211Gln + Gly215Ser + Thr217Asp + Arg218AspGly205Asn + Thr217Asp + Arg218Asp + Ile220GlnIle208Thr + Leu209Thr + Ser221Asp + Gly222GluGly203Asn + Gly205Asn + Ser221Asp + Gly222GluThr206Ser + Trp212Pro + Ser221Asp + Gly222AspIle208Pro + Thr211Ser + Gly222Asp + Thr223AspPro204Ser + Trp212Met + Gly222Glu + Thr223AspGly203Gln + Thr206Glu + Ser207Glu + Thr211GlnGly203Asn + Thr206Glu + Ser207Asp + Thr211AsnThr206Asp + Ser207Asp + Ile220Thr + Thr223ProGly203Gln + Thr217Ser + Ile220Glu + Ser221AspGly205Ser + Thr211Gln + Arg218Glu + Ser219AspLeu209Gly + Gly214Ser + Arg218Glu + Ser219GluIle208Gln + Gly214Pro + Ser216Asp + Thr217GluIle208Gly + Ile213Met + Gly215Glu + Ser216GluIle208Ser + Leu209Asp + Thr211Gly + Ser219AspPro204Gln + Ile208Thr + Leu209Asp + Ser219AspThr206Asp + Trp212Gln + Ser221Asp + Thr223GlyGly203Asn + Thr206Asp + Trp212Phe + Ser221GluPro204Gln + Thr206Glu + Leu209Ile + Ser221GluPro204Gln + Gly205Gln + Ser207Glu + Ser221GluSer207Asp + Ile208Leu + Trp212Ile + Ser221GluSer207Glu + Leu209Met + Ile220Glu + Thr223SerThr217Asn + Arg218Asp + Ser219Asp + Ile220AspPro204Asn + Ser207Asp + Ser219Glu + Gly222GlnPro204Gly + Gly205Pro + Ser207Glu + Ser219GluGly205Gln + Ser207Asp + Trp212Val + Ser219GluSer207Glu + Ile208Ser + Ser219Glu + Thr223GlyThr206Asp + Thr217Ser + Ile220Asp + Ser221GluTrp212Gln + Gly214Asp + Ser216Glu + Ile220ValGly214Asp + Gly215Gln + Ser216Glu + Thr223AsnGly203Gln + Leu209Asp + Thr211Asn + Arg218GluPro204Ser + Leu209Glu + Arg218Asp + Ile220CysSer207Asp + Ile208Gly + Ser221Glu + Gly222AspPro204Asn + Ser207Glu + Ser221Asp + Gly222AspPro204Ser + Gly205Ser + Ile213Asp + Ser216AspThr206Asn + Ser219Glu + Ser221Glu + Thr223AsnThr206Pro + Trp212Pro + Ser216Glu + Arg218GluGly203Ser + Gly215Asn + Ser216Asp + Arg218GluThr206Gln + Trp212Asn + Ser216Asp + Arg218GluLeu209Val + Trp212Ile + Ser216Asp + Arg218GluPro204Gly + Ile208Pro + Ser216Asp + Arg218AspIle208Gly + Thr211Asn + Ser216Glu + Arg218GluTrp212His + Ile213Asn + Thr217Glu + Ser219GluIle208Leu + Thr211Pro + Thr217Asp + Ser219GluGly203Asn + Thr217Asp + Ser219Glu + Ile220AlaThr206Pro + Thr217Glu + Ser219Glu + Ile220AlaGly203Gln + Gly215Glu + Thr217Glu + Ile220CysGly203Asn + Arg218Glu + Ser219Asp + Ser221GluLeu209Met + Ser219Asp + Ser221Glu + Gly222AspThr211Ser + Ile213Glu + Ser216Asp + Arg218AspThr211Asn + Ser216Asp + Arg218Asp + Ser219AspSer216Glu + Arg218Asp + Ser219Glu + Thr223SerGly203Pro + Thr206Glu + Leu209Asp + Ile220AspSer207Glu + Leu209Gln + Gly222Asp + Thr223SerGly205Gln + 5er207Glu + Ile213Gln + Gly222AspGly205Asn + Ser207Glu + Ile213Leu + Gly222AspGly205Gln + Ser207Glu + Gly222Asp + Thr223GluSer207Asp + Trp212Gly + Ser219Glu + Gly222GluTrp212Thr + Gly214Glu + Ser216Asp + Arg218AspSer207Asp + Arg218Asp + Ser221Asp + Gly222AsnSer207Asp + Trp212His + Arg218Glu + Ser221GluPro204Asn + Thr206Asp + Thr211Ser + Ser219AspThr206Asn + Ile213Pro + Gly214Glu + Thr217AspThr211Gln + Gly214Glu + Gly215Ser + Thr217GluLeu209Asp + Gly214Pro + Ser221Glu + Gly222AspSer207Glu + Leu209Ala + Ile213Ala + Arg218GluPro204Gln + Ser207Glu + Trp212Leu + Arg218AspPro204Gly + Ile213Glu + Arg218Glu + Ser219GluPro204Gly + Thr206Glu + Leu209Asp + Thr223Gly表26環(huán)區(qū)6-五替換突變體Pro204Gln + Gly205Gln + Ile208Ala + Ser216Asp +Gly222ProPro204Gln + Ser207Glu + Ile208Ser + Thr211Gly +Ile220AlaGly205Gln + Trp212Phe + Gly214Pro + Gly215Asp +Thr217GlyGly203Asn + Ile208Gln + Leu209Gln + Thr211Pro +Thr223AspGly203Ser + Ile208Cys + Gly214Gln + Ser219Glu +Ile220ThrTrp212Gln + Ile213Ser + Gly214Asn + Thr217Glu +Ile220SerGly205Ser + Ile208Pro + Trp212Tyr + Gly214Gln +Ser221AspGly205Gln + Thr206Glu + Ile208Thr + Leu209Thr +Trp212CysGly203Asn + Gly205Pro + Ile208Val + Gly214Asn +Ser219GluGly203Gln + Gly205Gln + Ile208Gly + Thr217Asn +Gly222SerIle208Met + Thr211Gly + Ile213Cys + Gly214Pro +Ile220LeuThr211Pro + Ile213Ser + Thr217Asp + Ile220Pro +Gly222GlnPro204Asn + Gly205Ser + Thr211Gly + Ile220Thr +Gly222AsnGly203Asn + Gly205Asn + Leu209His + Ile220His +Thr223GlnPro204Gly + Ile208Asn + Thr217Gln + Gly222Glu +Thr223GlnGly205Gln + Ser207Asp + Ile208Met + Ile213Cys +Gly214ProGly205Asn + Thr211Gln + Trp212Met + Thr217Glu +Gly222AsnGly205Gln + Thr206Pro + Leu209Glu + Ile213Thr +Ile220GlyThr211Asn + Gly214Gln + Gly215Ser + Thr217Ser +Gly222SerPro204Gly + Gly205Gln + Thr211Gly + Ser221Glu +Thr223GlnGly203Ser + Ile208Thr + Ile220Val + Gly222Pro +Thr223GluGly205Asn + Thr206Pro + Ser216Glu + Thr217Gln +
Gly222SerGly203Asn + Pro204Gln + Ile208Met + Gly215Gln +Ser219AspGly205Gln + Thr206Pro + Trp212Ser + Ile213Gln +Ser221GluLeu209Gln + Gly214Gln + Thr217Ser + Ser221Asp +Thr223AsnSer207Glu + Trp212Tyr + Ile213Gly + Gly214Pro +Ile220ProGly203Pro + Trp212Pro + Gly214Glu + Gly215Pro +Gly222ProPro204Ser + Thr206Asn + Thr211Gly + Trp212Gln +Thr223GlnPro204Gly + Thr206Pro + Trp212Val + Gly214Pro +Arg218GluGly203Gln + Thr211Gln + Gly215Aan + Thr217Glu +Ile220ValGly203Gln + Gly205Pro + Leu209Val + Thr211Gln +Gly214AspPro204Asn + Ile213Thr + Gly215Gln + Ser219Asp +Ile220ProPro204Gln + Thr206Asn + Ile208Met + Gly215Glu +Ile220AlaGly203Ser + Gly205Pro + Ile208Asn + Leu209Glu +Thr211GlnPro204Gly + Gly205Gln + Thr211Ser + Trp212Met +Thr217ProGly205Ser + Thr206Gly + Ile208Val + Gly214Asp +Thr223ProSer207Glu + Ile208Ala + Gly215Pro + Thr217Gln +Thr223ProGly203Ser + Gly205Gln + Trp212Asn + Ile213Ser +Thr223ProPro204Gln + Ile208Ala + Leu209Gln + Thr211Ser +Gly214SerPro204Gln + Ile208His + Thr211Gln + Gly215Pro +Ser216AspPro204Gln + Leu209Ile + Thr211Ser + Gly214Asp +Ile220CysLeu209Ile + Thr211Gln + Trp212Asn + Ile213Val +Thr217AspPro204Asn + Ile208His + Leu209Val + Thr211Asn +Gly222SerGly203Gln + Pro204Asn + Gly205Asn + Ile213Ala +Gly214AspSer207Asp + Thr211Gln + Trp212Gly + Gly222Gln +Thr223GlyGly205Gln + Ile208Gly + Thr211Gln + Ile220Ser +Gly222AspGly205Pro + Ile208Pro + Leu209Val + Ile220Leu +Gly222GluPro204Gly + Thr206Glu + Leu209Cys + Ile213His +Ile220MetLeu209Gly + Trp212Val + Ser216Asp + Ile220Cys +Thr223SerGly205Gln + Leu209Val + Thr211Asn + Gly214Pro +Gly222AspIle208Ala + Leu209His + Gly215Asn + Ile220Val +Gly222GluSer207Glu + Ile208Pro + Thr211Asn + Ile213His +Gly222SerIle208His + Leu209Cys + Thr217Pro + Ile220Glu +Gly222GlnPro204Gly + Gly205Ser + Ile208Ala + Trp212Leu +Thr223AsnGly203Pro + Gly205Pro + Trp212Ala + Gly215Ser +Arg218GluPro204Ser + Trp212Ala + Ile213Thr + Gly215Asn +Ser216GluThr211Asn + Trp212Thr + ser216Glu + Gly222Gln +Thr223ProThr206Gln + Ile208Pro + Thr211Gly + Ile213Leu +Gly215AspGly203Asn + Ile220Met + Ser221Glu + Gly222Gln +Thr223AsnPro204Gln + Thr206Gly + Trp212Gly + Gly215Asp +Ile220CysGly203Asn + Pro204Gln + Trp212Pro + Ile220Gly +Thr223AspPro204Ser + Thr206Ser + Ile208Leu + Thr211Ser +Gly222ProGly203Ser + Ile208Thr + Leu209His + Thr211Gln +Ile220GluPro204Asn + Thr211Pro + Gly214Gln + Gly215Glu +Thr217SerPro204Asn + Thr211Pro + Thr217Asp + Ile220Val +Thr223GlnGly203Ser + Ile208Gln + Leu209Asn + Thr211Ser +Trp212AsnPro204Asn + Ile208His + Thr211Gln + Ile220Gly +Gly222AspGly205Pro + Trp212Val + Gly215Pro + Ser216Asp +Thr217ProGly203Gln + Ile208Ala + Thr211Pro + Gly214Ser +Arg218GluGly203Asn + Pro204Gln + Leu209Met + Trp212Ser +Thr217GluGly205Pro + Ser207Glu + Ile208Met + Leu209Met +Ile220AsnThr211Gly + Trp212Asn + Ser219Glu + Ile220Asp +Thr223ProIle208Met + Ile213Thr + Thr217Asp + Arg218Glu +Thr223GlyGly203Ser + Ile208Gln + Thr217Glu + Arg218Asp +Gly222ProGly205Gln + Thr217Glu + Arg218Glu + Ile220His +
Thr223ProGly203Ser + Ile213Gln + Thr217Asp + Arg218Asp +Gly222GlnGly203Pro + Pro204Gly + Ile208Gly + Ser221Asp +Gly222AspGly203Asn + Thr211Gln + Ile213Asp + Gly214Glu +Gly222GlnThr206Glu + Ser207Glu + Leu209Asn + Thr211Gln +Thr217AsnGly203Gln + Leu209Met + Thr211Pro + Ile220Glu +Ser221GluIle208Val + Leu209His + Thr211Gln + Arg218Glu +Ser219GluGly203Pro + Pro204Asn + Leu209His + Arg218Asp +Ser219AspIle208Ala + Trp212Gln + Arg218Glu + Ser219Glu +Ile220ThrLeu209Gly + Thr211Pro + Gly215Asn + Ser216Glu +Thr217AspIle208Asn + Leu209Thr + Ile213His + Ser216Glu +Thr217GluLeu209Asn + Gly215Glu + Ser216Asp + Ile220Asn +Thr223AsnGly203Ser + Pro204Gly + Gly215Glu + Ser216Asp +Thr223SerPro204Gln + Ile208Cys + Ile213Cys + Gly215Glu +Ser216GluPro204Gln + Gly205Asn + Thr206Asp + Ser207Glu +Ser221GluGly203Gln + Pro204Asn + Thr206Asp + Ile208Cys +Ser221GluPro204Gly + Thr206Glu + Ile208Gln + Trp212His +Ser221GluThr206Asp + Ile208Met + Trp212Met + Ile213Met +Ser221GluSer207Asp + Thr211Gln + Trp212Ala + Ile213Pro +Ser221AspThr206Gln + Ser207Asp + Leu209Val + Thr211Gly +Ser221AspPro204Ser + Ser207Glu + Ile208Thr + Trp212Ile +Ser221AspSer207Glu + Leu209Ala + Thr217Gly + Ile220Ala +Ser221AspSer207Asp + Thr211Pro + Trp212Cys + Gly215Pro +Ser221GluGly205Pro + Ser207Glu + Trp212Asn + Ser221Glu +Thr223SerGly205Gln + Ser207Glu + Thr211Gly + Thr217Gly +Ser221GluThr206Gln + Ser207Glu + Ile208Ala + Leu209Cys +Ile220GluThr211Gln + Arg218Glu + Ser219Asp + Ile220Asp +Gly222SerGly205Asn + Thr206Pro + Ser207Asp + Ser219Glu +Gly222GlnPro204Ser + Ser207Asp + Ser219Asp + Ile220Met +Thr223ProThr206Asn + Ser207Glu + Ile208Thr + Ile213Gly +Ser219GluGly205Gln + Leu209Asn + Thr217Asp + Arg218Glu +Ser219AspPro204Gly + Ile208Val + Ile213Asp + Gly214Glu +Ser216AspPro204Ser + Ile220His + Ser221Glu + Gly222Glu +Thr223GluThr206Asp + Trp212Val + Ile220Glu + Ser221Asp +Thr223ProThr206Glu + Ser207Asp + Gly214Asn + Ile220Asp +Thr223AsnIle208Leu + Leu209Val + Thr211Ser + Gly214Asp +Ser216GluThr211Ser + Ile213His + Gly214Asp + Ser216Asp +Ile220GlnGly205Pro + Thr206Ser + Leu209Glu + Thr217Asn +Arg218GluGly205Ser + Leu209Asp + Gly214Ser + Gly215Ser +Arg218AspSer207Glu + Leu209Gly + Trp212Phe + Ser219Glu +Ser221GluGly203Pro + Pro204Asn + Thr211Ser + Ile213Glu +Ser216GluThr206Glu + Ser207Asp + Ile208Ala + Gly215Pro +Ser219GluThr206Glu + Leu209Ser + Trp212Ile + Ile220Thr +Gly222GluThr206Asp + Ser207Asp + Ile208Leu + Ile213Pro +Gly222GluGly203Ser + Thr206Asp + Ser207Glu + Ile220Gln +Gly222GluThr211Gly + Ile213Ala + Gly214Glu + Ser216Glu +Thr217AspSer207Asp + Thr211Gln + Arg218Glu + Ser219Glu +Ile220ValSer207Glu + Leu209Glu + Gly214Gln + Ile220Val +Gly222GlnSer207Glu + Leu209Glu + Thr211Gln + Gly214Gln +Thr217GlnGly205Gln + Ser207Glu + Thr211Ser + Arg218Asp +Ile220GluLeu209Glu + Thr217Pro + Ser219Asp + Ser221Glu +Gly222SerThr211Gly + Gly214Asn + Ser219Glu + Ile220Leu +Ser221AspGly203Pro + Ile208Gln + Gly214Pro + Ser219Asp +Ser221GluPro204Asn + Leu209Ile + Ser219Glu + Ser221Asp +
Gly222SerThr211Ser + Trp212Gln + Gly214Gln + Ser219Asp +Ser221AspLeu209Ala + Gly214Pro + Ser219Glu + Ile220Met +Ser221GluPro204Gly + Gly205Gln + Ile213Val + Ser216Asp +Arg218GluPro204Gly + Thr206Pro + Thr211Asn + Ser216Glu +Arg218GluThr206Asp + Ile208Thr + Leu209Ala + Thr217Asn +Ile220AspPro204Gln + Thr206Glu + Ile208Pro + Trp212His +Ile220GluThr206Gln + Thr211Ser + Thr217Asp + Ser219Asp +Thr223ProGly203Ser + Pro204Ser + Gly205Pro + Gly215Asp +Thr217GluGly203Pro + Pro204Gln + Gly215Glu + Thr217Glu +Gly222GlnPro204Ser + Leu209Met + Gly215Glu + Thr217Asp +Thr223AsnPro204Gly + Trp212Leu + Gly214Ser + Ser221Asp +Thr223AspLeu209Ser + Thr211Asn + Arg218Asp + Ile220Glu +Ser221GluIle208Gly + Leu209Asn + Ser219Asp + Ser221Glu +Gly222AspPro204Gly + Ile208Leu + Gly215Asp + Ser216Glu +Arg218AspPro204Gln + Ile208Gly + Gly215Asp + Ser216Glu +Arg218GluThr206Gly + Ile213Ser + Ser216Glu + Arg218Asp +Ser219GluPro204Gln + Thr206Glu + Ser207Asp + Thr211Asn +Thr223GluPro204Gly + Gly205Ser + Ser207Glu + Arg218Asp +Ser221AspThr206Asp + Ile213Thr + Gly214Ser + Ser219Asp +Gly222GluGly203Asn + Thr206Asn + Ile213Pro + Gly214Glu +Thr217AspPro204Ser + Ile213Gly + Gly214Glu + Thr217Asp +Ile220AlaSer207Glu + Thr217Asp + Arg218Asp + Ile220Ala +Thr223SerSer207Asp + Leu209Ser + Trp212Val + Gly215Ser +Arg218GluPro204Asn + Ser207Glu + Leu209Val + Trp212Phe +Arg218AspGly205Ser + Ser207Glu + Gly215Gln + Arg218Glu +Thr223AsnGly205Ser + Leu209Asp + Ile213Pro + Gly215Asn +Ser221AspGly205Pro + Ser207Glu + Leu209Glu + Thr217Ser +Gly222AspSer207Asp + Leu209Asp + Thr217Gly + Gly222Asp +Thr223SerGly203Asn + Ile208Ser + Leu209Asp + Ser216Glu +Ser219AspLeu209Ala + Trp212Ser + Gly214Glu + Gly215Glu +Arg218GluGly205Gln + Thr206Glu + Ile213Gln + Arg218Glu +Ser221GluPro204Gly + Leu209Asn + Gly215Pro + Thr217Asp +Ile220AspThr206Pro + Trp212Ala + Arg218Asp + Ser219Glu +Gly222AspGly203Asn + Gly205Pro + Trp212Asn + Arg218Glu +Ser221AspThr206Gly + Ile208Ala + Trp212Tyr + Arg218Asp +Ser221GluGly205Gln + Thr211Ser + Arg218Asp + Ser221Glu +Thr223ProGly205Pro + Thr211Pro + Trp212Pro + Arg218Glu +Ser221GluGly203Ser + Arg218Asp + Ile220Asn + Ser221Asp +Thr223SerTrp212His + Ile213Asn + Gly214Glu + Thr217Glu +Ser219GluGly205Pro + Ile213Pro + Gly215Asp + Arg218Glu +Ser219AspPro204Ser + Gly215Asp + Arg218Glu + Ser219Glu +Gly222AsnSer207Glu + Gly214Gln + Ser216Asp + Arg218Glu +Gly222GlnSer207Asp + Gly215Pro + Ser216Glu + Thr217Asn +Arg218AspGly205Ser + Ile208Cys + Leu20gAla + Gly215Asp +Arg218AspGly203Asn + Ser207Asp + Ser216Glu + Ser219Asp +Gly222ProSer207Glu + Leu209Ile + Ser216Glu + Thr217Gly +Ser219GluLeu209Asp + Trp212Pro + Gly214Glu + Ser216Glu +Ile220CysGly203Gln + Thr206Gly + Leu209Ser + Ser216Glu +Ser219GluIle208Leu + Gly214Asn + Ser216Glu + Thr217Ser +Ser219GluLeu209Ser + Ser216Glu + Thr217Ser + Ser219Glu +Ile220HisPro204Gln + Leu209Pro + Gly214Ser + Ser216Glu +Ser219AspThr206Gly + Ile208Met + Gly215Asp + Arg218Asp +Ile220Glu
表27環(huán)區(qū)6-六替換變異體Gly203Asn + Thr206Asn + Ile208Asn + Trp212Asn +Ile213Leu + Ser221GluPro204Gln + Gly205Gln + Ile208Ala + Leu209Pro +Ser216Asp + Gly222ProGly203Gln + Gly205Asn + Gly215Ser + Thr217Ser +Ile220Met + Ser221AspIle208Pro + Leu209Ala + Gly215Gln + Arg218Asp +Ile220Val + Thr223SerGly203Ser + Leu209Cys + Trp212Tyr + Ser216Asp +Thr217Asn + Ile220ValGly203Gln + Gly205Ser + Leu209Ala + Ile213Met +Gly215Asn + Gly222AsnGly203Pro + Gly205Ser + Leu209Ala + Trp212Ala +Gly214Gln + Ser219AspGly203Pro + Leu209Ile + Thr211Pro + Ile213Val +Gly215Pro + Ile220ProThr206Ser + Ile208Asn + Thr211Gln + Ile213Gly +Ile220Leu + Ser221AspPro204Ser + Gly205Pro + Ile208Thr + Gly215Asp +Ile220His + Thr223GlyGly203Pro + Pro204Gln + Leu209Met + Thr211Pro +Ser219Asp + Ile220CysPro204Asn + Thr206Asn + Ser207Asp + Thr211Gln +Gly214Ser + Ile220ProGly203Ser + Leu209Pro + Trp212Met + Ile213Cys +Thr217Pro + Arg218GluPro204Asn + Gly205Ser + Ile208Ala + Ile213Pro +Gly215Ser + Ile220GlyGly203Ser + Gly205Asn + Ser207Glu + Thr211Ser +Trp212Val + Thr217AsnGly203Gln + Pro204Asn + Ile208His + Leu209Pro +Ile213Leu + Gly222AsnGly205Asn + Thr206Gly + Ile208Pro + Thr211Pro +Trp212Ile + Thr223AsnGly205Gln + Thr206Ser + Ile208Gln + Ser221Asp +Gly222Gln + Thr223ProIle208Leu + Trp212Asn + Ile213Thr + Gly214Ser +Thr217Gln + Ser219GluGly203Gln + Pro204Gln + Leu209His + Ser219Glu +Gly222Asn + Thr223GlnPro204Gln + Gly205Asn + Ile208Cys + Leu209Ile +Ile220Asn + Thr223GluGly203Pro + Gly205Gln + Thr206Gly + Thr211Gln +Trp212Pro + Gly215GluThr206Gln + Ile208Leu + Thr211Gln + Gly214Asn +Ile220Asn + Thr223AsnIle208Val + Leu209Cys + Thr211Ser + Ile213Gly +Gly214Asp + Thr223GlyPro204Ser + Gly205Asn + Leu209Ser + Thr217Gly +Arg218Glu + Gly222SerPro204Ser + Gly205Gln + Leu20gHis + Thr211Gln +Gly214Ser + Ser221GluIle208His + Leu209Cys + Thr211Ser + Trp212Ser +Gly214Glu + Gly222AsnIle208Gln + Leu209Gly + Trp212Phe + Thr217Pro +Ile220Thr + Gly222GluGly205Pro + Ser207Glu + Ile208Met + Leu209Met +Trp212Cys + Ile220AsnPro204Ser + Thr206Asn + Ile208Gln + Trp212Phe +Ser219Glu + Thr223GlyGly205Pro + Ile208Cys + Trp212Ile + Thr217Gln +Ile220Leu + Gly222ProPro204Ser + Gly205Ser + Ile208Cys + Ile213Pro +Ser221Asp + Thr223GlnPro204Ser + Thr206Gln + Trp212Met + Gly214Ser +Ser221Asp + Thr223SerGly205Ser + Ile208Gln + Ile213Cys + Gly215Pro +Thr217Gln + Thr223AspPro204Asn + Gly205Pro + Ile208Ser + Ile213Asp +Ile220Thr + Gly222GlnPro204Asn + Ile208Ala + Leu209Ser + Thr211Gly +Gly215Ser + Ser219GluGly203Gln + Gly205Gln + Thr206Gln + Ser207Glu +Ile208Ala + Ile220AlaLeu209Ser + Thr211Gly + Trp212Tyr + Ile213His +Ser216Asp + Thr223ProPro204Gln + Thr206Ser + Ile208Met + Leu209Cys +Thr211Ser + Gly214AspGly203Asn + Leu209Gln + Thr211Ser + Gly215Pro +Ile220Cys + Gly222ProLeu209His + Thr211Asn + Trp212Ile + Ile213Ala +Ser219Glu + Thr223GlyThr206Gln + Ile208His + Trp212Gln + Gly215Ser +Ser221Asp + Thr223GlnIle208Cys + Leu209Met + Trp212Ser + Ile213Gln +Ser216Glu + Thr223ProGly205Ser + Leu209Met + Trp212Gly + Arg218Glu +Ile220Gly + Gly222AsnPro204Asn + Trp212Gln + Gly215Ser + Ser216Glu +Thr217Asn + Ile220AsnGly203Pro + Ile208Val + Leu209Asp + Trp212Ala +Ile220Gln + Thr223GlyGly203Gln + Gly205Pro + Ile208Gly + Trp212Ser +Thr217Asp + Gly222AsnGly205Ser + Leu209Thr + Thr217Gln + Ile220Met +Gly222Gln + Thr223GluGly203Asn + Pro204Ser + Gly205Pro + Trp212Pro +Gly215Gln + Thr217GluPro204Ser + Gly205Ser + Leu209Asn + Ser216Glu +Ile220Leu + Gly222AsnGly203Gln + Leu209Thr + Trp212Leu + Ile213Gly +
Ser219Glu + Ile220HisIle208Gln + Trp212Gly + Ile213Asn + Thr217Pro +Gly222Ser + Thr223AspPro204Ser + Ile208His + Thr211Gly + Ile213Met +Gly214Gln + Ser216AspGly203Asn + Gly205Pro + Leu209Glu + Trp212Ile +Ile213Val + Gly215ProGly203Asn + Pro204Gln + Leu209Met + Trp212Ser +Thr217Glu + Arg218GluLeu209Gln + Trp212Gln + Gly214Asn + Gly215Ser +Gly222Glu + Thr223GluGly205Pro + Thr211Gly + Trp212Val + Arg218Glu +Ser219Asp + Ile220ProGly203Asn + Pro204Gln + Gly205Gln + Gly214Pro +Arg218Glu + Ser219AspGly205Pro + Thr211Gln + Gly215Asn + Thr217Ser +Arg218Glu + Ser219GluPro204Asn + Gly205Ser + Ile208Gly + Thr217Ser +Arg218Glu + Ser219AspThr206Pro + Gly214Ser + Thr217Gln + Arg218Glu +Ser219Glu + Thr223AsnThr206Gln + Leu209Glu + Thr211Asn + Trp212Ile +Gly215Ser + Ser219AspLeu209Glu + Thr211Gln + Ile213Val + Gly215Gln +Ser219Glu + Gly222ProPro204Gln + Ser207Glu + Ile208Pro + Thr211Gly +Ile220Pro + Ser221AspPro204Asn + Thr206Gln + Ser207Glu + Ile208Ala +Leu209Cys + Ile220GluIhr206Glu + Thr211Gln + Ile213Pro + Gly214Asn +Ser221Asp + Gly222AspGly203Asn + Pro204Gln + Leu209Asp + Gly215Pro +Ser219Asp + Ile220AspSer207Glu + Ile208Ala + Thr211Gly + Trp212Ser +Ser219Glu + Ile220GlnSer207Glu + Leu209Gly + Thr211Pro + Gly215Asn +Ser219Glu + Ile220ValSer207Glu + Ile208Asn + Leu209Cys + Gly215Asn +Ser219Glu + Thr223SerGly203Asn + Gly205Ser + Ser207Asp + Thr217Ser +Ser219Asp + Ile220LeuGly203Asn + Pro204Asn + Ser207Asp + Leu209Thr +Ser219Asp + Thr223GlnGly203Pro + Ile208Pro + Thr211Ser + Ser221Asp +Gly222Asp + Thr223AspGly205Ser + Ser207Glu + Leu209Glu + Thr217Ser +Ser219Glu + Gly222AsnThr206Pro + Leu209Ser + Thr211Pro + Ile213Glu +Gly215Glu + Ile220AlaGly203Ser + Ile208Ser + Leu209His + Trp212Gly +Gly214Asp + Ser216GluGly203Ser + Gly205Ser + Trp212Ala + Gly214Glu +Gly215Ser + Ser216AspGly205Gln + Ser207Glu + Ile208Ser + Leu209Gly +Ser219Asp + Ser221GluThr206Pro + Ser207Glu + Ile208His + Trp212Ala +Ser219Glu + Ser221AspGly205Ser + Ser207Asp + Leu209Cys + Thr217Pro +Ser221Asp + Gly222GluSer207Glu + Leu209Asp + Thr211Gln + Trp212Gln +Thr217Gln + Ile220GluLeu209Asp + Thr211Gln + Gly214Pro + Thr217Glu +Ser219Glu + Thr223GlnGly203Ser + Thr206Glu + Ser207Glu + Gly215Ser +Ser219Asp + Ile220ThrGly205Ser + Thr206Glu + Ser207Glu + Thr211Gly +Ile220Val + Gly222GluThr206Glu + Ser207Asp + Leu209Ser + Trp212Ile +Ile220Thr + Gly222GluGly203Pro + Ser207Glu + Leu209Asn + Thr217Gln +Arg218Asp + Ser219AspGly205Gln + Thr206Gln + Ser207Asp + Arg218Glu +Ser219Asp + Ile220ProPro204Ser + Gly205Gln + Ser207Asp + Thr217Asn +Arg218Asp + Ser219AspGly203Pro + Thr206Pro + Ser207Glu + Ile208Asn +Leu209Asp + Thr217SerGly203Asn + Pro204Gln + Thr206Asp + Trp212Phe +Ser221Glu + Thr223AspPro204Gln + Gly205Gln + Ser207Glu + Leu209Glu +Trp212Met + Ser221GluGly203Asn + Trp212Phe + Gly214Ser + Ser219Asp +Ser221Glu + Thr223GlnGly205Gln + Thr206Gln + Ile208Pro + Thr217Pro +Ser219Asp + Ser221AspGly203Asn + Thr211Asn + Gly215Pro + Ser219Asp +Ser221Asp + Gly222AsnPro204Ser + Trp212His + Gly214Gln + Ser219Glu +Ser221Glu + Gly222GlnGly203Pro + Thr211Gln + Ile213Met + Ser219Glu +Ser221Asp + Gly222SerIle208Leu + Thr211Gly + Gly214Pro + Gly215Asn +Ser216Glu + Arg218AspIle208Ala + Leu209Pro + Trp212Pro + Ser216Glu +Arg218Asp + Thr223ProGly205Gln + Ile208Asn + Gly215Ser + Ser216Glu +Arg218Glu + Thr223AsnGly203Ser + Thr206Gly + Gly214Pro + Gly215Gln +Ser216Asp + Arg218AspGly203Ser + Thr206Pro + Trp212Pro + Gly215Asn +Ser216Glu + Arg218GluGly203Asn + Gly205Asn + Leu209Val + Ile220Glu +Gly222Glu + Thr223SerPro204Gly + Gly205Pro + Leu209Ile + Ile213Met +Ser221Asp + Thr223GluGly205Ser + Trp212Tyr + Gly214Pro + Arg218Glu +
Ser219Asp + Ser221AspGly205Gln + Ile208Ala + Leu209Cys + Arg218Glu +Ser219Asp + Ser221AspGly205Gln + Leu209Val + Trp212Cys + Ile220Asp +Ser221Glu + Thr223AspPro204Gln + Thr211Gln + Gly215Glu + Thr217Asp +Arg218Glu + Thr223AsnGly203Gln + Leu209Asn + Ser216Asp + Arg218Glu +Ser219Asp + Thr223ProIle208Ala + Leu209Pro + Gly214Ser + Ser216Asp +Arg218Glu + Ser219AspPro204Asn + Ile208Gly + Thr211Gln + Ser216Asp +Arg218Glu + Ser219GluGly203Ser + Ile208Ala + Trp212Ser + Ser216Glu +Arg218Asp + Ser219GluGly205Pro + Thr211Ser + Ser216Glu + Thr217Asp +Ser219Asp + Gly222AsnPro204Gln + Leu209Ala + Gly215Gln + Ser216Glu +Thr217Asp + Ser219AspPro204Asn + Ser207Glu + Thr211Gln + Gly214Ser +Gly222Glu + Thr223AsnGly205Asn + Ser207Asp + Trp212His + Gly214Ser +Gly222Glu + Thr223GluGly203Ser + Ser207Glu + Thr211Ser + Ile213Ala +Arg218Asp + Ser221AspGly203Gln + Thr206Glu + Trp212Ile + Ile213Asn +Ser219Asp + Gly222GluIle208Ala + Leu209Met + Thr211Pro + Ile213Thr +Gly214Glu + Thr217GluGly203Ser + Pro204Ser + Gly205Ser + Gly214Asp +Gly215Pro + Thr217GluGly203Asn + Ile208Met + Ile213Cys + Gly214Asp +Thr217Glu + Thr223AsnPro204Asn + Leu209Ile + Trp212Val + Ile213Asp +Gly215Asp + Arg218GluSer207Asp + Ile208Met + Thr211Pro + Trp212Ala +Gly215Asn + Arg218GluPro204Asn + Thr206Gln + Ser207Glu + Gly214Asn +Gly215Gln + Arg218GluGly203Gln + Gly205Pro + Ser207Asp + Ile208Gly +Arg218Glu + Gly222GlnGly205Pro + Thr206Asn + Ser207Asp + Trp212Tyr +Arg218Asp + Ile220ThrPro204Asn + Gly205Pro + Ser207Glu + Ile208Gln +Thr217Pro + Arg218AspThr206Gln + Ser207Glu + Trp212Ser + Thr217Asn +Arg218Glu + Ile220AlaGly205Pro + Thr206Glu + Leu209Thr + Gly214Gln +Arg218Asp + Ser219GluGly203Ser + Pro204Asn + ser207Asp + Gly215Gln +Thr217Asp + Ile220AspIle208Gly + Thr211Gln + Ile213Glu + Arg218Glu +Gly222Ser + Thr223GlnGly203Pro + Thr206Gly + Gly214Gln + Ser219Glu +Ser221Asp + Thr223AspThr211Gly + Trp212Phe + Ser219Asp + Ile220Pro +Ser221Glu + Thr223GluLeu209Ala + Ile213Ala + Ser219Asp + Ile220Gln +Ser221Asp + Thr223AspPro204Gln + Leu209Glu + Trp212Met + Gly214Ser +Gly215Asp + Thr217GluIle208Met + Ile213Ser + Thr217Glu + Ile220Asp +Ser221Asp + Thr223GlnLeu209Asp + Thr211Asn + Ile213Asp + Arg218Glu +Ile220Leu + Gly222SerGly203Pro + Gly205Gln + Thr217Asp + Arg218Glu +Ile220Ala + Ser221AspTrp212Cys + Ile213Gly + Arg218Glu + Ser219Glu +Ile220Pro + Gly222GluThr206Gln + Ile208Gly + Leu209Ile + Arg218Glu +Ser219Glu + Gly222AspGly203Gln + Ile208Met + Ser219Glu + Ile220Glu +Gly222Pro + Thr223GluGly203Gln + Gly205Asn + Thr211Gln + Ser219Asp +Ile220Glu + Thr223GluPro204Asn + Gly205Pro + Leu209Ser + Gly214Ser +Arg218Glu + Ser221GluIle208Thr + Leu209Asn + Trp212Asn + Arg218Asp +Ser221Glu + Gly222AsnGly203Ser + Pro204Gln + Gly205Asn + Thr211Gly +Arg218Asp + Ser221GluPro204Asn + Thr206Pro + Gly214Pro + Arg218Glu +Ser221Glu + Thr223AsnThr206Asp + Ile213Gln + Gly215Asn + Thr217Asp +Ser219Asp + Thr223SerIle213Met + Gly214Asp + Gly215Ser + Thr217Glu +Ser219Asp + Ile220SerPro204Asn + Gly205Gln + Ser207Asp + Gly215Pro +Ser219Glu + Thr223GluPro204Ser + Thr206Gln + Ser207Glu + Thr217Gly +Ser219Asp + Thr223AspPro204Gln + Trp212Leu + Gly215Glu + Thr217Pro +Arg218Asp + Ser219AspGly203Asn + Thr211Gly + Gly214Asn + Gly215Pro +Ser219Asp + Gly222AspGly203Pro + Trp212Ile + Gly214Asn + Ser219Glu +Ile220Pro + Gly222GluThr206Glu + Leu209Ile + Gly215Asn + Ser219Asp +Ile220Cys + Thr223AspGly203Asn + Ser207Asp + Gly214Gln + Ser216Glu +Arg218Asp + Ile220ThrSer207Glu + Ile208Cys + Ile213His + Gly214Ser +Ser216Asp + Arg218GluThr206Gln + Ile208Ser + Leu209Asp + Gly215Glu +Arg218Asp + Ile220AlaGly205Ser + Ile213Cys + Gly214Asp + Arg218Glu +
Ser219Asp + Gly222GlnPro204Asn + Gly214Asp + Arg218Asp + Ser219Asp +Gly222Pro + Thr223GlnPro204Gly + Thr211Asn + Ile213Ala + Gly215Asp +Arg218Glu + Ile220GlySer207Glu + Leu209Ile + Ser216Glu + Thr217Gly +Ser219Asp + Ile220GlyGly205Ser + Ser207Asp + Thr211Gly + Ser216Glu +Ser219Asp + Ile220AsnPro204Gln + Gly205Ser + Thr206Asn + Leu209Glu +Trp212Gly + Ser216AspGly205Pro + Trp212Gly + Ile213His + Gly214Asp +Ser216Glu + Ser219GluPro204Ser + Thr206Asn + Ile213Ala + Ser216Asp +Ser219Glu + Thr223GlnIle208Asn + Trp212Asn + Gly215Asn + Ser216Glu +Ser219Glu + Ile220SerIle208Leu + Thr211Gly + Gly214Gln + Ser216Glu +Ser219Glu + Gly222ProPro204Ser + Thr206Ser + Ser216Asp + Thr217Pro +Ser219Glu + Thr223AsnLeu209Val + Trp212Cys + Gly214Ser + Arg218Glu +Ile220Glu + Thr223AspGly205Gln + Trp212Met + Gly215Asp + Arg218Glu +Ile220Glu + Gly222SerGly205Pro + Thr206Ser + Ser207Glu + Leu209Gly +Gly214Ser + Thr223GluThr206Asp + Leu209Glu + Thr211Gln + Thr217Gln +Ile220Met + Thr223GluPro204Gly + Ile208Cys + Thr217Ser + Arg218Glu +Ser221Glu + Thr223AspSer207Glu + Leu209Val + Ile213Gly + Ser216Glu +Thr217Asp + Gly222GlnIle208Thr + Ser216Glu + Ser219Glu + Ile220Leu +Ser221Glu + Thr223ProGly203Gln + Gly205Gln + Ile213Ala + Ser216Glu +Ser219Asp + Ser221AspGly203Gln + Pro204Gln + Thr206Asp + Leu209Asn +Arg218Glu + Gly222GluGly205Gln + Ile213Ala + Gly215Glu + Ser216Glu +Ile220Glu + Gly222AsnThr206Asn + Ile208Ala + Leu209Asp + Ile213Glu +Gly214Glu + Gly222SerGly203Ser + Pro204Ser + Ile208Cys + Ile213Glu +Gly215Glu + Ser219GluThr206Pro + Leu209Cys + Ile213Glu + Gly215Glu +Thr217Ser + Ser219Glu表28環(huán)區(qū)6-七替換突變體Pro204Asn + Leu209Thr + Thr211Gln + Trp212His+
Gly215Asp + Ile220Val + Thr223SerPro204Gly + Gly205Asn + Ile208Leu + Thr211Asn +Trp212Gly + Gly214Pro + Arg218AspGly203Pro + Thr206Gly + Ile208Pro + Thr211Gln +Thr217Glu + Ile220Met + Thr223AsnPro204Gln + Ile208Ala + Leu209Gln + Thr211Ser +Gly214Ser + Ser219Glu + Thr223ProPro204Gly + Gly205Pro + Thr206Glu + Ile208Pro +Leu209Val + Ile213His + Ile220LeuGly205Gln + Thr206Ser + Ile208Leu + Thr211Ser +Gly214Asn + Gly222Pro + Thr223GluPro204Gly + Gly205Ser + Ile208Ala + Trp212Leu +Thr217Gln + Arg218Asp + Thr223AsnIle208Asn + Thr211Asn + Trp212Val + Ile213Asp +Ile220Ala + Gly222Pro + Thr223ProPro204Gly + Ile208Cys + Trp212Cys + Ile213Gly +Thr217Gln + Ser219Asp + Ile220AlaGly205Pro + Ile208Asn + Thr211Pro + Trp212Phe +Ile220Met + Ser221Glu + Gly222SerGly203Gln + Pro204Ser + Thr206Ser + Ile208Val +Trp212Tyr + Ile220Gly + Ser221GluPro204Ser + Gly205Pro + Leu209Met + Trp212Leu +Ile213Asp + Gly215Gln + Gly222SerGly205Pro + Ile208Pro + Leu209Thr + Thr211Ser +Trp212Asn + Ser216Glu + Ile220AlaGly203Gln + Gly205Ser + Thr206Gly + Trp212Ala +Gly215Pro + Thr217Ser + Ser221GluGly203Ser + Ile208Cys + Leu209Met + Trp212Ser +Gly215Pro + Ser216Glu + Thr223ProGly205Ser + Thr206Gly + Trp212Gly + Thr217Gly +Arg218Glu + Ile220Gly + Gly222AsnGly203Ser + Gly205Pro + Thr206Gly + Gly214Gln +Ser216Asp + Ile220Gly + Thr223SerPro204Asn + Trp212Gln + Ile213Thr + Gly215Ser +Ser216Glu + Thr217Asn + Ile220AsnGly203Asn + Pro204Gly + Thr206Asn + Ile208Leu +Thr211Pro + Ile220Leu + Gly222ProGly203Asn + Gly205Asn + Thr211Ser + Trp212Met +Ile213Glu + Gly214Asn + Thr223AsnPro204Asn + Gly205Pro + Ile208Ser + Thr211Gln +Ile213Gln + Gly215Gln + Arg218GluGly203Gln + Gly205Gln + Thr206Ser + Ile208Val +Trp212Gln + Thr217Pro + Ile220GluPro204Gly + Gly205Pro + Ile208Met + Thr211Ser +Ile213Gly + Gly215Pro + Ser216GluGly203Gln + Gly205Ser + Thr206Ser + Ile208Ala +Ile213Ala + Ile220Met + Thr223GluPro204Gly + Thr206Gln + Leu209Ala + Thr211Gln +Thr217Asn + Arg218Glu + Ile220ProGly203Gln + Pro204Gly + Gly205Ser + Ser207Asp +Leu209Asn + Gly214Ser + Gly215ProGly203Gln + Gly205Gln + Leu209Met + Trp212Thr +Gly214Gln + Ile220Asn + Gly222GluGly203Ser + Thr206Asp + Ile208Leu + Thr211Pro +Gly214Ser + Gly215Ser + Gly222GlnPro204Gln + Thr206Gly + Ile208Asn + Trp212Gly +Ile213Pro + Thr217Pro + Arg218AspGly205Asn + Ile208Thr + Thr211Gly + Ile213His +Thr217Gly + Arg218Glu + Thr223AsnGly205Asn + Thr206Pro + Ile208Ala + Leu209Ala +Trp212Leu + Gly215Pro + Gly222AspGly203Pro + Thr206Gln + Ile208Asn + Leu209Cys +Gly215Glu + Gly222Pro + Thr223SerPro204Gln + Ile208Ala + Trp212Asn + Ile213Ser +Gly215Ser + Ser219Asp + Ile220AspGly203Ser + Thr211Ser + Trp212Met + Arg218Asp +Ser219Asp + Gly222Asn + Thr223AsnGly203Ser + Gly205Pro + Thr206Pro + Ile208Leu +Arg218Glu + Ser219Glu + Ile220AlaPro204Gly + Thr211Asn + Gly214Pro + Ser216Asp +Thr217Glu + Ile220Gln + Gly222ProGly203Asn + Gly205Pro + Leu209Val + Thr211Pro +Gly215Asn + Ser216Glu + Thr217AspPro204Gly + Gly205Gln + Thr211Ser + Trp212Met +Gly215Asp + Ser216Glu + Thr217ProPro204Asn + Thr206Gly + Ile208Met + Leu209Asp +Ser219Glu + Ile220His + Thr223ProGly205Gln + Thr206Glu + Ile208Thr + Leu209Thr +Thr211Gln + Trp212Cys + Ser221AspPro204Gly + Thr206Glu + Leu209Ala + Trp212Met +Ile220Val + Ser221Asp + Gly222SerGly205Gln + Thr206Glu + Thr211Asn + Trp212Ser +Thr217Gln + Ser221Asp + Thr223GlnSer207Glu + Leu209Thr + Ile213Gln + Gly214Gln +Gly215Asn + Ser219Glu + Ile220GluThr206Gln + Ser207Glu + Leu209Ile + Ile213Ala +Gly214Ser + Ile220Thr + Ser221AspPro204Asn + Thr206Gln + Ser207Glu + Ile208Ala +Leu209Cys + Thr211Gly + Ile220GluThr206Pro + Ser207Glu + Thr211Gln + Gly214Pro +Gly215Ser + Thr217Gly + Ser219GluPro204Asn + Ser207Glu + Leu209Gly + Thr211Asn +Gly215Asn + Ser219Glu + Ile220ValPro204Gln + Gly205Ser + Leu209Ser + Trp212Gly +Thr217Glu + Arg218Glu + Ser219AspGly203Gln + Ser207Asp + Leu209His + Gly214Ser +Gly215Gln + Ser219Asp + Ser221AspPro204Gln + Gly205Asn + Ser207Glu + Leu209Gln +Thr211Gly + Ser219Glu + Ser221AspGly203Asn + Thr206Glu + Ile208Gln + Leu209Gln +Thr211Pro + Trp212Ile + Gly222GluGly203Ser + Thr206Glu + Ser207Asp + Thr211Ser +Trp212Gly + Ile220Gly + Gly222AspGly205Pro + Thr206Asp + Ser207Asp + Ile213Pro +Gly214Ser + Gly222Glu + Thr223AsnGly203Gln + Thr206Asp + Leu209Met + Trp212Leu +
Gly214Asn + Gly222Asp + Thr223GluPro204Asn + Thr206Gln + Leu209Ser + Ser216Glu +Thr217Asp + Arg218Glu + Ser219AspPro204Asn + Thr206Gly + Ser207Glu + Ile208Thr +Gly215Gln + Ile220Asp + Gly222GluGly203Pro + Gly205Gln + Thr206Ser + Leu209Asp +Thr211Ser + Ile213Cys + Thr217AspGly205Gln + Ser207Glu + Thr211Ser + Trp212Ser +Ile213Gln + Arg218Asp + Ile220GluGly203Ser + Pro204Gly + Thr206Glu + Ser207Glu +Ile208Ser + Leu209Asp + Trp212AlaGly203Ser + Ile208Thr + Leu209Glu + Trp212Tyr +Thr217Gln + Ser219Asp + Ser221GluGly203Ser + Gly205Ser + Thr211Ser + Gly215Gln +Thr217Pro + Ser219Glu + Ser221AspGly205Gln + Thr206Gly + Leu209Ser + Ile213Gly +Ser219Glu + Ser221Asp + Thr223GlnPro204Gln + Ser207Asp + Ile208Ser + Ile213Leu +Arg218Glu + Ser219Glu + Ser221GluGly203Asn + Trp212Gln + Gly214Pro + Ser216Glu +Arg218Glu + Ile220Leu + Thr223ProPro204Asn + Gly205Gln + Thr206Gly + Ile213His +Gly214Ser + Ser216Asp + Arg218GluIle208Val + Ile213Met + Gly214Ser + Ser216Asp +Thr217Gly + Arg218Asp + Ile220MetPro204Ser + Gly205Asn + Ile208Leu + Leu209His +Ser216Asp + Arg218Glu + Thr223ProGly203Gln + Ile208Cys + Thr211Pro + Trp212Ser +Gly215Ser + Ser216Glu + Arg218GluGly203Pro + Gly205Ser + Leu209Ile + Thr211Asn +Thr217Glu + Ser219Glu + Ile220HisGly205Ser + Leu209Cys + Thr211Asn + Gly215Gln +Thr217Glu + Ser219Asp + Gly222ProGly203Gln + Ile208Ala + Trp212Ile + Gly215Gln +Thr217Glu + Ser219Asp + Gly222ProGly203Ser + Thr206Glu + Ser207Glu + Ile208Pro +Ile213Thr + Gly222Glu + Thr223AspGly203Ser + Ile208Gln + Trp212Ser + Ile213Leu +Arg218Asp + Ser219Glu + Ser221AspGly203Gln + Gly205Asn + Trp212Asn + Ile213Glu +Ser216Glu + Thr217Pro + Arg218AspThr206Asn + Leu209Asp + Ile213Met + Ser216Glu +Thr217Glu + Ser219Glu + Ile220GlyGly205Gln + Thr211Pro + Trp212Val + Gly214Ser +Ser216Asp + Arg218Glu + Ser219GluThr206Asn + Ser207Asp + Thr211Ser + Ile213Cys +Ile220Met + Ser221Asp + Thr223AspGly203Asn + Ile213Ser + Thr217Asp + Arg218Glu +Ser219Asp + Ser221Asp + Thr223GlnThr206Asn + Ser207Glu + Leu20gCys + Trp212Ser +Arg218Glu + Ser221Asp + Gly222ProGly203Gln + Ser207Asp + Gly214Pro + Gly215Pro +Arg218Asp + Ser221Asp + Gly222SerGly203Asn + Thr206Glu + Leu209Gln + Arg218Glu +Ser219Asp + Ser221Glu + Gly222ProGly203Pro + Ser207Glu + Trp212Ser + Gly214Pro +Ser219Glu + Ser221Asp + Thr223AspGly205Gln + Thr211Gln + Ile213Glu + Ser216Glu +Arg218Asp + Ser219Asp + Ile220AlaGly203Asn + Pro204Ser + Ser207Glu + Gly215Gln +Thr217Asp + Ile220Asp + Ser221AspGly203Gln + Gly205Asn + Ser207Asp + Leu209Ile +Thr211Gly + Thr217Asp + Arg218GluPro204Gly + Trp212Met + Gly214Asp + Ser216Asp +Thr217Glu + Ser219Glu + Thr223GlnSer207Asp + Trp212Tyr + Gly215Asn + Ser216Asp +Arg218Glu + Ser219Glu + Thr223ProThr206Gly + Ser207Asp + Ile208Gln + Ser216Glu +Arg218Glu + Ser219Asp + Ile220GlnSer207Glu + Leu209Gln + Thr211Pro + Gly215Pro +Ser216Asp + Arg218Glu + Ser219AspGly205Asn + Ser207Asp + Ile208Val + Gly214Pro +Ser216Asp + Arg218Glu + Ser219GluThr206Asp + Ile208His + Thr211Ser + Trp212Asn +Arg218Asp + Ser219Glu + Gly222ProIle208Gly + Trp212Met + Ile213Gln + Ser216Glu +Arg218Asp + Ser219Asp + Ser221AspGly203Asn + Thr206Glu + Leu209Asp + Thr217Pro +Ile220Ser + Gly222Glu + Thr223AspGly203Asn + Thr206Asp + Ile208His + Thr211Ser +Trp212Phe + Arg218Asp + Ser221GluPro204Asn + Gly214Asn + Gly215Pro + Ser216Glu +Thr217Asp + Arg218Asp + Ser221AspPro204Gln + Gly205Pro + Thr211Asn + Gly215Gln +Ser219Glu + Ser221Asp + Thr223GluSer207Asp + Leu209Val + Trp212Cys + Gly214Ser +Arg218Glu + Ile220Glu + Thr223AspThr206Asn + Ser207Asp + Ile208Ala + Gly215Pro +Arg218Glu + Ser221Glu + Thr223AspPro204Gln + Gly205Asn + Thr206Gln + Leu209Cys +Thr217Asp + Arg218Glu + Ser221GluGly203Pro + Thr206Ser + Ile213His + Ser219Glu +Ile220Cys + Gly222Glu + Thr223AspGly205Ser + Thr211Ser + Trp212Gln + Gly214Ser +Ser219Asp + Ile220Glu + Thr223AspSer207Asp + Leu209Gln + Ile213Gly + Ser216Glu +Ser219Asp + Ile220Ser + Ser221GluSer207Asp + Thr211Pro + Trp212Gln + Ile213Pro +Ser216Asp + Ser219Asp + Ser221AspPro204Gly + Gly205Pro + Ile208Gly + Leu209Glu +Ile213Thr + Thr217Asp + Ser221GluThr206Gln + Ser207Asp + Ile208Cys + Leu209Thr +Arg218Asp + Ser219Asp + Thr223AspGly203Pro + Ile208Asn + Thr211Gln + Ile213Met +Thr217Gln + Arg218Glu + Ser221AspGly203Asn + Gly205Asn + Thr206Gln + Thr211Gly +
Trp212Gln + Arg218Glu + Ser221GluGly203Ser + Ile208Pro + Gly215Pro + Arg218Asp +Ile220Asn + Ser221Glu + Thr223AsnThr206Pro + Thr211Pro + Gly214Ser + Gly215Pro +Arg218Asp + Ser221Glu + Thr223SerThr206Asp + Ser207Asp + Trp212Thr + Ile213Cys +Ser216Glu + Thr217Pro + Ser219AspGly205Asn + Thr206Asp + Ser207Asp + Ile213Thr +Ser216Glu + Ser219Glu + Ile220LeuThr206Ser + Ile213Met + Gly214Asp + Gly215Ser +Thr217Glu + Ser219Asp + Ile220GluPro204Asn + Thr206Pro + Leu209Glu + Thr217Gly +Arg218Glu + Ser219Asp + Thr223AspPro204Gly + Ser207Glu + Thr211Pro + Gly214Asp +Arg218Asp + Ser219Asp + Thr223GlnGly205Pro + Leu209Glu + Thr211Asn + Gly214Asp +Thr217Asp + Ile220Met + Gly222AsnPro204Ser + Trp212His + Ile213Asn + Gly214Glu +Thr217Glu + Ser219Glu + Gly222AsnGly203Gln + Ser207Glu + Ile208Cys + Gly215Pro +Ser219Glu + Ile220Asn + Thr223GluSer207Glu + Ile208Gly + Ile213Pro + Arg218Asp +Ile220Met + Gly222Asp + Thr223GluThr206Asp + Ser207Glu + Ile208Gly + Thr211Gln +Ser216Asp + Ser221Glu + Thr223AsnGly203Asn + Ser207Glu + Trp212Ser + Ile213Asp +Gly215Asn + Ser216Glu + Arg218GluGly203Asn + Leu209Ser + Ile213Val + Thr217Pro +Ser219Asp + Ile220Ala + Gly222GluIle208Met + Thr211Gly + Trp212Leu + Gly214Pro +Gly215Pro + Ser219Asp + Gly222GluSer207Glu + Leu209Ser + Ile213Ser + Gly214Asn +Ser216Asp + Arg218Glu + Ser221AspGly203Ser + Ser207Asp + Ile208Asn + Thr211Asn +Gly214Glu + Ser219Asp + Ile220AspSer207Asp + Thr211Asn + Ile213Ala + Gly215Asp +Thr217Ser + Ser219Glu + Ile220GluPro204Ser + Gly205Asn + Thr211Pro + Thr217Glu +Ser221Asp + Gly222Glu + Thr223GluSer207Glu + Leu209Ala + Gly214Pro + Ser216Glu +Thr217Ser + Arg218Glu + Ile220LeuIle208Val + Leu209Glu + Trp212Ser + Gly215Glu +Ser219Asp + Ile220Asp + Gly222ProGly203Gln + Ile213Ser + Gly215Glu + Ser216Asp +Ser219Asp + Gly222Ser + Thr223GlnGly205Pro + Leu209Val + Gly214Asn + Ser216Glu +Ser219Asp + Ile220Glu + Gly222AspPro204Asn + Leu209Asp + Ile213Met + Gly214Pro +Thr217Glu + Ser221Glu + Gly222AspGly203Ser + Ser207Glu + Trp212Pro + Gly214Asp +Ser216Asp + Thr217Asp + Ile220MetThr206Glu + Thr211Gln + Ile213Pro + Gly214Asp +Thr217Gly + Ser221Asp + Gly222AspIle208Cys + Leu209Ser + Thr211Asn + Ile213Ala +Gly215Asp + Arg218Glu + Ile220GlyIle208Thr + Leu209His + Ile213Met + Gly214Glu +Ser221Glu + Gly222Asp + Thr223AspThr206Gln + Gly214Gln + Gly215Glu + Thr217Gly +Arg218Glu + Ser219Glu + Ser221AspPro204Ser + Thr206Ser + Ile213Cys + Gly215Glu +Ser216Glu + Thr217Glu + Thr223AspThr206Gln + Ile208Met + Trp212Gly + Gly214Asp +Ser216Asp + Thr217Pro + Ser219AspSer207Glu + Ile208Thr + Thr211Gln + Trp212Gln +Gly215Asp + Ser219Asp + Ser221AspGly203Ser + Pro204Gln + Ser207Glu + Ile208Cys +Gly214Glu + Ser219Asp + Ser221GluSer207Asp + Ile208Thr + Thr211Asn + Gly215Glu +Ser219Glu + Ile220Met + Ser221GluPro204Ser + Ser207Glu + Gly214Ser + Gly215Asp +Ser219Asp + Ile220Met + Ser221GluSer207Asp + Ile208Leu + Trp212Ile + Ile213Asp +Thr217Asn + Arg218Glu + Ser221GluThr206Glu + Trp212Ser + Ile213Cys + Thr217Gly +Arg218Glu + Ser219Glu + Thr223GluThr206Asp + Ser207Glu + Trp212Cys + Ile213Cys +Gly215Asn + Ser216Asp + Arg218GluThr206Glu + Ser207Glu + Gly214Gln + Gly215Pro +Ser216Asp + Thr217Ser + Arg218AspPro204Ser + Gly205Ser + Trp212Pro + Ser216Asp +Ser219Asp + Ile220Val + Thr223SerGly203Gln + Leu209Gln + Trp212Ala + Gly215Asn +Arg2l8Glu + Ile220Glu + Thr223GluGly203Gln + Leu209Asp + Thr211Asn + Gly214Glu +Arg218Glu + Ile220Leu + Gly222SerGly203Ser + Pro204Gln + Thr211Pro + Gly215Asp +Arg218Glu + Ile220Glu + Thr223AsnThr206Asn + Ile208Gln + Ser216Glu + Thr217Gly +Arg218Asp + Ile220Gly + Ser221AspPro204Ser + Ile208His + Gly214Pro + Ser216Glu +Arg218Glu + Ser221Glu + Thr223SerPro204Gln + Leu209Ile + Trp212Ala + Ser216Asp +Thr217Ser + Arg218Asp + Ser221AspGly203Pro + Pro204Asn + Ser207Asp + Ile208Cys +Thr217Gly + Ile220Gly + Thr223GluPro204Ser + Ser207Asp + Leu209His + Thr211Ser +Gly215Asn + Gly222Ser + Thr223GluIle208His + Thr211Pro + Trp212Leu + Thr217Ser +Arg218Asp + Ser221Asp + Thr223GluGly205Asn + Thr206Asn + Ser207Asp + Ile213Thr +Ser216Asp + Ser219Asp + Gly222GluThr206Asp + Ser207Glu + Ile208Leu + Trp212Gly +Ser216Glu + Thr217Gln + Gly222AspGly205Asn + Thr206Asn + Ser207Asp + Ile208Gln +Trp212Cys + Ser216Glu + Thr217GluSer207Glu + Ile208Asn + Trp212Thr + Ile213Gln +
Ser216Asp + Thr217Glu + Thr223GlySer207Asp + Ile208His + Thr211Asn + Gly215Asn +Ser216Asp + Thr217Glu + Thr223GlyGly203Ser + Pro204Ser + Ile213Thr + Ser216Glu +Thr217Gly + Ser219Asp + Ser221GluGly203Asn + Ile208Ser + Thr211Asn + Ser216Asp +Ser219Glu + Ser221Asp + Thr223GlyGly203Ser + Thr206Pro + Thr211Gly + Trp212Thr +Ser216Glu + Ser219Glu + Ser221AspTrp212Ser + Gly214Pro + Gly215Asp + Thr217Asp +Ser219Glu + Ser221Asp + Thr223ProThr206Ser + Ser207Asp + Ile208Pro + Leu209Glu +Gly215Asp + Ile220Thr + Ser221GluGly203Asn + Trp212Tyr + Gly214Asp + Gly215Ser +Arg218Glu + Ile220Pro + Thr223SerPro204Ser + Ile208Thr + Thr211Gly + Trp212His +Gly214Asp + Thr217Pro + Arg218GluLeu209Glu + Thr21lPro + Ile213Cys + Gly214Glu +Arg218Asp + Ile220Asn + Ser221AspGly205Gln + Thr211Asn + Gly214Asp + Gly215Glu +Thr217Asp + Ile220Ala + Thr223GluGly205Gln + Thr211Gln + Ile213Asn + Gly215Asp +Thr217Glu + Ile220Glu + Ser221AspPro204Asn + Ile213Asp + Thr217Glu + Arg218Glu +Ile220Ser + Gly222Pro + Thr223GluGly205Gln + Ser207Glu + Ile208Cys + Gly214Asp +Ser216Glu + Ser219Asp + Thr223GlyGly203Ser + Ser207Glu + Gly215Asn + Ser216Asp +Thr217Gly + Arg218Glu + Gly222GluThr206Gln + Leu209Met + Gly214Glu + Gly215Asn +Ser219Glu + Ser221Asp + Gly222GluLeu209Met + Thr211Ser + Trp212Cys + Ser216Glu +Thr217Pro + Ile220Glu + Ser221GluThr206Gly + Thr211Gln + Trp212Cys + Ser216Asp +Ile220Glu + Ser221Asp + Thr223GlnGly203Ser + Pro204Asn + Ser207Glu + Ile213Asp +Thr217Ser + Ser219Glu + Gly222AsnGly205Pro + Thr206Asp + Trp212Leu + Gly214Asn +Ser216Glu + Ser219Asp + Gly222SerGly203Asn + Pro204Ser + Gly205Gln + Ser207Asp +Trp212Phe + Gly215Asp + Arg218Glu表29多環(huán)區(qū)雙突變變異體Leu 96Gly + Leu209ProTyr104Cys + Ile220GlnLeu133Ile + Ser216GluLeu 96Ala + Asn162GluGln103Asp + Ile220ThrTyr169Ser + Ser190AspVa1 95Asp + Gly205GlnAsp 65Glu + Ile213AsnGly100Ser + Ile220GluGly100Glu + Asn163GlnAsn162Ser + Ser207AspSer170Glu + Ile220SerSer140Glu + Ile220LeuGln103Glu + Thr106ProLeu133Pro + Leu209AsnGly203Asn + Gly222AspSer207Glu + Ile213LeuSer101Asp + Thr206GlyGly203Ser + Ser207GluAsn 99Glu + Trp212AsnGly102Pro + Ser216AspSer219Asp + Ile220MetGly 70Asp + Leu133SerGly136Asn + Ser216AspArg167Glu + Leu209ProAsn194Glu + Leu209GlyLeu209Gln + Ser216GluGly 68Asn + Ser221GluAsp 65Glu + Ilel07CysArg218G1u + Ile220AlaAsp 65Glu + Gly135ProGly 68Asp + Phe192HisGly 66Asn + Leu209SerAsn168Ser + Gly214ProThr 71Gly + Leu 96GlyGly 70Gln + Ile220GlyTyr169His + Leu209IleThr217Pro + Ser219AspGly 70Pro + Ile220AspLeu 96Gln + Asn 99GluAsp 65Glu + Gly136SerAsn 99Glu + Ile213MetGly205Pro + Ser207AspAsn 99Glu + Leu209GlnVal 95Gln + Ser207AspVal 95Gln + Ser219AspAsn 67Ser + Thr217GluGly160Gln + Leu209AspGly136Asn + Gly160AsnAsn163Gln + Thr223AsnLeu209Glu + Thr217AsnGly102Pro + Trp212GlnArg 64Asp + Gly203AsnAsn 67Gln + Thr211AsnAsp 65Glu + Tyr137ProGln103Asp + Leu133IleLeu 96Glu + Ile213GlnAsn161Gln + Arg218GluLeu209Asn + Gly214ProGly 68Ser + Thr206AsnArg167Asp + Phe192IleAsp 65Glu + Thr211SerSer207Asp + Thr223GlySer207Asp + Gly222SerTyr104Leu + Arg167GluAsp 98Glu + Ile220AsnArg 64Glu + Gly215AsnAsn 67Asp + Asn163SerGly160Ser + Thr206GlnIle213Ser + Thr223AspGly215Gln + Ser219AspPhe202Leu + Ser219GluSer207Asp + Ile220CysTyr137Ser + Ser219AspGly100Asn + Leu209ThrTyr137His + Ser170AspSer170Asp + Ile220ThrThr106Gln + Ser207AspArg167Asp + Leu209ProSer105Asp + Gly136AsnSer139Asp + Phe192ThrSer190Glu + Pro204SerAsn 67Glu + Leu209AlaTrp212Ser + Ser216GluTyr104Ala + Thr206AspAsn161Gln + Gly222AsnVal 95Thr + Leu133SerGly160Ser + Asp165GluAsn163Ser + Leu209ValGly136Glu + Ile220ProSer191Asp + Pro204SerThr217Glu + Ile220AsnLeu209Met + Gly214AspThr 71Pro + Ser191GluSer101Glu + Trp212PheGly 70Ser + Ser219GluGly 66Gln + Asn 99SerAsn163Gln + Ser190GluIle107Ser + Ser190AspIle208Thr + Ser219AspSer216Asp + Ile220LeuSer140Asp + Ala164HisAsp 65Glu + Val 95SerPhe202Met + Leu209SerAsn 67Gln + Tyr169GlyGly136Ser + Asn162AspGly160Pro + Ser219AspGly102Pro + Ser207AspVal 95Asn + Ser207AspAsp 97Glu + Ile107GlnThr206Glu + Ile220ProSer216Glu + Thr223GlyAsp 97Glu + Leu133CysGly134Gln + Gly135AspThr206Gln + Ile220HisGly100Asn + Thr223AspGly 66Pro + Ile220ValGly 66Asn + Asn168GluSer190Asp + Ile213ValGly135Pro + Asn162GluLeu133Ile + Gly134ProAsn163Ser + Arg167GluGly134Gln + Ser216GluAsn194Gln + Leu209GlyAsn 99Gln + Ile213MetLeu 96Ile + Gly160AspArg167Glu + Ile220AlaVal 95Ala + Ser139AspGln103Ser + Gly214SerAsn 67Glu + Thr 71GlnGly135Pro + Ser191GluIle107Ala + Ile220GluGly214Gln + Ser219GluGly160Asp + Phe192MetLeu 96Ala + Asp 97GluAsn168Gln + Leu209GluGln103Glu + Gly136AsnGly215Asp + Gly222SerSer207Asp + Thr223AsnSer216Asp + Ile220CysVal 95Ser + Thr206GluGly 70Asn + Ser216AspLeu209Ser + Ile220AspAla164Pro + Ile208ThrTyr137Thr + Leu209GluThr106Asp + Tyr137ThrThr106Gly + Asp165GluAsn 99Asp + Ile213GlyAsn161Glu + Ile220SerGly160Asp + Ile220MetGly135Asp + Asn163GlnTyr169Ser + Ser216GluSer140Asp + Leu209AsnSer140Asp + Leu209GlnSer170Glu + Gly222ProIle213His + Ser216AspLeu 96Thr + Gly222AsnAsp 65Glu + Asn168GlnTyr104Leu + Ser219GluThr106Pro + Gly134ProAsn 99Asp + Gly215AsnThr206Glu + Ile220ValGly 70Pro + Leu 96GluThrl06G1y + Ser219GluGly100Glu + Ile107GlyGly102Gln + Tyr169GlyArg 64Glu + Pro204GlyLeu133Gln + Ser207AspGly100Gln + Leu133HisThr106Asp + Leu209AlaGly 68Glu + Ile220GlnAsn 67Asp + Gly 68GlnLeu 96Glu + Ile220LeuThr206Gln + Ser219AspAla164Gln + Gly222GlnGly 66Glu + Gly 68ProSer140Asp + Ala166AsnAsn 67Ser + Gly100AspSer219Asp + Ile220ThrSer170Glu + Gly215SerThr 71Gly + Phe192IleGly100Ser + Ser219AspGly215Pro + Ser219AspLeu209Met + Ser221AspAsn162Asp + Tyr169MetTyr104His + Asn162GluAsn 67Gln + Leu 96SerLeu209Asp + Ile220AlaGly 70Ser + Ile220GlnGly 66Ser + Gly 70GluTyr169His + Thr206GlyAsn194Gln + Thr206GlyIle107Glu + Leu209AsnGly 66Ser + Gly222GluLeu133Ile + Ser190GluSer105Asp + Ile220HisGly102Asp + Asn194GlnLeu 96Met + Ser216AspAsn161Asp + Gly203GlnGly136Gln + Asn161AspPhe202Ile + Ser219AspGln103Asp + Ile107ThrGly100Gln + Ser216AspPhe192Ala + Ser207GluGly136Pro + Ser138AspAsp 98Glu + Leu209ThrAsn194Ser + Ser216GluVal 95Asp + Gly222GlnThr211Pro + Ile220MetArg167Glu + Tyr169AlaGly 66Asn + Ser138AspAsn 67Glu + Leu209ProGly 70Glu + Tyr169ProGly102Pro + Leu209AsnGly 68Ser + Ser207AspSer101Glu + Ile220SerTyr137Val + Ser191GluThr211Gly + Arg218AspAsn161Gln + Asn194GlnThrl06Asn + Leu209GlnVal 95Ser + Gly134AspSer191Asp + Ile220SerAsn163Ser + Thr223GluLeu209Thr + Arg218AspGly 70Asp + Val 95GlyLeu209Met + Ser216AspThr206Ser + Arg218GluLeu209Ile + Thr217AspIle213Pro + Ser219GluGly205Ser + Thr206AspPhe192Gln + Thr206GluGly 68Asp + Leu209SerGly160Gln + Thr206GlyGly214Asp + Ile220LeuGly 70Asn + Arg218AspGly 70Glu + Gly136AsnLeu133Glu + Gly203ProGly135Gln + Ser207GluIle107Gly + Ile213GlnSer140Asp + Ile220LeuSer191Asp + Gly214GlnLeu209Ser + Gly214AspAsn 67Glu + Ile220GlnAla166Pro + Ser219AspGly102Ser + Ile220ValGln103Ser + Ser140GluGly102Asp + Ile220GlyTyr104Ile + Gly134GlnSer101Glu + Ile220CysAsn162Gln + Ser207AspVal 95Pro + Ser207AspAsn168Gln + Leu209AlaGly100Gln + Gly136AsnPhe192Pro + Asn194GluAsn 99Ser + Thr211GlnGly215Pro + Ile220AspArg 64Glu + Leu133IleSer207Glu + Ile220CysAsp 97Glu + Thr206AsnThr106Asp + Tyr137MetSer138Glu + Thr223ProSer138Asp + Ile220AlaSer191Asp + Leu209AsnLeu133Ile + Ser170GluIle107Cys + Ser139GluAsn161Gln + Ser207AspThr206Asn + Ser216GluAsn 67Gln + Ser207AspLeu209Met + Ser219GluGly 68Ser + Asp 97GluGly136Glu + Gly222AsnThr106Pro + Leu209ValGln103Glu + Ile213AlaLeu 96Gln + Ser219GluIle107Asp + Ala164GlnTyr169Asp + Thr211ProLeu133Glu + Leu209ValAsp 97Glu + Asn161SerIle213Gly + Ser216AspThr206Pro + Ser207AspAla164Gln + Leu209CysSer170Glu + Leu209AlaGly160Pro + Leu209CysAsn 67Asp + Asn163GlnThr206Gly + Ser216AspLeu133Thr + Ser216GluAsn163Asp + Leu209ThrVal 95Met + Phe202SerGly136Ser + Gly222SerLeu209Met + Ile220MetGln103Ser + Asn163GluIle107His + Arg218AspPhe192Ala + Gly215GlnLeu209Thr + Ser216GluTyr169Glu + Thr217SerSer101Asp + Leu209AlaLeu209Ala + Ser221AspGly 66Asp + Thr106GlnVal 95Glu + Ile213CysSer105Asp + Thr211GlnAsp165Glu + Asn168SerGly 66Asn + Ile220ThrSer219Glu + Ile220ProGly 68Glu + Ile220MetVal 95His + Leu209MetGly 66Ser + Leu209MetSer140Glu + Gly214GlnGly160Asn + Leu209AspThr206Asp + Ile220MetGly102Asn + Thr206GluGly134Pro + Phe192ProLeu133Asp + Phe202SerAsn162Glu + Ile220HisLeu133Gly + Leu209MetAsn194Ser + Ser219AspGly 70Gln + Thr217ProAsp 97Glu + Thr217GlnGly100Asp + Leu209CysSer216Asp + Ile220ThrArg167Glu + Asn194SerGly 66Gln + Ser170GluSer101Asp + Tyr137AlaAsn 67Gln + Asn161AspAsn168Asp + Leu209IleAsn 67Gln + Gly 68GluAsn161Asp + Thr217ProThr206Asp + Leu209MetAsp 97Glu + Leu209MetAsp 97Glu + Tyr104HisAsp 98Glu + Gly102ProTyr104Ala + Ser191GluGly100Ser + Arg167GluSer207Asp + Leu209SerAsn194Ser + Ser207AspLeu 96Val + Ile220CysAsn163Gln + Ser191AspAsn162Gln + Asp165GluGly 66Gln + Thr 71AsnTyr137Leu + Phe192AsnGly 66Asn + Ser207GluAsp 97Glu + Leu133SerThr206Gln + Ser216AspLeu 96Pro + Ile213GluAsp165Glu + Gly214AsnSer190Glu + Thr217GlyTyr104Met + Ile220GluAsp 65Glu + Thr223SerGly100Asn + Ser170GluGly134Ser + Leu209CysAla164Gly + Ser207AspThr 71Asn + Ile220AspIle107Cys + Ser219AspThr106Gly + Asn162AspAsn161Glu + Leu209ThrGly214Gln + Ser221AspAla164Gln + Ser207GluAsn 67Ser + Ser216AspLeu209Ala + Thr217SerAla166Gln + Ser216AspLeu209Gln + Ile220Glu表33多環(huán)區(qū)三突變變異體Leu 96Gly + Leu209Pro + Ser216GluArg 64Glu + Tyr104Cys + Ile220GlnTyr169Ser + Ser190Asp + Ile220ThrGly100Glu + Asn162Ser + Asn163GlnGln103Glu + Leu133Pro + Leu209AsnGly 70Asp + Leu133Ser + Gly136AsnGly 68Asn + Leu209Gln + Ser216GluAsp 65Glu + Gly135Pro + Ile220AlaGly 66Asn + Asn 67Glu + Leu209SerThr 71Gly + Leu 96Gly + Gly214ProGly 70Gln + Leu209Ile + Ile220GlyTyr169His + Thr217Pro + Ser219AspAsn 67Ser + Val 95Gln + Thr217GluGly136Asn + Gly160Gln + Leu209AspGly136Glu + Asn163Gln + Thr223AsnArg 64Asp + Gly203Asn + Trp212GlnGly 68Ser + Leu209Asn + Gly214ProArg167Asp + Phe192Ile + Thr206AsnSer207Asp + Gly222Ser + Thr223GlyGly160Ser + Asn163Ser + Thr206GlnGly100Asn + Tyr137Ser + Ser219AspAsn 67Asp + Gly100Ser + Tyr137HisSer105Asp + Gly136Asn + Phe192ThrVal 95Thr + Leu133Ser + Asn161GlnSer140Glu + Asn163Ser + Leu209ValPro204Ser + Thr217Glu + Ile220AsnIle107Ser + Ile208Thr + Ser219AspAsp 65Glu + Val 95Ser + Leu209SerAsn 67Gln + Tyr169Gly + Phe202MetGly136Ser + Gly160Pro + Asn162AspVal 95Asn + Ile107Gln + Ser207AspGly135Asp + Thr206Gln + Ile220HisGly 66Pro + Gly100Asn + Ile220ValGly135Pro + Asn162Glu + Ile213ValLeu133Ile + Gly134Pro + Arg167GluGly134Gln + Asn163Ser + Ser216GluAsn194Gln + Ser207Glu + Leu209GlyArg 64Glu + Asn 99Gln + Ile213MetTyr104Ser + Arg167Glu + Ile220AlaVal 95Ala + Gln103Ser + Ser139AspIle107Ala + Gly214Gln + Ile220GluGln103Glu + Gly136Asn + Asn168GlnThr206Pro + Ser216Glu + Ile220CysAsn 67Asp + Ala164Pro + Ile208ThrAsp 65Glu + Asn168Gln + Gly222AsnTyr104Leu + Leu209Ser + Ser219GluIle107Pro + Asn194Glu + Thr206GlyLeu133Asn + Leu209Ile + Ile213AspGly 70Pro + Thr106Gly + Ser219GluAsp 65Glu + Gly102Gln + Tyr169GlyGly100Gln + Leu133Gln + Gly160AspAla164Ser + Ser216Asp + Thr217GlyGly 68Glu + Leu209Ala + Ile220GlnGly 66Glu + Ala164Gln + Gly222GlnThr 71Gly + Phe192Ile + Gly215SerAsn 67Gln + Leu 96Ser + Leu209AspArg 64Glu + Gly 70Ser + Ile220GlnGly 66Ser + Tyr169His + Thr206GlySer170Glu + Thr206Gly + Gly215SerGly 66Ser + Ile107Glu + Leu209AsnSer105Asp + Asn194Gln + Ile220HisLeu 96Met + Asn161Asp + Gly203GlnGly136Gln + Asn161Asp + Phe202IleGly100Gln + Phe192Ala + Ser216AspThr211Pro + Ile220Met + Gly222GlnGly 68Ser + Thr106Gln + Ser207AspAsn161Gln + Thr211Gly + Arg218AspThr106Asn + Asn194Gln + Leu209GlnAsp 65Glu + Tyr169Val + Ile220ValGly 70Asp + Val 95Gly + Leu209MetGly205Ser + Thr206Asp + Ile213ProPhe192Gln + Thr206Glu + Leu209SerGly160Gln + Thr206Gly + Gly214AspGly 70ASn + Arg218Asp + Ile220LeuGly 70Glu + Gly136Asn + Gly203ProIle107Gly + Ile213Gln + Ser219AspGly102Ser + Ala166Pro + Ile220ValTyr104Ile + Gly134Gln + Ile220CysVal 95Pro + Asn162Gln + Ser207AspGly100Gln + Gly136Asn + Asn194GluAsn 99Ser + Thr211Gln + Ile220AspArg 64Glu + Leu133Ile + Ile220SerAsp 97Glu + Thr206Asn + Ile220CysIle107Asn + Thr206Asn + Ser207GluAsn 67Gln + Leu 96Gln + Ile213AspGly 68Ser + Leu209Met + Ser219GluTyr169Asp + Leu209Val + Thr211ProThr206Pro + Ile213Gly + Ser216AspAla164Gln + Ser207Asp + Leu209CysAsn 67Asp + Gly160Pro + Leu209CysAsn163Gln + Thr206Gly + Ser216AspVal 95Met + Phe202Ser + Leu209ThrGly136Ser + Ile220Met + Gly222SerGln103Ser + Asn163Glu + Leu209MetIle107His + Phe192Ala + Arg218AspSer101Asp + Leu209Ala + Thr217SerGly 66Asn + Asp165Glu + Asn168SerGly 66Ser + Leu209Met + Gly214GlnGly134Pro + Ser140Glu + Phe192ProLeu133Gly + Ser207Glu + Leu209MetGly 70Gln + Asn163Asp + Asn194SerGly100Asp + Leu209Cys + Thr217GlnGly 66Gln + Ser101Asp + Tyr137AlaAsn 67Gln + Asn168Asp + Leu209IleAsn161Asp + Leu209Met + Thr217ProGly100Ser + Ser207Asp + Leu209SerLeu 96Val + Asn194Ser + Ser207AspAsn163Gln + Ser191Asp + Ile220CysGly 66Gln + Thr 71Asn + Ser216AspGly 66Asn + Tyr137Leu + Phe192AsnAsp 97Glu + Leu133Ser + Ile220MetLeu 96Pro + Thr206Gln + Ile213GluAsp165Glu + Gly214Asn + Thr217GlyAsp 65Glu + Gly100Asn + Thr223SerGly134Ser + Ser170Glu + Leu209CysThr 71Asn + Ile107Cys + Ser219AspThr106Gly + Asn162Asp + Leu209ThrAsn 67Ser + Ala164Gln + Ser207GluAla166Gln + Leu209Ala + Ser216AspLeu 96Gly + Leu133Ser + Ser190GluSer207Asp + Leu209Thr + Ile220ProTyr137Thr + Ser139Glu + Gly215ProThr206Gly + Leu209Asp + Thr211AsnThr206Asp + Ile208Val + Ile220LeuLeu 96Cys + Ser207Glu + Ile220LeuIle107Leu + Gly136Glu + Ile220GlyThr106Asn + Ser139Glu + Ala164ThrSer191Glu + Leu209Ser + Ile220GlySer140Glu + Gly215Ser + Gly222ProLeu 96Ser + Ala164Gly + Ile220HisAsp 97Glu + Tyr104Asn + Tyr137GlnGly 70Gln + Asp 98Glu + Leu133ThrGly 66Ser + Thr 71Gly + Phe202SerSer101Asp + Thr206Gly + Ile213GlyTyr169Ile + Ser170Glu + Thr211GlyGly160Ser + Ile213Val + Ser219GluLeu209Gly + Ile220Gly + Thr223ProThr106Gln + Ala164Gln + Phe192GluAsn 99Glu + Leu209Gln + Ile220MetGly160Gln + Asn194Glu + Gly215GlnSer105Asp + Phe202Pro + Leu209GlnGly 66Glu + Leu133Thr + Leu209AsnAla164Thr + Leu209Met + Ile220ThrThr 71Gly + Gly214Asp + Gly222AsnVal 95Pro + Thr206Gly + Ile213AspLeu 96Asn + Gly100Asn + Asn194SerArg 64Glu + Gly100Asn + Ile220MetGly 66Ser + Arg218Asp + Gly222SerAsp 65Glu + Asn162Gln + Ile220ThrAsn 99Asp + Thr206Ser + Ile220SerLeu 96Ser + Tyr169His + Ser216AspAsp 98Glu + Tyr169His + Gly215AsnTyr104Leu + Ser207Asp + Leu209SerAsn161Ser + Phe192His + Thr206ProLeu 96Ser + Ser170Asp + Leu209IleThr106Gln + Asn163Gln + Ser207AspGly 70Gln + Asn 99Ser + Asn161SerAsn 99Ser + Ala164Asp + Thr217ProSer139Glu + Thr206Asn + Ile213LeuLeu 96Asn + Thr217Asn + Ile220HisGln103Ser + Leu209Pro + Ser219GluAsp 65Glu + Leu 96Cys + Gln103AsnAsn 67Gln + Ser207Asp + Gly214AsnGly 68Pro + Thr211Ser + Thr223AspTyr137Gln + Ser138Glu + Ile220AsnAsp 65Glu + Phe192Val + Gly215SerThr 71Gln + Gly203Gln + Thr206AspArg 64Asp + Asn 99Ser + Thr106SerGly135Gln + Asn168Ser + Phe192AspLeu133Ala + Leu209Ala + Ser219GluAla164Thr + Asn168Ser + Ile213ProSer207Glu + Gly214Gln + Ile220AsnPhe192Tyr + Leu209Pro + Gly215GlnAsn163Gln + Thr206Gly + Leu209AsnLeu 96Pro + Asp 98Glu + Asn163GlnSer101Asp + Asn168Gln + Tyr169ValGly136Pro + Asn162Glu + Ile220ThrLeu209Cys + Gly214Gln + Ile220CysArg 64Asp + Leu209Ile + Ile220HisGly134Pro + Gly135Ser + Ser219GluAla164Thr + Leu209Thr + Ser219GluTrp212Phe + Ser219Glu + Ile220GluLeu 96Glu + Asp 97Glu + Ile220LeuLeu 96Glu + Asp 97Glu + Gly222ProAsn161Asp + Asn162Glu + Gly214SerGly160Asp + Asn161Glu + Ile220SerTyr137Val + Thr206Glu + Ser207AspGly 68Pro + Arg218Glu + Ser219GluLeu209Thr + Arg218Glu + Ser219AspTyr104Gln + Ser216Asp + Thr217AspLeu209Glu + Thr217Asn + Ser219AspLeu209Asp + Ser219Glu + Ile220SerAsn194Gln + Ser207Asp + Ser221GluAla164Gly + Ser207Asp + Ile220AspLeu 96Val + Ser207Asp + Ile220AspLeu 96Glu + Gly102Glu + Ile213GlnGly 68Asn + Ser207Glu + Ser219GluSer207Asp + Ser219Glu + Ile220CysLeu 96Gln + Ser207Glu + Ser219GluGly102Pro + Ser207Asp + Ser219AspGln103Ser + Ser207Asp + Ser219GluVal 95Gln + Ser207Asp + Ser219AspSer207Asp + Ile213Pro + Ser219GluAsp 65Glu + Asn 67Asp + Ala164HisAsp 65Glu + Asn 67Glu + Gly136SerAsn162Gln + Asp165Glu + Arg167GluLeu209Met + Gly214Asp + Ser216GluSer138Asp + Ser170Asp + Ile220MetSer138Glu + Asn168Gln + Ser170GluArg 64Glu + Gly 66Asp + Thr217ProAsp165Glu + Asn168Asp + Thr211AsnLeu 96Glu + Asp 98Glu + Gly222SerThr206Ser + Ser216Asp + Arg218GluAla164Ser + Ser216Glu + Arg218GluAsn161Asp + Asn162Asp + Thr223AspLeu209Ile + Thr217Asp + Ser219GluSer101Asp + Gln103Asp + Leu133IleAsn 67Glu + Asp 97Glu + Leu209AlaLeu133Ile + Ser190Glu + Gly222GluSer207Asp + Ser219Glu + Gly222AspAla164Asp + Asn168Glu + Leu209ProAsp 65Glu + Gly 68Glu + Asp 97GluAsn163Ser + Thr206Asp + Ser219GluArg 64Glu + Asn 67Asp + Gly215AsnTyr137Asp + Ser139Glu + Arg167AspSer140Asp + Ser170Glu + Gly222SerTyr137Glu + Ser140Asp + Leu209GlnArg 64Glu + Asp 97Glu + Tyr104HisVal 95Glu + Ser105Asp + Thr211GlnGly100Asn + Asn161Glu + Ser221AspAsp 65Glu + Asn 99Glu + Trp212AsnAsp 65Glu + Asn 99Asp + Thr217SerSer190Glu + Ser207Glu + Ile220GluGly 68Glu + Gly136Pro + Ser216GluVal 95Glu + Asp 98Glu + Gly102ProAsn162Asp + Asp165Glu + Ser191GluVal 95Cys + Ser138Asp + Arg167GluLeu209Asp + Ser216Glu + Ile220AlaGly100Glu + Gln103Asp + Ile107GlyGly100Asp + Gly102Pro + Gln103GluSer216Asp + Ser219Glu + Ile220ThrGly102Pro + Ser216Asp + Ser219AspTyr104Gln + Ser216Glu + Ser219GluAsn 99Gln + Ser216Glu + Ser219AspThr206Gln + Ser216Asp + Ser219AspSer207Asp + Ser216Asp + Thr217AspGly134Asp + Asn162Glu + Phe192GluGly102Glu + Ser105Asp + Leu133GluSer216Asp + Ser219Asp + Ser221AspAsn163Asp + Ser170Glu + Asn194AspTyr137Glu + Asn162Glu + Arg167GluSer105Asp + Gly135Asp + Tyr169AspVal 95Asp + Asp 98Glu + Ser105GluGly136Asp + Ser140Asp + Gly203GlnSer207Asp + Ser216Glu + Ile220AspThr106Gly + Tyr137Asp + Asp165GluAsp 65Glu + Gly100Glu + Gln103AspSer101Glu + Arg167Glu + Asn168GluAsn 67Asp + Ser191Asp + Phe192GluAsp 98Glu + Asn 99Glu + Ser216AspAsp 98Glu + Asn 99Asp + Ser216AspArg 64Glu + Asp 65Glu + Asp165GluGly102Glu + Gln103Glu + Ser221GluLeu133Glu + Thr206Asp + Ser207GluSer139Asp + Ser216Asp + Thr217AspAsp165Glu + Gly215Asp + Ser216AspAsp 97Glu + Leu209Glu + Ser219GluSer191Glu + Leu209Glu + Ser219GluThr106Asp + Leu209Asp + Ser219AspAsn 99Glu + Ile213Asp + Ser216GluSer207Glu + Leu209Glu + Ile213AspAsn 67Glu + Asp 98Glu + Leu209AspSer101Asp + Ser207Asp + Ser221AspGly135Asp + Ser207Asp + Ser219GluAsn162Glu + Ser207Asp + Ser219GluSer139Glu + Ser207Glu + Ser219GluGly134Glu + Ser170Glu + Ser216AspAsp165Glu + Arg167Glu + Ser207AspAsp165Glu + Arg167Asp + Ser219AspAsp 65Glu + Ser138Glu + Ser140AspAsp 97Glu + Arg167Glu + Tyr169AspSer207Glu + Ile213Glu + Ser216GluThr 71Pro + Ser191Glu + Ser207GluSer191Asp + Ser207Asp + Gly214GlnGly 66Glu + Gly 70Glu + Ser216GluAsn163Asp + Ser191Asp + Ile220GluAsp 97Glu + Ser207Glu + Leu209GluTyr104Asp + Ser207Glu + Leu209GluSer190Asp + Thr206Glu + Ser216AspAsp 97Glu + Leu133Glu + Asn161SerAsn168Asp + Ser170Asp + Ser190AspAsp 98Glu + Ser221Asp + Thr223GluAsn 67Asp + Asp 97Glu + Gly135GluAsn 67Asp + Asp 97Glu + Ser219AspSer105Glu + Gly160Asp + Gly222AspAsp 98Glu + Gly160Asp + Asn163GluSer139Glu + Ser170Glu + Arg218AspGln103Glu + Ser207Asp + Gly222GluSer207Glu + Ser216Asp + Gly222AspGly135Glu + Gly160Glu + Ser216GluTyr104Glu + Asn163Asp + Ser190GluSer190Glu + Asn194Asp + Ser219AspTyr104Met + Ser190Glu + Ile220GluAsn 67Asp + Ser216Asp + Ser219GluLeu133Asp + Asn162Glu + Phe202SerAsn162Glu + Ser191Asp + Leu209GluAsp 65Glu + Leu 96Asp + Asn162AspArg 64Asp + Gly136Glu + Arg167AspAsn 67Asp + Gly 68Gln + Ser216GluAsn 67Glu + Ser216Glu + Ile220CysSer101Glu + Thr206Asp + Leu209GluAsp 65Glu + Leu209Glu + Ser216GluSer101Glu + Ile107Glu + Ser219GluGly 68Asp + Ser170Glu + Ser216AspGly136Asp + Ser139Glu + Ser216GluGly135Asp + Leu209Ser + Thr223AspLeu133Glu + Asn161Glu + Gly214GluArg 64Glu + Ser191Glu + Ser221AspAsn 67Glu + Gly214Asp + Ser219GluThr106Glu + Ser219Glu + Gly222GluAsp 98Glu + Ile220Asp + Thr223GluArg167Asp + Gly215Glu + Arg218AspTyr104Asp + Ser139Glu + Ser219AspIle107Gly + Asn161Asp + Ile220AspGly160Glu + Ser216Asp + Ser219AspAsp165Glu + Ser216Glu + Ser219AspAsp 98Glu + Ser207Asp + Thr223AspSer139Asp + Asn168Glu + Ser207GluSer105Asp + Ser138Glu + Ile220GluSer105Glu + Ser138Asp + Ser219AspSer105Asp + Ser138Asp + Gly222AspIle213Pro + Ser216Glu + Ile220GluSer101Glu + Gly214Glu + Arg218GluGly136Glu + Asp165Glu + Ser190AspAsp 65Glu + Leu209Cys + Ser216AspSer138Asp + Asn161Glu + Thr206GluGly136Asp + Ser140Asp + Ser219GluGly 68Glu + Gly134Glu + Ser219AspGly 68Asp + Asn194Glu + Ser219AspGly 68Glu + Asn161Glu + Ser219AspGly 68Asp + Ile107Asp + Ser219GluGly 68Glu + Ser139Asp + Ser219AspAsp 98Glu + Gly135Asp + Ser139GluGly135Glu + Ser139Glu + Leu209GluAla164Glu + Ser190Asp + Ser219GluGln103Asp + Tyr137Asp + Gly160SerGly 70Glu + Gly102Asp + Tyr169ProGly 70Glu + Ser219Glu + Gly222AsnGly 70Glu + Leu209Met + Ser221AspIle213Ser + Ser219Asp + Thr223AspGly135Asp + Ser140Glu + Gly215AspGly102Glu + Ser170Glu + Thr206AsnGly102Glu + Gly135Asp + Thr206GluGly 70Glu + Asp165Glu + Ile220AspPhe192Asp + Ser207Asp + Ile213GluAla164Glu + Arg218Asp + Thr223AspGly135Asp + Ser190Glu + Ile220GluAsn 99Glu + Leu133Asp + Asp165GluAsp165Glu + Ser219Glu + Thr223AspAsn 67Glu + Asn162Asp + Ser216Glu表34多環(huán)區(qū)四突變變異體Tyr104Cys + Leu133Ile + Ser216Glu + Ile220GlnGly102Asn + Gly160Ser + Ala164Gly + Arg218AspAsn 67Glu + Thr206Gln + Leu209Met + Ile220MetGln103Glu + Thr106Pro + Leu133Pro + Leu209AsnVal 95His + Gly136Asn + Gly160Gln + Gly222GluArg 64Asp + Gly102Pro + Gly203Asn + Trp212GlnVal 95Thr + Leu133Ser + Asn161Gln + Gly222AsnGly 66Gln + Gly 70Ser + Asn 99Ser + Ser219GluAsp 65Glu + Gly 68Asn + Tyr169Val + Ile220ValPhe192Gln + Gly205Ser + Thr206Glu + Leu209SerLeu133Val + Asn162Asp + Leu209Val + Ile220AlaLeu 96Thr + Leu209Ile + Ile220Val + Thr223SerGly100Glu + Gly102Asn + Gly134Ser + Ile208ProGly100Ser + Asn194Gln + Ser207Glu + Ile220ThrAsn 67Gln + Ala164Gln + Tyr169Asp + Ile220ThrGly160Ser + Ser191Glu + Ile220Ser + Gly222SerThr206Pro + Ser207Asp + Leu209Cys + Ile213GlyVal 95Met + Gly136Ser + Phe202Ser + Gly222SerGln103Ser + Asn163Glu + Leu209Met + Ile220MetIle107His + Phe192Ala + Gly215Gln + Arg218AspGly 66Asn + Asp165Glu + Asn168Ser + Ile220ThrGly100Asn + Ser140Glu + Leu209Met + Gly214GlnGly 70Gln + Asn163Asp + Asn194Ser + Thr217ProAsn161Ser + Phe192His + Thr206Pro + Ser219AspVal 95Cys + Leu 96Ser + Ser138Asp + Leu209IleGly 68Gln + Ser101Asp + Thr106Pro + Ile220MetGly100Pro + Gly102Gln + Leu209Cys + Gly215AspGly100Asn + Gln103Ser + Ser207Asp + Leu209ValThr106Gln + Ile107Asn + Phe192Val + Thr223GlyTyr137Gln + Ser138Asp + Leu209Val + Trp212AsnLeu133Ala + Asn162Asp + Tyr169Val + Leu209IleGly102Ser + Gly134Gln + Gly136Asn + Ser207GluGly100Gln + Gly134Pro + Gly160Asn + Ser219GluTyr137Met + Asn194Gln + Ser207Asp + Gly215ProAla164Asn + Ser190Asp + Phe202Ser + Ile213AsnGly 68Pro + Asp 98Glu + Ala166His + Asn168SerIle107Pro + Gly136Pro + Ser207Glu + Gly222GlnArg 64Asp + Tyr169Gly + Phe192Pro + Gly205SerVal 95Thr + Tyr104Cys + Gly134Asp + Ile208AsnIle107His + Gly135Glu + Phe192Gly + Thr217ProIle107Gly + Ser191Glu + Leu209Met + Thr223GlnGly 68Gln + Val 95Ala + Ser207Glu + Leu209AsnGly 66Asn + Asp 97Glu + Asn161Ser + Gly214AsnTyr137Met + Leu209Ser + Ser216Asp + Ile220ProVal 95Asn + Leu209Val + Ser219Asp + Thr223ProGly135Asn + Ser140Glu + Ala164Thr + Ile220LeuGly 70Ser + Gly135Asp + Thr217Gln + Ile220ThrGly 66Ser + Ser140Asp + Leu209Pro + Ile213LeuVal 95Asn + Leu 96Ile + Thr217Pro + Ile220GlyLeu 96Met + Gly203Asn + Ser219Glu + Ile220GlyTyr104Cys + Tyr169Ser + Leu209Thr + Ser216GluGly 70Gln + Ser138Asp + Thr206Asn + Ile220GlnAsn163Gln + Gly214Pro + Ile220Asn + Thr223GluGly 68Asn + Ser138Asp + Asn168Ser + Ile220MetAsn161Gln + Phe192His + Thr217Asp + Ile220AlaGln103Ser + Phe192Asn + Arg218Glu + Ile220ThrAla164Gly + Asn168Gln + Ser207Asp + Ile208MetThr106Gly + Ser139Asp + Phe192Thr + Gly215AsnLeu209His + Ile213Ser + Gly214Asn + Ser219GluGly 66Asn + Asn 99Ser + Ser101Glu + Asn194GlnTyr137Glu + Thr206Pro + Trp212Thr + Ile220HisGly135Glu + Tyr169Cys + Thr206Gln + Ile220SerThr206Pro + Leu209Ser + Ser216Glu + Ile220ValGly 66Asn + Gly 68Asn + Ser105Asp + Leu209ProSer170Glu + Thr206Gly + Leu209Gln + Ile220CysTyr104Thr + Asn163Ser + Leu209His + Gly222AsnThr106Glu + Tyr137Gln + Asn162Ser + Leu209AlaLeu 96Thr + Ser207Asp + Gly214Ser + Ile220GlyAsn 99Ser + Phe192Pro + Trp212Val + Ser216AspGly136Ser + Tyr137Asp + Leu209Val + Ile220GlyGly 66Ser + Gly203Asn + Ser219Glu + Ile220MetAla164Thr + Thr206Asn + Thr217Ser + Ser219GluGly102Pro + Thr106Asn + Asn161Glu + Tyr169SerThr106Gly + Gly134Gln + Thr206Asn + Ser207GluGly 66Asn + Gln103Asn + Thr106Gln + Ile220GlnArg 64Asp + Asn 67Gln + Ile107Ala + Asn162SerThr 71Gly + Thr106Ser + Asn163Ser + Ile220AsnAsn 67Gln + Ala164Ser + Ser207Asp + Ile220ThrGly 68Ser + Asn163Gln + Arg167Asp + Ile208ThrTyr137Ser + Asn168Glu + Leu209Asn + Ile213AlaGly134Ser + Gly135Pro + Ala164His + Asn168SerAsn163Gln + Ala164Asp + Leu209Gly + Gly215ProGly102Pro + Gly136Pro + Leu209Ser + Ile220ProTyr104Cys + Gly136Asn + Ala164Asn + Leu209HisAsp 65Glu + Leu 96Val + Gly136Asn + Tyr169ProAsn 67Asp + Gly100Pro + Tyr104Leu + Leu209AlaVal 95His + Gly135Gln + Ser191Glu + Ile220AlaAsn163Ser + Asn194Ser + Ser207Glu + Leu209SerGly 66Glu + Leu 96Ile + Tyr104Thr + Ile213AlaGln103Asn + Ser138Glu + Gly160Ser + Phe202CysGly 68Pro + Thr106Ser + Asn194Asp + Thr211SerGly100Gln + Tyr169Thr + Thr211Pro + Thr217SerGly 66Asn + Val 95Cys + Ser139Asp + Asn194GlnGly135Pro + Asn161Glu + Asn162Ser + Leu209AlaGly 66Ser + Thr 71Ser + Val 95Thr + Ile107AsnAsn168Gln + Leu209Pro + Ser219Glu + Ile220GlnGly 66Pro + Tyr137Asn + Tyr169Ile + Thr223AspSer101Glu + Ile107Pro + Leu133Cys + Leu209ProAsn 67Asp + Gly 68Ser + Asn161Ser + Thr223SerAsn 99Gln + Gly102Asn + Ile107Met + Trp212GlyTyr137Ala + Asn194Gln + Ile213Asp + Gly214AsnLeu 96Ala + Leu209Met + Gly222Asn + Thr223GlnLeu 96Val + Asn162Ser + Asn163Gln + Ser170GluAsn 67Ser + Leu133Gln + Leu209Gln + Ser221AspVal 95Gln + Ala164Thr + Ser207Glu + Leu209HisGly100Gln + Asn168Gln + Ser219Asp + Ile220AspAsp 97Glu + Asp 98Glu + Ile213Ala + Thr217GlyGln103Asn + Gly135Ser + Gly215Glu + Ser216GluAsn194Ser + Leu209Gly + Gly215Glu + Ser216GluGly 70Gln + Tyr104Cys + Leu209Glu + Ser219GluIle107Val + Thr206Asp + Ser207Glu + Ser221GluLeu133Gly + Ser207Glu + Leu209Met + Ser221GluVal 95Thr + Tyr137His + Ser207Glu + Ser219GluGly 68Gln + Asn162Ser + Ser207Asp + Ser219GluTyr137Ala + Ser207Glu + Ile213Pro + Ser219AspGly100Asn + Tyr137Ser + Ser207Asp + Ser219AspTyr137Gly + Ser207Glu + Gly215Asn + Ser219GluAsp 65Glu + Asn 67Asp + Phe202Ser + Thr206GlyGly160Ser + Asn161Glu + Asn194Glu + Thr217AsnAsn 67Gln + Ser207Asp + Ser219Asp + Ser221GluGly102Asn + Gly134Gln + Ser138Glu + Ser170AspAsp 65Glu + Gly 68Asp + Gly135Pro + Phe192HisPhe192Asn + Thr206Glu + Ser207Glu + Ser219GluGly160Pro + Thr206Asp + Ser207Glu + Ser219AspLeu 96Asn + Thr206Glu + Ser207Asp + Gly222GluAsp 65Glu + Asp 98Glu + Phe192Leu + Ile213GlnGly 66Gln + Asn162Ser + Ser207Glu + Leu209AspGly 66Ser + Gly136Gln + Ser190Asp + Thr206AspAsn 67Gln + Ile107His + Asp165Glu + Asn168AspPhe202Thr + Ser207Asp + Ile220Glu + Gly222GluAsn 67Ser + Val 95Gln + Leu209Asp + Thr217GluAsn163Asp + Ser191Asp + Asn194Glu + Ile220MetSer207Glu + Leu209Glu + Ile213Met + Ser221AspAsn 67Glu + Leu 96Asp + Asp 97Glu + Leu209AlaThr206Asp + Leu209Asp + Ser219Asp + Ile220ProSer216Glu + Thr217Glu + Ser219Glu + Gly222GlnSer139Glu + Ser170Glu + Phe192His + Leu209GlnGly102Asn + Pro204Ser + Ser207Asp + Gly222GluThr106Gln + Ser207Asp + Leu209Ser + Gly222AspGly100Ser + Ser207Asp + Ile220Asn + Gly222AspThr106Asn + Ser190Glu + Ser207Asp + Ser221GluLeu209Met + Gly214Asp + Thr217Glu + Ile220AsnAla164Gln + Ser207Asp + Arg218Asp + Ile220ValTyr169Gly + Leu209Asp + Ser216Glu + Ser219GluArg167Glu + Asn194Asp + Leu209Ser + Ile220ProGly102Pro + Ile213Asp + Arg218Asp + Ser219GluAsp 98Glu + Gly102Asp + Asn161Gln + Leu209SerIle107Gly + Gly134Asp + Ser138Asp + Tyr169SerGly 68Glu + Gly134Pro + Phe192Pro + Ser216GluAsp 65Glu + Gly214Glu + Ile220Thr + Gly222ProSer190Asp + Ser207Asp + Ile208Met + Ile220ValGly134Asn + Phe202His + Arg218Asp + Ser221AspLeu 96Ala + Ile213Glu + Ser216Glu + Ser219AspGly160Gln + Ser207Asp + Arg218Glu + Gly222AspGly 70Glu + Ile107His + Leu133Glu + Phe192ThrGly136Asp + Ser170Asp + Asn194Asp + Gly222SerTyr104Glu + Leu133Asp + Ile213Ser + Gly215GlnGly100Gln + Ser207Glu + Ser216Asp + Ser219GluGly100Asn + Tyr137Ala + Leu209Asp + Ser216GluAsn162Ser + Ser216Glu + Ser219Asp + Ile220AsnVal 95Ser + Leu 96Pro + Ser216Asp + Ser219AspGly102Pro + Ser216Asp + Ser219Asp + Ile220MetGly135Glu + Gly136Gln + Arg167Asp + Asn194GluAsn 99Gln + Ser207Glu + Ser216Asp + Thr217AspAsp 65Glu + Ser101Glu + Gly102Glu + Leu133ThrAsn163Gln + Ser216Glu + Ser219Asp + Ser221AspGly 66Gln + Asn163Asp + Ser170Glu + Asn194AspGly 66Pro + Gly134Asp + Asn161Glu + Ser191AspLeu133Thr + Asn161Asp + Ser219Asp + Ile220AspSer105Asp + Gly135Asp + Gly136Ser + Tyr169AspAsn 67Gln + Asn163Glu + Asn194Glu + Ser221AspSer207Glu + Ile213Pro + Ser216Glu + Ile220GluGly 68Asp + Leu 96Glu + Thr206Gly + Gly214GluAsn 99Glu + Ser101Glu + Gly136Glu + Asn163GlnSer138Glu + Ser139Asp + Asn163Asp + Ile220MetSer101Glu + Trp212Phe + Ser219Glu + Ile220GluAla164Glu + Asn168Gln + Ser219Glu + Ile220AspSer101Glu + G1y160Gln + Arg167Glu + Asn168GluArg167Glu + Asn168Glu + Asn194Ser + Ser207AspArg167Glu + Asn168Asp + Asn194Ser + Ile220GluLeu 96Thr + Arg167Asp + Asn168Glu + Ser216GluThr106Asp + Leu209Asn + Ser221Asp + Gly222AspAsp 65Glu + Tyr137His + Asn161Glu + Asn162AspAsn 67Asp + Ser191Asp + Phe192Glu + Pro204SerArg 64Asp + Gly 68Ser + Gly222Asp + Thr223GluSer139Glu + Ser140Glu + Arg218Asp + Thr223ProLeu 96Met + Asp 98Glu + Asn 99Asp + Ser219AspArg 64Asp + Asp 65Glu + Gln103Asp + Leu209IleTyr137Glu + Ser138Glu + Ser216Asp + Thr217AsnGly136Asp + Thr206Asp + Ser207Glu + Ile220MetSer101Asp + Ala164Ser + Thr206Asp + Ser207GluGly 66Gln + Tyr104Asp + Ser105Asp + Ser219AspSer138Asp + Thr217Gln + Arg218Glu + Ser219AspArg 64Asp + Leu209Gln + Ser216Glu + Thr217GluSer105Glu + Leu209Asp + Ser219Glu + Thr223GlnSer101Asp + Leu209Asp + Ser219Glu + Ile220SerAsn163Asp + Phe192Leu + Asn194Glu + Ser207AspAsn163Asp + Asn194Glu + Ser207Glu + Trp212HisAla164Asp + Asn194Glu + Ser207Asp + Ile220LeuAla164Glu + Asn194Glu + Ser207Glu + Ile220SerGly100Asp + Ala164Glu + Asn194Asp + Thr206SerLeu 96Pro + Ser207Glu + Ser216Glu + Ser221AspVal 95Asp + Phe192Tyr + Ser207Asp + Ser221AspAla164Asp + Ser207Asp + Gly215Gln + Ser221AspAsp 65Glu + Leu209Gln + Ser216Asp + Arg218AspIle107Cys + Ser139Asp + Ser207Glu + Ser219AspTyr137Glu + Ser207Asp + Ser219Glu + Ile220SerVal 95Gln + Tyr104Asp + Ser207Asp + Ser219AspAsn163Glu + Ser207Glu + Ser219Glu + Gly222SerAsn168Glu + Ser207Glu + Ser219Glu + Gly222AsnGly135Asp + Gly203Ser + Ser207Glu + Ser219GluLeu 96Thr + Ser139Glu + Ser207Asp + Ser219AspGly100Glu + Ser207Asp + Ser219Glu + Ile220CysGln103Glu + Tyr104Pro + Gly136Glu + Ile220AsnGln103Glu + Ser105Glu + Ser219Asp + Ile220CysGln103Glu + Ser105Asp + Tyr137Thr + Thr206GluGly160Asn + Ser207Asp + Gly214Glu + Ser216AspAsp 97Glu + Ser101Asp + Ser207Asp + Ile220ThrThrl06Asp + Tyr137Met + Leu209Glu + Arg218GluGly136Gln + Ser138Asp + Ser170Glu + Ser216GluGly 70Asp + Leu133Ser + Ser138Asp + Ser170AspAsn163Glu + Tyr169Cys + Ser219Asp + Gly222AspArg 64Glu + Gly 66Asp + Asn168Gln + Arg218AspSer105Asp + Gly136Asn + Ser139Asp + Phe192ThrTyr137Leu + Ser191Glu + Ser207Glu + Gly222SerSer191Asp + Ser207Asp + Leu209His + Ile220ThrAsn 99Ser + Ser191Glu + Ser207Asp + Ile220AsnGly 66Asp + Asn 99Asp + Thr106Gln + Ser216AspAsp 97Glu + Phe202Thr + Arg218Glu + Ile220AspGly 70Gln + Asn 99Asp + Ser207Glu + Leu209GluThr106Glu + Ser207Glu + Leu209Asp + Gly214GlnAsp 65Glu + Tyr137Pro + Asp165Glu + Asn168AspAsn 99Gln + Asp165Glu + Asn168Asp + Gly215GluAsn 67Glu + Asn161Glu + Ile220Ala + Gly222GluGly 66Asp + Leu 96Gly + Leu209Glu + Ile220AspAsp 98Glu + Leu209Ala + Ser219Glu + Ser221AspAsn 67Glu + Ala164Gln + Ser219Glu + Ser221AspGlyl00Glu + Gly136Asn + Ser216Asp + Arg218GluIle107Asp + Thr206Ser + Ser216Asp + Arg218GluAla164Gly + Asn194Asp + Thr217Asp + Ser219GluAsp 98Glu + Asn163Asp + Asp165Glu + Trp212TyrSer139Glu + Gly215Glu + Thr217Asp + Gly222ProSer101Glu + Ser105Glu + Phe192Val + Leu209HisTyr104Asn + Leu209Glu + Ser216Asp + Gly222AspGly 70Glu + Tyr104Ala + Thr206Asp + Ser219GluLeu 96Glu + Asn 99Asp + Ala164Gln + Gly214GluSer139Asp + Asn161Glu + Ala164Glu + Ile220GlyAsn 67Ser + Gly 70Asp + Leu 96Glu + Ser221GluAsn 67Asp + Asp 97Glu + Asn163Ser + Ser221GluSer170Glu + Ser207Asp + Ile220Ser + Gly222AspLeu 96Gly + Asn163Asp + Thr206Asp + Ser219GluAsn 67Asp + Ser190Asp + Gly215Ser + Gly222AspTyr137Cys + Arg167Asp + Ser170Asp + Ser219AspGly135Glu + Ser138Asp + Gly214Glu + Gly222SerAsp 65Glu + Asp 97Glu + Asn194Glu + Gly205AsnGly102Ser + Serl05Asp + Leu133Glu + Thr223AspGly 70Asp + Asp 98Glu + Leu209Met + Ser216AspSer138Glu + Ala164Ser + Thr206Glu + Ser219GluGly 68Pro + Asp 97Glu + Thr206Asp + Ser219AspSer105Glu + Ser140Glu + Arg218Glu + Thr223GlnAsn 99Asp + Gly102Glu + Ile213Val + Ser219GluAsn 99Asp + Gly102Asp + Gly136Ser + Ser138AspGly 70Glu + Val 95Glu + Gly100Asn + Gly222GluAsn 67Glu + Ser216Glu + Ser219Asp + Ile220CysAsn 67Asp + Ala164Pro + Ser216Glu + Ser219AspAsn 99Asp + Asn161Glu + Ser190Asp + Gly215AsnVal 95Asp + Asnl61Gln + Ser190Glu + Ser221AspGly 68Asp + Gly100Glu + Gly222Ser + Thr223GluGly134Glu + Tyr169Leu + Ser207Glu + Arg218GluThr106Asp + Ser207Glu + Arg218Asp + Ile220CysArg 64Glu + Asn163Ser + Ser207Asp + Arg218AspSer139Asp + Ser207Asp + Arg218Asp + Ile220ThrLeu133Asp + Gly134Pro + Asn162Asp + Tyr169AsnVal 95Asp + Gly134Glu + Asn194Glu + Thr211GlyIle107Asn + Ser140Glu + Ser170Glu + Ser219AspTyr137Cys + Ser140Glu + Ser170Asp + Ser219GluTyr137Thr + Ser140Glu + Ser170Glu + Thr206GluTyr137Asn + Gly160Glu + Asp165Glu + Ser216GluAsn 99Glu + Gly160Asp + Asp165Glu + Leu209GlnAsp 97Glu + Tyr104Glu + Leu133Ser + Ile220LeuAsn 67Glu + Val 95Gly + Pro204Ser + Ser216AspAsn 99Asp + Asn162Gln + Ser191Asp + Gly222GluGly100Glu + Thr106Gly + Asn162Asp + Tyr169GluAsp 65Glu + Asn 99Asp + Gly135Asn + Gly160AspAsp 65Glu + Asn 99Glu + Trp212Asn + Ser219AspGly134Glu + Asn161Asp + Thr217Pro + Ser219AspAsp 97Glu + Gln103Glu + Asn163Gln + Ile220GluAsn161Glu + Asp165Glu + Ser207Asp + Thr223GlnSer140Glu + Ala164His + Thr217Asp + Ile220AspGly 70Ser + Gly102Asp + Leu133Asp + Ser191AspSer101Glu + Ile107Glu + Thr217Pro + Ser219GluTyr137Glu + Asn162Glu + Ser190Asp + Ile213GlnGly 68Asp + Ser170Glu + Ser216Asp + Ile220ValGln103Asp + Gly135Asp + Leu209Met + Ser216AspTyr104Val + Gly136Glu + Ser139Glu + Ser190AspGly102Pro + Asn161Glu + Asp165Glu + Gly214GluGly 70Glu + Tyr169Cys + Ser190Glu + Ser207GluAsn168Glu + Ser190Glu + Pro204Ser + Ser207GluSer190Asp + Ser207Asp + Leu209Ser + Ser216GluAsn 67Glu + Ser101Glu + Tyr137Asp + Gly215GlnArg 64Glu + Ser191Glu + Ile220His + Ser221AspArg167Asp + Ser191Asp + Ile213Ala + Ser221GluArg167Glu + Thr206Ser + Ser219Asp + Gly222GluSer101Glu + Asn161Gln + Asn162Asp + Ser190AspSer101Asp + Gly136Pro + Ser216Asp + Ser219GluTyr104Gln + Thr106Glu + Ser216Glu + Ser219GluGly 68Pro + Leu133Glu + Ser216Glu + Ser219GluGly160Glu + Leu209Val + Ser216Glu + Ser219GluSer191Glu + Leu209Gln + Ser216Asp + Ser219AspAsn 67Gln + Arg167Glu + Ser216Glu + Ser219AspAsn 99Gln + Ser170Asp + Ser216Glu + Ser219AspSer138Glu + Asn162Gln + Ser216Glu + Ser219GluGly135Gln + Asn168Glu + Ser216Asp + Ser219AspGly100Glu + Thr206Ser + Ser207Glu + Thr223GluAsn163Gln + Gly214Asn + Ser216Glu + Ile220AspGly100Pro + Thr211Asn + Ser216Asp + Ile220GluThr206Pro + Leu209Thr + Ser216Glu + Ile220AspGlyl02Asp + Gly160Asp + Ser190Glu + Trp212SerLeu133Cys + Gly134Asp + Asn162Asp + Ser216AspAsp 65Glu + Thr106Gln + Ser216Glu + Gly222GlnAsp 98Glu + Gly100Ser + Asn168Asp + Ile213GluSer101Asp + Tyr137Ala + Asn161Glu + Thr206GluGly 70Asp + Gly136Glu + Ser140Glu + Ile220ProGly 68Glu + Tyr104Glu + Leu209Val + Ser219AspGly 68Asp + Gly160Gln + Asn194Glu + Ser219AspAsn163Gln + Ser190Glu + Ser216Glu + Ile220GluArg 64Asp + Gln103Asn + Leu133Asp + Gly214GluArg 64Glu + Tyr104Ser + Leu209Glu + Gly214AspGly 70Asp + Ser101Asp + Ser140Glu + Ile220ValGly 70Asp + Leu 96Thr + Leu209Asn + Ser216GluIle107Asp + Ser138Glu + Ala166Gln + Ser219AspAsn161Glu + Ser207Asp + Leu209Ile + Thr217AspGly 68Asn + Asn163Glu + Arg167Glu + Ser219AspAsn 99Ser + Gln103Asp + Gly136Glu + Thr206GluGln103Glu + Leu133Gln + Gly136Glu + Ser216GluGlyl02Asp + Leu133Thr + Ser207Asp + Thr217GluAsp 97Glu + Ser207Asp + Thr217Glu + Thr223AsnSer101Glu + Ser191Asp + Ser207Glu + Leu209CysIle107Ser + Ser191Glu + Ser207Asp + Ser216AspThr 71Pro + Ser191Glu + Ser207Glu + Ser216GluAlal64Gln + Ser191Glu + Ser207Glu + Ser216AspSer170Glu + Asn194Asp + Ser207Glu + Ile220ValSer170Asp + Asn194Glu + Trp212Ser + Ser216GluArg 64Glu + Asn 99Glu + Ser170Asp + Leu209ThrAsp 97Glu + Leu133Glu + Asn161Ser + Ser216AspTyr137Pro + Ser140Asp + Phe192Asp + Ser207GluAsn 99Ser + Gln103Glu + Ser170Asp + Ser219AspGln103Glu + Asn163Glu + Ser170Glu + Ile220HisAsp 65Glu + Ash 99Ser + Ser101Glu + Ser191AspSer105Glu + Gly135Asp + Thr206Asp + Thr217GlnLeu 96Cys + Asn161Glu + Arg167Glu + Arg218AspArg 64Glu + Gly100Glu + Asn161Ser + Leu209GluVal 95Met + Asp 97Glu + Ser105Glu + Ile220AsnGly 68Glu + Val 95Glu + Asn168Ser + Leu209AlaAsn 67Glu + Asn161Asp + Ser216Asp + Gly222AsnAsn 67Asp + Leu133His + Arg167Glu + Ser216GluSer105Asp + Gly160Ser + Ser170Asp + Thr206GluGly 66Pro + Asn162Asp + Ser170Glu + Ser219GluGly100Glu + Gly102Asn + Tyr104Asp + Ser207AspGly100Glu + Tyr104Asp + Gly160Glu + Gly215Gln表35多環(huán)區(qū)五突變變異體Leu133Gln + Gly136Glu + Thr206Gln + Leu209Met +Ile220MetGly203Asn + Gly205Gln + Ser216Glu + Thr217Gly +Ile220GlyVal 95His + Ala164Ser + Ser170Glu + Phe192Met +Thr223GlyLeu133Asn + Asn161Gln + Asn163Gln + Leu209Met +Ile220LeuAsn 67Gln + Gly136Ser + Asn162Asp + Tyr169Gly +Phe202MetIle107Asn + Leu209Gln + Ile213Gly + Ser219Glu +Ile220HisGly 66Ser + Thr 71Gly + Tyr169His + Phe202Ser +Ile220AspGly135Ser + Ala166Ser + Ser207Glu + Ile213Gly +Gly222SerAsp 98Glu + Thr106Pro + Tyr137Cys + Trp212Ile +Ile220ValGly134Asn + Ala166Pro + Phe192Met + Thr206Gly +Ser219GluThr106Glu + Gly205Asn + Leu209Cys + Gly215Pro +Ile220ThrAsn163Gln + Asn168Gln + Phe192Cys + Thr206Gln +Leu209SerLeu 96Gly + Ile107Gly + Ser140Asp + Leu209Ala +
Thr223GlyAsn 67Ser + Val 95His + Gly135Gln + Ser191Glu +Ile220AlaIle107Gly + Tyr169Ser + Asn194Ser + Ser207Glu +Leu209SerTyr137His + Leu209Met + Ile220Val + Gly222Asn +Thr223GlnVal 95Ala + Tyr137Pro + Ala164Glu + Asn168Gln +Thr206AsnLeu 96Gln + Tyr137Leu + Tyr169His + Leu209Asp +Ile220ProGly 66Pro + Gly 70Asn + Leu133Pro + Phe202Pro +Ser207GluVal 95Ala + Leu 96Met + Tyr104Asn + Ile107Ala +Ser216AspGly 70Ser + Ile107Gly + Leu133Met + Phe192His +Asn194AspGly 68Pro + Val 95Ser + Gly100Asn + Tyr137Cys +Ile220ProGly 66Ser + Gly102Gln + Ala164Gln + Ser219Glu +Gly222AsnGly135Gln + Ser190Glu + Leu209Cys + Gly215Gln +Ile220ValGly 68Pro + Asn163Asp + Phe192Pro + Leu209Gly +Ile220ThrAsn 67Gln + Gly 70Asn + Ile107Leu + Gly136Asn +Asn162GluGly 70Pro + Ile107His + Ser190Glu + Pro204Asn +Leu209GlnLeu 96Ile + Asp165Glu + Phe202Gly + Leu209Asn +Thr211GlyGly 66Pro + Gly 70Asp + Tyr169Asn + Leu209His +Gly215SerTyr104Met + Asn194Glu + Leu209Cys + Thr217Pro +Ile220GlnGly 70Asn + Leu 96Ile + Tyr104His + Asn161Asp +Ile220GlnVal 95Thr + Gly102Pro + Thr206Ser + Gly214Glu +Gly222SerThr106Gln + Asn163Ser + Ile213Pro + Ser216Glu +Ile220SerLeu 96Cys + Gly136Asn + Ser170Glu + Thr206Gln +Ile220GlnGly 66Gln + Ser105Glu + Gly135Pro + Gly205Asn +Thr206SerGly102Pro + Tyr104Ile + Thr211Gly + Ser216Glu +Thr223GlnTyr104Ala + Gly203Asn + Leu209Gly + Arg218Asp +Ile220CysGly 66Pro + Val 95Thr + Asn161Ser + Asn162Gln +Ile220CysGly102Asn + Gly136Gln + Phe202Met + Ile208Val +Ile213GluGly 66Ser + Gly 68Ser + Gly134Gln + Ala164Gly +Asp165GluGly 66Pro + Ala164Asp + Tyr169Ser + Thr206Gln +Thr217AsnLeu 96Ile + Gly136Gln + Asn161Ser + Thr206Gln +Ser207AspAsn 67Asp + Tyr104Asn + Gly135Gln + Tyr169Ile +Leu209HisGly 70Pro + Gly136Pro + Ser190Asp + Leu209Ser +Gly214ProAsp 65Glu + Tyr137Val + Ala164Asn + Phe192Ala +Thr206SerLeu 96Thr + Gln103Ser + Tyr104Asn + Asp165Glu +Ile220HisGly 68Asp + Gly 70Pro + Asn 99Ser + Gly214Ser +Thr223SerLeu 96Ser + Leu133Ala + Tyr137Met + Leu209Ile +Ser221AspGly134Gln + Asn194Gln + Thr206Ser + Leu209Met +Ile213LeuGly 70Glu + Leu133Cys + Gly135Gln + Thr206Gly +Gly222AsnGly134Ser + Ala166Gln + Tyr169Cys + Thr206Gly +Ser221AspGly160Ser + Thr206Asp + Ile213Gln + Ile220Pro +Thr223GlyTyr104Asn + Gly135Pro + Leu209Ile + Ser219Glu +Ile220ValAsn 67Gln + Thrl06Asn + Gly160Glu + Tyr169Asn +Leu209AlaVal 95Glu + Gly100Pro + Thr206Asn + Leu209Thr +Ile220AlaAsp 98Glu + Gly102Ser + Ala164Gln + Thr206Ser +Ile220AsnGln103Asn + Gly136Pro + Ser138Asp + Leu209Gly +Ile220GlnAsn 67Gln + Thr106Asn + Gly136Gln + Thr206Ser +Ile220AsnAsn 67Gln + Gly134Gln + Tyr169Cys + Ser219Asp +Ile220AspAla166His + Thr206Glu + Ser207Asp + Ile213Cys +Ile220HisGly 70Pro + Ile107Gly + Leu209Gln + Ile220Glu +Ser221AspAsn 99Ser + Gly160Ser + Gly215Glu + Ser216Glu +Thr217GlnGly102Gln + Ile107Cys + Ala164Ser + Leu209Asp +Ser219AspGly134Asn + Tyr137Gln + Leu209Glu + Ser219Asp +Ile220MetGly102Gln + Gly160Gln + Leu209Asp + Arg218Asp +Ser219GluGly 70Asn + Ser207Glu + Leu209Val + Ser219Glu +
Ile220GluAla164Asn + Ser207Asp + Thr217Pro + Ser219Glu +Ile220AspVal 95Thr + Ala164Pro + Ser207Glu + Ile213Val +Ser221AspVal 95Asn + Gly102Pro + Gly160Pro + Ser207Asp +Ser219AspAsn 67Gln + Ser207Glu + Leu209Val + Ser219Asp +Ile220AlaAsn 67Gln + Gly136Gln + Ser138Glu + Ser140Asp +Leu209MetTyr104Asn + Ser138Glu + Ser140Asp + Leu209Thr +Ile220ValAsp 97Glu + Asn 99Asp + Gly102Gln + Tyr137Ser +Ile220CysAsn161Asp + Asn163Gln + Tyr169Ala + Asn194Asp +Leu209AlaThr206Glu + Ser207Asp + Leu209His + Ser219Asp +Thr223GlyGly102Ser + Gly136Asn + Thr206Glu + Leu209Thr +Gly222AspThr 71Gly + Gly102Asn + Ser219Asp + Ile220Cys +Ser221GluThr106Ser + Gly160Asn + Gly205Asn + Ser219Asp +Ser221AspVal 95Gln + Leu133Gln + Gly215Asn + Thr217Asp +Ser219AspLeu209Ser + Ser219Glu + Ile220Gly + Ser221Glu +Gly222AspGln103Ser + Gly203Pro + Ser216Glu + Arg218Glu +Ser219AspVal 95Ser + Gly135Gln + Ser138Asp + Tyr169Glu +Ile213ThrAsn 67Gln + Gly160Pro + Ser207Glu + Leu209Cys +Gly222GluGly135Asp + Ser138Glu + Gly203Gln + Ile213Met +Gly222AsnAsn162Gln + Ser207Asp + Leu209Pro + Ser219Glu +Gly222AspTyr137Asn + Ala164Asp + Arg167Asp + Leu209Ile +Ile220MetLeu 96Gln + Gly102Asn + Gly135Asp + Asn168Asp +Ile220AlaVal 95Pro + Asn 99Asp + Gly102Asp + Gly160Gln +Leu209AsnAsn 99Ser + Ile107Gly + Asn162Gln + Leu209Asp +Ser221GluGly 66Pro + Asn162Asp + Ser191Asp + Thr206Asn +Thr223AsnGln103Asp + Tyr104Asn + Ser105Glu + Ser140Asp +Leu209CysLeu 96Ser + Gly136Ser + Asn168Glu + Asn194Asp +Thr206AsnGly 68Glu + Val 95Ala + Gly136Asn + Gly203Asn +Ser216AspAsp 65Glu + Thr106Asn + Gly134Gln + Gly214Asp +Ser216GluGly 68Gln + Val 95Pro + Asn162Gln + Ser190Glu +Ser207AspAla166Thr + Ser190Glu + Ser207Glu + Leu209Gln +Thr217GlyGly100Pro + Gly160Gln + Ser190Glu + Ser207Asp +Leu209ProAsn 67Glu + Ser101Glu + Tyr104His + Phe192Ser +Leu209ThrSer191Glu + Phe202His + Leu209Met + Ser221Asp +Thr223GlnGly134Asp + Ser139Asp + Ser140Glu + Ile213Met +Thr223AsnTyr137Asn + Tyr169Ile + Thr206Asp + Ser219Asp +Thr223AspTyr104Cys + Leu133Ile + Ser207Glu + Ser216Glu +Arg218AspGly 68Asn + Tyr137Ser + Ser207Glu + Ser216Asp +Ser219GluTyr137Ser + Asn168Ser + Gly214Asp + Ser216Asp +Ser219GluIle107His + Asn162Ser + Ser216Glu + Ser219Asp +Ile220AsnAsn 67Gln + Asn163Ser + Ser216Asp + Ser219Glu +Thr223SerLeu 96Asn + Leu133Pro + Thr206Gln + Ser216Asp +Ser219AspSer191Glu + Thr206Asp + Gly214Asn + Ser219Asp +Thr223AsnGly 66Ser + Ser105Glu + Thr106Pro + Ser138Glu +Leu209IleGly136Asp + Gly160Glu + Asn163Ser + Ile220Ser +Gly222AspIle107Met + Asn162Asp + Ser190Asp + Ser221Asp +Gly222SerGly 70Gln + Gly214Ser + Ser216Glu + Ser219Glu +Ser221GluGly136Glu + Asn162Glu + Gly214Ser + Gly215Asn +Ile220LeuGly 68Asp + Leu209Thr + Arg218Glu + Ser221Asp +Thr223ProGly100Ser + Phe192Glu + Gly215Asn + Ser219Asp +Ile220GluGly134Gln + Phe192Glu + Ile213Gly + Ser219Asp +Ile220GluThr106Gly + Asn162Ser + Asn194Asp + Thr206Asp +Ser221AspGln103Asn + Asnl61Asp + Ser207Asp + Thr217Ser +Ile220GluGly 68Asn + Phe192Asp + Ser207Asp + Leu209Asp +
Ile220AsnGly 68Asp + Phe192Tyr + Leu209Gly + Ser219Asp +Ile220GlnArg 64Asp + Asp 98Glu + Tyr137Val + Phe192Val +Gly214AspTyr104Gly + Ile107Glu + Gly160Glu + Ser170Glu +Leu209CysVal 95Thr + Ile107Gly + Ser191Glu + Ser219Glu +Ile220AspAsp 65Glu + Gly 66Asp + Gly100Asn + Gly102Ser +Ser190GluAsp 65Glu + Gly 66Asp + Gly134Gln + Ile220Asp +Thr223GlnAsp 65Glu + Gly 66Asp + Leu209Glu + Ile213Ala +Ile220AlaAsn 99Gln + Serl90Glu + Serl91Asp + Ser219Glu +Ile220HisGly 70Asp + Ser101Asp + Tyr137Cys + Gly160Ser +Ile220HisTyr137Ser + Ser138Asp + Ser139Glu + Ile213Met +Ser216AspGly135Ser + Ser138Glu + Asn194Ser + Ser219Glu +Ile220GluThr 71Pro + Asp 97Glu + Thr106Ser + Ser219Glu +Ile220GluArg 64Asp + Gly 66Gln + Thr206Gly + Ser219Glu +Ile220AspVal 95Ala + Gln103Ser + Ser139Asp + Arg167Glu +Ile220AlaSer101Asp + Gly102Glu + Ile213Val + Thr217Ser +Thr223GluAsn 99Asp + Gly100Glu + Tyr137Thr + Asn163Glu +Gly215GlnGly 66Ser + Ile107Glu + Leu209Asn + Ser221Glu +Gly222GluAsn 67Glu + Thr 71Gln + Asnl61Asp + Asn162Glu +Gly214SerGly100Asp + Ser101Glu + Thr106Gln + Ile213Cys +Ser221AspArg 64Asp + Asp 65Glu + Gly 68Ser + Leu 96Gln +Asn168AspGly160Pro + Thr206Glu + Ser207Glu + Gly215Glu +Gly222ProGly102Ser + Tyr137Asp + Tyr169His + Thr206Glu +Ser207AspGly 70Asp + Asn 99Ser + Tyr104Glu + Ser105Asp +Thr217GlnLeu 96Glu + Gly135Ser + Thr217Ser + Ile220Glu +Ser221GluAsp 97Glu + Asp 98Glu + Gly205Pro + Gly215Ser +Ser219GluGly 68Gln + Ser138Asp + Leu209Ile + Arg218Asp +Ser219AspAsn 67Ser + Gly136Asp + Gly215Pro + Ser216Glu +Thr217AspAsn 99Gln + Ser101Glu + Asn161Gln + Asn162Asp +Asn163AspTyr137Gln + Tyr169Thr + Ser207Asp + Gly215Asp +Ser216AspLeu 96Cys + Ser190Asp + Gly215Glu + Ser216Glu +Ile220CysGly160Pro + Phe192Asn + Ser207Glu + Gly215Asp +Ser216GluThr 71Asn + Phe192Val + Ser207Asp + Gly215Asp +Ser216AspGly134Glu + Asn168Asp + Trp212Ile + Ile220Asn +Thr223GluGln103Asp + Gly134Ser + Ser139Glu + Asn162Ser +Ser170AspGln103Asp + Thr106Ser + Ser138Glu + Leu209Val +Gly215GlnASn 67Gln + Gln103Asp + Gly135Gln + Ser138Glu +Leu209AsnAsp 97Glu + Ile107Gln + Gly136Ser + Ser207Asp +Ser221AspTyr137Ile + Ser138Glu + Asn194Ser + Ser207Asp +Ser221AspGln103Asp + Gly135Asn + Gly136Pro + Ser207Glu +Ile220GluGly102Gln + Gly135Glu + Gly203Ser + ser207Glu +Ile220GluTyr104Ile + Asn163Asp + Arg167Glu + Tyr169Cys +Ile208CysGly 66Glu + Asp 97Glu + Tyr104Glu + Gly136Gln +Leu209MetGly102Ser + Ile107Cys + Arg167Asp + Ser207Asp +Ser219AspGly100Asn + Gly134Asp + Ser207Glu + Gly214Ser +Ser219AspArg 64Asp + Val 95Ser + Thr206Ser + Ser207Asp +Ser219GluAsn194Asp + Ser207Glu + Ser219Asp + Ile220Met +Thr223AsnSerl40Glu + Ser207Glu + Leu209Ala + Gly215Gln +Ser219GluTyr104Ser + Gly160Glu + Ser207Glu + Gly215Ser +Ser219AspAsp 65Glu + Thr206Ser + Ser207Glu + Gly215Ser +Ser219AspGln103Ser + Asn168Glu + Pro204Gln + Ser207Glu +Ser219AspLeu133Thr + Ser140Glu + Asn161Ser + Ser207Asp +Ser219GluLeu 96Pro + Ser140Glu + Ser207Glu + Leu209Thr +Ser219GluAsn161Asp + Asn168Gln + Ser170Asp + Ser191Asp +
Phe192GlnGln103Glu + Thr106Glu + Leu209Gly + Ile213His +Ser219GluAsp 65Glu + Asn 67Asp + Thr106Asn + Asn194Glu +Ile208AsnAsp 65Glu + Asn 67Glu + Leu 96Ala + Gly102Ser +Tyr169AspAsn 99Ser + Asp165Glu + Arg167Asp + Ile213Gly +Ser219AspTyr104Ile + Tyr137Ile + Asp165Glu + Arg167Glu +Ser219AspSer190Glu + Phe192Glu + Leu209Cys + Ser219Glu +Ile220AlaSer138Glu + Ser140Glu + Thr206Gly + Ile220His +Ser221GluVal 95Pro + Ile107Ser + Ser138Asp + Ser140Glu +Ser219AspAsp 65Glu + Gly102Ser + Ser138Glu + Ser140Asp +Ala166GlyVal 95Cys + Ser101Glu + Ser138Glu + Ser140Asp +Phe192SerAsp 97Glu + Asn 99Asp + Ser139Asp + Ile213Thr +Thr223GlyAsp 97Glu + Asn 99Asp + Ser140Glu + Ala166Gln +Phe192ThrLeu 96Met + Gly136Glu + Asn161Asp + Asn168Gln +Ile220ProGln103Asp + Ser105Glu + Asn162Ser + Ser207Glu +Thr223GlnArg 64Glu + Gln103Glu + Ser105Asp + Asn163Gln +Leu209CysIyr137Ala + Ser170Glu + Phe192Met + Gly214Glu +Ser216AspGly 70Ser + Ser105Glu + Gly214Glu + Ser216Glu +Thr223AsnGln103Ser + Asn168Asp + Gly205Ser + Gly214Asp +Ser216AspGly135Glu + Tyr169Glu + Ser207Asp + Leu209Met +Ile220CysAsn 99Gln + Gly136Asp + Gly160Ser + Ser170Asp +Ser216AspGly136Asp + Tyr169Ser + Ser170Asp + Ile213Glu +Thr217AsnAsp 97Glu + Ser101Asp + Ser139Glu + Asn162Ser +Ile220ValAsp 97Glu + Ser101Glu + Ile213Met + Thr217Gly +Arg218AspThr106Asn + Ile107Pro + Asn194Glu + Ser219Asp +Ser221GluLeu 96Met + Asn 99Asp + Ser101Glu + Ser139Asp +Leu209ThrAsn 99Asp + Ser101Glu + Gln103Ser + Ser139Asp +Ile220ProGly 68Glu + Asp 97Glu + Gly160Asn + Asn163Ser +Ile220AspArg 64Asp + Gly 66Asp + Ile107Gly + Ser207Glu +Ile220ValGly 70Gln + Gln103Asp + Leu133Ile + Ser140Asp +Leu209ThrAsn 67Glu + Gly 70Glu + Asn 99Gln + Tyr104Met +Leu209AspTyr104Gln + Ile107His + Ser191Asp + Thr206Glu +Arg218AspAsn161Glu + Ala164Gln + Ile213Asp + Ser216Asp +Thr217AsnThr106Gln + Gly136Pro + Thr206Ser + Ser207Glu +Thr217GluAsp 65Glu + Leu 96Gly + Asp 98Glu + Thr106Asp +Ile220GlnAsp 65Glu + Asp 98Glu + Ile107Gly + Gly135Asn +Gly136AspGly136Asp + Ser138Asp + Ser207Glu + Leu209Asn +Gly222AsnThr106Gln + Gly136Asp + Ser138Asp + Leu209Cys +Ser216GluVal 95Ala + Asp 97Glu + Asn168Ser + Ser207Asp +Leu209GluIle107Glu + Thr206Ser + Ser207Glu + Leu209Glu +Thr211GlyGly 70Pro + Leu 96Val + Gly102Glu + Ser207Glu +Leu209AspVal 95His + Asn194Asp + Ser207Asp + Leu209Asp +Thr217AsnSer140Asp + Ala164Pro + Ser207Asp + Leu209Asp +Ile220GlyGln103Asp + Tyr104Gln + Gly160Ser + Asp165Glu +Asn168AspAsp 65Glu + Gly 66Ser + Gly 70Glu + Gly136Asn +Ser207AspGly 68Asp + Phe192Asn + Gly205Asn + Thr217Asp +Ser221AspGly134Asp + Gly136Asp + Ser207Glu + Leu209Ile +Ile220CysGly100Asn + Gly136Glu + Gly160Gln + Ser219Asp +Ser221GluGly102Ser + Ser170Glu + Gly215Asn + Ser219Glu +Ser221GluGly 66Ser + Asn163Gln + Arg167Glu + Ser219Glu +Ser221AspGly100Glu + Gly136Asn + Leu209Ser + Ser219Glu +Ser221AspLeu133Ala + Gly134Asp + Ile213Cys + Ser219Glu +Ser221GluGly102Asn + Gln103Glu + Thr206Ser + Ser216Glu +Arg218AspAsp 98Glu + Thr206Gly + Ile213Ala + Ser216Asp +
Arg218GluAsn163Asp + Ile208Asn + Ile213Asn + Ser216Asp +Arg218AspGly 66Ser + Gly 70Glu + Tyr169His + Thr206Gly +Ile220AspArg 64Glu + Asn 67Glu + Ile107G1y + Leu209Val +Ile220GluGln103Glu + Gly134Ser + Ser138Glu + Arg167Asp +Thr223AsnGly100Pro + Tyr104Pro + Leu133Gln + Phe192Asp +Ser207GluGly 68Asn + Ser138Glu + Asn163Glu + Arg167Asp +Thr206GlyGly 70Ser + Gln103Asp + Asn162Ser + Ser170Asp +Leu209MetAsp 98Glu + Gly100Ser + Ser101Asp + Asn161Ser +Ile220AspTyr137Pro + Ser139Asp + Asn161Glu + Ala164Glu +Ile220GlyGly 66Asp + Asn 99Glu + Tyr104Met + Ser207Glu +Leu209ProGly 66Asp + Asp 97Glu + Tyr104Gln + Ile208Val +Leu209AspIle107Asp + Phe192Met + Ser207Asp + Gly222Glu +Thr223GlnArg 64Glu + Leu 96Ala + Ala164Asn + Ser207Glu +Gly222AspLeu 96His + Ser207Glu + Ser216Glu + Thr217Asn +Gly222GluTyr169His + Ser170Glu + Ser207Asp + Ile220Ser +Gly222AspGly 68Pro + Leu133Glu + Leu209Asp + Gly214Gln +Ser221GluSer101Asp + Arg167Asp + Ser170Glu + Gly215Asn +Thr223GlyAsp 65Glu + Asp 97Glu + Ser207Asp + Gly222Ser +Thr223SerAsp 65Glu + Asp 97Glu + Asn 99Gln + Gly135Glu +Thr206GlyTyr104Met + Gly160Asp + Ala164Glu + Leu209Asp +Ile220ThrLeu 96Ser + Asn162Glu + Asp165Glu + Tyr169Cys +Arg218GluAsp 65Glu + Ile107Thr + Asn162Glu + Asp165Glu +Thr217ProVal 95Glu + Ser101Asp + Asn162Glu + Phe192Thr +Gly215GlnGln103Asn + Tyr104His + Thr206Glu + Ser216Glu +Thr223GluGly135Asp + Asn168Asp + Tyr169Asn + Ser191Glu +Gly205ProGly102Glu + Ser105Glu + Asn161Gln + Ser207Glu +Ile220MetLeu133Glu + Phe192Pro + Thr206Glu + Thr217Pro +Ser219GluAsn 99Asp + Ser105Glu + Leu133Val + Ser140Glu +Gly214SerAsn 99Glu + Gly102Asp + Asn162Gln + Phe192Pro +Ser219AspIle107Val + Ser190Glu + Asn194Glu + Leu209Thr +Ser216GluGly100Glu + Leu133Glu + Ala166Thr + Ser216Asp +Thr217ProArg 64Asp + Ala164Gln + Ser191Asp + Thr206Glu +Leu209CysLeu 96Val + Gly134Asp + Gly136Pro + Ser191Asp +Thr206GluTyr104Asn + Asp165Glu + Phe192Asp + Ser207Asp +Ile220GlnSer101Asp + Ala164Ser + Thr206Pro + Ser207Glu +Arg218AspAsp165Glu + Ser207Glu + Leu209Asn + Trp212Gly +Arg218AspGly100Asn + Tyr104Pro + Ser138Glu + Ser207Glu +Arg218GluSer140Glu + Ser170Glu + Thr211Gln + Gly215Asn +Ser221AspGly100Glu + Ser140Asp + Ser170Asp + Thr206Ser +Leu209HisSer140Glu + Tyr169Pro + Ser170Glu + Arg218Asp +Ile220ProGln103Ser + Tyr104Asp + Leu209Glu + Ser221Asp +Gly222SerAsp 97Glu + Ile107Cys + Asn162Asp + Ser191Asp +Leu209AlaAsp 97Glu + Tyr104Glu + Ile107Gly + Leu133Ser +Ile220LeuLeu 96Glu + Leu133Asp + Gly134Asn + Gly215Ser +Ser216GluAsn 67Glu + Leu 96Glu + Thr106Pro + Ile107Cys +Ser207GluArg 64Glu + Asp 97Glu + Gly102Asn + Asp165Glu +Phe192TyrGly 66Ser + Gly134Asp + Asn161Asp + Ser207Glu +Thr217GlyAsp 65Glu + Gly 70Asn + Gly100Pro + Leu209Asp +Ser216AspGly 68Glu + Ser101Asp + Leu209Thr + Trp212Ala +Ser219AspThr106Gly + Asn163Asp + Leu209Pro + Ser219Glu +Thr223GluAsp 65Glu + Gln103Ser + Gly134Glu + Gly136Asn +Ser138GluGln103Ser + Ile107His + Asn161Asp + Asp165Glu +Ser207GluGly 66Pro + Ser140Glu + Ala164His + Thr217Asp +
Ile220AspArg 64Asp + Ser101Asp + Tyr104Leu + Ile107Glu +Ile220AsnArg 64Glu + Val 95Met + Gly136Ser + Ser190Glu +Ile213AspGly135Glu + Gly160Pro + Leu209Gln + Ile220Leu +Thr223GluAsp 65Glu + Val 95Gln + Ser170Glu + Phe192Met +Gly214GluGly100Asn + Ser190Asp + Ser207Asp + Leu209Ser +Ser216GluVal 95Gly + Arg167Asp + Ser190Asp + Ser207Glu +Ile220AsnAsn 67Glu + Ser101Glu + Leu133Ser + Asn163Asp +Thr223SerAsn 67Glu + Asn 99Ser + Ser101Glu + Tyr104Ala +Ser207GluGly134Asn + Ser138Asp + Arg218Asp + Ile220His +Ser221GluGly 66Gln + Val 95Pro + Ser138Glu + Arg218Glu +Ser221AspAsn 67Asp + Thr 71Ser + Gly135Glu + Asn161Asp +Ala164HisAsp 65Glu + Gly 66Pro + Ser191Glu + Ile213Ser +Ser221GluGly 70Pro + Gly134Pro + Ser191Asp + Ser216Asp +Ser221AspGly100Glu + Tyr104Glu + Asn163Gln + Ile213Pro +Ile220SerGly134Glu + Gly136Pro + Asn162Gln + Ser216Asp +Thr223AspThr 71Asn + Ser105Asp + Leu209Met + Ser219Asp +Gly222GluGly 66Asn + Gly102Asn + Tyr169Asp + Ser216Asp +Ser219AspGly136Pro + Asn161Glu + Ser216Glu + Ser219Glu +Ile220SerSer140Glu + Ile208Met + Ser216Glu + Ser219Glu +Ile220AsnGly160Asp + Thr206Gln + Gly214Ser + Ser216Glu +Ser219GluAsn 99Ser + Ser190Glu + Ser216Glu + Ser219Glu +Ile220HisSer139Asp + Thr206Gln + Ser216Glu + Ser219Asp +Gly222AsnGly102Asn + Ser190Asp + Ser216Glu + Ser219Asp +Gly222GlnGln103Ser + Asn163Asp + Trp212Met + Ser216Asp +Ser219AspThr 71Gly + Gly136Glu + Asn194Gln + Ser216Asp +Ser219GluAsp 97Glu + Leu209Met + Ser216Glu + Ser219Asp +Ile220ValAsn 99Gln + Asp165Glu + Leu209Met + Ser216Glu +Ser219GluThr106Glu + Asn168Gln +Leu209Met + Ser216Asp +Ser219GluLeu 96Gln + Asn161Gln + Tyr169Asp + Ser216Asp +Ser219GluLeu 96Met + Asn161Asp + Gly203Gln + Ser216Asp +Ser219GluAsn 99Glu + Thr206Ser + Ser207Glu + Leu209Val +Thr223GluSer207Glu + Leu209Met + Ser216Asp + Ile220Leu +Thr223AspVal 95Ala + Gly214Asn + Ser216Asp + Ile220Asp +Gly222ProSer101Asp + Tyr104Gln + Gly136Asn + Gly160Glu +Ser190AspGly 70Asp + Gly135Glu + Arg167Asp + Asn168Gln +Ile220GlnAsp 65Glu + Leu 96Met + Gly136Glu + Asn162Asp +Leu209AlaVal 95Asp + Asn 99Glu + Asn194Ser + Gly214Pro +Thr223AspGly136Glu + Ala164Ser + Asp165Glu + Tyr169Pro +Ser216AspArg 64Glu + Gly135Asn + Asn162Gln + Ser19lAsp +Gly214AspArg 64Asp + Asn162Gln + Thr206Gln + Ser207Glu +Gly214GluLeu 96Ala + Gln103Ser + Asn162Glu + Arg167Asp +Gly215GluAsn161Ser + Asn162Glu + Arg167Glu + Thr206Pro +Ser219AspAla164His + Asp165Glu + Tyr169Pro + Ser170Asp +Gly215GluVal 95Ala + Gln103Glu + Ser138Asp + Gly160Asn +Ser219AspIle107Glu + Leu133Asn + Ser138Asp + Ala164Gly +Ser191GluIle107Glu + Leu133Ser + Asn163Glu + Arg167Glu +Leu209HisSer101Glu + Leu209Asp + Gly215Gln + Ile220Asn +Gly222GluSer139Asp + Asn162Gln + Tyr169Gln + Leu209Asp +Gly222AspThr106Glu + Gly136Ser + Ser170Glu + Asn194Glu +Thr206GlnTyr104Ala + Leu133Asn + Asn162Asp + Ser207Asp +Thr217AspTyr169Cys + Ser191G1u + Ser207Glu + Ser216Asp +Gly222GlnVal 95Gln + Tyr104Leu + Tyr137Glu + Ser191Glu +Ser207GluGly 66Asp + Gly100Asn + Ser101Glu + Ser139Glu +
Tyr169GlnGly 66Glu + Val 95Ser + Ser101Glu + Asn163Ser +Ser191AspArg 64Glu + Val 95Ala + Asn 99Glu + Ala164Glu +Gly214SerArg 64Glu + Asn 99Asp + Ser105Asp + Gly160Pro +Leu209GlyAsn 67Asp + Val 95Asp + Gly102Pro + Ser207Glu +Gly214SerSer101Asp + Ser105Asp + Asn162Glu + Tyr169Gln +Ile220SerSer139Asp + Gly160ser + Ala166Thr + Phe192Glu +Ser207AspVal 95Gly + Gly136Pro + Asn161Glu + Arg167Asp +Ser207AspAsp 65Glu + Ser101Glu + Gly102Asn + Leu133Thr +Ser170AspGly136Gln + Asn161Asp + Arg167Asp + Asn194Ser +Ser216GluGly135Asp + Ala164Asp + Ser216Glu + Ile220Leu +Gly222AsnAsp 97Glu + Asn 99Gln + Gln103Asn + Ser105Glu +Ala166SerAsn 99Gln + Asn163Glu + Ile213Leu + Ile220His +Ser221AspGly135Glu + Asp165Glu + Phe192Gln + Ser207Glu +Gly215SerAsn162Asp + Ser170Glu + Asn194Ser + Leu209Gln +Ser219GluGly100Asp + Thr106Asp + Ala164Glu + Phe192Thr +Asn194GlnSer105Glu + Asn162Glu + Leu209His + Ser221Glu +Gly222GlnGly 68Asn + Gly135Glu + Gly160Gln + Ser216Glu +Ile220AspAsp 97Glu + Ala164Gly + Leu209Met + Ser216Asp +Ile220GluGly 68Asp + Tyr137Asp + Asn194Gln + Leu209Cys +Ser221AspIle107Asp + Gly136Gln + Ser139Asp + Ser216Asp +Ile220ThrGly100Pro + Ser101Glu + Gly136Glu + Ser191Asp +Phe192AlaGly 70Glu + Gly160Pro + Ala164Thr + Ser170Glu +Ser216GluArg 64Asp + Tyr104His + Asp165Glu + Leu209Gln +Thr223GluLeu133Ala + Ser138Asp + Leu209Cys + Thr217Glu +Ser221AspSer140Glu + Gly203Asn + Thr217Glu + Ile220Met +Ser221AspAsp 97Glu + Thr106Asn + Gly134Glu + Asp165Glu +Thr211GlnGly102Pro + Ser140Asp + Asn194Asp + Gly215Pro +Ser221AspArg167Asp + Leu209Asn + Ser219Asp + Ile220Val +Thr223GluGly 68Asp + Gly102Asp + Gly136Ser + Ser170Asp +Phe192Leu表36多環(huán)區(qū)六突變變異體Val 95His + Ala164Ser + Ser170Glu + Phe192Met +Leu209Thr + Ile220ProLeu133Asn + Asn161Gln + Asn163Gln + Leu209Met +Ile220Leu + Thr223GlyGly134Asn + Tyr169Ala + Thr211Pro + Ser216Asp +Ile220Met + Gly222GlnGly136Glu + Gly203Gln + Thr206Pro + Leu209Thr +Gly215Pro + Ile220MetGly100Ser + Gly135Asn + Ala164Thr + Gly214Glu +Thr217Gln + Ile220LeuThr106Ser + Thr206Pro + Ile208Cys + Leu209Ser +Ser216Asp + Ile220SerGly 68Asn + Leu 96His + Gly102Asn + Leu209Cys +Trp212Ala + Ile220ValGly 70Pro + Val 95Gln + Gly102Gln + Asn162Asp +Gly214Gln + Ile220HisAsn 67Gln + Leu 96Val + Leu133Ala + Ala164Thr +Leu209Ala + Ser219AspGly100Asn + Leu133Asp + Asnl61Gln + Thr206Gly +Ile213Leu + Gly222GlnLeu 96Asn + Gly100Gln + Thr106Asn + Ser140Glu +Tyr169His + Ile220AsnGly 70Pro + Asn 99Ser + Gly135Gln + Tyr169Gly +Thr206Ser + Ser216AspSer101Glu + Gly102Glu + Tyr169Met + Thr206Asn +Leu209Ile + Ile220GlnVal 95Ala + Asn168Gln + Gly214Asp + Gly215Glu +Ser216Glu + Ile220ProLeu 96Pro + Thr106Gly + Ile107Gln + Asn163Glu +Phe192Glu + Leu209GlyAsn194Ser + Phe202Gly + Ser207Glu + Arg218Glu +Ser219Asp + Ile220AspAsp 65Glu + Asp 97Glu + Asp 98Glu + Asn168Ser +Tyr169Thr + Thr211SerAsn 67Gln + Gly215Ser + Ser219Glu + Ile220Pro +Ser221Glu + Gly222GlnGly102Asn + Gln103Ser + Tyr169Ile + Thr206Gly +Ser216Glu + Arg218AspAsn 67Glu + Gly 70Glu + Leu 96Pro + Asn 99Ser +Asn162Gln + Phe192ProAsn 67Asp + Gly 68Gln + Gly 70Asp + Leu133Val +Leu209Gly + Thr217AsnGly 68Ser + Leu133Glu + Gly135Asp + Ser170Glu +Ile213Asn + Ile220SerVal 95Asp + Asp 97Glu + Gly100Asp + Gly135Gln +Leu209Gln + Thr223SerLeu 96Cys + Gly134Asp + Gly160Glu + Asn161Glu +Asn162Glu + Leu209ProAsp 65Glu + Leu 96Glu + Asp 97Glu + Asn 99Asp +Ile220Ser + Gly222ProVal 95Pro + Leu209Ser + Ser219Glu + Ile220Gly +Ser221Glu + Gly222AspGly 66Asn + Asp 98Glu + Ser101Asp + Tyr104Cys +Asn194Gln + Ile220AsnIle107Thr + Tyr137His + Phe202Leu + Ser216Glu +Arg216Asp + Ser219GluGly134Pro + Tyr137Pro + Ser207Glu + Leu209Cys +Ser219Asp + Gly222GluLeu133Thr + Asn163Ser + Ser207Asp + Ile213Met +Arg218Asp + Ser221AspArg 64Asp + Asp 98Glu + Thr106Ser + Gly160Asn +Ile213Pro + Ile220AsnGly 70Gln + Leu 96Asn + Tyr169Met + Thr206Glu +Leu209Cys + Thr223AspGly 68Pro + Ala164Glu + Arg167Glu + Pro204Asn +Thr206Gln + Ile220SerAsn 99Ser + Gly135Asp + Tyr137Met + Ser138Glu +Asn168Asp + Thr211AsnGly 68Pro + Tyr104Glu + Ser138Glu + Thr217Ser +Ile220Gln + Thr223ProGly 66Ser + Asn163Gln + Thr206Asp + Leu209Gly +Ser219Asp + Ile220MetGly 68Asp + Thr106Pro + Thr206Gly + Leu209Glu +Ser219Glu + Ile220AspTyr137Leu + Tyr169Cys + Phe192Asn + Ser207Asp +Arg218Glu + Ile220LeuGly 68Asn + Val 95Glu + Gly100Glu + Leu133Cys +Gly134Pro + Thr217SerGly 68Asp + Gly100Gln + Thr206Pro + Ser219Glu +Ile220Glu + Ser221GluTyr137Asn + Ala164Gln + Ser207Glu + Leu209Glu +Arg218Asp + Gly222GluGly135Pro + Ser191Asp + Asn194Gln + Thr206Glu +Ser207G1u + Ser219GluVal 95Asp + Ser105Asp + Gly160Asn + Leu209Gly +Gly215Pro + Ile220AlaGly 68Asp + Gly100Ser + Gly134Asn + Ser207Glu +Ser219Glu + Ser221GluAsn 67Gln + Gln103Ser + Thr206Glu + Leu209Glu +Ser216Glu + Ser219AspTyr104Ile + Thr206Glu + Ser216Asp + Arg218Asp +Ser219Glu + Thr223SerGly 68Gln + Val 95Pro + Leu133Ala + Asn162Gln +Ser190Glu + Ser207AspAsn163Ser + Thr206Asp + Ser207Glu + Leu209Ser +
Ser216Glu + Ser219AspAsn161Ser + Thr206Ser + Gly214Asp + Gly215Asp +Thr217Glu + Ile220GluAsn 99Ser + Ser138Asp + Ser139Asp + Ser140Asp +Ile213Gly + Ser216AspGly 70Glu + Leu 96Asn + Thr206Asn + Ser207Glu +Ser219Asp + Ser221AspTyr104His + Asn161Asp + Ser170Asp + Phe192His +Asn194Asp + Gly222ProLeu 96Glu + Asp 97Glu + Ser101Asp + Leu209Thr +Ser219Asp + Ile220AsnSer207Asp + Ile208Ala + Leu209Thr + Ser216Asp +Ser219Glu + Ile220LeuGly100Glu + Leu133Glu + Gly135Glu + Asn161Glu +Ala164Gly + Leu209ThrLeu 96Asn + Gly136Ser + Leu209His + Gly214Glu +Ser216Asp + Ser219GluTyr104Leu + Ile107Glu + Tyr137Glu + Ser138Asp +Asn168Gln + Leu209IleTyr137Glu + Phe192Met + Ser207Glu + Ser219Asp +Ser221Glu + Thr223ProTyr137Asp + Asn168Asp + Ser170Glu + Phe192Asn +Ser207Asp + Ile220AsnVal 95Ser + Gly102Ser + Asn162Gln + Leu209Ser +Ser216Glu + Ser219AspGly 66Ser + Gly102Gln + Ser216Glu + Ser219Asp +Ile220Val + Gly222AsnAsp 97Glu + Asp 98Glu + Ser101Glu + Gly134Pro +Asn162Glu + Thr206GlnVal 95Pro + Ser101Asp + Gly135Asn + Ser207Asp +Leu209Asp + Ile220AspAsp 98Glu + Gly203Ser + Ser207Glu + Leu209Pro +Arg218Glu + Ser219AspThr106Pro + Gly135Asp + Ser140Asp + Asn162Glu +Ser170Glu + Gly214GlnGly 68Gln + Ile107Asn + Asn162Ser + Ser191Asp +Ser207Asp + Ser219GluGly102Ser + Gly136Asn + Thr206Glu + Leu209Thr +Arg218Asp + Gly222AspAsp 65Glu + Gln103Ser + Ser207Glu + Leu209Gln +Arg218Asp + Ile220GluGly 66Pro + Asn 99Gln + Gly136Asp + Arg218Asp +Ile220Glu + Ser221AspTyr104Asp + Gly134Glu + Gly135Asp + Thr211Ser +Ser219Glu + Ile220ValGly100Gln + Tyr104Ser + Ser139Glu + Ser191Glu +Phe192Asp + Thr206GluSer138Glu + Thr206Pro + Leu209Thr + Ser216Glu +Arg218Asp + Ser219AspAsp 97Glu + Leu209Gln + Ser216Asp + Arg218Asp +Ser219Glu + Ile220AlaGly102Asp + Leu133Thr + Asn163Gln + Ser216Glu +Thr217Glu + Ser219AspAsp 98Glu + Asn 99Asp + Gly102Asp + Thr106Ser +Ile107Gln + Ser138AspAsn 67Asp + Gly 70Glu + Ser170Asp + Phe192Pro +Leu209Thr + Ile213GluGln103Asp + Gly134Asn + Asn162Glu + Phe192Glu +Ile220Leu + Gly222AspAsp 65Glu + Asp 97Glu + Gly100Asp + Tyr169Met +Ser191Glu + Ile220CysAsn 67Asp + Asp 98Glu + Ala164Pro + Ile208Thr +Leu209Ser + Ile220AspGly136Asn + Ser191Asp + Thr206Pro + Arg218Asp +Ser219Asp + Gly222AspAsp 98Glu + Gly136Asp + Ser138Glu + Ser140Asp +Gly203Gln + Ile220MetLeu133Ile + Gly134Pro + Gly135Pro + Asn162Glu +Arg167Glu + Ile213ValGly102Asn + Ser140Asp + Asn161Glu + Asn162Asp +Ser190Glu + Gly205AsnSer139Glu + Gly160Glu + Asn161Gln + Thr206Pro +Ser221Glu + Gly222GluGly 66Asp + Asn 67Asp + Ile107Gly + Asn194Gln +Ser207Asp + Ile220GluTyr104Gly + Gly136Asp + Ser170Asp + Ser190Glu +Asn194Asp + Gly222SerGly 68Asp + Ile107Asp + Gly135Asn + Gly215Glu +Ser216Glu + Ile220GlySer101Glu + Ser207Asp + Leu209Met + Gly215Pro +Thr217Glu + Ser219GluAsp 65Glu + Gly100Glu + Gln103Asp + Asn161Gln +Phe192His + Ile220SerGly 68Gln + Ser138Asp + Ser139Glu + Ala164Gly +Asn194Glu + Phe202GlnAsn 99Glu + Thr106Gln + Gly214Gln + Gly215Asn +Ser219Asp + Ile220GluGly 66Pro + Asn163Glu + Ala164Gln + Phe192His +Ser219Asp + Ile220AspGly100Gln + Tyr104Pro + Tyr137His + Arg167Asp +Asn168Asp + Ser207AspAsn 99Ser + Gly100Ser + Asn161Asp + Asn162Asp +Leu209Asn + Ser216GluGly100Asp + Ser101Glu + Asn168Gln + Leu209Met +Ile213Ser + Ser219AspVal 95His + Gly100Gln + Ser139Glu + Ser140Asp +Ser207Glu + Thr217SerGly100Asp + Gly134Gln + Ser139Asp + Ser140Asp +Asn161Ser + Leu209ValVal 95Cys + Gly100Glu + Thr106Asn + Ser139Asp +Ser140Glu + Gly222SerSer105Asp + Asn194Gln + Thr206Asp + Ser207Asp +Leu209Val + Gly215SerAsn163Glu + Phe192Pro + Trp212Val + Ser216Asp +Thr217Asp + Gly222ProVal 95Asn + Gln103Asn + Leu209Gln + Gly215Asp +
Ser216Glu + Ser221AspGly 66Gln + Gly134Glu + Ser170Asp + Leu209Thr +Ile220Asp + Gly222AspGly 66Gln + Thr106Glu + Ile107Glu + Asn163Asp +Asn194Glu + Pro204AsnGly102Glu + Gln103Asp + Phe192Ile + Ser207Glu +Leu209Pro + Ile220AspArg 64Glu + Asp 65Glu + Ala164Asn + Ser207Glu +Leu209His+ Ser219AspArg 64Asp + Asp 65Glu + Gly135Pro + Tyr137Met +Ser207Glu + Ser219AspTyr104Gly + Ser105Glu + Phe192Gln + Ser207Asp +Ser221Asp + Gly222GlnSer105Glu + Tyr137Ile + Ala164His + Thr206Asn +Ser207Glu + Ser221GluAsn 67Gln + Asp 98Glu + Asn161Gln + Asn168Ser +Ser207Glu + Ile220AspGly134Ser + Ser139Asp + Thr206Asp + Ile213Ala +Arg218Glu + Ser219AspAsp 98Glu + Gly100Gln + Gln103Asn + Gly134Asp +Ser139Glu + Ser170GluLeu 96Gly + Asp 98Glu + Thr106Asn + Leu209Glu +Ser216Asp + Ser219AspGly 70Glu + Asp 97Glu + Asn 99Glu + Gly135Ser +Asn194Glu + Leu209ThrThr106Gly + Arg167Glu + Ser207Glu + Gly214Gln +Gly215Pro + Ser219GluAla164Glu + Thr206Gly + Ser207Asp + Leu209His +Ser219Asp + Ile220GlnGly160Ser + Asn162Gln + Ser170Glu + Thr206Pro +Ser207Asp + Ser219AspTyr104Cys + Asn162Gln + Tyr169Asp + Ser207Glu +Leu209Ala + Ser219AspAsn 67Gln + Asp165Glu + Ser207Glu + Leu209Val +Ser219Asp + Ile220AlaGly 66Glu + Thr106Gln + Phe192Asp + Asn194Ser +Thr206Glu + Ser207AspGly134Asp + Ala164Ser + Ser170Asp + Ser207Asp +Ile208Met + Ser219AspGly 66Gln + Gly102Asn + Gly136Asp + Ser139Glu +Asn168Asp + Ser219AspAsp 97Glu + Asn 99Asp + Thr206Asn + Ser207Asp +Ser219Asp + Ile220MetIle107Val + Asn162Asp + Ser191Asp + Ser207Asp +Ser219Glu + Gly222ProGly 66Ser + Asn 67Glu + Ser138Asp + Ser140Glu +Leu209Cys + Ile213AlaGly 70Ser + Asp 98Glu + Gly100Asp + Asn162Gln +Ser207Glu + Ser219GluAsp 97Glu + Asn 99Glu + Tyr104Gly + Thr206Glu +Leu209Ala + Thr217GlnArg 64Glu + Gly 66Glu + Asn162Asp + Tyr169Ser +Leu209His + Gly214GluGly 70Gln + Leu133Asp + Gly136Glu + Asn194Asp +Ile213Cys + Gly222AspGly 70Gln + Gln103Asp + Ser105Glu + Thr106Pro +Ser219Asp + Ile220SerGln103Glu + Ser105Glu + Asn162Gln + Gly205Pro +Trp212Asn + Arg218GluAsp 97Glu + Asn 99Gln + Gly136Asp + Gly160Ser +Ser170Asp + Leu209MetSer105Glu + Ile107Glu + Ser207Glu + Ile213Thr +Gly215Ser + Ile220AsnSer190Asp + Phe192Ile + Ser216Glu + Ser219Glu +Ile220Glu + Gly222SerAsp 65Glu + Gly102Gln + Ser207Asp + Ile213Asp +Ser216Glu + Ile220AsnGln103Asp + Leu133Asn + Phe192Asp + Ile213Val +Ile220Asp + Ser221AspLeu 96Cys + Ile107Gly + Asn162Asp + Asn194Asp +Ser216Asp + Ile220HisSer101Asp + Ser105Glu + Thr106Asn + Ser138Asp +Ser170Glu + Leu209SerGly 70Ser + Tyr169Val + Phe192Asp + Ser216Glu +Ser2l9Glu + Ile220AspGly136Gln + Asn161Asp + Asn163Gln + Arg167Asp +Asn194Glu + Ser216GluAsp 97Glu + Asp 98Glu + Asn161Glu + Ala164Asp +Ile213Ala + Thr217GlyIle107Cys + Arg167Asp + Asn168Glu + Ser207Glu +Ile220Thr + Gly222GluArg167Asp + Tyr169His + Ser170Glu + Leu209Val +Ser219Asp + Ile220GluGly 70Gln + Phe192Asp + Ser216Glu + Arg218Glu +Ile220Val + Gly222GluArg 64Asp + Gly 70Asn + Phe192Ala + Ser207Asp +Leu209Glu + Thr223GlyGly 68Asn + Asp 98Glu + Ser207Asp + Leu209Asp +Ile220Asn + Thr223GlnGly100Gln + Leu133Ile + Asp165Glu + Ala166His +Tyr169Asp + Ser191GluAsn163Asp + Ser191Asp + Thr206Gly + Leu209Asp +Thr211Asn + Ile220GluAsn 99Gln + Gly160Glu + Tyr169Val + Ser191Asp +Ser216Asp + Gly222AspAsn 67Glu + Asp 97Glu + Ser105Asp + Ala164Thr +Thr206Ser + Ile220ValArg 64Asp + Gly 70Glu + Val 95Thr + Asp 97Glu +Asn162Asp + Thr223GlnLeu 96Ser + Gln103Glu + Ser105Glu + Gly134Asp +Asn194Ser + Ser219AspGly100Gln + Leu133Gln + Ala164Gln + Arg167Glu +Ser219Asp + Ser221GluVal 95Gln + Tyr137Asn + Ser140Glu + Pro204Asn +Ser219Asp + Ser221AspGly134Ser + Ser190Glu + Ser207Glu + Leu209Gln +
Thr217Glu + Ile220HisGly 70Ser + Gly134Ser + Asn162Ser + Ser170Asp +Ser216Glu + Arg218AspAsp 97Glu + Tyr137Thr + Tyr169Gln + Ser216Glu +Arg218Glu + Gly222GlnAsn 67Glu + Gly 70Glu + Asn 99Gln + Tyr104Met +Gly136Asp + Ala164AsnAsn 67Glu + Gly 68Asp + Gly 70Gln + Ser191Glu +Ile213Ser + Ile220GluThr106Gly + Asn162Glu + Gly215Glu + Thr217Asp +Ser219Glu + Ile220AsnArg 64Asp + Asp 65Glu + Ile208Gln + Leu209Asp +Ser216Asp + Ile220ValSer101Glu + Leu133Glu + Thr206Asn + Ser207Glu +Thr217Asn + Ser219AspTyr104Glu + Ser138Glu + Asn162Glu + Asn194Glu +Thr211Ser + Trp212SerAsp 98Glu + Asn 99Gln + Gly102Glu + Ser190Glu +Ser191Glu + Ile220ProVal 95Asp + Gly134Gln + Tyr169Pro + Phe192Asn +Ser221Glu + Thr223AspAsn 67Glu + Leu 96Glu + Gly160Ser + Asn162Ser +Thr206Gly + Thr217AspGly 68Asp + Val 95Glu + Asn 99Glu + Tyr169Ala +Gly215Asp + Thr217SerVal 95Ser + Gly135Ser + Asn161Asp + Ile213Asp +Ser216Asp + Ser219AspArg 64Glu + Asp 98Glu + Gly136Ser + Gly160Gln +Leu209Asp + Ile220AspAsp 65Glu + Gly 66Ser + Asn194Gln + Ser207Asp +Ser216Asp + Ser219AspAsn 99Ser + Ser101Asp + Tyr104Asp + Gly134Ser +Ser207Asp + Ile220AspArg 64Glu + Asp 98Glu + Ile107Asn + Thr206Ser +Ser219Asp + Ser221GluVal 95Pro + Ala164Asp + Asp165Glu + Ser170Asp +Ile213Asp + Ile220ValGly135Ser + Asn161Gln + Asn162Asp + Tyr169Asp +Gly215Gln + Ser221GluTyr137His + Ser140Asp + Asn161Gln + Ser170Asp +Ser207Asp + Leu209AspLeu 96Ile + Ser138Glu + Ala164His + Asn194Asp +Thr206Asp + Gly222GluAsn161Glu + Ser207Asp + Gly214Asn + Ser216Asp +Arg218Glu + Gly222GlnLeu 96Asn + Ser170Asp + Ile208Asn + Gly215Glu +Arg218Glu + Ser219AspGly134Asp + Ser139Asp + Ser140Glu + Thr206Asp +Ile213Met + Thr223AsnAsp 65Glu + Asp165Glu + Phe192Pro + Leu209Gly +Gly215Glu + Ser216AspAsn 99Asp + Ser101Glu + Ile107Glu + Ser191Glu +Asn194Gln + Ile220LeuAsp 65Glu + Ser190Glu + Asn194Gln + Ser207Glu +Gly215Pro + Ser219GluGly134Asn + Ser138Glu + Ser139Glu + Ser216Glu +Ser219Asp + Ile220ProTyr137Cys + Ala164Thr + Ser190Glu + Ile208His +Ser216Glu + Gly222GluAsp165Glu + Ser207Asp + Ser216Glu + Arg218Asp +Ile220Pro + Gly222ProVal 95Met + Ser139Glu + Ser140Asp + Ala164Asn +Ser216Asp + Ser219AspVal 95Gly + Gly102Ser + Ser105Asp + Ser138Glu +Asn163Glu + Ala164GluAsp 65Glu + Val 95Gly + Asp 97Glu + Gly160Ser +Asp165Glu + Ile220SerThr 71Gln + Ser101Asp + Thr106Glu + Tyr169Pro +Ser207Glu + Ser219AspTyr137Ile + Asp165Glu + Tyr169Cys + Gly214Asp +Arg218Glu + Ser219AspAsn 99Ser + Ala164Asp + Ser170Asp + Asn194Glu +Thr206Gln + Ser216AspAsp 65Glu + Gly134Asp + Gly160Asn + Asn161Glu +Asn168Asp + Ile220AsnAsp 65Glu + Phe192Ser + Ser216Glu + Ser219Glu +Ile220Met + Ser221GluGly 68Asn + Leu133Gln + Asn168Asp + Ser207Glu +Ser216Glu + Ser219AspGly135Glu + Ser207Glu + Ser216Asp + Thr217Asn +Ser219Glu + Ile220ProGly 68Asn + Asn163Glu + Ser207Asp + Ser216Glu +Ser219Glu + Ile220AsnGly102Glu + Tyr137Cys + Thr206Ser + Ser207Glu +Ser216Asp + Ser219AspLeu133Ser + Arg167Glu + Ser170Asp + Ser191Asp +Thr206Asp + Leu209HisGly 66Gln + Gly136Glu + Ser138Glu + Thr206Gln +Arg218Glu + Ser221AspAsn 67Glu + Ser139Asp + Ile208Val + Leu209Ser +Ser216Asp + Thr217AspArg 64Asp + Val 95His + Gly134Glu + Ser138Glu +Arg167Glu + Leu209CysArg 64Asp + Phe192Val + Thr206Asp + Leu209Ala +Ser219Glu + Gly222SerLeu 96Ala + Ser140Asp + Phe202Gln + Thr206Asp +Leu209Ala + Ser219GluLeu 96Ile + Gly102Pro + Gly134Asp + Thr206Asp +Leu209Ala + Ser219AspGly102Glu + Ser105Asp + Ile107Cys + Ser140Asp +Asn194Asp + Ile220AlaGly100Glu + Ser105Asp + Tyr137Pro + Ser140Glu +Gly203Asn + Gly222ProThr106Asn + Asn162Glu + Asn168Asp + Ser216Glu +Arg218Asp + Ile220AsnAsp 98Glu + Leu133Gly + Gly136Asp + Ser140Glu +
Asn168Glu + Phe192SerVal 95Ala + Asp165Glu + Asn168Ser + Ser207Asp +Leu209Glu + Ser216AspGly134Glu + Ala164Asp + Asn168Glu + Phe192Cys +Ser219Glu + Ile220ProGly 68Asn + Ser191Asp + Phe192Asn + Gly215Glu +Ser219Glu + Ser221GluGln103Glu + Ile107Asn + Ser140Glu + Ser190Glu +Ser191Glu + Leu209ThrTyr104Ala + Leu133Asn + Asn162Asp + Thr206Glu +Ser207Asp + Thr217AspGly160Asn + Asn194Asp + Ser207Asp + Ile213Val +Ser216Asp + Thr217AspLeu 96Ile + Ala164Gln + Asp165Glu + Ser207Glu +Arg218Glu + Ile220MetGly 66Glu + Ile107Ala + Gly160Asn + Phe192Val +Ser207Glu + Arg218GluGly100Gln + Tyr104Asp + Gly134Asp + Ala164Ser +Phe192Leu + Ser216AspArg167Glu + Leu209Thr + Ser216Glu + Ser219Glu +Ser221Glu + Gly222AsnGly 68Asn + Asp 98Glu + Leu209Gly + Ser216Asp +Ser219Asp + Ser221GluAsp 65Glu + Ser207Glu + Leu209Asp + Gly215Asp +Gly222Ser + Thr223AsnGly134Ser + Ser140Glu + Asn168Gln + Ser191Glu +Asn194Glu + Ser221AspLeu 96Ile + Thr106Glu + Gly160Asp + Ser219Glu +Gly222Glu + Thr223SerSer101Asp + Ser105Glu + Asn161Gln + Ser207Glu +Ile213Asn + Ser221GluGln103Ser + Gly135Asp + Ser190Glu + Thr206Ser +Ser219Glu + Ile220GluAsp 65Glu + Gly100Asp + Ser105Asp + Ser140Glu +Phe192Thr + Ile220HisAsp 65Glu + Ser101Glu + Ala164Thr + Ile213Glu +Thr217Asn + Ser219GluSer105Asp + Gly134Asp + Asn168Ser + Ser191Asp +Phe192Glu + Ile220CysLeu 96Asp + Ile107Asp + Gly214Ser + Ser216Glu +Ser219Glu + Ile220ValAsn162Gln + Asp165Glu + Arg218Asp + Ser219Glu +Ile220Thr + Thr223AspThr 71Gly + Asn161Asp + Asn168Asp + Ser207Glu +Leu209Asp + Thr223GlnAsp 65Glu + Asp 98Glu + Leu133Ser + Asp165Glu +Leu209Met + Gly215GluArg 64Asp + Asn 99Glu + Asp165Glu + Ala166Asn +Asn194Glu + Ile220CysAsn 99Ser + Gly136Asp + Asn163Asp + Asp165Glu +Phe202Cys + Ser216GluVal 95Pro + Leu133Thr + Gly136Glu + Asp165Glu +Ser216Asp + Arg218GluLeu133Pro + Gly134Asn + Asn194Asp + Thr206Glu +Gly214Glu + Ser221AspGly 70Asp + Asp 97Glu + Tyr104Asn + Pro204Gly +Leu209Asp + Ser216GluGln103Asp + Gly135Asp + Ser140Asp + Asn168Gln +Gly214Asp + Ile220LeuAsp 65Glu + Thr 71Ser + Asn 99Glu + Ser138Asp +Phe192Tyr + Leu209GlyTyr104Val + Ile107Glu + Thr206Asp + Ser216Asp +Ser219Glu + Ile220LeuLeu133Ala + Arg167Glu + Leu209Gln + Ser216Glu +Thr217Glu + Ser221GluAsn 67Glu + Asp 98Glu + Tyr137Ala + Asp165Glu +Thr206Pro + Ile220AspSer101Glu + Ile107Asp + Asn161Glu + Asp165Glu +Ile220Thr + Thr223GlnTyr104Asp + Thr106Asn + Tyr137Glu + Gly160Gln +Asn162Asp + Gly215SerAsn 67Asp + Asp 97Glu + Tyr104Thr + Leu133Asp +Ala164Glu + Ile220AlaAsp 97Glu + Asn 99Ser + Ser105Asp + Thr206Asp +Ser207Glu + Trp212ThrAsn 67Gln + Tyr104Ile + Ser105Glu + Asn163Glu +Ile220Glu + Ser221GluAsp 98Glu + Asn 99Gln + Leu133Asp + Phe192Glu +Asn194Glu + Gly205ProAsn 99Asp + Ile107Ser + Tyr137Met + Asn161Glu +Asp165Glu + Ile213AsnGly 70Asn + Gln103Ser + Ser138Glu + Ala164Asp +Ser170Glu + Ser219AspSer101Glu + Thr106Asn + Gly135Glu + Leu209Ser +Thr217Asn + Ser219GluGly 68Glu + Gly102Gln + Gly135Ser + Ser190Asp +Ser207Asp + Ile220AlaAsp 65Glu + Val 95Glu + Gly100Gln + Ser105Asp +Asn194Ser + Leu209GluAsp 65Glu + Tyr137His + Asn161Glu + Asn162Asp +Tyr169Thr + Ser207AspAsn 67Ser + Gly134Pro + Asn194Asp + Gly214Asp +Arg218Asp + Ser221GluAsp 65Glu + Tyr137Asn + Thr211Pro + Arg218Asp +Ile220Val + Ser221AspAsp 65Glu + Gly100Ser + Tyr104Ala + Ser207Glu +Thr217Asp + Gly222AspAsp 98Glu + Asn 99Gln + Gln103Glu + Gly136Ser +Leu209Asp + Arg218GluAsp 65Glu + Val 95Pro + Ser101Asp + Gln103Glu +Asn168Ser + Ser219GluTyr104Val + Gly134Pro + Tyr137Asp + Ala164Glu +Thr211Gly + Gly215SerSer101Asp + Gly135Pro + Ser190Asp + Ser191Asp +Ser216Asp + Gly222GlnAsn 99Glu + Ser101Glu + Gln103Ser + Gly160Pro +
Ile213Asp + Gly222GluGly 68Ser + Ser138Glu + Ser139Glu + Gly160Glu +Asn163Ser + Ser219GluArg 64Glu + Ser101Glu + Ser138Glu + Ser139Glu +Asn161Ser + Thr206AsnGly 66Asn + Gly 68Asp + Tyr104Leu + Ser138Asp +Ser139Asp + Ser207AspAsp 65Glu + Gln103Glu + Tyr137Cys + Thr206Asn +Ser219Glu + Ile220GluLeu 96Asp+ Tyr137Asp + Leu209Thr + Ile213Gly +Ser219Asp + Ile220GluLeu 96Asp + Asp 97Glu + Ser140Glu + Phe192Ala +Thr217Asp + Gly222ProSer101Glu + Leu133Cys + Arg167Asp + Asn168Glu +Leu209Ala + Ser216GluVal 95Gly + Thr106Glu + Arg167Asp + Asn168Glu +Ser191Asp + Ile220ValGly 66Ser + Asp 97Glu + Tyr169Thr + Ser170Asp +Thr217Glu + Arg218AspArg 64Asp + Ile107His + Gly135Asp + Gly136Asp +Ile213Thr + Ser219AspAsp 97Glu + Leu133Thr + Gly134Glu + Arg218Glu +Ser219Asp + Thr223SerAsp 98Glu + G1y102Ser + Leu133Met + Ser139Asp +Ser140Asp + Ser216GluTyr137Ile + Ser139Glu + Ser140Glu + Gly160Gln +Leu209Asp + Gly214AspLeu 96Ala + Asp 98Glu + Asn 99Glu + Asp165Glu +Leu209Thr + Ser216GluAsn 99Gln + Ser138Asp + Asn161Glu + Gly215Glu +Ser216Asp + Ile220GlySer105Asp + Thr106Pro + Gly136Pro + Gly160Glu +Leu209Asp + Ser219AspGln103Glu + Ala166Thr + Asn194Glu + Leu209Asp +Ser219Glu + Ile220CysTyr104Met + Thr106Ser + Ser139Asp + Thr206Pro +Ser219Asp + Gly222GluIle107Gln + Asp165Glu + Asn168Glu + Ser191Asp +Leu209Met + Ser219GluGly 68Ser + Thr106Glu + Ile107Gly + Asn162Glu +Ser207Asp + Ile220AspArg 64Asp + Gly160Asp + Ser207Glu + Ile208Gly +Ile213Leu + Ile220AspTyr137Asp + Ser170Asp + Phe192Ser + Ser207Glu +Leu209Pro + Gly214GluTyr104Cys + Gly135Asp + Ser138Asp + Thr206Asp +Gly214Gln + Arg218GluArg 64Asp + Asp 97Glu + Ser139Asp + Asn168Gln +Thr206Asn + Gly214AspAsp 65Glu + Leu133Asn + Ser170Glu + Ile208Thr +Ser216Glu + Arg218AspSer101Asp + Ser138Glu + Thr206Pro + Ser207Glu +Ser219Glu + Ile220CysGly134Glu + Ser207Glu + Leu209Ser + Gly214Glu +Ser219Glu + Ile220AlaArg 64Asp + Gly 68Asn + Gly135Asp + Gly136Gln +Ser207Asp + Ser219AspSer101Asp + Ser138Glu + Ser207Asp + Leu209Ile +Ser219Glu + Gly222GlnGly 66Asp + Ser105Asp + Ala164Gln + Phe192Ala +Ser207Glu + Ser219AspArg 64Glu + Gly 66Asn + Asn 99Ser + Ser138Glu +Ser207Asp + Ser219GluArg 64Asp + Ile107Thr + Ile213Gly + Ser216Asp +Arg218Asp + Gly222GluGln103Glu + Ile107His + Gly135Pro + Gly136Asp +Arg167Asp + Ser219GluGly 68Glu + Asp 97Glu + Thr106Glu + Phe192Met +Ser207Glu + Ile213AlaAla164Glu + Asn168Ser + Ser191Glu + Ser216Asp +Ser219Glu + Ile220ThrGly 68Gln + Asn162Glu + Ser190Asp + Leu209Thr +Ser219Asp + Gly222GlnVal 95Cys + Ile107Asp + Tyr137Ala + Asn194Glu +Gly214Asp + Ser216GluAsp 65Glu + Gly 70Gln + Leu209Gly + Ser216Glu +Ser221Asp + Thr223GluSer101Glu + Gly205Gln + Leu209Glu + Ser216Asp +Thr217Ser + Ile220ThrTyr137Thr + Ser139Glu + Leu209Asp + Gly215Pro +Ser216Glu + Thr217GlnAsn 99Glu + Gly136Pro + Ser140Glu + Ser170Glu +Asn194Glu + Ile220MetGly 66Gln + Asp 98Glu + Gly100Glu + Ser105Glu +Ile220Asp + Thr223AsnAsp 98Glu + Gly100Glu + Ser105Asp + Gly136Ser +Asn168Glu + Gly214ProVal 95Asn + Asp 97Glu + Ser101Asp + Asp165Glu +Phe192His + Ile220GluGly135Glu + Asn163Asp + Phe192Tyr + Asn194Gln +Ser219Glu + Ser221GluAsn 99Glu + Ser101Asp + Ser140Asp + Asn161Gln +Ser190Asp + Gly215AsnGly 68Gln + Gly100Asp + Gln103Asp + Tyr137Gly +Gly160Asp + Leu209CysGly100Asp + Gln103Asp + Gly160Gln + Leu209Asn +Trp212Ser + Ser221GluGly135Asp + Ser138Asp + Asn162Gln + Leu209Val +Ser216Glu + Thr223AspArg 64Asp + Gly 66Glu + Leu 96Thr + Thr106Ser +Asn161Glu + Arg218GluTyr137Ser + Asn161Asp + Ser170Asp + Ser190Glu +Leu209Asp + Gly215GlnLeu133Ala + Ser139Asp + Thr206Gln + Leu209Ala +Ser216Asp + Ser219GluAsn 67Ser + Val 95Thr + Ser140Glu + Thr206Pro +
Ser216Glu + Ser219AspVal 95Asn + Ser138Glu + Ile208Gln + Leu209Val +Ser216Glu + Ser219AspGly 70Asp + Asn 99Gln + Ser101Glu + Leu133Asn +Leu209Gln + Ser216AspTyr104Gln + Ile107His + Ser138Asp + Ser191Asp +Thr206Glu + Arg218AspThr106Glu + Ser138Glu + Thr206Gly + Ser219Glu +Ile220Thr + Ser221GluTyr137Glu + Asn161Glu + Leu209Thr + Ile213Asp +Ser216Asp + Thr217AsnArg 64Glu + Asn 67Glu + Ser170Asp + Trp212His +Ile213Ala + Arg218GluVal 95Asp + Ile107Gly + Gly134Asp + Ser138Asp +Asn161Gln + Ser190GluAsp 65Glu + Asp 98Glu + Gly160Ser + Asp165Glu +Phe192Gly + Ser219GluAsp 97Glu + Leu133His + Gly136Glu + Ser138Asp +Leu209Gly + Ser216AspAsp 65Glu + Ser138Glu + Tyr169Met + Ser207Asp +Leu209Glu + Gly215ProLeu 96Gln + Asp 97Glu + Ser138Glu + Thr206Pro +Ser207Glu + Leu209AspGly 70Gln + Asp 97Glu + Ser190Asp + Thr206Glu +Ser216Asp + Ile220AsnArg 64Glu + Gly 66Pro + Gly 70Ser + Asp165Glu +Tyr169Glu + Ser191AspGly136Asp + Asn163Asp + Phe192Pro + Ser216Glu +Arg218Asp + Ile220ValGly 66Asp + Tyr137Gly + Asn162Ser + Ala164Glu +Arg167Asp + Ser216GluGly100Asn + Gly102Asp + Tyr104His + Asp165Glu +Ser216Asp + Arg218AspGly102Glu + Leu133Val + Ser140Glu + Thr206Glu +Ile220Glu + Thr223ProSer105Glu + Leu133Asn + Ala164Asn + Asp165Glu +Thr217Glu + Ser219AspGly 70Asp + Val 95Thr + Gly134Asp + Ser139Glu +Asn161Glu + Asn194SerGly100Ser + Gly135Glu + Ser140Glu + Asn161Asp +Ile213Cys + Arg218GluGly136Asp + Tyr137Gly + Ser140Asp + Asn162Glu +Thr211Gly + Gly214AspVal 95Gly + Tyr104Ala + Ser138Glu + Asn163Asp +Arg167Asp + Ser219AspAsp 65Glu + Gly 70Asn + Val 95Ser + Ser191Asp +Thr206Glu + Ser216AspTyr104Leu + Leu133Cys + Gly134Asp + Asn162Asp +Tyr169Pro + Ser216AspVal 95Glu + Asn 99Asp + Tyr104Ala + Tyr137Ala +Leu209Asp + Ile220CysGly 66Glu + Thr106Ser + Leu209Gly + Gly215Asn +Ser216Asp + Ile220HisThr 71Pro + Gly102Glu + Gly134Asp + Ser139Asp +Ile220Pro + Ser221AspLeu 96Ser + Asp 98Glu + Ser101Asp + Gln103Ser +Gly160Asp + Ser216GluLeu 96Cys + Ala164Asn + Tyr169Pro + Thr217Asn +Arg218Asp + Gly222AspTyr104Asn + Gly136Glu + Tyr137His + Ser140Glu +Ser216Glu + Ile220CysAsp 98Glu + Gly135Asp + Ser138Glu + Asn163Glu +Phe192Cys + Ile220SerGly 66Glu + Asn 99Glu + Gly102Pro + Thr106Asn +Ser139Glu + Ser221AspGly 68Glu + Tyr104Ala + Ala164Gln + Asn168Asp +Ser219Glu + Thr223AsnAsh 99Glu + Gly102pro + Ser140Asp + Ser207Glu +Leu209Asn + Gly222AspSer105Asp + Thr106Gln + Gly160Asp + Ser191Asp +Phe192His + Gly203GlnAsp 98Glu + Tyr104Thr + Asn162Gln + Arg167Asp +Ser170Glu + Ser216GluAsn 99Asp + Gly102Ser + Tyr137Asp + Asp165Glu +Ile220Ala + Thr223GlyAsp 98Glu + Thr106Pro + Gly135Asp + Ser138Glu +Phe192Asp + Ile220GlnArg 64Glu + Val 95Ala + Leu 96Asn + Ala164Glu +Leu209Ala + Gly214GluArg 64Glu + Gly136Asn + Ala166Ser + Leu209His +Gly214Asp + Ser221GluAsn 67Asp + Gly102Asn + Ala164Glu + Asn168Glu +Ser207Asp + Ile220LeuGly135Glu + Gly136Gln + Ser139Asp + Gly160Asn +Ala164Ser + Thr206GluSer105Asp + Asn163Gln + Thr206Glu + Gly215Glu +Arg218Glu + Ile220CysGly 68Gln + Ser101Asp + Ser139Glu + Asn162Asp +Asp165Glu + Leu209GlnGly100Ser + Ser140Asp + Asn162Glu + Asp165Glu +Pro204Gln + Ser207GluThr106Glu + Ile107Ala + Gly136Glu + Ser140Asp +Asn168Gln + Ser219AspArg 64Glu + Val 95Thr + Asp 98Glu + Tyr104Thr +Ser140Glu + Ser191AspII.洗滌組合物在本發(fā)明的另一個(gè)實(shí)施方案中，在用于清潔需要除去含蛋白污漬的多種表面的組合物中含有有效量的一種或多種酶變異體。這些洗滌組合物包括用于清潔硬表面的洗滌劑組合物，形式不限(如液體和粒狀)；用于清潔織物的洗滌劑組合物，形式不限(如粒狀、液體和棒狀)；洗餐具用組合物(形式不限)；口腔清潔組合物，形式不限(如牙粉，牙膏和漱口藥制品)；假牙清潔組合物，形式不限(如液體，片狀)；和隱形眼鏡清潔組合物，形式不限(如液體，片狀)。
洗滌組合物中除了含有上文所述的蛋白酶K變異體外，還含有與這些蛋白酶相容的一種或多種洗滌組合物成份。這里用到的“洗滌組合物成份”指那些為所希望的清洗組合物的特定類(lèi)型和產(chǎn)品形式(如液體、粒狀、棒狀、噴霧型、桿狀、糊狀、膠狀)而選擇的任何液體、固體或氣體物質(zhì)，這些物質(zhì)也與組合物中所用到的蛋白酶K相容。洗滌組合物成份的具體選擇考慮到使用時(shí)(如通過(guò)洗滌劑使用)被清潔的表面材料和適合清潔條件的組合物形式而容易被作到。這里用到的“相容”指洗滌組合物成份不會(huì)將蛋白酶K變異體的蛋白水解活性降低到在正常使用情況下蛋白酶失去所需效果的程度。一些具體的洗滌組合物成份在后面詳細(xì)地舉例說(shuō)明。
這里用到的“酶變異體的有效量”指酶變異體在具體的洗滌組合物中達(dá)到需要的酶活性所需的量。這種有效量很容易為本領(lǐng)域的技術(shù)人員確定，其基于多種因素，如所用的具體的酶變異體、洗滌作用、洗滌組合物中特殊組份、是否需要濕或干(如粒狀、棒狀)的組合物等等。優(yōu)選的洗滌組合物包含約0.0001％-10％的本發(fā)明中的一種或多種酶變異體，更優(yōu)選約0.001％-1％，最優(yōu)選約0.01％-0.1％。其中加入了酶變異體的各種洗滌組合物的幾個(gè)實(shí)例將在后面更詳細(xì)地討論。這里所用到的所有份數(shù)、百分比和比率除非另外說(shuō)明，均按重量計(jì)。
這里用到的“非織物洗滌組合物”包括硬表面洗滌組合物、洗餐具用組合物、口腔清潔組合物、假牙清潔組合物和隱形眼鏡清潔組合物。A.用于硬表面、餐具和織物的洗滌組合物本發(fā)明中的酶變異體可用于多種洗滌組合物中，這些洗滌組合物具有豐富的泡沫和優(yōu)良的不溶底物去除能力。因此這些酶變異體可與各種常規(guī)組分合用得到全配制的硬表面清潔劑、餐具清洗組合物、洗滌織物用組合物等等。這些組合物可為液體、粒狀、棒狀等多種形式。這些組合物可配制為現(xiàn)代的“濃縮”洗滌劑形式，其中包含了高達(dá)30％-60％(重量)的表面活性劑。
這些洗滌組合物可任選地和優(yōu)選地包含各種陰離子型的、非離子型的、兩性離子型的等表面活性劑。這些表面活性劑在組合物中的典型含量約為5％-35％。
這里有用的表面活性劑的非限制性實(shí)例包括常規(guī)的C11-C18烷基苯磺酰鹽和伯和無(wú)規(guī)型烷基硫酸鹽，具有式CH3(CH2)x(CHOSO3)-M+)CH3和CH3(CH2)y(CHOSO3-M+)CH2CH3的C10-C18仲(2，3)烷基硫酸鹽，其中x和y+1為至少約7的整數(shù)，優(yōu)選至少約9的整數(shù)，M為水溶性的陽(yáng)離子，尤其是鈉離子，C10-C18烷基烷氧基硫酸鹽(特別是EO1-5乙氧基硫酸鹽)，C10-C18烷基烷氧基羧酸鹽(特別是EO1-5乙氧基羧酸鹽)，C10- C18烷基多苷及其相應(yīng)的硫酸化多苷，C12-C18α-磺化的脂肪酸酯，C12-C18烷基和烷基酚烷氧基化物(特別是乙氧基化物和混合的乙氧基/丙氧基化物)，C12-C18甜菜堿和磺化甜菜堿(“Sultaines”)，C10-C18胺氧化物等等。在此烷基烷氧基硫酸鹽(AES)和烷基烷氧基羧酸鹽(AEC)是優(yōu)選的。(根據(jù)配制者的需要，這些表面活性劑與上面的胺氧化物和/或甜菜堿或磺化甜菜堿表面活性劑聯(lián)合使用也是優(yōu)選的)。其它常規(guī)的有用的表面活性劑列于標(biāo)準(zhǔn)教科書(shū)中。特別有用的表面活性劑包括C10-C18N-甲基葡萄糖酰胺，其公開(kāi)于Connor等人的美國(guó)專(zhuān)利5,194,639，1993年3月16日授權(quán)，在此引入作為參考。
洗滌組合物中的多種其它有用組分也能包括在這里的組合物中，包括其它活性成份、載體、水溶助長(zhǎng)劑、加工助劑、染料或色素、用于液體配方中的溶劑等。如果需要另外增加泡沫，增泡劑如C10-C16烷醇酰胺可加到組合物中，一般為約1％-10％的量。
C10-C14單乙醇胺和二乙醇酰胺為典型的這類(lèi)增泡劑。這些增泡劑與多泡的附加表面活性劑如上面提到的胺氧化物、甜菜堿和磺化甜菜堿合用也有很好的效果。如果需要，可加入可溶的鎂鹽如MgCl2、MgSO4等，以提供更多的泡沫，加入的量一般為約0.1％-2％。
這里的液體洗滌組合物可以含水和其它溶劑作為載體。低分子量的伯或仲醇，如甲醇、乙醇、丙醇和異丙醇都是合適的。對(duì)可溶性表面活性劑，一元醇是優(yōu)選的，但多元醇如那些含大約2到6個(gè)碳原子并含大約2到6個(gè)羥基的多元醇(如1，3-丙二醇、乙二醇、甘油和1，2-丙二醇)也能使用。組合物中可含有約5％-90％的這些載體，一般約含10％-50％的載體。
這里的洗滌組合物優(yōu)選被配制成在水洗操作時(shí)，洗滌水的pH控制在約6.8-11.0之間。因而一般將最終產(chǎn)品配制在這個(gè)范圍內(nèi)。將pH控制在建議使用值的技術(shù)包括使用緩沖劑、堿、酸等，對(duì)本領(lǐng)域的技術(shù)人員來(lái)說(shuō)它們是熟知的。
當(dāng)配制本發(fā)明中的硬表面洗滌組合物和織物洗滌組合物時(shí)，配制者也許希望加入約5％-50％(重量)的各種助洗劑。一般的助洗劑包括1-10微米的沸石、多羧酸鹽如檸檬酸鹽和氧聯(lián)二琥珀酸鹽、層狀硅酸鹽、磷酸鹽等。其它常規(guī)助洗劑列于標(biāo)準(zhǔn)的配方書(shū)中。
另外，配制者可能也希望在組合物中使用各種附加酶，如纖維素酶、脂肪酶、淀粉酶和蛋白酶，常用量為約0.001％-1％(重量)。在洗衣洗滌劑領(lǐng)域，許多有清潔能力又能護(hù)理織物的酶已廣為人知。
許多漂白化合物，如過(guò)碳酸鹽和過(guò)硼酸鹽等也用于這些組合物中，一般含量為約1％-15％(重量)。如果需要，這些組合物中也可包含漂白活化劑，如四乙酰乙二胺、壬酰氧基苯磺酸鹽等，它們已為本領(lǐng)域所熟知。常用量為約1％-10％(重量)。
各種污垢解脫劑如陰離子型低聚酯、各種螯合劑，尤其是氨基膦酸鹽和乙二胺二琥珀酸鹽、各種粘土類(lèi)去污劑，尤其是乙氧基化四亞乙基戊胺、各種分散劑尤其是聚丙烯酸酯和聚天冬氨酸、各種增白劑尤其是陰離子型增白劑、各種抑泡劑尤其是聚硅氧烷和仲醇、各種織物柔軟劑尤其是綠土等都能用于這些組合物，其含量為約1％-35％(重量)。標(biāo)準(zhǔn)的配方書(shū)和已公開(kāi)的專(zhuān)利中均有這些常規(guī)物質(zhì)的全面詳細(xì)的描述。
酶穩(wěn)定劑也被用于這些洗滌組合物中。這些酶穩(wěn)定劑包括丙二醇(優(yōu)選約1％-10％)，甲酸鈉(優(yōu)選約0.1％-1％)和甲酸鈣(優(yōu)選約0.1％- 1％)。1.硬表面洗滌組合物這里所說(shuō)的“硬表面洗滌組合物”指用于清潔硬表面如地板、墻、浴室貼面等的液體和粒狀的洗滌組合物。本發(fā)明中的硬表面洗滌組合物包含有效量的一種或多種本發(fā)明的酶變異體，組合物中所含活性酶的重量?jī)?yōu)選約0.001-10％，更優(yōu)選約0.01％-5％，最優(yōu)選約0.05％-1％。除含一種或多種酶變異體外，這些硬表面洗滌組合物一般還包括表面活性劑和水溶性多價(jià)螯合助洗劑。在某些特殊的產(chǎn)品如窗戶(hù)清潔噴霧劑中，有時(shí)不用表面活性劑，因?yàn)樗鼈兛赡茉诓ＡП砻嫔袭a(chǎn)生薄膜狀或條紋狀的殘余物。
表面活性劑組分，如果有的話(huà)，只占本發(fā)明的組合物的0.1％，而通常的組合物將含約0.25％-10％，優(yōu)選約1％-5％。
通常組合物含有約0.5％-50％的洗滌助洗劑，優(yōu)選約1％-10％。
優(yōu)選的pH值應(yīng)在約8-12范圍內(nèi)，如果需要調(diào)整，可以使用常規(guī)的pH調(diào)整試劑如氫氧化鈉、碳酸鈉或鹽酸。
組合物中可以包含溶劑。常用的溶劑包括，但不僅限于乙二醇醚，如二乙二醇單己醚、二乙二醇單丁醚、乙二醇單丁醚、乙二醇單己醚、丙二醇單丁醚、二丙二醇單丁醚，和二元醇，如2，2，4-三甲基-1，3-戊二醇和2-乙基-1，3-己二醇。當(dāng)使用這些溶劑時(shí)，它們的含量約為0.5％-15％，優(yōu)選約3％-11％。
另外，高揮發(fā)性溶劑如異丙醇或乙醇也用于本發(fā)明的組合物中，當(dāng)組合物已完全應(yīng)用于表面而沒(méi)有沖掉時(shí)，它們可使之快速蒸發(fā)掉。當(dāng)使用時(shí)，揮發(fā)性溶劑常常在組合物中占約2％-12％。
本發(fā)明中硬表面洗滌組合物的實(shí)施方案在下面的實(shí)施例中列表說(shuō)明。
實(shí)施例7-12液體硬表面洗滌組合物實(shí)施例序號(hào)組份 7 8 9101112Asn67Ser 0.05 0.50 0.02 0.03 0.10 0.03Val95His - - - - 0.20 0.02Na2DIDA*EDTA**- - 2.90 2.90 - -檸檬酸鈉 - - - - 2.90 2.90C12烷基苯磺酸鈉 1.95 - 1.95 - 1.95 -C12烷基硫酸鈉- 2.20 - 2.20 - 2.20C12(乙氧基)***硫酸鈉- 2.20 - 2.20 - 2.20C12二甲胺氧化物 - 0.50 - 0.50 - 0.50枯烯磺酸鈉 1.30 - 1.30- 1.30 -己基卡必醇***6.30 6.30 6.30 6.30 6.30 6.30水****平衡到100％*N-二乙二醇 N，N-亞胺基二乙酸二鈉**乙二胺二乙酸四鈉***二乙二醇單己醚****所有配方調(diào)至pH7在實(shí)施例7-10中，用表2-36中列出的其它蛋白酶K變異體替換Asn 67 Ser具有大致相同的結(jié)果。
在實(shí)施例11-12中，表2-36中列出的蛋白酶K變異體的其它任何組合代替Asn67Ser和Val95His，具有大致相同的結(jié)果。
實(shí)施例13-18清潔硬表面和除去室內(nèi)霉菌的噴霧型組合物實(shí)施例序號(hào)組份131415161718Gly134Asn+Ser140Asp 0.50 0.05 0.60 0.30 0.20 0.30Leu96Gly+Leu209Pro - - - - 0.30 0.10辛基硫酸鈉2.00 2.00 2.00 2.00 2.00 2.00十二烷基硫酸鈉4.00 4.00 4.00 4.00 4.00 4.00氫氧化鈉 0.80 0.80 0.80 0.80 0.80 0.80硅酸鈉0.04 0.04 0.04 0.04 0.04 0.04香料 0.35 0.35 0.35 0.35 0.35 0.35水 平衡至100％產(chǎn)品pH約為7。
在實(shí)施例13-16中，用表2-36中列出的其它蛋白酶K變異體代替Gly134Asn+Ser140Asp，具有大致相同的結(jié)果。
在實(shí)施例17-18中，用表2-36中列出的其它蛋白酶K變異體的任何組合代替Gly134Asn+Ser140Asp和Leu96Gly+Leu209Pro，具有大致相同的結(jié)果。2.洗餐具用洗滌組合物在本發(fā)明的另一個(gè)實(shí)施方案中，洗餐具用洗滌組合物包含一種或多種本發(fā)明的酶變異體。這里用到的“洗餐具用組合物”指所有形式的用于洗滌餐具的組合物，包括但不限于粒狀和液體形式的洗滌劑。本發(fā)明中洗餐具用洗滌組合物的實(shí)施方案在下面的實(shí)施例中列表說(shuō)明。
實(shí)施例19-24洗餐具用洗滌組合物實(shí)施例序號(hào)192021222324Ala164Asp 0.05 0.50 0.02 0.40 0.10 0.03Ser191Asp+Phe192Met+Asn194Gln - - - - 0.40 0.02C12-C14N-甲基-葡糖酰胺 0.90 0.90 0.90 0.90 0.90 0.90C12乙氧基(1)硫酸鹽 12.00 12.00 12.00 12.00 12.00 12.002-甲基十一烷酸4.50 4.50 4.50 4.50 4.50 4.50C12乙氧基(2)羧酸鹽 4.50 4.50 4.50 4.50 4.50 4.50C12醇乙氧基化物(4) 3.00 3.00 3.00 3.00 3.00 3.00C12胺氧化物 3.00 3.00 3.00 3.00 3.00 3.00枯烯磺酰鈉2.00 2.00 2.00 2.00 2.00 2.00乙醇 4.00 4.00 4.00 4.00 4.00 4.00Mg++(如MgCl2) 0.20 0.20 0.20 0.20 0.20 0.20Ca++(如CaCl2) 0.40 0.40 0.40 0.40 0.40 0.40水 平衡至100％產(chǎn)品pH調(diào)至7。
在實(shí)施例19-22中，用表2-36列出的其它蛋白酶K變異體代替Ala164Asp，具有大致相同的結(jié)果。
在實(shí)施例23-24中，用表2-36列出的其它蛋白酶K變異體的任何組合代替A1al64Asp和Ser191Asp+Phe192Met+Asn194Gln，具有大致相同的結(jié)果。3.織物洗滌組合物本發(fā)明的另一個(gè)實(shí)施方案中，織物洗滌組合物包含本發(fā)明的一種或多種酶變異體。這里用到的“織物洗滌組合物”指所有形式的用于洗滌織物的洗滌組合物，包括但不限于粒狀、液體形式的和棒狀的洗滌劑。優(yōu)選的織物洗滌組合物是液體形式的。a.粒狀織物洗滌組合物本發(fā)明中的粒狀織物洗滌組合物含有有效量的本發(fā)明中的一種或多種酶變異體，優(yōu)選活性酶占組合物重量的大約0.001％-10％，更優(yōu)選占大約0.005％-5％，最優(yōu)選占大約0.01％-1％。除了一種或多種酶變異體外，粒狀織物洗滌組合物還常常含有至少一種表面活性劑，一種或多種助洗劑，在某些情況下包括漂白劑。
本發(fā)明中粒狀織物洗滌組合物的實(shí)施方案在下面的實(shí)施例中詳細(xì)說(shuō)明。
實(shí)施例25-28粒狀織物洗滌組合物實(shí)施例序號(hào)組份25 26 27 28Ile208Ala 0.100.200.030.05Trp212Phe- -0.020.05C13直鏈烷基苯磺酸鹽 22.00 22.00 22.00 22.00磷酸鹽(如三聚磷酸鈉) 23.00 23.00 23.00 23.00碳酸鈉 23.00 23.00 23.00 23.00硅酸鈉 14.00 14.00 14.00 14.00沸8.208.208.208.20螯合劑(二亞乙基三胺五乙酸)0.400.400.400.40硫酸鈉5.505.505.505.50水 平衡到100％在實(shí)施例25-26中，用表2-36列出的其它蛋白酶K變異體代替Ile208Ala，具有大致相同的結(jié)果。
在實(shí)施例27-28中，用表2-36列出的其它蛋白酶K變異體的任何組合代替Ile208Ala和Trp212Phe，具有大致相同的結(jié)果。
實(shí)施例29-32粒狀織物洗滌組合物實(shí)施例序號(hào)組份 29 30 31 32Asnl94Glu+Leu209Gly0.100.20 0.03 0.05Gly100Asn+Gln103Ser+Ser207Asp - - 0.02 0.05+Leu209ValC12烷基苯磺酸鹽 12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.004.00 4.00 4.00C12-C14仲(2，3)烷基硫酸鈉5.005.00 5.00 5.00熒光增白劑 0.100.10 0.10 0.10檸檬酸鈉 5.005.00 5.00 5.00硫酸鈉17.00 17.00 17.00 17.00水和次要成份 平衡到100％在實(shí)施例29-30中，用表2-36列出的其它蛋白酶K變異體代替Asn194Glu+Leu209Gly，具有大致相同的結(jié)果。
在實(shí)施例31-32中，用表2-36列出的其它蛋白酶K變異體的任何組合代替Asn194Glu+Leu209Gly和Gly100Asn+Gln103Ser+Ser207Asp+Leu209Valo，具有大致相同的結(jié)果。
實(shí)施例33-36粒狀織物洗滌組合物實(shí)施例序號(hào)成份 33 34 35 36Arg64Asp+Gly70Gln+Thr71Gly 0.10 0.20 0.03 0.05Phe192Asp+Asn194Ser- - 0.02 0.05C13直鏈烷基苯磺酸鹽 22.00 22.00 22.00 22.00磷酸鹽(如三聚磷酸鈉) 23.00 23.00 23.00 23.00碳酸鈉 23.00 23.00 23.00 23.00硅酸鈉 14.00 14.00 14.00 14.00沸石8.20 8.20 8.20 8.20螯合劑(二亞乙基三胺五乙酸) 0.40 0.40 0.40 0.40硫酸鈉 5.50 5.50 5.50 5.50水 平衡至100％在實(shí)施例33-34中，用表2-36列出的其它蛋白酶K變異體代替Arg64Asp+Gly70Gln+Thr71Gly，具有大致相同的結(jié)果。
在實(shí)施例35-36中，用表2-36列出的其它蛋白酶K變異體的任何組合代替Arg64Asp+Gly70Gln+Thr71Gly和Phe192Asp+Asn194Ser，具有大致相同的結(jié)果。
實(shí)施例37-40粒狀織物洗滌組合物實(shí)施例序號(hào)組份 37 38 39 40Ala164His+Ala166Gly0.10 0.20 0.03 0.05Ile213Pro+Ser216Glu+Ile220Glu - - 0.02 0.05C12烷基苯磺酸鹽 12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸，鈉鹽5.00 5.00 5.00 5.00檸檬酸鈉 5.00 5.00 5.00 5.00熒光增白劑 0.10 0.10 0.10 0.10硫酸鈉17.00 17.00 17.00 17.00水及次要成份 平衡至100％在實(shí)施例37-38中，用表2-36列出的其它蛋白酶K變異體代替Ala164His+Ala166Gly，具有大致相同的結(jié)果。
在實(shí)施例39-40中，用表2-36列出的其它蛋白酶K變異體的任何組合代替Ala164His+Ala166Gly和Ile213Pro+Ser216Glu+Ile220Glu，具有大致相同的結(jié)果。
實(shí)施例41-42粒狀織物洗滌組合物實(shí)施例序號(hào)組份 41 42直鏈烷基苯磺酸鹽 11.4 10.70牛油烷基硫酸鹽 1.802.40C14-C15烷基硫酸鹽 3.003.10C14-C15醇七倍乙氧化物 4.004.00牛油醇11倍乙氧化物 1.801.80分散劑 0.07 0.1聚硅氧烷流體 0.800.80檸檬酸三鈉 14.00 15.00檸檬酸 3.002.50沸石32.50 32.10馬來(lái)酸丙烯酸共聚物 5.005.00二亞乙基三胺五亞甲基膦酸 1.000.20Gly203Gln+Thr211Gly+Ile213Leu+Gly214Asn 0.300.30脂肪酶 0.360.40淀粉酶 0.300.30硅酸鈉 2.002.50硫酸鈉 3.505.20聚乙烯吡咯烷酮 0.300.50過(guò)硼酸鹽 0.5 1苯酚磺酸鹽0.1 0.2過(guò)氧化物酶0.1 0.1次要成份 加至100 加至100
實(shí)施例43-44粒狀織物洗滌組合物實(shí)施例序號(hào)組份 43 44直鏈C12烷基苯磺酸鈉6.58.0硫酸鈉 15.0 18.0沸石A 26.0 22.0次氮基三乙酸鈉 5.05.0聚乙烯吡咯烷酮 0.50.7四乙酰乙二胺3.03.0硼酸4.0 -過(guò)硼酸鹽0.5 1苯酚磺酸鹽 0.10.2Asp98Glu+Tyr104Leu 0.40.4填料(如硅酸鹽；碳酸鹽；香料；水) 加至100加至100
實(shí)施例45致密粒狀織物洗滌組合物組份 重量％烷基硫酸鹽 8.0烷基乙氧基硫酸鹽 2.0C25和C45醇3和7倍乙氧化物混合物 6.0多羥基脂肪酸酰胺 2.5沸石17.0層狀硅酸鹽/檸檬酸鹽 16.0碳酸鹽 7.0馬來(lái)酸丙烯酸共聚物 5.0污垢解脫聚合物 0.4羧甲基纖維素 0.4聚(4-乙烯基吡啶)-N-氧化物0.1乙烯基咪唑和乙烯基吡咯烷酮的共聚體 0.1PEG 2000 0.2Gly136Ser+Tyr137Met+Ser138Glu0.5脂肪酶 0.2纖維素酶 0.2四乙酰乙二胺 6.0過(guò)碳酸鹽22.0亞乙基二胺二琥珀酸 0.3抑泡劑 3.54，4′-雙(2-嗎啡代-4-苯胺基-S-三吖嗪-6-基 0.25氨基)芪-2，2′-二磺酸二鈉4，4′-雙(2-硫代styril)聯(lián)苯二鈉 0.05水，香料及次要組份 加至100
實(shí)施例46粒狀織物洗滌組合物組份 重量％直鏈烷基苯磺酸鹽 7.6C16-C18烷基硫酸鹽1.3C14-C15醇7倍乙氧化物 4.0椰子烷基二甲基羥乙基銨氯化物 1.4分散劑0.07聚硅氧烷流體 0.8檸檬酸三鈉 5.0沸4A 15.0馬來(lái)酸丙烯酸共聚體 4.0二亞乙基三胺五亞甲基膦酸 0.4過(guò)硼酸鹽 15.0四乙酰乙二胺 5.0綠土 10.0聚(氧乙烯)(MW 300,000) 0.3Ser219Asp 0.4脂肪酶 0.2淀粉酶 0.3纖維素酶 0.2硅酸鈉 3.0碳酸鈉10.0羧甲基纖維素 0.2增白劑 0.2水、香料和次要組份 加至100
實(shí)施例47粒狀織物洗滌組合物組份 重量％直鏈烷基苯磺酸鹽 6.92牛油烷基硫酸鹽2.05C14-C15醇7倍乙氧化物 4.4C12-C15烷基乙氧基硫酸鹽-3倍乙氧化物 0.16沸石 20.2檸檬酸鹽 5.5碳酸鹽15.4硅酸鹽 3.0馬來(lái)酸丙烯酸共聚物 4.0羧甲基纖維素酶0.31污垢解脫聚合物0.30Ser190Glu+Ser191Glu+Phe192Leu+Asn194Ser0.2脂肪酶0.36纖維素酶 0.13過(guò)硼酸鹽四水合物 11.64過(guò)硼酸鹽一水合物 8.7四乙酰乙二胺 5.0二亞乙基三胺五亞甲基膦酸 0.38硫酸鎂0.40增白劑0.19香料、聚硅氧烷、抑泡劑0.85次要組份 加至100b.液體織物洗滌組合物本發(fā)明的液體織物洗滌組合物含有有效量的本發(fā)明中的一種或多種酶變異體，活性酶的含量?jī)?yōu)選約占組合物重量的0.005％-5％，更優(yōu)選約占0.01％-1％。這些液體織物洗滌組合物一般還包含陰離子型表面活性劑、脂肪酸、水溶性洗滌助洗劑及水。
本發(fā)明中液體織物洗滌組合物的實(shí)施方案在下面的實(shí)施例中詳細(xì)說(shuō)明。
實(shí)施例48-52液體織物洗滌組合物實(shí)施例序號(hào)組份 48 49 50 51 52Ile220Asp 0.05 0.03 0.300.03 0.10Thr206Asp+Ile220Met - - -0.01 0.20C12-C14烷基硫酸鈉 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.005.00檸檬酸鈉 1.00 1.00 1.00 1.001.00C10醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00單乙醇胺 2.50 2.50 2.50 2.502.50水/丙二醇/乙醇(100∶1∶1) 平衡到100％在實(shí)施例48-50中，用表2-36列出的其它蛋白酶K變異體代替Ile220Asp，具有大致相同的結(jié)果。
在實(shí)施例51-52中，用表2-36列出的其它蛋白酶K變異體的任何組合代替Ile220Asp和Thr206Asp+Ile220Met，具有大致相同的結(jié)果。
實(shí)施例53-57液體織物洗滌組合物實(shí)施例序號(hào)組份 53 54 55 56 57Gly136Ser 0.05 0.03 0.30 0.03 0.10Gly215Pro - - - 0 01 0.20C12-C14烷基硫酸鈉 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00檸檬酸鈉 1.00 1.00 1.00 1.00 1.00C10醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00單乙醇胺 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1) 平衡至100％
在實(shí)施例53-55中，用表2-36列出的其它蛋白酶K變異體代替Gly136Ser，具有大致相同的結(jié)果。
在實(shí)施例56-57中，用表2-36列出的其它蛋白酶K變異體的任何組合代替Gly136Ser和Gly215Pro，具有大致相同的結(jié)果。
實(shí)施例58-59液體織物洗滌組合物實(shí)施例序號(hào)組份 58 59C12-C14鏈烯基琥珀酸 3.08.0檸檬酸一水合物 10.0 15.0C12-C15烷基硫酸鈉 8.08.0C12-C15醇2倍乙氧化物硫酸鈉 -3.0C12-C15醇7倍乙氧化物-8.0C12-C15醇5倍乙氧化物 8.0 -二亞乙基三胺五(亞甲基膦酸) 0.2 -油酸1.8 -乙醇4.04.0丙二醇 2.02.0Pro204Asn+Thr211Pro+Gly214Gln 0.20.2+Gly215Glu+Thr217Ser聚乙烯吡咯烷酮 1.02.0抑泡劑 0.15 0.15NaOH 調(diào)pH至7.5過(guò)硼酸鹽 0.5 1苯酚磺酸鹽 0.1 0.2過(guò)氧化物酶 0.4 0.1水及次要組份 加至100份在實(shí)施例58和59中，用表2-36列出的其它蛋白酶K變異體代替Pro204Asn+Thr211Pro+Gly214Gln+Gly215Glu+Thr217Ser，均具有大致相同的結(jié)果。
實(shí)施例60-62液體織物洗滌組合物實(shí)施例序號(hào)組份 60 61 62檸檬酸 7.103.003.00脂肪酸 2.00 -2.00乙醇 1.933.203.20硼酸 2.223.503.50單乙醇胺 0.711.091.091，2丙二醇 7.898.008.00枯烯磺酸鈉 1.803.003.00甲酸鈉 0.080.080.08NaOH 6.703.803.80硅抗泡劑 1.161.181.18Leu96Met+Asn99Ser+Ser105Asp0.0145 - -Thr211Ser+Gly222Asp - 0.0145 -Tyr169Ser+Ser190Asp+Ile220Thr - - 0.0145脂肪酶 0.200 0.200 0.200纖維素酶-7.507.50污垢解脫聚合物 0.290.150.15抗泡劑 0.06 0.085 0.085增白劑360.095 - -增白劑3 -0.050.05C12烷基苯磺酸 9.86 - -C12-C15烷基聚乙氧化物(2.5)硫酸鹽 13.80 18.00 18.00C12葡萄糖酰胺 -5.005.00C12-C13烷基聚乙氧化物(9) 2.002.002.00水、香料及次要組分平衡至100％C.棒狀織物洗滌組合物本發(fā)明中適用于手洗臟的織物的棒狀織物洗滌組合物含有有效量的本發(fā)明的一種或多種酶變異體，優(yōu)選占組合物重量的大致0.001％-10％，更優(yōu)選占大約0.01％-1％。
本發(fā)明中棒狀織物洗滌組合物的實(shí)施方案在下面的實(shí)施例中詳細(xì)說(shuō)明。
實(shí)施例63-66棒狀織物洗滌組合物實(shí)施例序號(hào)組份63 64 65 66Leu96Ala+Asp97Glu+Ser101Asp0.3 -0.1 0.02+Ile107AspSer193Asp- -0.4 0.03C12-C16烷基硫酸鈉 20.0 20.0 20.0 20.00C12-C14N-甲基葡萄糖酰胺 5.05.05.0 5.00C11-C13烷基苯磺酰鈉 10.0 10.0 10.0 10.00碳酸鈉25.0 25.0 25.0 25.00焦磷酸鈉 7.07.07.0 7.00三聚磷酸鈉 7.07.07.0 7.00沸石A(0.1-10μ)5.05.05.0 5.00羧甲基纖維素 0.20.20.2 0.20聚丙烯酸酯(MW 1400)0.20.20.2 0.20椰子單乙醇酰胺 5.05.05.0 5.00增白劑、香料 0.20.20.2 0.20CaSO41.01.01.0 1.00MgSO41.01.01.0 1.00水 4.04.04.0 4.00填料*平衡到100％*可從常用的材料如CaCO3、滑石粉、粘土、硅酸鹽等中選擇。
在實(shí)施例63-64中，用表2-36列出的蛋白酶K變異體代替Leu96Ala+Asp97Glu+Ser101Asp+Ile107Asp，具有大致相同的結(jié)果。
在實(shí)施例65-66中，用表2-36列出的其它蛋白酶K變異體的任何組合代替Leu96Ala+Asp97Glu+Ser101Asp+Ile107Asp和Ser193Asp，具有大致相同的結(jié)果。
實(shí)施例67-70棒狀織物洗滌組合物實(shí)施例序號(hào)組份 67 68 69 70Arg64Asp+Gly68Gln 0.3 - 0.10.02Tyr137Gln - 0.3 0.40.03C12-C16烷基硫酸鈉 20.020.020.0 20.00C12-C14N-甲基葡萄糖酰胺 5.0 5.0 5.05.00C11-C13烷基苯磺酸鈉10.010.010.0 10.00碳酸鈉 25.025.025.0 25.00焦磷酸鈉 7.0 7.0 7.07.00三聚磷酸鈉7.0 7.0 7.07.00沸石A(0.1-10μ) 5.0 5.0 5.05.00羧甲基纖維素 0.2 0.2 0.20.20聚丙烯酸酯(MW 1400) 0.2 0.2 0.20.20椰子單乙醇酰胺5.0 5.0 5.05.00增白劑、香料 0.2 0.2 0.20.20CaSO41.0 1.0 1.01.00MgSO41.0 1.0 1.01.00水4.0 4.0 4.04.00填料*平衡到100％*可從常規(guī)的材料如CaCO3、滑石粉、粘土、硅酸鹽等中選擇。
在實(shí)施例67中，用表2-36列出的其它蛋白酶K變異體代替Arg64Asp+Gly68Gln，具有大致相同的結(jié)果。
在實(shí)施例68中，用表2-36列出的其它蛋白酶K變異體代替Tyr137Gln，具有大致相同的結(jié)果。
在實(shí)施例69-70中，用表2-36列出的其它蛋白酶K變異體的任何組合代替Arg64Asp+Gly68Gln和Tyr137Gln，具有大致相同的結(jié)果。B.其它洗滌組合物除了上述已討論的硬表面，餐具和纖維洗滌組合物外，本發(fā)明中的一種或多種酶變異體也可摻入到需要水解不溶底物的多種其它清洗組合物中。這些其它的清洗組合物包括但不僅限于口腔清潔組合物、假牙清洗組合物和隱形眼鏡清洗組合物。1.口腔清潔組合物在本發(fā)明的另一個(gè)實(shí)施方案中，可藥用量的本發(fā)明的一種或多種酶變異體加入到組合物中，用以去除牙或假牙中的蛋白質(zhì)類(lèi)污垢。這里用到的“口腔清潔組合物”指洗牙水、牙膏、牙膠、牙粉、漱口藥、口腔噴霧劑、口膠、咀嚼膠、錠劑、香粉、藥片、生物膠、預(yù)防膏、假牙處理液等。優(yōu)選按重量計(jì)口腔清潔組合物中含約0.0001％-20％的本發(fā)明的一種或多種酶變異體，更優(yōu)選含約0.001％-10％，最優(yōu)選含約0.01％-5％，和一種可藥用的載體。這里用到的“可藥用”指那些用術(shù)語(yǔ)描述的藥品、藥物或惰性成份適合與人和較低等動(dòng)物的組織直接接觸使用而沒(méi)有不當(dāng)?shù)亩拘?、不適應(yīng)性、不穩(wěn)定性、刺激性、過(guò)敏反應(yīng)等，同時(shí)具有合理的益處/風(fēng)險(xiǎn)比。
通常，口腔清潔組合物中口腔清潔成份中的可藥用的口腔清潔載體成份通常占組合物重量的大約50％-99.99％，優(yōu)選占大約65％-99.99％，更優(yōu)選占大約65％-99％。
本發(fā)明中的口腔清潔組合物中可能包含的可藥用的載體和任選成份對(duì)本領(lǐng)域的技術(shù)人員來(lái)說(shuō)是熟知的。這些口腔清潔組合物中用到的多種組合物類(lèi)型、載體成份和任選成份，公開(kāi)于以下專(zhuān)利文獻(xiàn)中美國(guó)專(zhuān)利5,096,700，Seibel，1992年3月17日授權(quán)；美國(guó)專(zhuān)利5,028,414，Sampathkumar，1991年7月2日授權(quán)；和美國(guó)專(zhuān)利5,028,415,Benedict，Bush和Sunberg，1991年7月2日授權(quán)；所有這些在此引入作為參考。
本發(fā)明中口腔清潔組合物的實(shí)施方案在下面的實(shí)施例中詳細(xì)說(shuō)明。
實(shí)施例71-74洗牙水組合物實(shí)施例序號(hào)組份71 72 73 74Gly160Pro2.000 3.500 1.500 2.000山梨醇(70％水溶液) 35.00 35.000 35.000 35.000PEG-6*1.000 1.000 1.000 1.000二氧化硅牙磨粉**20.000 20.000 20.000 20.000氟化鈉 0.243 0.243 0.243 0.243二氧化鈦 0.500 0.500 0.500 0.500糖精鈉 0.286 0.286 0.286 0.286烷基硫酸鈉(27.9％水溶液) 4.000 4.000 4.000 4.000香料 1.040 1.040 1.040 1.040羥乙烯基聚合物***0.300 0.300 0.300 0.300藻膠****0.800 0.800 0.800 0.800水平衡至100％*PEG-6＝分子量為600的聚乙二醇**由J.M.Huber提供的確定為Zeodent119的沉淀二氧化硅。***Carbopol，由B.F.Goodrich化學(xué)公司提供。****Iota藻膠，由Hercules化學(xué)公司提供。
在實(shí)施例71-74中，用表2-36列出的其它蛋白酶K變異體代替Gly160Pro，具有大致相同的結(jié)果。
實(shí)施例75-78漱口水組合物實(shí)施例序號(hào)組份 75 76 77 78Ala166Gln+Ser216Asp3.007.501.005.00SDA 40醇 8.008.008.008.00香料 0.080.080.080.08乳化劑 0.080.080.080.08氟化鈉 0.050.050.050.05甘油 10.00 10.00 10.00 10.00甜味劑 0.020.020.020.02苯甲酸 0.050.050 050.05氫氧化鈉 0.200.200.200.20染料 0.040.040.040.04水 平衡至100％在實(shí)施例75-78中，用表2-36列出的其它蛋白酶K變異體代替Ala166Gln+Ser216Asp，具有大致相同的結(jié)果。
實(shí)施例79-82錠劑組合物實(shí)施例序號(hào)組份 79 80 81 82Gly70Ser+Ile107Gly+Leu133Met 0.01 0.03 0.10 0.02+Phe192His+Asn194Asp山梨醇17.50 17.50 17.50 17.50甘露醇17.50 17.50 17.50 17.50淀粉 13.60 13.60 13.60 13.60甜味劑 1.20 1.20 1.20 1.20香料 11.70 11.70 11.70 11.70色素 0.10 0.10 0.10 0.10玉米糖漿 平衡至100％
在實(shí)施例79-82中，用表2-36列出的其它蛋白酶K變異體代替Gly70Ser+Ile107Gly+Leu133Met+Phe192His+Asn194Asp，具有大致相同的結(jié)果。
實(shí)施例83-86口香糖組合物實(shí)施例序號(hào)組份83 84 85 86Gly214Asn 0.03 0.020.10 0.05山梨醇晶體 38.44 38.40 38.40 38.40Paloja-T膠基質(zhì)*20.00 20.00 20.00 20.00山梨醇(70％水溶液) 22.00 22.00 22.00 22.00甘露醇 10.00 10.00 10.00 10.00甘油 7.56 7.567.56 7.56香料 1.00 1.001.00 1.00*由L.A.Dreyfus公司提供。
在實(shí)施例83-86中，用表2-36列出的其它蛋白酶K變異體代替Gly214Asn，具有大致相同的結(jié)果。2.假牙清洗組合物在本發(fā)明的另一個(gè)實(shí)施方案中，用于在口腔外洗滌假牙的假牙清洗組合物中含有本發(fā)明的一種或多種酶變異體。這些假牙清洗組合物含有有效量的一種或多種酶變異體，優(yōu)選占組合物重量的大約0.0001％-50％，更優(yōu)選占約0.001％-35％，最優(yōu)選占約0.01％-20％，以及一種假牙洗滌載體。不同的假牙清洗組合物形式如起泡的藥片等在本領(lǐng)域眾所周知(參見(jiàn)Young的美國(guó)專(zhuān)利5,055,305，在此引入作為參考)，并通常適合摻入一種或多種酶變異體，以便從假牙上除去蛋白污垢。
本發(fā)明中假牙清洗組合物的實(shí)施方案在下面的實(shí)施例中詳細(xì)說(shuō)明。
實(shí)施例87-90雙層起泡假牙洗滌藥片實(shí)施例序號(hào)組份 87 88 89 90酸性層Tyr137Leu+Tyr169Cys+Phe192Asn 1.01.50.010.05+Ser207Asp+Arg218Glu+Ile220Leu酒石酸 24.0 24.0 24.00 24.00碳酸鈉 4.04.04.004.00磺胺酸 10.0 10.0 10.00 10.00PEG 20,000 4.04.04.004.00碳酸氫鈉 24.5 24.5 24.50 24.50過(guò)硫酸鉀 15.0 15.0 15.00 15.00焦磷酸鈉7.07.07.007.00焦化硅石2.02.02.002.00TAED*7.07.07.007.00蓖麻?；腔晁?.50.50.500.50香料1.01.01.001.00堿性層一水過(guò)硼酸鈉 32.0 32.0 32.00 32.00碳酸氫鈉 19.0 19.0 19.00 19.00EDTA3.03.03.003.00三聚磷酸鈉 12.0 12.0 12.00 12.00PEG 20,000 2.02.02.002.00過(guò)硫酸鉀 26.0 26.0 26.00 26.00碳酸鈉 2.02.02.002.00焦化硅石2.02.02.002.00染料/香料 2.02.02.002.00*四乙酰乙二胺在實(shí)施例87-90中，用表2-36列出的其它蛋白酶K變異體代替Tyr137Leu+Tyr169Cys+Phe192Ans+Ser207Asp+Arg218Glu-Ile220Leu，具有大致相同的結(jié)果。3.隱形眼鏡清洗組合物在本發(fā)明的另一個(gè)實(shí)施方案中，隱形眼鏡清洗組合物包含一種或多種本發(fā)明的酶變異體。這些隱形眼鏡清洗組合物中含有有效量的一種或多種酶變異體，優(yōu)選大約占組合物重量的0.01％-50％，更優(yōu)選約占0.01％-20％，最優(yōu)選約占1％-5％，以及一種隱形眼鏡洗滌載體。不同的隱形眼鏡清洗組合物形式如藥片、液體等在本領(lǐng)域中為人熟知(參見(jiàn)美國(guó)專(zhuān)利4,863,627,Davies，Meaken和Rees，1989年9月5日授權(quán)；美國(guó)專(zhuān)利4,609,493,Schfer，1986年9月2日授權(quán)；美國(guó)專(zhuān)利Re.32,672，Huth，Lam和Kirai，1988年5月24日授權(quán)；美國(guó)專(zhuān)利4,690,793,Ogunbiyi和Simth，1987年9月1日授權(quán)；美國(guó)專(zhuān)利4,614,549，Ogunbiyi，Riedhammer和Smith，1986年9月30日授權(quán)；和美國(guó)專(zhuān)利4,285,738，Ogata，1981年8月25日授權(quán)；所有這些在此引入作為參考)。并且通常適合摻入一種或多種本發(fā)明的酶變異體，以便從隱形眼鏡上除去蛋白質(zhì)性的污垢。
本發(fā)明中隱形眼鏡清洗組合物的實(shí)施方案在下面的實(shí)施例中詳細(xì)說(shuō)明。
實(shí)施例91-94加酶隱形眼鏡洗滌溶液實(shí)施例序號(hào)組份 91929394Gly102Gln 0.01 0.5 0.1 2.0葡萄糖 50.00 50.0 50.0 50.0非離子型表面活性劑(聚氧乙烯 2.00 2 0 2.0 2.0-聚氧丙烯共聚物)陰離子表面活性劑(聚氧乙烯- 1.00 1.0 1.0 1.0烷基苯醚硫酸鈉)氯化鈉 1.00 1.0 1.0 1.0硼砂0.30 0.3 0.3 0.3水平衡至100％在實(shí)施例91-94中，用表2-36列出的其它蛋白酶K變異體代替Gly102Gln，具有大致相同的結(jié)果。
盡管本發(fā)明僅敘述了具體實(shí)施方案，但很顯然對(duì)本領(lǐng)域的技術(shù)人員來(lái)說(shuō)，可以在不超出本發(fā)明的精神和范圍的情況下對(duì)本發(fā)明作各種改變和修改。本發(fā)明意在后附的權(quán)利要求中包括在本發(fā)明范圍之內(nèi)的所有這些修改。
序列表(1)一般信息(i)申請(qǐng)人BRODE，PHILIP FBARNETT，BOBBY LRUBINGH，DONN N(ii)發(fā)明題目能降低吸附作用并提高水解作用的蛋白酶K變異體(iii)序列數(shù)1(iv)通訊地址(A)收件人THE PROCTER & GAMBLE COMPANY(B)街道11810 EAST MIAMIRIVER ROAD(C)城市ROSS(D)州OH(E)國(guó)家US(F)郵編45061(v)計(jì)算機(jī)可讀形式(A)介質(zhì)類(lèi)型軟盤(pán)(B)計(jì)算機(jī)IBM PC兼容(C)操作系統(tǒng)PC-DOS/MS-DOS(D)軟件PatentIn Release#1.0，Version#1.30(vi)現(xiàn)申請(qǐng)資料(A)申請(qǐng)?zhí)?B)申請(qǐng)日(C)分類(lèi)(viii)代理人/事務(wù)所資料(A)姓名HAKE，RICHARD A(B)注冊(cè)號(hào)37，343(C)參考/摘要號(hào)5605(ix)電信信息(A)電話(huà)(513)-627-0087(B)傳真(513)-627-0260(2)SEQ ID NO1信息(i)序列特征(A)長(zhǎng)度279氨基酸(B)類(lèi)型氨基酸(C)鏈型單鏈(D)拓?fù)鋵W(xué)線(xiàn)性(xi)序列表述SEQ ID NO1Ala Ala Gln Thr Asn Ala Pro Trp Gly Leu Ala Arg Ile Ser Ser Thr1 5 10 15Ser Pro Gly Thr Ser Thr Tyr Tyr Tyr Asp Glu Ser Ala Gly Gln Gly20 25 30Ser Cys Val Tyr Val Ile Asp Thr Gly Ile Glu Ala Ser His Pro Glu35 40 45Phe Glu Gly Arg Ala Gln Met Val Lys Thr Tyr Tyr Tyr ser Ser Arg50 55 60Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr Val Gly Ser Arg65 70 75 80Thr Tyr Gly Val Ala Lys Lys Thr Gln Leu Phe Gly Val Lys Val Leu85 90 95Asp Asp Asn Gly Ser Gly Gln Tyr Ser Thr Ile Ile Ala Gly Mer Asp100 105 110Phe Val Ala Ser Asp Lys Asn Asn Arg Asn Cys Pro Lys Gly Val Val115 120 125Ala Ser Leu Ser Leu Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala130 135 140Ala Ala Arg Leu Gln Ser Ser Gly Val Met Val Ala Val Ala Ala Gly145 150 155 160Asn Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser165 170 175Val Cys Thr Val Gly Ala Ser Asp Arg Tyr Asp Arg Arg Ser Ser Phe180 185 190Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Gly Pro Gly Thr Ser Ile195 200 205Leu Ser Thr Trp Ile Gly Gly Ser Thr Arg Ser Ile Ser Gly Thr Ser210 215 220Met Ala Thr Pro His Val Ala Gly Leu Ala Ala Tyr Leu Met Thr Leu225 230 235 240Gly Lys Thr Thr Ala Ala Ser Ala Cys Arg Tyr Ile Ala Asp Thr Ala245 250 255Asn Lys Gly Asp Leu Ser Asn Ile Pro Phe Gly Thr Val Asn Leu Leu260 265 270Ala Tyr Asn Asn Tyr Gln Ala27權(quán)利要求
1.一種對(duì)野生型蛋白酶K的氨基酸序列進(jìn)行改良的蛋白酶K變異體，野生型氨基酸序列中包含第一環(huán)區(qū)、第二環(huán)區(qū)、第三環(huán)區(qū)、第四環(huán)區(qū)、第五環(huán)區(qū)和第六環(huán)區(qū)，其中改良的氨基酸序列在一個(gè)或多個(gè)環(huán)區(qū)中的一個(gè)或多個(gè)位置上有替換；其中A.當(dāng)替換發(fā)生在第一環(huán)區(qū)時(shí)，替換發(fā)生在64、65、66、68、70或71位中的一個(gè)或多個(gè)上；其中a.當(dāng)替換發(fā)生在64位時(shí)，替代氨基酸為Asp或Glu；b.當(dāng)替換發(fā)生在65位時(shí)，替代氨基酸為Glu；c.當(dāng)替換發(fā)生在66位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；d.當(dāng)替換發(fā)生在68位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；e.當(dāng)替換發(fā)生在70位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；f.當(dāng)替換發(fā)生在71位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；B.當(dāng)替換發(fā)生在第二環(huán)區(qū)時(shí)，替代發(fā)生在95、96、97、98、100、102、103、104、106或107位中一個(gè)或多個(gè)上；其中a.當(dāng)替換發(fā)生在95位時(shí)，替代氨基酸為Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr；b.當(dāng)替換發(fā)生在96位時(shí)，替代氨基酸為Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val；c.當(dāng)替換發(fā)生在97位時(shí)，替代氨基酸為Glu；d.當(dāng)替換發(fā)生在98位時(shí)，替代氨基酸為Glu；e.當(dāng)替換發(fā)生在100位時(shí)，替代氨基酸為Asn、Gln、Pro或Ser；f.當(dāng)替換發(fā)生在102位時(shí)，替代氨基酸為Asn、Gln、Glu、Pro或Ser；g.當(dāng)替換發(fā)生在103位時(shí)，替代氨基酸為Asn或Ser；h.當(dāng)替換發(fā)生在104位時(shí)，替代氨基酸為Asp、His、Ile、Leu、Met、Pro或Val；i.當(dāng)替換發(fā)生在106位時(shí)，替代氨基酸為Asn、Gln、Gly、Pro或Ser；和j.當(dāng)替換發(fā)生在107位時(shí)，替代氨基酸為Ala、Asn、Cys、Gln、Gly、His、Leu、Met、Pro、Ser、Thr或Val；C.當(dāng)替換發(fā)生在第三環(huán)區(qū)時(shí)，替代發(fā)生在133、134、136、137、138、139或140位中一個(gè)或多個(gè)上；其中a.當(dāng)替換發(fā)生在133位時(shí)，替代氨基酸為Ala、Asn、Cys、Gln、Gly、His、Met、Pro、Ser、Thr或Val；b.當(dāng)替換發(fā)生在134位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；c.當(dāng)替換發(fā)生在136位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；d.當(dāng)替換發(fā)生在137位時(shí)，替代氨基酸為Asp、His、Ile、Leu、Met或Pro；e.當(dāng)替換發(fā)生在138位時(shí)，替代氨基酸為Asp或Glu；f.當(dāng)替換發(fā)生在139位時(shí)，替代氨基酸為Asp或Glu；和g.當(dāng)替換發(fā)生在140位時(shí)，替代氨基酸為Asp或Glu；D.當(dāng)替換發(fā)生在第四環(huán)區(qū)時(shí)，替換發(fā)生在161、162、163、164、165、166、167、168、169或170位中一個(gè)或多個(gè)上；其中a.當(dāng)替換發(fā)生在161位時(shí)，替代氨基酸為Gln；b.當(dāng)替換發(fā)生在162位時(shí)，替代氨基酸為Asp、Gln、Glu或Ser；c.當(dāng)替換發(fā)生在163位時(shí)，替代氨基酸為Asp、Gln、Glu或Ser；d.當(dāng)替換發(fā)生在164位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；e.當(dāng)替換發(fā)生在165位時(shí)，替代氨基酸為Glu；f.當(dāng)替換發(fā)生在166位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；g.當(dāng)替換發(fā)生在167位時(shí)，替代氨基酸為Asp或Glu；h.當(dāng)替換發(fā)生在168位時(shí)，替代氨基酸為Asp、Gln、Glu或Ser；i.當(dāng)替換發(fā)生在169位時(shí)，替代氨基酸為Asp、His、Ile、Leu、Met或Pro；和j.當(dāng)替換發(fā)生在170位時(shí)，替代氨基酸為Asp或Glu；E.當(dāng)替換發(fā)生在第五環(huán)區(qū)時(shí)，替換發(fā)生在190、192、193或194位中的一個(gè)或多個(gè)上，其中a.當(dāng)替換發(fā)生在190位時(shí)，替代氨基酸為Asp或Glu；b.當(dāng)替換發(fā)生在192位時(shí)，替代氨基酸為Asn、Cys、Gln、His、Ile、Met、Pro、Thr、Tyr或Val；c.當(dāng)替換發(fā)生在193位時(shí)，替代氨基酸為Asp或Glu；和d.當(dāng)替換發(fā)生在194位時(shí)，替代氨基酸為Asp、Gln、Glu或Ser；和F.當(dāng)替換發(fā)生在第六環(huán)區(qū)時(shí)，替換發(fā)生在203、204、205、206、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222或223位中的一個(gè)或多個(gè)上；其中a.當(dāng)替換發(fā)生在203位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；b.當(dāng)替換發(fā)生在204位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Gly或Ser；c.當(dāng)替換發(fā)生在205位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；d.當(dāng)替換發(fā)生在206位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Gly、Pro或Ser；e.當(dāng)替換發(fā)生在208位時(shí)，替代氨基酸為Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val；f.當(dāng)替換發(fā)生在209位時(shí)，替代氨基酸為Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val；g.當(dāng)替換發(fā)生在210位時(shí)，替代氨基酸為Asp或Glu；h.當(dāng)替換發(fā)生在211位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；i.當(dāng)替換發(fā)生在212位時(shí)，替代氨基酸為Asp或Glu；j.當(dāng)替換發(fā)生在213位時(shí)，替代氨基酸為Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val；k.當(dāng)替換發(fā)生在214位時(shí)，替代氨基酸為Asn、Gln、Pro或Ser；l.當(dāng)替換發(fā)生在215位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；m.當(dāng)替換發(fā)生在216位時(shí)，替代氨基酸為Asp或Glu；n.當(dāng)替換發(fā)生在217位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Gly、Pro或Ser；o.當(dāng)替換發(fā)生在218位時(shí)，替代氨基酸為Asp或Glu；p.當(dāng)替換發(fā)生在219位時(shí)，替代氨基酸為Asp或Glu；q.當(dāng)替換發(fā)生在220位時(shí)，替代氨基酸為Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val；r.當(dāng)替換發(fā)生在221位時(shí)，替代氨基酸為Asp或Glu；s.當(dāng)替換發(fā)生在222位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；t.當(dāng)替換發(fā)生在223位時(shí)，替代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；因此與野生型蛋白酶K相比，蛋白酶K變異體降低了對(duì)不溶底物的吸附作用而提高了對(duì)不溶底物的水解作用。
2.權(quán)利要求1的蛋白酶K變異體，其中在第一環(huán)區(qū)發(fā)生了一個(gè)或多個(gè)替換。
3.權(quán)利要求1的蛋白酶K變異體，其中在第二環(huán)區(qū)發(fā)生了一個(gè)或多個(gè)替換。
4.權(quán)利要求1的蛋白酶K變異體，其中在第三環(huán)區(qū)發(fā)生了一個(gè)或多個(gè)替換。
5.權(quán)利要求1的蛋白酶K變異體，其中在第四環(huán)區(qū)發(fā)生了一個(gè)或多個(gè)替換。
6.權(quán)利要求1的蛋白酶K變異體，其中在第五環(huán)區(qū)發(fā)生了一個(gè)或多個(gè)替換。
7.權(quán)利要求1的蛋白酶K變異體，其中在第六環(huán)區(qū)發(fā)生了一個(gè)或多個(gè)替換。
8.一種選自硬表面洗滌組合物、餐具洗滌組合物、口腔清潔組合物、假牙清洗組合物、隱形眼鏡清洗組合物以及織物洗滌組合物的清洗組合物，其特征在于該清洗組合物含有權(quán)利要求1-7中任一項(xiàng)中的蛋白酶K變異體以及一種洗滌組合物載體；優(yōu)選洗滌組合物為硬表面洗滌組合物或織物洗滌組合物；優(yōu)選該組合物至少含有占總重約5％的表面活性劑和至少約5％的助洗劑；優(yōu)選該組合物還包含選自以下的洗滌組合物成份溶劑、緩沖劑、酶、污垢解脫劑、粘土類(lèi)去污劑、分解劑、增白劑、抑泡劑、織物柔軟劑、發(fā)泡劑、酶穩(wěn)定劑、漂白劑、染料、香料及其混合物。
9.編碼權(quán)利要求1-7中任一項(xiàng)中的蛋白酶K變異體的DNA序列。
全文摘要
本發(fā)明涉及蛋白酶K的變異體,它們對(duì)野生型蛋白酶K的氨基酸序列進(jìn)行了改良,野生型氨基酸序列包含第一環(huán)區(qū)、第二環(huán)區(qū)、第三環(huán)區(qū)、第四環(huán)區(qū)、第五環(huán)區(qū)和第六環(huán)區(qū);其中改良的氨基酸序列是在一個(gè)或多個(gè)環(huán)區(qū)的特定位置上含有與野生型蛋白酶K不同的氨基酸(即替換),因此,與野生型蛋白酶K相比,蛋白酶K變異體降低了對(duì)不溶底物的吸附作用,并提高了對(duì)不溶底物的水解作用。本發(fā)明也涉及編碼這些蛋白酶K變異體的DNA序列。本發(fā)明還涉及包含這些蛋白酶K變異體的用于清潔多種表面的組合物。
文檔編號(hào)C12N9/50GK1183809SQ96193792
公開(kāi)日1998年6月3日 申請(qǐng)日期1996年3月6日 優(yōu)先權(quán)日1995年3月9日
發(fā)明者P·F·布羅德三世, B·L·巴爾內(nèi)特, D·N·魯賓 申請(qǐng)人:普羅格特-甘布爾公司