專利名稱：間日瘧原蟲(chóng)和鐮狀瘧原蟲(chóng)紅細(xì)胞結(jié)合蛋白的結(jié)合區(qū)的制作方法
背景技術(shù)：
每年有200-400百萬(wàn)人感染瘧疾，引起1-2百萬(wàn)人死亡，因此瘧疾是世界上現(xiàn)存的最嚴(yán)重要的感染性疾病之一。在非洲鄉(xiāng)村，年齡在一到四歲之間的兒童總死亡數(shù)中有大約25％由瘧疾引起。由于該疾病作為一種世界研究性健康問(wèn)題的嚴(yán)重性，人們花費(fèi)了相當(dāng)大的努力來(lái)鑒定和研究瘧疾疫苗。
人類的瘧疾由寄生瘧原蟲(chóng)屬的四個(gè)種引起鐮狀瘧原蟲(chóng)，間日瘧原蟲(chóng)，P.knowlesi和三日瘧原蟲(chóng)。人類瘧疾的主要起因是鐮狀瘧原蟲(chóng)，它每年感染200百萬(wàn)到400百萬(wàn)人，導(dǎo)致1到4百萬(wàn)人死亡。
間日瘧原蟲(chóng)(人類的四種感染性瘧原蟲(chóng)之一)不能在體外培養(yǎng)，而P.knowlesi(在東半球猴中發(fā)現(xiàn)的瘧疾品種，也侵入人紅細(xì)胞)和鐮狀瘧原蟲(chóng)能。盡管間日瘧原蟲(chóng)與P.knowle-si在系統(tǒng)發(fā)育上基本相似，但兩個(gè)品種在許多重要方面不同。例如間日瘧原蟲(chóng)對(duì)許多猿猴種類不感染且在其它種類中不易形成感染，而P.knowlesi不易感染人類但容易感染許多猿猴種類。
抗瘧疾的各種有效疫苗的基礎(chǔ)鑒別是通過(guò)了解該寄生蟲(chóng)的生存周期來(lái)實(shí)現(xiàn)的。當(dāng)幼齡瘧疾寄生蟲(chóng)或“子孢子”被蚊注射進(jìn)人的血液中時(shí)感染人類。注射后，寄生蟲(chóng)定位于肝細(xì)胞。大約一周后，寄生蟲(chóng)或“裂殖子”釋放進(jìn)血液中。寄生蟲(chóng)進(jìn)入血液后開(kāi)始“紅細(xì)胞”期。每個(gè)寄生蟲(chóng)進(jìn)入紅血細(xì)胞以便生長(zhǎng)和發(fā)育。當(dāng)裂殖子在紅血細(xì)胞中成熟時(shí)，將它稱為滋養(yǎng)體，滋養(yǎng)體進(jìn)行幾輪核分裂(裂殖生殖)直到使紅細(xì)胞破裂，釋放6到24個(gè)裂殖子。經(jīng)過(guò)幾個(gè)無(wú)性裂殖生殖循環(huán)后，有些寄生蟲(chóng)發(fā)育成稱為“配子母細(xì)胞”的形態(tài)學(xué)上明顯不同的形式，其壽命長(zhǎng)且進(jìn)行有性發(fā)育以代替通過(guò)有性繁殖產(chǎn)生的裂殖體。
瘧原蟲(chóng)的有性發(fā)育涉及雌性或“大配子母細(xì)胞”和雄性瘧原蟲(chóng)或“小配子母細(xì)胞”。這些配子母細(xì)胞在人體內(nèi)不進(jìn)行進(jìn)一步的發(fā)育。當(dāng)配子母細(xì)胞攝入到蚊體內(nèi)后，在蚊中腸開(kāi)始復(fù)雜的有性循環(huán)。10到20分鐘后紅血細(xì)胞在紋中腸分裂。小配子母細(xì)胞通過(guò)小配子形成繼續(xù)發(fā)育并釋放8個(gè)鞭毛豐富的小配子母細(xì)胞。受精通過(guò)大配子母細(xì)胞和小配子母細(xì)胞的融合實(shí)現(xiàn)。受精的瘧原蟲(chóng)稱為受精卵，由它發(fā)育成“動(dòng)合子”。動(dòng)合子包埋于蚊中腸，在那里轉(zhuǎn)化成卵母細(xì)胞，形成許多小孢子。在包埋于中腸之前，動(dòng)合子必須首先穿透圍食膜，圍食膜明顯充當(dāng)攝入的瘧原蟲(chóng)入侵的屏障。卵母細(xì)胞破裂成小孢子，經(jīng)血淋巴遷移到蚊唾液腺。一旦進(jìn)入蚊唾液，瘧原蟲(chóng)可被注射進(jìn)宿主體內(nèi)。
瘧原蟲(chóng)屬生活史的紅細(xì)胞期與疫苗發(fā)展特別相關(guān)，因?yàn)樗拗鳢懠驳呐R床和病理特征歸因于這一階段。在間日瘧原蟲(chóng)和P.knowlesi中，存在于Duffy陽(yáng)性紅細(xì)胞上的Duffy血型決定簇是侵入人紅細(xì)胞所必需的(Miller et al.，Science 189561-563，(1975)；Miller et al.，N.Engl.J.Med.295302-304，(1976))。在鐮狀瘧原蟲(chóng)中，裂殖子侵入紅細(xì)胞似乎取決紅細(xì)胞上血型糖蛋白唾液酸的結(jié)合(Miller，et al.，J.Exp.Med.146277-281，(1971)；Pasvol，et al.，Lancet.，ii947-950(1982)；Pasvol，et al.，Nature，27964-66(1982)；Perkins，J.Exp.Med.160788-798(1984))。以猴瘧原蟲(chóng)P.knowlesi進(jìn)行的研究使人們能更清楚地了解侵入過(guò)程中發(fā)生的多個(gè)事件。很可能盡管間日瘧原蟲(chóng)和鐮狀瘧原蟲(chóng)分別與Duffy抗原和唾液酸結(jié)合，但它們互相間及與P.knowlesi以相同的方式進(jìn)行侵入。
對(duì)于P.knowlesi在侵入過(guò)程中，裂殖子首先以裂殖子的任一表面附著于紅細(xì)胞上，然后重新調(diào)整方向以便其頂端與紅細(xì)胞接觸(Dvorak et al.，Science 187748-750(1975)。裂殖子的附著和重新取向在Duffy陽(yáng)性和Duffy陰性細(xì)胞上能同等順利地進(jìn)行。然后在裂殖子頂端和Duffy陽(yáng)性紅細(xì)胞間形成連接，隨后形成液泡，裂殖子進(jìn)入液泡(Aikawa etal.，J.Cell Biol.7772-82(1978))。在Duffy陰性細(xì)胞上不發(fā)生連接形成和裂殖子進(jìn)入紅細(xì)胞(Miller et al.，J.Exp.Med.149172-184(1979)，認(rèn)為Duffy決定簇特異性受體與頂端連接形成而不是起始附著有關(guān)。
以3種細(xì)胞器的存在定義裂殖子的頂端rhopteries，密集顆粒和微絲體。在液泡形成時(shí)rhopteries和密集顆粒釋放其內(nèi)含物(Ladda et al.，1969；Aikawa et al.，J.Cell Biol.，7772-82(1978)；Torn et al.，Infection and Immunity 573230-3233(1989)；Bannister and Dluzewski，Blood Cell 16257-292(1990)。到目前為止微絲體的功能尚不清楚。然而，微絲體的位置表明它們與侵入過(guò)程有關(guān)。Duffy抗原結(jié)合蛋白(DABP)和唾液酸結(jié)合蛋白(SABP)已分別定位于P.knowlesi和鐮狀瘧原蟲(chóng)的微絲體上(Adams et al.，cell63141-153(1990)；sim et al.，Mol.Biochem.Parasitol.51157-160(1992))。
DABP和SABP是可溶蛋白質(zhì)，在感染的紅細(xì)胞釋放裂殖子后它們出現(xiàn)在培養(yǎng)物上清液中。免疫化學(xué)資料表明DABP和SABP分別是間日瘧原蟲(chóng)和鐮狀瘧原蟲(chóng)在紅細(xì)胞上Duffy和唾液酸受體的配體，在可溶或膜結(jié)合形式下具有同樣的結(jié)合特異性。
DABP是135KDa的蛋白質(zhì)，它與Duffy血型決定簇特異性地結(jié)合(Wertheimer et al.，Exp.Parasitol.69340-350(1989)；Barnwell，et al.，J.Exp.Med.1691795-1805(1989))。因此，DABP與人Duffy陽(yáng)性紅細(xì)胞的結(jié)合是具有特異性的。人紅細(xì)胞有4種主要Duffy表型Fy(a)，F(xiàn)y(b)，F(xiàn)y(ab)和Fy(陰性)，以抗Fya和抗-Fyb血清來(lái)定義(Hadley et al.，In Red Cell A ntigens and Antibodies.G.Garrat-ty，ed.(Arlingten，VaAmerican Association of Blood Banks)PP.17-33(1986)。DABP同等地與Fy(a)和Fy(b)紅細(xì)胞結(jié)合(兩類紅細(xì)胞對(duì)間日瘧原蟲(chóng)的感染同樣敏感)而不與Fy(陰性)紅細(xì)胞結(jié)合。
至于SABP，它是175KDa的蛋白質(zhì)，與紅細(xì)胞上血型糖蛋白的唾液酸殘基特異性地結(jié)合(Camus and Hadley，Science230553-556(1985)；Orlandi，et al.，J.Cell Biol.116901-909(1992))。因此，神經(jīng)氨酸苷酶處理(裂解唾液酸殘基)使紅細(xì)胞對(duì)鐮狀瘧原蟲(chóng)的侵入具有免疫力。
結(jié)合的特異性和與瘧原蟲(chóng)感染的相關(guān)性表明DABP和SABP是間日瘧原蟲(chóng)和鐮狀瘧原蟲(chóng)的與唾液酸和紅細(xì)胞上的Duffy抗原相互作用的蛋白質(zhì)。編碼兩種蛋白質(zhì)的基因已被克隆、已測(cè)定了該DNA和預(yù)期的蛋白質(zhì)序列(B.Kim Leesim，et al.，J.Cell Biol.1111877-1884(1990)；Fang.X.，et al.，Mol.Biochem Parasitol，44125-132(1992))。
盡管全世界做出了相當(dāng)大的研究努力，但由于瘧原蟲(chóng)的復(fù)雜性和其與宿主的相互作用，還不可能發(fā)現(xiàn)用于阻止或抵抗瘧疾血液階段的令人滿意的溶液。由于瘧疾是一個(gè)巨大的世界性健康問(wèn)題，因此需要抵抗該疾病影響的方法。本發(fā)明提供了抗瘧原蟲(chóng)屬感染的有效的防止和治療措施。
本發(fā)明概述本發(fā)明提供了包含分離的DABP結(jié)合區(qū)多肽和/或分離的SABP結(jié)合區(qū)多肽的組合物。DABP結(jié)合區(qū)多肽優(yōu)選包含在大約200到大約300之間的氨基酸殘基，而SABP結(jié)合區(qū)多肽優(yōu)選包含在大約200到大約600之間的氨基酸殘基。優(yōu)選的DABP結(jié)合區(qū)多肽具有SEQ ID NO2中所示的含有

圖1所示的蛋白質(zhì)的半胱氨酸富集部分的大約325個(gè)氨基酸殘基。優(yōu)選的SABP結(jié)合區(qū)多肽具有SEQ ID NO4的含有圖1所示的蛋白質(zhì)的半胱氨酸富集部分的大約616的氨基酸殘基。
本發(fā)明還包括包含藥用學(xué)上可接受的載體和以在生物體內(nèi)足以誘導(dǎo)抗間日瘧原蟲(chóng)裂殖子的保護(hù)性免疫反應(yīng)的量的分離的DABP結(jié)合區(qū)多肽之藥用組合物。而且以足以誘導(dǎo)抗鐮狀瘧原蟲(chóng)保護(hù)性免疫反應(yīng)的量的分離的SABP結(jié)合區(qū)多肽可加入藥用組合物中。
還提供了包含藥學(xué)上可接受的載體和分離的SABP結(jié)合區(qū)多肽的藥用組合物，其中多肽的量是以在生物體內(nèi)誘導(dǎo)抗鐮狀瘧原蟲(chóng)的保護(hù)性免疫反應(yīng)。另外，分離的DABP結(jié)合區(qū)多肽能以足以誘導(dǎo)抗間日瘧原蟲(chóng)的保護(hù)性免疫反應(yīng)的量加入藥用組合物中。
還公開(kāi)了編碼DABP結(jié)合區(qū)多肽或SABP結(jié)合區(qū)多肽的分離的多聚核苷酸。另外，本發(fā)明包括含有編碼DABP結(jié)合區(qū)多肽的多聚核苷酸的重組細(xì)胞。
本發(fā)明進(jìn)一步包括在病人體內(nèi)誘導(dǎo)抗瘧原蟲(chóng)屬裂殖子的保護(hù)性免疫反應(yīng)的方法。該方法包括給病人施用免疫上有效量的藥用組合物，該組合物包含藥學(xué)上可接受的載體和分離的DABP結(jié)合區(qū)多肽，SABP結(jié)合區(qū)多肽或其組合。
本說(shuō)明書(shū)還提供來(lái)自紅細(xì)胞結(jié)合配體(EBL)家族其它鐮狀瘧原蟲(chóng)基因的DNA序列。該家族具有與鐮狀原蟲(chóng)175KD和間日瘧原蟲(chóng)135KD結(jié)合蛋白保守的區(qū)域。
定義本文所用的“DABP結(jié)合區(qū)多肽”或“SABP結(jié)合區(qū)多肽”是分別與來(lái)自Duffy抗原結(jié)合蛋白(DABP)富含半胱氨酸的氨基末端區(qū)域或唾液酸結(jié)合蛋白(SABP)的序列基本相同(定義如下)的多肽。該多肽能結(jié)合Duffy抗原或血型糖蛋白上的唾液酸殘基。特別是DABP結(jié)合區(qū)多肽由與含有圖1所示的蛋白質(zhì)的半胱氨酸富集部分的SABP結(jié)合區(qū)內(nèi)的序列基本相似的氨基酸殘基組成。SABP結(jié)合區(qū)多肽由與含有圖1所示的蛋白質(zhì)的半胱氨酸富集部分的DABP結(jié)合區(qū)內(nèi)的序列基本相似的殘基組成。
由EBL家族基因編碼的結(jié)合區(qū)多肽由與上面定義的DABP和SABP結(jié)合區(qū)的序列基本相同的那些殘基組成。EBL家族包含與DABP和SABP保守區(qū)基本相似的序列。它們包括本文以EBL-e1(SEQ ID Nos 5和6)，E31a(SEQ IDNos 7和8)，EBL-e2(SEQ ID Nos 9和10)和Proj3(SEQ IDNos 11和12)報(bào)道的那些序列。
本發(fā)明的多肽可由結(jié)合區(qū)的全長(zhǎng)或其片段組成。典型的DABP結(jié)合區(qū)多肽由以大約50到大約325的殘基組成，優(yōu)選在大約75和300之間，更優(yōu)選的是在大約100到大約250之間的殘基。SABP結(jié)合區(qū)多肽由從大約50到大約616的殘基組成，優(yōu)選大約75到300之間，更優(yōu)選的是大約100到大約250之間的殘基。
本發(fā)明特別優(yōu)選的多肽是那些位于結(jié)合區(qū)內(nèi)的多肽，該結(jié)合區(qū)在SABP和EBL家族之間保守。這些保守的區(qū)域內(nèi)的殘基如下面圖1所示。
當(dāng)進(jìn)行最大相關(guān)性序列對(duì)比時(shí)，如果兩個(gè)序列中的核苷酸或氨基酸殘基序列相同時(shí)兩個(gè)多聚核苷酸或多肽說(shuō)成是“相同的”?？捎镁植客葱怨?Smith and Waterman，Adv.Appl.Math.2487(1981)，同源性排列公式(Needleman andWunsch，J.Mol.Biol.48443(1970))相似性尋找方法(Pearson and Lipman，Proc Natl Acad.Sci(U.S.A)852444(1988)，這些公式計(jì)算機(jī)化的程序(GAP，BESTFIT，F(xiàn)ASTA，and TFASTA in the Wisconsin Genetics Software Packag，Genet-ics Computer Group，575 Science Dr.，Madison，WI)或經(jīng)檢查來(lái)進(jìn)行序列比較的最佳排列。本文引用這些文獻(xiàn)以供參考。
術(shù)語(yǔ)“基本上相同”指多肽包含的序列與對(duì)照序列相比(通過(guò)比較大約20個(gè)殘基至大約600個(gè)殘基，典型的是大約50到大約500個(gè)殘基，通常是大約250到300個(gè)殘基)具有至少80％的序列相同性，優(yōu)選90％，更優(yōu)選的是95％或更多。使用上面程序測(cè)定相同百分?jǐn)?shù)的值。本發(fā)明特別優(yōu)選的多肽包含的序列中至少存在在DABP和SABP中保守的半脫氨酸殘基的70％。另外，該多肽包含的序列至少有在DABP和SABP保守的色氨酸殘基的50％存在。考慮到在DABP，SABP和EBL家族的序列之間發(fā)現(xiàn)的那些保守的氨基酸殘基，本文還具體定義了術(shù)語(yǔ)基本相似。這些保守的氨基酸主要由在本文報(bào)道的所有序列中保守的色氨酸和半脫氨酸殘基組成。其它保守的氨基酸殘基包括苯丙氨酸殘基，它可用酪氨酸取代。本領(lǐng)域的技術(shù)人員使用上面所列方法進(jìn)行序列對(duì)比后可測(cè)定這些氨基酸殘基是保守的。
多肽序列基本相同的另一解釋是一種蛋白質(zhì)是否與產(chǎn)生的抗其它蛋白質(zhì)的抗體發(fā)生免疫反應(yīng)。因此，本發(fā)明的多肽包括與產(chǎn)生的抗SABP結(jié)合區(qū)，DABP結(jié)合區(qū)或產(chǎn)生的抗EBL家族保守區(qū)的抗體發(fā)生免疫反應(yīng)的多肽。
核苷酸序列基本相同的另一解釋是2種分子是否在嚴(yán)格條件下互相雜交。嚴(yán)格條件具有序列依賴性并在不同的環(huán)境下會(huì)有差別。一般來(lái)說(shuō)，嚴(yán)格條件的選擇是在限定離子強(qiáng)度和ph下列入下比特定序列的熱熔點(diǎn)(Tm)大約約5℃。Tm是50％的靶序完全匹配的探針雜交的溫度(在限定的離子強(qiáng)度和ph下)。典型的嚴(yán)格條件是在pH7下鹽濃度至少為大約0.02摩爾，溫度至少為大約60℃。
短語(yǔ)“分離的”或“生物學(xué)純的”指物質(zhì)基本上或必須沒(méi)有在基天然狀態(tài)下通常與其共存的成份。因此，本發(fā)明的結(jié)合區(qū)多肽不含正常情況下與其原位環(huán)境相聯(lián)的物質(zhì)，例如，裂殖子膜的其它蛋白質(zhì)。然而，即使蛋白質(zhì)經(jīng)PAGE分離成均質(zhì)或占優(yōu)勢(shì)的帶，可能仍有5-10％范圍的與所需蛋白質(zhì)共同純化的天然蛋白質(zhì)的微量污染。本發(fā)明分離的多肽不含這類內(nèi)源性共同純化的蛋白質(zhì)。
蛋白質(zhì)的純度或勻質(zhì)性可用許多本領(lǐng)域公知的方法來(lái)表示，如蛋白質(zhì)樣品的聚丙烯酰胺凝膠電泳，接著經(jīng)染色進(jìn)行肉眼觀察。對(duì)于某些用途需要高分辯率，可使用HPLC或相似的純化方法。
術(shù)語(yǔ)“殘基”指以酰胺鍵或酰胺鍵類似物摻入寡肽的氨基酸(D或L)或氨基酸類似物。本發(fā)明的酰胱鍵類似物包括本領(lǐng)域的技術(shù)人員公知的肽骨架修飾。
附圖的簡(jiǎn)要描述圖1顯示了DABP結(jié)合區(qū)(間日瘧原蟲(chóng))，2個(gè)同源SABP區(qū)(SABP F1和SABP F2)和EBL基因家族測(cè)序的成員(eb1-e1，E31a，EBL-e2和三個(gè)同源Proj3區(qū))預(yù)期氨基酸序列的序列對(duì)比。
圖2顯示PRE4克隆載體的圖示。
圖3顯示了在分離編碼本發(fā)明保守區(qū)的序列中有用的引物。
優(yōu)選實(shí)施例方案的描述裂殖子和裂殖體紅細(xì)胞的結(jié)合是由裂殖子或裂殖體表面的特異性結(jié)合蛋白介導(dǎo)的，它對(duì)紅細(xì)胞的感染是必需的。對(duì)于鐮狀瘧原蟲(chóng)，這種結(jié)合涉及紅細(xì)胞上唾液酸血型糖蛋白殘基與裂殖子或裂殖體表面的唾液酸結(jié)合蛋白(SABP)之間特異性的相互作用。純化的SABP結(jié)合具有化學(xué)或酶修飾的唾液酸殘基的紅細(xì)胞的能力與鐮狀瘧原蟲(chóng)感染這些紅細(xì)胞的能力相平行。而且，唾液酸缺陷型紅細(xì)胞既不結(jié)合SABP，也不能被鐮狀瘧原蟲(chóng)感染。還克隆和側(cè)序了編碼鐮狀瘧原蟲(chóng)SABP的DNA。
在間日瘧原蟲(chóng)中，與紅細(xì)胞的特異性結(jié)合涉及紅細(xì)胞上Duffy血型抗原與裂殖子上Duffy抗原結(jié)合蛋白(DABP)之間的相互作用。Duffy結(jié)合蛋白在生物學(xué)上定義為感染的紅細(xì)胞釋放裂殖子后在培養(yǎng)物上清液中出現(xiàn)的那些可溶性蛋白質(zhì)，它與人Duffy陽(yáng)性紅細(xì)胞結(jié)合，而不與人Duffy陰性紅細(xì)胞結(jié)合。顯示了間日瘧原蟲(chóng)DABP蛋白質(zhì)與Duffy陰性紅細(xì)胞的結(jié)合被抗Duffy血型決定簇的抗血清抑制。純化的Duffy血型抗原也抑制與紅細(xì)胞的結(jié)合。Western印跡也顯示DABP與Duffy血型決定簇結(jié)合。
人紅細(xì)胞上的Duffy陽(yáng)性血型決空簇對(duì)間日瘧原蟲(chóng)引起的人紅細(xì)胞的感染是必需的。間日瘧原蟲(chóng)裂殖子的附著和重新取向在Duffy陽(yáng)性和陰性紅細(xì)胞上同等順利地發(fā)生。然后在裂殖子頂端和Duffy陽(yáng)性紅細(xì)胞之間形成聯(lián)接，接著形成液泡且裂殖子進(jìn)入液泡。在Duffy陰性細(xì)胞上不發(fā)生連接的形成和裂殖子進(jìn)入紅細(xì)胞，這表明Duffy決定簇特異性的受體與頂端連接的形成而不是與起始附著有關(guān)。克隆和側(cè)序了編碼間日瘧原蟲(chóng)和P.knonlesi DABP的DNA序列。
間日瘧原蟲(chóng)的紅細(xì)胞感染絕對(duì)需要Duffy血型抗原。然而，鐮狀瘧原蟲(chóng)分離物的感染對(duì)唾液酸的依賴性不同。建立了一些以正常頻率感染唾液酸缺陷型紅細(xì)胞的鐮狀瘧原蟲(chóng)克隆。這表明鐮狀瘧原蟲(chóng)的一些菌株能與紅細(xì)胞上的其它配體相互作用，因此可能有多種具有不同特異性的紅細(xì)胞結(jié)合蛋白。
本發(fā)明的基礎(chǔ)是在DABP和SABP上發(fā)現(xiàn)了結(jié)合區(qū)。DABP和SABP預(yù)期的蛋白質(zhì)序列比較揭示了在兩種蛋白質(zhì)之間具有高度相似性，在兩種蛋白質(zhì)中有一個(gè)氨基末端富含半胱氨酸的區(qū)域。DABP氨基末端的富含半胱氨酸的區(qū)域含大約325個(gè)氨基酸，而SABP氨基末端的富含半胱氨酸的區(qū)域含大約616個(gè)氨基酸。這是由于在SABP蛋白質(zhì)的氨基末端半胱氨酸富集區(qū)有明顯的重復(fù)。在SABP和DABP的2個(gè)區(qū)域之間半胱氨酸殘基周圍的氨基酸和許多芳香族氨基酸殘基也一樣。氨基末端的半胱氨酸富集區(qū)和靠近羧基末端的另一半胱氨酸富集區(qū)表明在DABP和SABP蛋白質(zhì)之間最相似。這2個(gè)半胱氨酸富集區(qū)之間的氨基酸序列區(qū)域表明在DABP和SABP之間僅有有限的相似性。
鑒定了其它具有與SABP和DABP結(jié)合區(qū)基本相同的區(qū)域的鐮狀瘧原蟲(chóng)開(kāi)架閱讀框和基因。這些序列在瘧原蟲(chóng)基因組中存在多個(gè)拷貝表明它們?cè)谒拗鳌懺x(chóng)的相互作用中具有重要活性。從染色體7的亞片段文庫(kù)中克隆了這些序列的一個(gè)家族(EBL家族)，該文庫(kù)在氯奎抗性基因座的遺傳研究中構(gòu)建(Wellems et.al.，PNAS 883382-3386(1991))。EBL序列的對(duì)比鑒定了鐮狀瘧原蟲(chóng)175KD蛋白質(zhì)中高度保守的區(qū)域；這些保守區(qū)接著用于鑒定其中之一(eb1-e1)位于染色體13的基因(eb1-e1，eb1-e2)。連鎖遺傳研究將該基因定位在染色體13影響瘧原蟲(chóng)感染人紅血細(xì)胞的區(qū)域上(Wellems et al.，Cell 49633-642(1987))。
使用來(lái)自這些開(kāi)架閱讀框的探針進(jìn)行的Southern雜交實(shí)驗(yàn)表明這些保守序列有額外的拷貝位于基因組的其它位置。染色體7上最大的開(kāi)架閱讀框是8千堿基，含4個(gè)與SABP和DABP N端半胱氨酸富集單位同源的串聯(lián)重復(fù)。
圖1代表EBL家族與DABP結(jié)合區(qū)和2個(gè)SABP同源在(F1和F2)的序列比較。將EBL家族分成2個(gè)亞家族以獲得最佳序列比較。保守的半胱氨酸殘基以黑體字顯示，保守的芳香族殘基下面劃線。
本發(fā)明的多肽可用于產(chǎn)生SABP，DABP結(jié)合區(qū)域EBL基因家族保守區(qū)特異性的單克隆抗體。該抗體可用于瘧疾感染的診斷或作為治療試劑抑制裂殖子與紅細(xì)胞的結(jié)合。抗所需抗原的單克隆抗體的生產(chǎn)對(duì)本領(lǐng)域的技術(shù)人員而言是公知的，在本文不再詳細(xì)描述。
本領(lǐng)域的技術(shù)人員，可得到的用于生產(chǎn)和控制各種免疫球蛋白”和“抗體”，指由一種或多種基本上由免疫球蛋白分子的許多技術(shù)可輕易地用來(lái)控制結(jié)合。本文所用的術(shù)語(yǔ)“免疫球蛋白基因編碼的多肽組成的蛋白質(zhì)。除抗體外，免疫球蛋白能以各種形式存在，例如包括Fv，F(xiàn)ab，和F(ab)2以及以單鏈的形式存在。免疫球蛋白結(jié)構(gòu)和功能的一般知識(shí)見(jiàn)基礎(chǔ)免疫學(xué)(第二版，W.E.Paul編輯，Ravens出版，紐約(1989))。
與本發(fā)明的多肽結(jié)合的抗體可用各種方法來(lái)生產(chǎn)。非人類單克隆抗體(例如鼠、兔形目、馬等)的生產(chǎn)是公知的并且可用(例如)以含該多肽的制品免疫動(dòng)物來(lái)完成。使從免疫的動(dòng)物獲得的抗體生產(chǎn)細(xì)胞成為永生細(xì)胞并進(jìn)行篩選，或者先篩選以生產(chǎn)控制裂殖子和紅細(xì)胞結(jié)合的抗體，然后使成的永生細(xì)胞。單克隆抗體生產(chǎn)一般方法的討論見(jiàn)Harlow和Lane的《抗體，實(shí)驗(yàn)室手冊(cè)》Cold Spring Harbor出版，紐約(1988)。
因此，本發(fā)明能達(dá)到保護(hù)性免疫反應(yīng)的目的或得到單克隆抗體以側(cè)序在SABP、DABP和EBL基因家族編碼區(qū)之間保守的結(jié)合區(qū)。這些蛋白質(zhì)結(jié)合區(qū)的鑒定有助于形成疫苗，因?yàn)樗谷藗兊靡詫⒕性诖蠓肿拥墓δ軉卧?。從結(jié)合區(qū)內(nèi)特定序列優(yōu)選出在進(jìn)化中保守的關(guān)鍵區(qū)域的目標(biāo)，于是優(yōu)選用作抗瘧原蟲(chóng)的疫苗。
EBL家族的基因(以前尚來(lái)側(cè)序)可用作標(biāo)記來(lái)檢測(cè)病人中鐮狀瘧原蟲(chóng)的存在。使用癥狀性病人的組織或血液與EBL家族基因片段互補(bǔ)的寡核酸進(jìn)行PCR反應(yīng)，以本領(lǐng)域的實(shí)踐者熟知的方法可完成這種檢測(cè)。而且，測(cè)序的EBL家族為熟練的實(shí)踐者提供了生產(chǎn)在各種運(yùn)用中用作遺傳標(biāo)記的限定的探針的一種方法。
另外，本發(fā)明定義了一個(gè)存在于(但不限于)參與宿主寄生作用的Apicomplexa亞門其它成員中的保守區(qū)?？墒褂脠D3所示的合成寡聚核苷酸引物以聚合酶鏈?zhǔn)椒磻?yīng)在瘧原蟲(chóng)種類和其它寄生的原生動(dòng)物中鑒定該區(qū)域。PCR方法在下文中詳細(xì)描述。從DABP，SABP和EBL家族中顯示出最高保守程序的保守部位的區(qū)域設(shè)計(jì)這些引物。圖3顯示了這些區(qū)域和來(lái)自它們的相一致的氨基酸序列。
A.一般方法本申請(qǐng)中所需的許多命名法和一般實(shí)驗(yàn)程序可參見(jiàn)Sambrook等的《分子克隆實(shí)驗(yàn)手冊(cè)》(第2版)(Vo.l 1-3)紐約、冷泉港，冷泉港實(shí)驗(yàn)室，1989)，該手冊(cè)下文稱為“Sam-brook等”。
B.分離編碼SABP、DABP和EBL結(jié)合區(qū)的方法本發(fā)明的核酸組合物，不論是RNA，cDAN，基因組DNA，還是各種組合的雜種，可從天然來(lái)源分離或在體外合成。要求保護(hù)的核酸可存在于轉(zhuǎn)化的或轉(zhuǎn)染的完整細(xì)胞中，在轉(zhuǎn)化的或轉(zhuǎn)染的細(xì)胞的溶胞產(chǎn)物中或以部分純化或基本上純的形式存在。
編碼本發(fā)明結(jié)合區(qū)的基因的核酸操作技術(shù)，如將編碼多肽的核酸序列亞克隆到表達(dá)載體上，標(biāo)記探針，DNA雜交和類似操作一般在Sambrook等中已有描述，本文引用以供參考。
可使用重組DNA技術(shù)生產(chǎn)結(jié)合區(qū)多肽。一般來(lái)說(shuō)，首先克隆編碼SABP和DABP結(jié)合區(qū)的DNA或以適合物連熱鬧到表達(dá)載體上的形式分離。連接后，含DNA片段或插入物的載體導(dǎo)入合適的宿主細(xì)胞以表達(dá)重組結(jié)合區(qū)。然后從宿主細(xì)胞中分離多肽。
有許多方法分離編碼SABP，DABP和EBL結(jié)合區(qū)的DNA序列。典型，使用DNA中序列特異性的標(biāo)記的寡核苷酸探針從基因組或cDNA文庫(kù)中分離DNA。限制性核酸內(nèi)切酶消化含合適基因的基因組DNA或cDNA可用來(lái)分離編碼這些蛋白質(zhì)結(jié)合區(qū)的DNA。由于已知SABP和DABP基因的DNA序列，可設(shè)計(jì)一組限制性核酸內(nèi)切酶在所需區(qū)域裂解該DNA。限制性核酸內(nèi)切酶消化后，經(jīng)過(guò)其與核酸探針雜交的能力(例如在Soutern印跡上)來(lái)鑒定編碼SABP結(jié)合區(qū)域DABP結(jié)合區(qū)的DNA，使用本領(lǐng)域的技術(shù)人員熟悉的標(biāo)準(zhǔn)方法(見(jiàn)Sambrook，et al)分離這些DNA區(qū)域。
也可用聚合酶鏈?zhǔn)椒磻?yīng)制備DABP，SABP和EBL結(jié)合區(qū)DNA。聚合酶鏈?zhǔn)椒磻?yīng)技術(shù)(PCR)用于直接從mRNA，cD-NA基因組文庫(kù)或cDNA文庫(kù)中擴(kuò)增DABP和SABP結(jié)合區(qū)的核酸序列。對(duì)這一方法而言，圖3所示的引物是特別優(yōu)選的。
用于擴(kuò)增SABP和DABP結(jié)合區(qū)DNA的合適引物和探針從DNA序列分析中產(chǎn)生。簡(jiǎn)單地說(shuō)，合成與所要擴(kuò)增的DNA區(qū)域2個(gè)3′端互補(bǔ)的寡核苷酸引物。然后使用這2個(gè)引物進(jìn)行聚合酶鏈?zhǔn)椒磻?yīng)。見(jiàn)PCR Proto colsA Guide toMethods and Appeicarions Innis，M.Gelfand.D.，Sninsky，J.and White.T.，eds.，Academic Press，San Diego(1990)。根據(jù)需要，可選擇引物來(lái)擴(kuò)增全部DABP區(qū)域或擴(kuò)增DABP和SABP結(jié)合區(qū)的小片段。
根據(jù)Beaucage，S.L和Caruthers.M.H.首先描述的固相phosphoramidite三酯方法(Tetrahedron Letts.，22(20)1859-1862，1981)，使用自動(dòng)合成儀化學(xué)合成用作探針的寡核苷酸，如Needham-VanDevanter，D.R.，et al.1984，NucleicAcids Res.，126159-6168中所述。寡核苷酸的純化經(jīng)過(guò)天然丙烯酰胺凝膠電泳或經(jīng)過(guò)陰離子交換HPLC完成，如Pearson.J.D.and Regnier.F.E.，1983.J.Chrom.，255137-149中所述。
使用Maxam.A.M和Gilbert的化學(xué)降解方法(見(jiàn)W.，Grossman.L.and Moldave，D.，eds.Academic Press，NewYork，Methods in Enzymology，65499-560)可證實(shí)合成的寡核苷酸序列。
本領(lǐng)域的技術(shù)人員已知的其它方法也可用于分離編碼SABP或DABP結(jié)合區(qū)的全部或部分的DNA。見(jiàn)Sambrook等。
C.DABP.SABP和EBL結(jié)合區(qū)多肽的表達(dá)一旦分離和克隆出結(jié)合區(qū)DNA，可在重組構(gòu)建的細(xì)胞如細(xì)菌、酵母，昆蟲(chóng)(特別是使用桿狀病毒載體)和哺乳動(dòng)物細(xì)胞中表達(dá)所需的多肽。按期望本領(lǐng)域的技術(shù)人員知道許多可用于表達(dá)編碼DABP和SABP結(jié)合區(qū)的DNA的表達(dá)系統(tǒng)。因此不再詳細(xì)描述已知的在原核或真核細(xì)胞中用于表達(dá)蛋白質(zhì)的各種方法。
作為簡(jiǎn)要的概述，編碼結(jié)合區(qū)的天然或合成的核酸的表達(dá)典型地可經(jīng)過(guò)將DNA或cDNA有效連接到啟動(dòng)子上(啟動(dòng)子可以是結(jié)構(gòu)性的或是誘導(dǎo)性的)，接著摻入表達(dá)載體中來(lái)實(shí)現(xiàn)。載體可適合于在原核生物或真核生物中復(fù)制和整合。典型的表達(dá)載體含有對(duì)調(diào)節(jié)編碼結(jié)合區(qū)的DNA的表達(dá)有用的轉(zhuǎn)錄和翻譯終止子，起始序列和啟動(dòng)子。為了獲得克隆基因的高水平表達(dá)，所需構(gòu)建的表達(dá)質(zhì)粒最少含有一個(gè)指導(dǎo)轉(zhuǎn)錄的強(qiáng)啟動(dòng)子，一個(gè)用于翻譯起始的核糖體結(jié)合位點(diǎn)和一個(gè)轉(zhuǎn)錄/翻譯終止子。
1.在原核生物中的表達(dá)在E.coli中適于該目的的調(diào)節(jié)區(qū)例子是Yanofsky，C.，1984，J.Bacteriol.，1581018-1024中所述的E.coli色氨酸生物合成途徑的啟動(dòng)子和操縱子區(qū)域和Herskowintz，I.and Hagen，D.，1980，Ann，Rev Genet.，14399-445所述的N噬菌體(PL)的左向啟動(dòng)子。轉(zhuǎn)化進(jìn)E.coli中的DNA載體包含有選擇標(biāo)記也是有用的。這類標(biāo)記的例子包括編碼氨芐青霉素，四環(huán)素或氯霉素抗性的基因。見(jiàn)Sambrook等關(guān)于在E.coli中使用的選擇標(biāo)記的詳細(xì)描述。
載體的選擇應(yīng)使之能導(dǎo)入合適的宿主細(xì)胞。細(xì)菌載體是典型的質(zhì)?；蚴删w來(lái)源。用噬菌體載體顆粒感染或用裸露的噬菌體載體DNA轉(zhuǎn)染合適的細(xì)菌細(xì)胞。如果使用質(zhì)粒載體，用質(zhì)粒載體DNA轉(zhuǎn)染細(xì)菌細(xì)胞。
可使用E.coli.Bacillus sp.(Palva，I et al.，1983，Gene22229-235；Mosbach，K.et al.Nature，302543-545)和沙門氏菌優(yōu)選E.coli系統(tǒng)。
原核細(xì)胞產(chǎn)生的結(jié)合區(qū)多肽可能不會(huì)正確地折疊。在從E.coli中純化的過(guò)程中，表達(dá)的多肽可能光變性，然后再?gòu)?fù)性。這可經(jīng)過(guò)將細(xì)菌產(chǎn)生的蛋白質(zhì)溶于離液劑如鹽酸胍中并用還原劑如β-巰基乙醇還原全部半胱氨酸殘基來(lái)實(shí)現(xiàn)。然后經(jīng)緩慢透折或凝膠過(guò)濾使多肽復(fù)性。見(jiàn)美國(guó)專利號(hào)4511503。
表達(dá)抗原的檢測(cè)經(jīng)過(guò)本領(lǐng)域已知的方法如放射免疫試驗(yàn)，Western印跡技術(shù)或免疫沉淀法來(lái)實(shí)現(xiàn)。從E.coli中的純化可按美國(guó)專利號(hào)4511503所述的程序進(jìn)行。
2.SABP，DABP和EBL結(jié)合區(qū)在真核生物中的合成本領(lǐng)域的技術(shù)人員已知各種真核表達(dá)系統(tǒng)，如酵母、昆蟲(chóng)細(xì)胞和哺乳動(dòng)物細(xì)胞。正如下面所作的簡(jiǎn)要描述，DABP和SABP結(jié)合區(qū)也可在這些真核系統(tǒng)中表達(dá)。
a.在酵母中的表達(dá)異源蛋白質(zhì)在酵母中的合成是人們所熟知的且有較好的描述。Methods in Yeast Genetics，Sherman，F(xiàn).，et a1.，Coldspring Harbor Laboratory，(1982)是一部較好的論著，描述了在酵母中生產(chǎn)結(jié)合區(qū)可用的各種方法。
用于酵母的啟動(dòng)子例子包括GAL.，10(Johnson，M.，and Davies，R.W.，1984，Mol.and Cell，Biol.，41440-1448)ADH2(Russell，D.，et al.1983，J.Biol，Chem.，2582674-2682).PHO5(EMBO J.6675-680，1982)和MFal(Herskowitz，Iand Oshima，Y.，1982 in The Molecular Bi-ology of the Yeast Saccharomyces，(eds，Strathern，J.N.Jones，E.W.，and Broach，J.R.，Cold Spring Harbor Lab.，Cotd Sp[ring Harbor.N.Y.，PP 181-209).也可使用具有選擇性標(biāo)記的多拷貝質(zhì)粒，如Leu-2，URA-3，Trp-1，和His-3。
許多酵母表達(dá)質(zhì)粒(如YEp6，YEpb，YEp)可用作載體。可將目的基因與各種酵母載體中的任一啟動(dòng)子融合。上述質(zhì)粒在文獻(xiàn)中已做了充分描述(Botstein，et al.，1979，Gene，817-24；Broach，et al.，1979，Gene，8121-133)。
在轉(zhuǎn)化酵母細(xì)胞中使用2種方法。在一種情況下，首先使用Zymolyase，溶細(xì)胞酶或glusulase將酵母細(xì)胞轉(zhuǎn)化為原生質(zhì)體，接著加入DNA和聚乙二醇(PEG)。然后在選擇性條件下在3％的瓊脂培養(yǎng)基中再生PEG處理的原生質(zhì)體。在文章(J.D.Beggs，1978，Nature(London).275104-109；和Hinnen，A.，et al.，1978，Proc.Natl.Acad.Sci.USA，751929-1933)中給出了該方法的細(xì)節(jié)。第二種方法不涉及去掉細(xì)胞壁。而是將細(xì)胞用氯化鋰或乙酸及PEG處理并涂布在選擇性培養(yǎng)板上(Ito，H.，et al.，1983.J.Bact.，153163-168)。
經(jīng)過(guò)溶融細(xì)胞并對(duì)溶胞產(chǎn)物運(yùn)用標(biāo)準(zhǔn)蛋白質(zhì)分離技術(shù)可以酵母中分離結(jié)合區(qū)。經(jīng)過(guò)使用Western印跡技術(shù)或其它標(biāo)準(zhǔn)免疫測(cè)定技術(shù)的放免試驗(yàn)可實(shí)現(xiàn)對(duì)純化過(guò)程的監(jiān)測(cè)。
b.在哺乳動(dòng)物和昆蟲(chóng)細(xì)胞培養(yǎng)物中的表達(dá)對(duì)生產(chǎn)結(jié)合區(qū)有用的細(xì)胞培養(yǎng)物的例子有昆蟲(chóng)或哺乳動(dòng)物起源的細(xì)胞。哺乳動(dòng)物細(xì)胞系統(tǒng)常以細(xì)胞單層的形式使用，盡管也可使用哺乳動(dòng)物細(xì)胞懸液。哺乳動(dòng)物細(xì)胞糸的例子包括VERO和Hela細(xì)胞，中國(guó)倉(cāng)鼠卵巢(CHO)細(xì)胞系，W138，BHK.Cos-7或MDCK細(xì)胞糸。
如上面所述，用于轉(zhuǎn)化宿主細(xì)胞的載體(例如質(zhì)粒)優(yōu)選含起動(dòng)轉(zhuǎn)錄的DNA序列和控制抗原基因序列翻譯的序列。這些序列稱為表達(dá)控制序列。當(dāng)宿主細(xì)胞是昆蟲(chóng)或哺乳動(dòng)物起源的細(xì)胞時(shí)，表達(dá)控制序列的例子是得自SV-40啟動(dòng)子(Science，222524-527，1983)，CMV I.E.啟動(dòng)子(Proc.Natl.Acad.Sci.81659-663，1984)或金屬硫蛋白啟動(dòng)子(Nature 29639-42，1982)的序列。使用限制性酶裂解含表達(dá)控制序列的克隆載體，如果必要或需要的話調(diào)節(jié)其大小并以本領(lǐng)域熟知的方法與編碼SABP或DABP多肽的DNA連接。
與酵母一樣，當(dāng)使用更高等動(dòng)物的宿主細(xì)胞時(shí)，需要將已知哺乳動(dòng)物基因的多聚腺苷化或轉(zhuǎn)錄終止子序列摻入載體中。終止子序列的例子是牛生長(zhǎng)激素基因的多聚腺苷酸化序列。還應(yīng)包括對(duì)轉(zhuǎn)錄子進(jìn)行精確剪接的序列。剪接序列的例子是SV40的VP1內(nèi)含子(sprague J.et al.，1983，J.Vi-rol.45773-781)。
另外，可向載體中摻入在宿主細(xì)胞中控制復(fù)制的基因序列，例如在牛乳頭狀瘤病毒類型載體中發(fā)現(xiàn)的序列(Saveria-Campo.M.，1985，“Bovine Papilloma viras DNA a EudargoticCloning Veitor”in DNA Cloniry Vol.II a praitical Approach Ed.D.M.Glover.IRL Press.Arlington.Virginia pp.213-238)。
為了進(jìn)行轉(zhuǎn)化，宿主細(xì)胞應(yīng)是感受態(tài)或經(jīng)過(guò)各種方法使其成為感受態(tài)。有一些熟知的方法誘導(dǎo)DNA進(jìn)入動(dòng)物細(xì)胞。這些磷酸鈣沉淀法，將受體細(xì)胞含DNA的細(xì)菌原生質(zhì)體融合，用含DAN的脂質(zhì)體處理受體細(xì)胞，DEAE葡聚糖，電穿孔和將DNA直接顯微注射進(jìn)細(xì)胞。
以本領(lǐng)域，已知的方法培養(yǎng)轉(zhuǎn)化的細(xì)胞(BiochenicalMethods in Cell Culture and Virologg，Kuchler，R.J.Dowden，Hutchinson and Ross.Inc.(1977)。從以懸液或單層生長(zhǎng)的細(xì)胞中分離表達(dá)的DABP和SABP結(jié)合區(qū)多肽。對(duì)于單層的回收使用熟知的機(jī)械，化學(xué)或酶方法。
c.在重組牛痘病毒或腺病毒感染的細(xì)胞中表達(dá)除了在重組表達(dá)系統(tǒng)中使用外，分離的結(jié)合區(qū)DNA序列也可用于轉(zhuǎn)化在病人中的轉(zhuǎn)染宿主細(xì)胞的病毒?；畹臏p毒病毒(如牛痘或腺病毒)是疫苗的常規(guī)選擇，因?yàn)樗鼈兩a(chǎn)成本低且易于運(yùn)輸和施用。在免疫方法中有用的牛痘載體和方法已有描述，例如美國(guó)專利號(hào)422848，本文引用以供參考。
用于本發(fā)明的合適的病毒包括(但不限于)痘病毒(如金絲雀痘和牛痘病毒)，牛痘病毒，α病毒，腺病毒和其它動(dòng)物病毒。可使用本領(lǐng)域熟知的方法生產(chǎn)重組病毒，例如使用同源重組或連接工種質(zhì)粒的方法。例如，重組金絲雀痘或奶牛痘病毒的制備可經(jīng)過(guò)將編碼DABP和SABP結(jié)合區(qū)多肽的DNA插入質(zhì)粒，使其兩端以病毒序列為側(cè)翼。然后通過(guò)同源重組將編碼結(jié)合區(qū)的DNA插入病基因組中。
例如，重組腺病毒的生產(chǎn)可經(jīng)過(guò)將各含大約50％病毒序列的2種質(zhì)粒與編碼紅細(xì)胞結(jié)合區(qū)多肽的DNA序列連接起來(lái)。重組RNA病毒(如α病毒)的制備可使用本領(lǐng)域已知的方法經(jīng)CDNA介導(dǎo)制備。
到于牛痘病毒(例如WR株)，經(jīng)過(guò)包括同源重組的許多方法，使用轉(zhuǎn)移載體pTKgpt-OFIS可將編碼結(jié)合區(qū)的DNA序列插入基因組中，如Kaslow.et al.，Science 2521310-1313(1991)中所述，本文引用以供參考。
作為選擇，編碼SABPS和DABP結(jié)合區(qū)的DNA可插入另一設(shè)計(jì)的質(zhì)粒(例如PGS62)中以生產(chǎn)重組中痘(Langford，C.L.，et al.，1980，Mol.Cell.Biol.63191-3199)。這種質(zhì)粒由用于插入外源基因的克隆位點(diǎn)，指導(dǎo)插入基因合成的痘P7.5啟動(dòng)子和在外源基因兩端側(cè)翼的牛痘TK基因組成。
可使用編碼DABP和SABP結(jié)合區(qū)多肽的cDNA經(jīng)DNA雜交和使用表達(dá)結(jié)合區(qū)多肽特異性的抗體經(jīng)免疫檢測(cè)技術(shù)來(lái)進(jìn)行重組病毒生產(chǎn)的證實(shí)。病毒種子的制備可經(jīng)過(guò)感染細(xì)胞(例如HELA S3旋轉(zhuǎn)細(xì)胞)并收獲病毒子代實(shí)現(xiàn)。
本發(fā)明的重組病毒可用于哺乳動(dòng)物(如小鼠或人類)中誘導(dǎo)抗-SABP和抗-DABP結(jié)合區(qū)抗體。另外，可經(jīng)過(guò)體外感染宿主細(xì)胞，該宿主細(xì)胞隨后表達(dá)多肽來(lái)將重組病毒用于生產(chǎn)SABP和DABP結(jié)合區(qū)(見(jiàn)上面SABP和DABP結(jié)合區(qū)在真核細(xì)胞中的表達(dá)部分)。
本發(fā)明還涉及用重組病毒感染的宿主細(xì)胞。本發(fā)明的宿主細(xì)胞優(yōu)選哺乳動(dòng)物，如BSC-1細(xì)胞。以重組病毒感染的宿主細(xì)胞在其細(xì)胞表面表達(dá)DABP和SABP結(jié)合區(qū)。另外，當(dāng)用于接種或加強(qiáng)預(yù)先接種的哺乳動(dòng)物時(shí)，感染細(xì)胞的膜提取物誘導(dǎo)保護(hù)性抗體。
D.SABP，DABP和EBL結(jié)合區(qū)多肽的純化重組DNA技術(shù)產(chǎn)生的結(jié)合區(qū)多肽可使用本領(lǐng)域的技術(shù)人員熟知的標(biāo)準(zhǔn)技術(shù)來(lái)純化。重組產(chǎn)生的結(jié)合區(qū)多肽可直接表達(dá)或以融合蛋白質(zhì)的形式表達(dá)。然后將細(xì)胞溶合(例如聲處理)和親合色譜結(jié)合使用來(lái)純化蛋白質(zhì)。對(duì)于融全產(chǎn)物，隨后用合適的蛋白裂解酶消化融合蛋白以釋放所需的SABP和DABP結(jié)合區(qū)。
本發(fā)明的多肽可用本領(lǐng)域熟知的標(biāo)準(zhǔn)技術(shù)純化成基本純的產(chǎn)品，這些技術(shù)包括用諸如硫酸銨的物質(zhì)進(jìn)行選擇性沉淀，柱色譜免疫純化方法和其它方法。例如，見(jiàn)R.Scopes.Pro-tein PurifieationPrinciples and Practice，Springer-VelagNewYork(1982)，本文引用以供參考。
E.以蛋白質(zhì)化學(xué)技術(shù)生產(chǎn)結(jié)合區(qū)可用大量各種各樣的方法結(jié)合成制備本發(fā)明的多肽。例如大小相當(dāng)短的多肽可按常規(guī)技術(shù)在溶液中或在固體支持物上合成。各種自動(dòng)合成儀可以商品的形式買到并根據(jù)已知的方法使用，例如見(jiàn)Stewart and Young，Solid Pharse PeptideSynthesis，2d.ed.，Pierce Chemical Co.(1984)。
作為選擇，可蛋白裂解酶處理純化的和分離的SABP，DABP或EBL家族蛋白質(zhì)以生產(chǎn)結(jié)合區(qū)多肽。例如，重組DABP和SABP蛋白質(zhì)可用于該目的。然后可分析DABP和SABP蛋白質(zhì)的序列以選擇蛋白裂解酶，用于生產(chǎn)含DABP和SABP結(jié)合區(qū)的所需區(qū)域的多肽。然后經(jīng)過(guò)使用蛋白質(zhì)和肽純化的標(biāo)準(zhǔn)技術(shù)純化所需多肽。至于標(biāo)準(zhǔn)技術(shù)，可見(jiàn)Meth-ods in Enzymology，″Guide to Protein Purification″M.Deutscher，ed.Vol.182(1990)，Page619-626，本文引用以供參考。
F.核酸和多肽序列的修飾用于轉(zhuǎn)染宿主細(xì)胞以生產(chǎn)重組結(jié)合區(qū)多肽的核苷酸序列可標(biāo)準(zhǔn)技術(shù)進(jìn)行修飾以產(chǎn)生是有各種所需特征的結(jié)合區(qū)多肽。本發(fā)明的結(jié)合區(qū)多肽經(jīng)使用本領(lǐng)域的技術(shù)人員熟知的各種重組DNA技術(shù)可較容易的設(shè)計(jì)和生產(chǎn)。例如可以氨基酸插入，取代，缺失和類似技術(shù)從天然存在的序列在一級(jí)結(jié)構(gòu)水平上改變結(jié)合區(qū)多肽。可使用這些修飾的許多組合以生產(chǎn)最終的修飾的蛋白質(zhì)鏈。
可據(jù)各種主觀目的制備氨基酸序列的變異體，包括利于純化和制備的重組多肽。修飾的多肽對(duì)于改變血槳半衰期，提高治療效力和降低治療使用中副作用的嚴(yán)重性或發(fā)生率也是有用的。氨基酸序列變異體通常是預(yù)先確定的變異體，它在自然是中不存在但與天然存在的多肽表現(xiàn)出相同的免疫活性。例如，可生產(chǎn)僅包含一級(jí)結(jié)構(gòu)一部分(通常是至少大約60-80％，典型的是90-95％)的多肽片段。為用作疫苗，典型的多肽片段的長(zhǎng)度應(yīng)優(yōu)選至少保留一個(gè)能誘導(dǎo)封閉抗體生產(chǎn)的抗原決定基。
一般來(lái)說(shuō)編碼結(jié)合區(qū)多肽序列的修飾應(yīng)以各種熟知的技術(shù)易于完成，如用定點(diǎn)突變技術(shù)(見(jiàn).Giliman and Smith，Gene 881-97(1979)and Roberts.S.et al.，Nature 328；731-734(1987))。一個(gè)普通技術(shù)人員將感到許多突變的效果難以預(yù)料。因此大多數(shù)修飾在爭(zhēng)對(duì)所需特征的合適試驗(yàn)中經(jīng)常規(guī)篩選來(lái)評(píng)價(jià)。例如，多肽免疫特征的改變可用合適的競(jìng)爭(zhēng)性結(jié)合試驗(yàn)來(lái)檢測(cè)。其它特征的修飾，如氧化還原或熱穩(wěn)定性，疏水性，對(duì)蛋白裂解的敏感性或凝集傾向性都可根據(jù)標(biāo)準(zhǔn)技術(shù)來(lái)測(cè)定。
G.診斷和篩選試驗(yàn)本發(fā)明的多肽可用于在生物樣品中裂殖子檢測(cè)的診斷學(xué)應(yīng)用。使用一些較好識(shí)別的以免疫結(jié)果為基礎(chǔ)的特異性結(jié)合試驗(yàn)可檢測(cè)寄生物的存在(見(jiàn)美國(guó)專利436624143761104517288和4837168，本文引用以供參考)。例如，抗本發(fā)明多肽的標(biāo)記的單克隆抗體可用于檢測(cè)生物學(xué)樣品中的裂殖子。作為選擇，本發(fā)明標(biāo)記的多肽可用于檢測(cè)生物學(xué)樣品中抗SABP或DABP抗體的存在。至于診斷性免疫試驗(yàn)中的一般方法，也見(jiàn)Basic and Clinical Immunology tth Edition(CD.Sti es and A.Terr edy)1991，本文引用以供參考。
另外，修飾的多肽，抗體或其它能抑制SABP或DABP與紅細(xì)胞間相互作用的化合物可測(cè)定生物活性。例如，多肽可在細(xì)胞表面以重組形式進(jìn)行表達(dá)且可按下面所述測(cè)定細(xì)胞結(jié)合紅細(xì)胞的能力。作為選擇，可試驗(yàn)肽或抗體抑制紅細(xì)胞與裂殖子或SABP和DABP之間的結(jié)合的能力。
也可使用無(wú)細(xì)胞試驗(yàn)測(cè)定DABP和SABP多肽與分離的Duffy抗原或血型糖蛋白多肽的結(jié)合。例如，紅細(xì)胞蛋白質(zhì)可被固著于固體表面并可測(cè)定標(biāo)記的SABP或DABP多肽的結(jié)合。
許多試驗(yàn)方法使用標(biāo)記的試驗(yàn)成份。標(biāo)記系統(tǒng)可以是各種形式?？筛鶕?jù)本領(lǐng)域熟知的方法將標(biāo)記物與試驗(yàn)所需的成份直接或間接偶聯(lián)。可使用大范圍的各種標(biāo)記物。成份可用一些方法中的任意一種進(jìn)行標(biāo)記。最常用的檢測(cè)方法是使用對(duì)3H，125I，35S，14C或32P標(biāo)記的成份或類似物進(jìn)行放射自顯影。非放射活性標(biāo)記包括與標(biāo)記的抗體，熒光團(tuán)，化學(xué)發(fā)光劑，酶和能用作標(biāo)記配體的特異性結(jié)合對(duì)成員的抗體相結(jié)合的配體。標(biāo)記的選擇取決于所需的靈敏性，與化合物連接的容易程度，穩(wěn)定性需要和可得到的儀器。
另外，本發(fā)明的多肽可使用本領(lǐng)域的技術(shù)人員熟知的動(dòng)物模型來(lái)試驗(yàn)。用于鐮狀瘧原蟲(chóng)的體內(nèi)模型包括Aotus sp。猴或黑猩猩；用于間日瘧原蟲(chóng)的體內(nèi)模型Saimiri猴。
H.含有結(jié)合區(qū)多肽的藥用組合物本發(fā)明的多肽在治療瘧疾的治療學(xué)和預(yù)防學(xué)應(yīng)用中有用。本發(fā)明的藥用組合物適用于各種藥品傳遞系統(tǒng)。用于本發(fā)明的合適配方見(jiàn)Remington′s Pharmaceutical Sciences，Mack Publishing Company，Philadelphia，PA，17th ed.(1985)，本文引用以供參考。至于藥品傳遞方法的簡(jiǎn)要描述見(jiàn)Langer，Science 2491527-1533(1990)，本文引用以供參考。
本發(fā)明的多肽可以藥用和疫苗組合物使用，它們?cè)谥委煵溉閯?dòng)物，特別是人類中是有用的。多肽可在某些情況下一起施用(例如在很可能同時(shí)被鐮狀瘧原蟲(chóng)和間日瘧原蟲(chóng)感染的情況下)。因此，單個(gè)藥用組合物就可用于或預(yù)防由兩種瘧原蟲(chóng)引起的瘧疾。
組合物適用于一次施用或系列地施用。當(dāng)以系列給藥時(shí)，在起始用藥后依次進(jìn)行接種以加強(qiáng)免疫反應(yīng)，典型地稱為加強(qiáng)接種。
本發(fā)明的藥用組合物打算用于腸胃外，局部，口服或定點(diǎn)用藥。優(yōu)選的是藥用組合物經(jīng)腸胃外施用，例如經(jīng)靜脈內(nèi)，皮下，皮內(nèi)或肌肉內(nèi)用藥。因此，本發(fā)明提供的經(jīng)腸胃外施用的組合物包含上述試劑溶于或懸浮于一種可接受的載體(優(yōu)選一種含水載體)中的溶液?？墒褂酶鞣N含水載體，例如，水，緩沖水，0.4％鹽水，0.3％甘氨酸，透明質(zhì)酸和類似物。這些組合物可用常規(guī)熟知的滅菌技術(shù)來(lái)滅菌或可過(guò)濾滅菌。所得水溶液可包裝備用或經(jīng)凍干。施用前將凍干制品與無(wú)溶液混合。按照挖近似生理?xiàng)l件的需要組合物可含藥用上可接受的輔助物質(zhì)，如pH調(diào)節(jié)和緩沖劑，張力調(diào)節(jié)劑，潤(rùn)濕劑和類似物。例如醋酸鈉、乳酸鈉、氯化鈉、氯化鉀、氯化鈣、山梨聚糖單月桂酸酯，三乙醇胺油酸酯等。
至于固態(tài)組合物，可使用常規(guī)無(wú)毒固態(tài)載體，例如包括藥用級(jí)的甘露醇、乳糖、淀粉、硬脂酸鎂、糖精鈉、滑石、纖維素、葡萄糖、蔗糖、碳酸鎂和類似物。對(duì)于口服用藥，藥用上可接受的無(wú)毒組合物經(jīng)摻入任意一種通常使用的賦形劑(如前面列出的那些載體)和通常10-95％的活性成份(更優(yōu)選25％-75％的濃度)來(lái)形成。
對(duì)于以煙霧劑進(jìn)行的用藥，優(yōu)選使用以細(xì)小的分離形式與表面活性劑和推進(jìn)劑一起的多肽。當(dāng)然，表面活性劑必須是無(wú)毒的并優(yōu)選可溶于推進(jìn)劑。這種試劑的代表是酯或部分含6到22個(gè)碳原子的脂肪酸酯，如己酸、辛酸、月桂酸、軟脂酸、硬脂酸、亞油酸、亞麻酸、olesteric和油酸的脂肪族多羥基醇酯或其環(huán)酐。可使用混合的酯，如混合的或天然的甘油酯。如果需要，也可包括一種載體，例如以鼻內(nèi)用藥時(shí)可含卵磷脂。
在一些實(shí)施例中，可用SABP或DABP多肽或其它特異性抑制(例如單克隆抗體)治療瘧疾病人以阻止瘧原蟲(chóng)屬裂殖子和裂殖體與紅細(xì)胞表面的結(jié)合。
病人用藥量的范圍取決于施用何種藥物，病人的狀態(tài)和用藥方式。在治療應(yīng)用中，給已患有瘧疾的病人施用的組合物的量應(yīng)足以抑制瘧原蟲(chóng)通過(guò)紅細(xì)胞的傳播并因此治愈或至少部分控制該疾病和其并發(fā)癥的癥狀。是以實(shí)現(xiàn)該目的的量定義為“治療有效量”。對(duì)這一用途有效的量取決于疾病的嚴(yán)重性和病人的體重和一般狀態(tài)。但一般范圍是每天大約1mg到大約5gm，對(duì)于70kg的病人，優(yōu)選大約100mg。
作為選擇，本發(fā)明的多肽可在預(yù)防上用作疫苗。本發(fā)明的疫苗含作為有效成份的免疫有效量的結(jié)合區(qū)多肽或本文所述的重組病毒。免疫應(yīng)答可包括抗體的產(chǎn)生，抗存在來(lái)自本發(fā)明SABP、DABP或EBL序列編碼肽的多肽的細(xì)胞之細(xì)胞毒T淋巴細(xì)胞(CFL)的激活，或本領(lǐng)域已知的其它機(jī)制。例如見(jiàn)Paul Funp damental Immunology Second Edition Pub-lished by Roven press New York(本文引用以供參考)關(guān)于免疫應(yīng)答的描述。有用的載體是本領(lǐng)域所熟知的，例如它們包括甲狀腺球蛋白，諸如人血清白蛋白的白蛋白，破傷風(fēng)類毒素。多聚氨基酸如聚(D-賴氨酸D-谷氨酸)，流感、肝炎B病毒核心蛋白質(zhì)，肝炎B病毒重組疫苗。疫苗也可含有生理耐受性(可接受的)稀釋劑，如水，磷酸鹽緩沖液，或鹽水，典型地還含有佐劑。諸如不完全Freund氏佐劑，磷酸鋁，氫氧化鋁，或明礬的佐劑是本領(lǐng)域熟知的物質(zhì)。
編碼SABP或DABP結(jié)合區(qū)和EBL基因家族基序的DNA或RNA可導(dǎo)入病人以獲得對(duì)核酸編碼的多肽的免疫應(yīng)答。Wolff et，al.，Science2471465-1468(1990)(本文引用以供參考)描述了核酸在引起該核酸編碼的基因表達(dá)中的使用。
含本發(fā)明的多肽，核酸或病毒的疫苗組合物施用給病人以誘導(dǎo)抗該多肽的保護(hù)性免疫應(yīng)答?！氨Ｗo(hù)性免疫應(yīng)答”指阻止或抑制瘧原蟲(chóng)通過(guò)紅細(xì)胞的擴(kuò)散，從而至少部分阻止該疾病和其并發(fā)瘧的癥狀。是以達(dá)到這一目的的量定義的“免疫有效劑量”。在這一用途中有效的量取決于所用的組合物，用藥方式，病人的體重和一般健康狀態(tài)，以及處方醫(yī)生的判斷。對(duì)于多肽組合物，開(kāi)始免疫的一般范圍(治療或預(yù)防用藥)用于70kg的病人是從大約10μg到大約1gm的多肽，隨后的加強(qiáng)劑量按照從幾周到幾月(取決于病人的反應(yīng)和狀態(tài)，例如，經(jīng)測(cè)量病人血液中的瘧原蟲(chóng)水平)的加強(qiáng)方式從大約100μg到大約1gm的多肽。對(duì)于核酸，典型地將30-1000μg的核酸注射入70kg的病人，更典型地將大約50-150μg的核酸注射入70kg的病人，接著如果合適就注射加強(qiáng)劑量。
下面以說(shuō)明的方式而不是限制的方式提供實(shí)施例。
實(shí)施例作為紅細(xì)胞結(jié)合區(qū)的SABP和DABP氨基末端，半脫氨酸富集區(qū)的鑒定1.SABP結(jié)合區(qū)多肽在Cos細(xì)胞表面的表達(dá)為了證實(shí)SABP蛋白質(zhì)的氨基末端，半胱氨酸富集區(qū)是唾液酸結(jié)合區(qū)，在哺乳動(dòng)物Cos細(xì)胞表面體外表達(dá)蛋白質(zhì)的這一區(qū)域。這一DNA序列是SABP DNA序列(SEQ ID No3)上從位置1到位置1848。聚合酶鏈?zhǔn)椒磻?yīng)技術(shù)(PCR)用于直接從克隆基因中擴(kuò)增SABP DNA的這一區(qū)域。
將與Pvu II或Apa I的限制性內(nèi)切酶位點(diǎn)相對(duì)應(yīng)的序列摻入用于PCR擴(kuò)增的寡核苻酸序列中以利于將PCR擴(kuò)增區(qū)域插入pRE4載體中(見(jiàn)下面)。合成特異性的寡核苷酸5′-ATCGATCAGCTGGGAAGAAATACTTCATCT-3′。這些寡核甘酸用作PCR擴(kuò)增編碼SABP蛋白半胱氨酸富集的氨基末端區(qū)的DNA序列區(qū)的引物。
PCR條件以Saiki，et al.，Science239487-491(1988)描述的標(biāo)準(zhǔn)為基礎(chǔ)。從經(jīng)過(guò)剪接并以單個(gè)開(kāi)架閱讀框片段再克隆的編碼SABP的基因克隆片段提供模板DNA。
用于在Cos細(xì)胞中表達(dá)的載體pRE4如圖1所示。該載體有一個(gè)SV40復(fù)制起，一個(gè)氨芐青霉素抗性標(biāo)記和在Rous肉瘤病毒長(zhǎng)末端重復(fù)(RSV LTR)下游克隆的單純皰疹病毒糖蛋白D基因(HSV glyd)。使用HSV glyd上的Pvu II和ApaI位點(diǎn)切除HSV glyd基因的部分細(xì)胞外區(qū)域。
如上面所述，PCR寡核苷酸引物含Pvu II或Apa I限制性位點(diǎn)，用限制性酶Pvu II和Apa I消化上面得到的PCR擴(kuò)增的DNA片段并克隆到載體pRE4的Pvu II和Apa I位點(diǎn)上。設(shè)計(jì)這些構(gòu)建體來(lái)表達(dá)以在N末端與HSV glyd信號(hào)序列及在C末端與HSV glyd跨膜和胞質(zhì)區(qū)嵌合的蛋白質(zhì)形式出現(xiàn)的SABP蛋白質(zhì)區(qū)域。HSV glyd信號(hào)區(qū)將這些嵌合蛋白質(zhì)定位于Cos細(xì)胞表面，HSV glyd跨膜片段將這些嵌合蛋白質(zhì)錨著于Cos細(xì)胞表面。
根據(jù)標(biāo)準(zhǔn)技術(shù)以磷酸鈣沉淀法用含PCR擴(kuò)增的SABPDNA區(qū)域的pRE4構(gòu)建體轉(zhuǎn)染哺乳動(dòng)物Cos細(xì)胞。
2.DABP結(jié)合區(qū)多肽在Cos細(xì)胞表面的表達(dá)為了證實(shí)DABP蛋白質(zhì)的氨基末端半胱氨酸富集區(qū)是結(jié)合區(qū)，在Cos細(xì)胞表面表達(dá)這一區(qū)域。首先從位置1-975經(jīng)PCR擴(kuò)增DNA序列的這一區(qū)域(SEQ ID NO.1)。
將與Pvu II和Apa I的限制性核酸內(nèi)切酶位點(diǎn)相對(duì)應(yīng)的序列摻入用于PCR擴(kuò)增的寡核苷酸探針中。以利于隨后將擴(kuò)增的DNA插入pRE4載體中，如上所述。合成寡核苷酸5′-TCTCGTCAGCTGACGATCTCTAGTGCTATT-3′和5′-ACGAGTGGGCCCTGTCACAACTTCCTGAGT-3′。這些寡核苷酸用作引物，使用與上面所述相同的條件直接從克隆的DABP基因中擴(kuò)增編碼DABP蛋白質(zhì)半胱氨酸富集的氨基末端區(qū)的DABP DNA序列區(qū)域。
也使用與上面在Cos細(xì)胞表達(dá)SABP區(qū)域部分中所述相同的pRE4載體作為DABP DNA區(qū)域的載體。
3.與紅細(xì)胞的結(jié)合試驗(yàn)為了證實(shí)它們與人紅細(xì)胞結(jié)合的能力，將轉(zhuǎn)染的表達(dá)DABP和SABP結(jié)合區(qū)的Cos細(xì)胞與紅細(xì)胞在培養(yǎng)基(DMEM/10％FBS)中37℃下培養(yǎng)2小時(shí)。用磷酸鹽緩沖液洗滌5次去掉未附著的紅細(xì)胞用光學(xué)顯微鏡觀察結(jié)合的細(xì)細(xì)胞。在其表面表達(dá)氨基末端半胱氨酸富集的SABP多肽的Cos細(xì)胞與未處理的人紅細(xì)胞結(jié)合，但不與神經(jīng)氨酸苷酶處理的紅細(xì)胞(即在其表面缺乏唾液酸殘基，資料未顯示)結(jié)合。在其表面表達(dá)SABP蛋白質(zhì)其它區(qū)域的Cos細(xì)胞不結(jié)合人紅細(xì)胞(資料未顯示)。這些結(jié)果將SABP的氨基末端半胱氨酸富集區(qū)鑒定為紅細(xì)胞結(jié)合區(qū)，表明表達(dá)這些區(qū)域的Cos細(xì)胞與人紅細(xì)胞的結(jié)合是特異性的。而且，表達(dá)區(qū)與紅細(xì)胞的結(jié)合與在結(jié)合紅細(xì)胞方面可信的SABP-175分子所見(jiàn)的結(jié)合方式相同。
同樣地，在其表面表達(dá)DABP氨基末端半胱氨酸富集區(qū)的Cos細(xì)胞結(jié)合Duffy陽(yáng)性人紅細(xì)胞，但不結(jié)合Duffy陰性人紅細(xì)胞(即缺乏Duffy血型抗原的紅細(xì)胞，資料未顯示)。在其表面表達(dá)DABP蛋白質(zhì)其它區(qū)域的Cos細(xì)胞不結(jié)合人紅細(xì)胞(資料未顯示)。這些結(jié)果將DABP氨基末端半胱氨酸富集區(qū)鑒定為紅細(xì)胞結(jié)合區(qū)并表明Cos細(xì)胞的結(jié)合是特異性的。
序列描述(1)一般資料(I)申請(qǐng)人(A)名稱美國(guó)，國(guó)有健康與人類服務(wù)部(B)街道6011 Executive Blvd.，Suite 325(C)城市Rockville(D)州馬里蘭(E)國(guó)家美國(guó)(F)郵編20852(G)電話(301)496-7056(H)電傳(301)402-0220(I)電信(II)發(fā)明題目間日瘧原蟲(chóng)和鐮狀瘧原蟲(chóng)紅細(xì)胞結(jié)合蛋白的結(jié)合區(qū)(III)序列數(shù)16(IV)計(jì)算機(jī)可讀形式(A)媒體類型Floppy盤(B)計(jì)算機(jī)IBM PC兼容性(C)操作系統(tǒng)PC-DOS/MS-DOS(D)軟件Patent In Realease #1.0，Version #1.25(V)最近申請(qǐng)資料(A)申請(qǐng)?zhí)朠CT/US94/10230(B)申請(qǐng)日1994年9月7日(C)分類(VI)優(yōu)先權(quán)申請(qǐng)資料(A)申請(qǐng)?zhí)朥S 08/119,677(B)申請(qǐng)日1993年9月10日(2)SEQ ID NO1資料(I)序列特征(A)長(zhǎng)度4084堿基對(duì)(B)類型核酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型DNA(基因組)(III)假定非(VI)原始來(lái)源(A)生物間日瘧原蟲(chóng)(XI)序列描述SEQ ID NO1AAGCTTTTAA AAATAGCAAC AAAATTTCGA AACATTGCCA CAAAAATTTT ATGTTTTACA 60TATATTTAGA TTCATACAAT TTAGGTGTAC CCTGTTTTTT GATATATGCG CTTAAATTTT120TTTTTCGCTC ATATGTTTAG TTATATGTGT AGAACAACTT GCTGAATAAA TTACGTACAC180TTTCTGTTCT GAATAATATT ACCACATACA TTTAATTTTA AATACTATGA AAGGAAAAAA240CCGCTCTTTA TTTGTTCTCC TAGTTTTATT ATTGTTACAC AAGGTATCAT ATAAGGATGA300TTTTTCTATC ACACTAATAA ATTATCATGA AGGAAAAAAA TATTTAATTA TACTAAAAAG360AAAATTAGAA AAAGCTAATA ATCGTGATGT TTGCAATTTT TTTCTTCATT TCTCTCAGGT420AAATAATGTA TTATTAGAAC GAACAATTGA AACCCTTCTA GAATGCAAAA ATGAATATGT480GAAAGGTGAA AATGGTTATA AATTAGCTAA AGGACACCAC TGTGTTGAGG AAGATAACTT540AGAACGATGG TTACAAGGAA CCAATGAAAG AAGAAGTGAG GAAAATATAA AATATAAATA600TGGAGTAACG GAACTAAAAA TAAAGTATGC GCAAATGAAT GGAAAAAGAA GCAGCCGCAT660TTTGAAGGAA TCAATTTACG GGGCGCATAA CTTTGGAGGC AACAGTTACA TGGAGGGAAA720AGATGGAGGA GATAAAACTG GGGAGGAAAA AGATGGAGAA CATAAAACTG ATAGTAAAAC780TGATAACGGG AAAGGTGCAA ACAATTTGGT AATGTTAGAT TATGAGACAT CTAGCAATGG840CCAGCCAGCG GGAACCCTTG ATAATGTTCT TGAATTTGTG ACTGGGCATG AGGGAAATTC900TCGTAAAAAT TCCTCGAATG GTGGCAATCC TTACGATATT GATCATAAGA AAACGATCTC960TAGTGCTATT ATAAATCATG CTTTTCTTCA AAATACTGTA ATGAAAAACT GTAATTATAA 1020GAGAAAACGT CGGGAAAGAG ATTGGGACTG TAACACTAAG AAGGATGTTT GTATACCAGA 1080TCGAAGATAT CAATTATGTA TGAAGGAACT TACGAATTTG GTAAATAATA CAGACACAAA 1140TTTTCATAGG GATATAACAT TTCGAAAATT ATATTTGAAA AGGAAACTTA TTTATGATGC 1200TGCAGTAGAG GGCGATTTAT TACTTAAGTT GAATAACTAC AGATATAACA AAGACTTTTG 1260CAAGGATATA AGATGGAGTT TGGGAGATTT TGGAGATATA ATTATGGGAA CGGATATGGA 1320AGGCATCGGA TATTCCAAAG TAGTGGAAAA TAATTTGCGC AGCATCTTTG GAACTGATGA 1380AAAGGCCCAA CAGCGTCGTA AACAGTGGTG GAATGAATCT AAAGCACAAA TTTGGACAGC 1440AATGATGTAC TCAGTTAAAA AAAGATTAAA GGGGAATTTT ATATGGATTT GTAAATTAAA 1500TGTTGCGGTA AATATAGAAC CGCAGATATA TAGATGGATT CGAGAATGGG GAAGGGATTA 1560CGTGTCAGAA TTGCCCACAG AAGTGCAAAA ACTGAAAGAA AAATGTGATG GAAAAATCAA 1620TTATACTGAT AAAAAAGTAT GTAAGGTACC ACCATGTCAA AATGCGTGTA AATCATATGA 1680TCAATGGATA ACCAGAAAAA AAAATCAATG GGATGTTCTG TCAAATAAAT TCATAAGTGT 1740AAAAAACGCA GAAAAGGTTC AGACGGCAGG TATCGTAACT CCTTATGATA TACTAAAACA 1800GGAGTTAGAT GAATTTAACG AGGTGGCTTT TGAGAATGAA ATTAACAAAC GTGATGGTGC 1860ATATATTGAG TTATGCGTTT GTTCCGTTGA AGAGGCTAAA AAAAATACTC AGGAAGTTGT 1920GACAAATGTG GACAATGCTG CTAAATCTCA GGCCACCAAT TCAAATCCGA TAAGTCAGCC 1980TGTAGATAGT AGTAAAGCGG AGAAGGTTCC AGGAGATTCT ACGCATGGAA ATGTTAACAG 2040TGGCCAAGAT AGTTCTACCA CAGGTAAAGC TGTTACGGGG GATGGTCAAA ATGGAAATCA 2100GACACCTGCA GAAAGCGATG TACAGCGAAG TGATATTGCC GAAAGTGTAA GTGCTAAAAA 2160TGTTGATCCG CAGAAATCTG TAAGTAAAAG AAGTGACGAC ACTGCAAGCG TTACAGGTAT 2220TGCCGAAGCT GGAAAGGAAA ACTTAGGCGC ATCAAATAGT CGACCTTCTG AGTCCACCGT 2280TGAAGCAAAT AGCCCAGGTG ATGATACTGT GAACAGTGCA TCTATACCTG TAGTGAGTGG 2340TGAAAACCCA TTGGTAACCC CCTATAATGG TTTGAGGCAT TCGAAAGACA ATAGTGATAG 2400CGATGGACCT GCGGAATCAA TGGCGAATCC TGATTCAAAT AGTAAAGGTG AGACGGGAAA 2460GGGGCAAGAT AATGATATGG CGAAGGCTAC TAAAGATAGT AGTAATAGTT CAGATGGTAC 2520CAGCTCTGCT ACGGGTGATA CTACTGATGC AGTTGATAGG GAAATTAATA AAGGTGTTCC 2580TGAGGATAGG GATAAAACTG TAGGAAGTAA AGATGGAGGG GGGGAAGATA ACTCTGCAAA 2640TAAGGATGCA GCGACTGTAG TTGGTGAGGA TAGAATTCGT GAGAACAGCG CTGGTGGTAG 2700CACTAATGAT AGATCAAAAA ATGACACGGA AAAGAACGGG GCCTCTACCC CTGACAGTAA 2760ACAAAGTGAG GATGCAACTG CGCTAAGTAA AACCGAAAGT TTAGAATCAA CAGAAAGTGG 2820AGATAGAACT ACTAATGATA CAACTAACAG TTTAGAAAAT AAAAATGGAG GAAAAGAAAA 2880GGATTTACAA AAGCATGATT TTAAAAGTAA TGATACGCCG AATGAAGAAC CAAATTCTGA 2940TCAAACTACA GATGCAGAAG GACATGACAG GGATAGCATC AAAAATGATA AAGCAGAAAG 3000GAGAAAGCAT ATGAATAAAG ATACTTTTAC GAAAAATACA AATAGTCACC ATTTAAATAG 3060TAATAATAAT TTGAGTAATG GAAAATTAGA TATAAAAGAA TACAAATACA GAGATGTCAA 3120AGCAACAAGG GAAGATATTA TATTAATGTC TTCAGTACGC AAGTGCAACA ATAATATTTC 3180TTTAGAGTAC TGTAACTCTG TAGAGGACAA AATATCATCG AATACTTGTT CTAGAGAGAA 3240AAGTAAAAAT TTATGTTGCT CAATATCGGA TTTTTGTTTG AACTATTTTG ACGTGTATTC 3300TTATGAGTAT CTTAGCTGCA TGAAAAAGGA ATTTGAAGAT CCATCCTACA AGTGCTTTAC 3360GAAAGGGGGC TTTAAAGGTA TGCAGAAAAA GATGCTGAAT AGAGAAAGGT GTTGAGTAAA 3420TTAAAAAGGA ATTAATTTTA GGAATGTTAT AAACATTTTT GTACCCAAAA TTCTTTTTGC 3480AGACAAGACT TACTTTGCCG CGGCGGGAGC GTTGCTGATA CTGCTGTTGT TAATTGCTTC 3540AAGGAAGATG ATCAAAAATG AGTAACCAGA AAATAAAATA AAATAACATA AAATAAAATA 3600AAAACTAGAA TAACAATTAA AATAAAATAA AATGAGAAAT GCCTGTTAAT GCACAGTTAA 3660TTCTAACGAT TCCATTTGTG AAGTTTTAAA GAGAGCACAA ATGCATAGTC ATTATGTCCA 3720TGCATATATA CACATATATG TACGTATATA TAATAAACGC ACACTTTCTT GTTCGTACAG 3780TTCTGAAGAA GCTACATTTA ATGAGTTTGA AGAATACTCT GATAATATTC ACAGAATCCC 3840TCTGATGCCT AACAGTAATT CAAATTTCAA GAGCAAAATT CCATTTAAAA AGAAATGTTA 3900CATCATTTTG CGTTTTTCTT TTTTTCTTTT TTTTTTCTTT TTTAGATATT GAACACATGC 3960AGCCATCAAC CCCCCTGGAT TATTCATGAT GCTACTTTGG TAAGTAAAAG CAATTCTGAT 4020TGTAGTGCTG ATGTAATTTT AGTCATTTTG CTTGCTGCAA TAAACGAGAA AATATATCAA 4080GCTT4084(2)SEQ ID NO2資料(I)序列特征(A)長(zhǎng)度1115氨基酸(B)類型氨基酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型蛋白質(zhì)(III)假定的非(VI)原始來(lái)源(A)生物間日瘧原蟲(chóng)(XI)序列描述SEQ ID NO2Met Lys Gly Lys Asn Arg Ser Leu Phe Val Leu Leu Val Leu Leu Leu1 5 10 15Leu His Lys Val Ser Tyr Lys Asp Asp Phe Ser Ile Thr Leu Ile Asn20 25 30Tyr His Glu Gly Lys Lys Tyr Leu Ile Ile Leu Lys Arg Lys Leu Glu35 40 45Lys Ala Asn Asn Arg Asp Val Cys Asn Phe Phe Leu His Phe Ser Gln50 55 60Val Asn Asn Val Leu Leu Glu Arg Thr Ile Glu Thr Leu Leu Glu Cys65 70 75 80Lys Asn Glu Tyr Val Lys Gly Glu Asn Gly Tyr Lys Leu Ala Lys Gly85 90 95His His Cys Val Glu Glu Asp Asn Leu Glu Arg Trp Leu Gln Gly Thr100 105 110Asn Glu Arg Arg Ser Glu Glu Asn Ile Lys Tyr Lys Tyr Gly Val Thr115 120 125Glu Leu Lys Ile Lys Tyr Ala Gln Met Asn Gly Lys Arg Ser Ser Arg130 135 140Ile Leu Lys Glu Ser Ile Tyr Gly Ala His Asn Phe Gly Gly Asn Ser145 150 155 160Tyr Met Glu Gly Lys Asp Gly Gly Asp Lys Thr Gly Glu Glu Lys Asp165 170175Gly Glu His Lys Thr Asp Ser Lys Thr Asp Asn Gly Lys Gly Ala Asn180 185 190Asn Leu Val Met Leu Asp Tyr Glu Thr Ser Ser Asn Gly Gln Pro Ala195 200 205Gly Thr Leu Asp Asn Val Leu Glu Phe Val Thr Gly His Glu Gly Asn210 215 220Ser Arg Lys Asn Ser Ser Asn Gly Gly Asn Pro Tyr Asp Ile Asp His225 230 235 240Lys Lys Thr Ile Ser Ser Ala Ile Ile Asn His Ala Phe Leu Gln Asn245 250 255Thr Val Met Lys Asn Cys Asn Tyr Lys Arg Lys Arg Arg Glu Arg Asp260 265 270Trp Asp Cys Asn Thr Lys Lys Asp Val Cys Ile Pro Asp Arg Arg Tyr275 280 285Gln Leu Cys Met Lys Glu Leu Thr Asn Leu Val Asn Asn Thr Asp Thr290 295 300Asn Phe His Arg Asp Ile Thr Phe Arg Lys Leu Tyr Leu Lys Arg Lys305 310 315 320Leu Ile Tyr Asp Ala Ala Val Glu Gly Asp Leu Leu Leu Lys Leu Asn325 330 335Asn Tyr Arg Tyr Asn Lys Asp Phe Cys Lys Asp Ile Arg Trp Ser Leu340 345 350Gly Asp Phe Gly Asp Ile Ile Met Gly Thr Asp Met Glu Gly Ile Gly355 360 365Tyr Ser Lys Val Val Glu Asn Asn Leu Arg Ser Ile Phe Gly Thr Asp370 375 380Glu Lys Ala Gln Gln Arg Arg Lys Gln Trp Trp Asn Glu Ser Lys Ala385 390 395 400Gln Ile Trp Thr Ala Met Met Tyr Ser Val Lys Lys Arg Leu Lys Gly405 410 415Asn Phe Ile Trp Ile Cys Lys Leu Asn Val Ala Val Asn Ile Glu Pro420 425 430Gln Ile Tyr Arg Trp Ile Arg Glu Trp Gly Arg Asp Tyr Val Ser Glu435 440 445Leu Pro Thr Glu Val Gln Lys Leu Lys Glu Lys Cys Asp Gly Lys Ile450 455 460Asn Tyr Thr Asp Lys Lys Val Cys Lys Val Pro Pro Cys Gln Asn Ala465 470 475 480Cys Lys Ser Tyr Asp Gln Trp Ile Thr Arg Lys Lys Asn Gln Trp Asp485 490 495Val Leu Ser Asn Lys Phe Ile Ser Val Lys Asn Ala Glu Lys Val Gln500 505 510Thr Ala Gly Ile Val Thr Pro Tyr Asp Ile Leu Lys Gln Glu Leu Asp515 520 525Glu Phe Asn Glu Val Ala Phe Glu Asn Glu Ile Asn Lys Arg Asp Gly
530 535 540Ala Tyr Ile Glu Leu Cys Val Cys Ser Val Glu Glu Ala Lys Lys Asn545 550 555 560Thr Gln Glu Val Val Thr Asn Val Asp Asn Ala Ala Lys Ser Gln Ala565 570 575Thr Asn Ser Asn Pro Ile Ser Gln Pro Val Asp Ser Ser Lys Ala Glu580 585 590Lys Val Pro Gly Asp Ser Thr His Gly Asn Val Asn Ser Gly Gln Asp595 600 605Ser Ser Thr Thr Gly Lys Ala Val Thr Gly Asp Gly Gln Asn Gly Asn610 615 620Gln Thr Pro Ala Glu Ser Asp Val Gln Arg Ser Asp Ile Ala Glu Ser625 630 635 640Val Ser Ala Lys Asn Val Asp Pro Gln Lys Ser Val Ser Lys Arg Ser645 650 655Asp Asp Thr Ala Ser Val Thr Gly Ile Ala Glu Ala Gly Lys Glu Asn660 665 670Leu Gly Ala Ser Asn Ser Arg Pro Ser Glu Ser Thr Val Glu Ala Asn675 680 685Ser Pro Gly Asp Asp Thr Val Asn Ser Ala Ser Ile Pro Val Val Ser690 695 700Gly Glu Asn Pro Leu Val Thr Pro Tyr Asn Gly Leu Arg His Ser Lys705 710 715 720Asp Asn Ser Asp Ser Asp Gly Pro Ala Glu Ser Met Ala Asn Pro Asp725 730 735Ser Asn Ser Lys Gly Glu Thr Gly Lys Gly Gln Asp Asn Asp Met Ala740 745 750Lys Ala Thr Lys Asp Ser Ser Asn Ser Ser Asp Gly Thr Ser Ser Ala755 760 765Thr Gly Asp Thr Thr Asp Ala Val Asp Arg Glu Ile Asn Lys Gly Val770 775 780Pro Glu Asp Arg Asp Lys Thr Val Gly Ser Lys Asp Gly Gly Gly Glu785 790 795 800Asp Asn Ser Ala Asn Lys Asp Ala Ala Thr Val Val Gly Glu Asp Arg805 810 815Ile Arg Glu Asn Ser Ala Gly Gly Ser Thr Asn Asp Arg Ser Lys Asn820 825 830Asp Thr Glu Lys Asn Gly Ala Ser Thr Pro Asp Ser Lys Gln Ser Glu835 840 845Asp Ala Thr Ala Leu Ser Lys Thr Glu Ser Leu Glu Ser Thr Glu Ser850 855 860Gly Asp Arg Thr Thr Asn Asp Thr Thr Asn Ser Leu Glu Asn Lys Asn865 870 875 880Gly Gly Lys Glu Lys Asp Leu Gln Lys His Asp Phe Lys Ser Asn Asp885 890 895Thr Pro Asn Glu Glu Pro Asn Ser Asp Gln Thr Thr Asp Ala Glu Gly900 905 910His Asp Arg Asp Ser Ile Lys Asn Asp Lys Ala Glu Arg Arg Lys His915 920 925Met Asn Lys Asp Thr Phe Thr Lys Asn Thr Asn Ser His His Leu Asn930 935 940Ser Asn Asn Asn Leu Ser Asn Gly Lys Leu Asp Ile Lys Glu Tyr Lys945 950 955 960Tyr Arg Asp Val Lys Ala Thr Arg Glu Asp Ile Ile Leu Met Ser Ser965 970 975Val Arg Lys Cys Asn Asn Asn Ile Ser Leu Glu Tyr Cys Asn Ser Val980 985 990Glu Asp Lys Ile Ser Ser Asn Thr Cys Ser Arg Glu Lys Ser Lys Asn995 10001005Leu Cys Cys Ser Ile Ser Asp Phe Cys Leu Asn Tyr Phe Asp Val Tyr101010151020Ser Tyr Glu Tyr Leu Ser Cys Met Lys Lys Glu Phe Glu Asp Pro Ser1025103010351040Tyr Lys Cys Phe Thr Lys Gly Gly Phe Lys Ile Asp Lys Thr Tyr Phe104510501055Ala Ala Ala Gly Ala Leu Leu Ile Leu Leu Leu Ile Ala Ser Arg Lys106010651070Met Ile Lys Asn Asp Ser Glu Glu Ala Thr Phe Asn Glu Phe Glu Glu107510801085Tyr Cys Asp Asn Ile His Arg Ile Pro Leu Met Pro Asn Asn Ile Glu109010951100His Met Gln Pro Ser Thr Pro Leu Asp Tyr Ser110511101115(2)SEQ ID NO3資料(I)序列特征(A)長(zhǎng)度4507堿基對(duì)(B)類型核酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型DNA(基因組)(III)假定的非(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO3TATATATATA TATATATATA GATAATAACA TATAAATATA TTCAATGTGC ATACAATGAA 60ATGTAATATT AGTATATATT TTTTTGCTTC CTTCTTTGTG TTATATTTTG CAAAAGCTAG120GAATGAATAT GATATAAAAG AGAATGAAAA ATTTTTAGAC GTGTATAAAG AAAAATTTAA180TGAATTAGAT AAAAAGAAAT ATGGAAATGT TCAAAAAACT GATAAGAAAA TATTTACTTT240TATAGAAAAT AAATTAGATA TTTTAAATAA TTCAAAATTT AATAAAAGAT GGAAGAGTTA300TGGAACTCCA GATAATATAG ATAAAAATAT GTCTTTAATA AATAAACATA ATAATGAAGA360AATGTTTAAC AACAATTATC AATCATTTTT ATCGACAAGT TCATTAATAA AGCAAAATAA420ATATGTTCCT ATTAACGCTG TACGTGTGTC TAGGATATTA AGTTTCCTGG ATTCTAGAAT480TAATAATGGA AGAAATACTT CATCTAATAA CGAAGTTTTA AGTAATTGTA GGGAAAAAAG540GAAAGGAATG AAATGGGATT GTAAAAAGAA AAATGATAGA AGCAACTATG TATGTATTCC600TGATCGTAGA ATCCAATTAT GCATTGTTAA TCTTAGCATT ATTAAAACAT ATACAAAAGA660GACCATGAAG GATCATTTCA TTGAAGCCTC TAAAAAAGAA TCTCAACTTT TGCTTAAAAA720AAATGATAAC AAATATAATT CTAAATTTTG TAATGATTTG AAGAATAGTT TTTTAGATTA780TGGACATCTT GCTATGGGAA ATGATATGGA TTTTGGAGGT TATTCAACTA AGGCAGAAAA840CAAAATTCAA GAAGTTTTTA AAGGGGCTCA TGGGGAAATA AGTGAACATA AAATTAAAAA900TTTTAGAAAA GAATGGTGGA ATGAATTTAG AGAGAAACTT TGGGAAGCTA TGTTATCTGA960GCATAAAAAT AATATAAATA ATTGTAAAAA TATTCCCCAA GAAGAATTAC AAATTACTCA1020ATGGATAAAA GAATGGCATG GAGAATTTTT GCTTGAAAGA GATAATAGAT CAAAATTGCC1080AAAAAGTAAA TGTAAAAATA ATACATTATA TGAAGCATGT GAGAAGGAAT GTATTGATCC1140ATGTATGAAA TATAGAGATT GGATTATTAG AAGTAAATTT GAATGGCATA CGTTATCGAA1200AGAATATGAA ACTCAAAAAG TTCCAAAGGA AAATGCGGAA AATTATTTAA TCAAAATTTC1260AGAAAACAAG AATGATGCTA AAGTAAGTTT ATTATTGAAT AATTGTGATG CTGAATATTC1320AAAATATTGT GATTGTAAAC ATACTACTAC TCTCGTTAAA AGCGTTTTAA ATGGTAACGA1380CAATACAATT AAGGAAAAGC GTGAACATAT TGATTTAGAT GATTTTTCTA AATTTGGATG1440TGATAAAAAT TCCGTTGATA CAAACACAAA GGTGTGGGAA TGTAAAAACC CTTATATATT1500ATCCACTAAA GATGTATGTG TACCTCCGAG GAGGCAAGAA TTATGTCTTG GAAACATTGA1560TAGAATATAC GATAAAAACC TATTAATGAT AAAAGAGCAT ATTCTTGCTA TTGCAATATA1620TGAATCAAGA ATATTGAAAC GAAAATATAA GAATAAAGAT GATAAAGAAG TTTGTAAAAT1680CATAAATAAA ACTTTCGCTG ATATAAGAGA TATTATAGGA GGTACTGATT ATTGGAATGA1740TTTGAGCAAT AGAAAATTAG TAGGAAAAAT TAACACAAAT TCAAAATATG TTCACAGGAA1800TAAAAAAAAT GATAAGCTTT TTCGTGATGA GTGGTGGAAA GTTATTAAAA AAGATGTATG1860GAATGTGATA TCATGGGTAT TCAAGGATAA AACTGTTTGT AAAGAAGATG ATATTGAAAA1920TATACCACAA TTCTTCAGAT GGTTTAGTGA ATGGGGTGAT GATTATTGCC AGGATAAAAC1980AAAAATGATA GAGACTCTGA AGGTTGAATG CAAAGAAAAA CCTTGTGAAG ATGACAATTG2040TAAAAGTAAA TGTAATTCAT ATAAAGAATG GATATCAAAA AAAAAAGAAG AGTATAATAA2100ACAAGCCAAA CAATACCAAG AATATCAAAA AGGAAATAAT TACAAAATGT ATTCTGAATT2160TAAATCTATA AAACCAGAAG TTTATTTAAA GAAATACTCG GAAAAATGTT CTAACCTAAA2220TTTCGAAGAT GAATTTAAGG AAGAATTACA TTCAGATTAT AAAAATAAAT GTACGATGTG2280TCCAGAAGTA AAGGATGTAC CAATTTCTAT AATAAGAAAT AATGAACAAA CTTCGCAAGA2340AGCAGTTCCT GAGGAAAACA CTGAAATAGC ACACAGAACG GAAACTCCAT CTATCTCTGA2400AGGACCAAAA GGAAATGAAC AAAAAGAACG TGATGACGAT AGTTTGAGTA AAATAAGTGT2460ATCACCAGAA AATTCAAGAC CTGAAACTGA TGCTAAAGAT ACTTCTAACT TGTTAAAATT2520AAAAGGAGAT GTTGATATTA GTATGCCTAA AGCAGTTATT GGGAGCAGTC CTAATGATAA2580TATAAATGTT ACTGAACAAG GGGATAATAT TTCCGGGGTG AATTCTAAAC CTTTATCTGA2640TGATGTACGT CCAGATAAAA AGGAATTAGA AGATCAAAAT AGTGATGAAT CGGAAGAAAC2700TGTAGTAAAT CATATATCAA AAAGTCCATC TATAAATAAT GGAGATGATT CAGGCAGTGG2760AAGTGCAACA GTGAGTGAAT CTAGTAGTTC AAATACTGGA TTGTCTATTG ATGATGATAG2820AAATGGTGAT ACATTTGTTC GAACACAAGA TACAGCAAAT ACTGAAGATG TTATTAGAAA2880AGAAAATGCT GACAAGGATG AAGATGAAAA AGGCGCAGAT GAAGAAAGAC ATAGTACTTC2940TGAAAGCTTA AGTTCACCTG AAGAAAAAAT GTTAACTGAT AATGAAGGAG GAAATAGTTT3000AAATCATGAA GAGGTGAAAG AACATACTAG TAATTCTGAT AATGTTCAAC AGTCTGGAGG3060AATTGTTAAT ATGAATGTTG AGAAAGAACT AAAAGATACT TTAGAAAATC CTTCTAGTAG3120CTTGGATGAA GGAAAAGCAC ATGAAGAATT ATCAGAACCA AATCTAAGCA GTGACCAAGA3180TATGTCTAAT ACACCTGGAC CTTTGGATAA CACCAGTGAA GAAACTACAG AAAGAATTAG3240TAATAATGAA TATAAAGTTA ACGAGAGGGA AGATGAGAGA ACGCTTACTA AGGAATATGA3300AGATATTGTT TTGAAAAGTC ATATGAATAG AGAATCAGAC GATGGTGAAT TATATGACGA3360AAATTCAGAC TTATCTACTG TAAATGATGA ATCAGAAGAC GCTGAAGCAA AAATGAAAGG3420AAATGATACA TCTGAAATGT CGCATAATAG TAGTCAACAT ATTGAGAGTG ATCAACAGAA3480AAACGATATG AAAACTGTTG GTGATTTGGG AACCACACAT GTACAAAACG AAATTAGTGT3540TCCTGTTACA GGAGAAATTG ATGAAAAATT AAGGGAAAGT AAAGAATCAA AAATTCATAA3600GGCTGAAGAG GAAAGATTAA GTCATACAGA TATACATAAA ATTAATCCTG AAGATAGAAA3660TAGTAATACA TTACATTTAA AAGATATAAG AAATGAGGAA AACGAAAGAC ACTTAACTAA3720TCAAAACATT AATATTAGTC AAGAAAGGGA TTTGCAAAAA CATGGATTCC ATACCATGAA3780TAATCTACAT GGAGATGGAG TTTCCGAAAG AAGTCAAATT AATCATAGTC ATCATGGAAA3840CAGACAAGAT CGGGGGGGAA ATTCTGGGAA TGTTTTAAAT ATGAGATCTA ATAATAATAA3900TTTTAATAAT ATTCCAAGTA GATATAATTT ATATGATAAA AAATTAGATT TAGATCTTTA3960TGAAAACAGA AATGATAGTA CAACAAAAGA ATTAATAAAG AAATTAGCAG AAATAAATAA4020ATGTGAGAAC GAAATTTCTG TAAAATATTG TGACCATATG ATTCATGAAG AAATCCCATT4080AAAAACATGC ACTAAAGAAA AAACAAGAAA TCTGTGTTGT GCAGTATCAG ATTACTGTAT4140GAGCTATTTT ACATATGATT CAGAGGAATA TTATAATTGT ACGAAAAGGG AATTTGATGA4200TCCATCTTAT ACATGTTTCA GAAAGGAGGC TTTTTCAAGT ATGATATTCA AATTTTTAAT4260AACAAATAAA ATATATTATT ATTTTTATAC TTACAAAACT GCAAAAGTAA CAATAAAAAA4320AATTAATTTC TCATTAATTT TTTTTTTCTT TTTTTCTTTT TAGGTATGCC ATATTATGCA4380GGAGCAGGTG TGTTATTTAT TATATTGGTT ATTTTAGGTG CTTCACAAGC CAAATATCAA4440AGGTTAGAAA AAATAAATAA AAATAAAATT GAGAAGAATG TAAATTAAAT ATAGAATTCG4500AGCTCGG 4507(2)SEQ ID NO4資料(I)序列特征(A)長(zhǎng)度1435個(gè)氨基酸(B)類型氨基酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型蛋白質(zhì)(III)假定的非(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO4Met Lys Cys Asn Ile Ser Ile Tyr Phe Phe Ala Ser Phe Phe Val Leu1 5 10 15Tyr Phe Ala Lys Ala Arg Asn Glu Tyr Asp Ile Lys Glu Asn Glu Lys20 25 30Phe Leu Asp Val Tyr Lys Glu Lys Phe Asn Glu Leu Asp Lys Lys Lys35 40 45Tyr Gly Asn Val Gln Lys Thr Asp Lys Lys Ile Phe Thr Phe Ile Glu50 55 60Asn Lys Leu Asp Ile Leu Asn Asn Ser Lys Phe Asn Lys Arg Trp Lys65 70 75 80Ser Tyr Gly Thr Pro Asp Asn Ile Asp Lys Asn Met Ser Leu Ile Asn85 90 95Lys His Asn Asn Glu Glu Met Phe Asn Asn Asn Tyr Gln Ser Phe Leu100 105110Ser Thr Ser Ser Leu Ile Lys Gln Asn Lys Tyr Val Pro Ile Asn Ala115 120 125Val Arg Val Ser Arg Ile Leu Ser Phe Leu Asp Ser Arg Ile Asn Asn130 135 140G1y Arg Asn Thr Ser Ser Asn Asn Glu Val Leu Ser Asn Cys Arg Glu145 150 155 160Lys Arg Lys Gly Met Lys Trp Asp Cys Lys Lys Lys Asn Asp Arg Ser165 170 175Asn Tyr Val Cys Ile Pro Asp Arg Arg Ile Gln Leu Cys Ile Val Asn180 185 190Leu Ser Ile Ile Lys Thr Tyr Thr Lys Glu Thr Met Lys Asp His Phe195 200 205Ile Glu Ala Ser Lys Lys Glu Ser Gln Leu Leu Leu Lys Lys Asn Asp210 215 220Asn Lys Tyr Asn Ser Lys Phe Cys Asn Asp Leu Lys Asn Ser Phe Leu225 230 235 240Asp Tyr Gly His Leu Ala Met Gly Asn Asp Met Asp Phe Gly Gly Tyr245 250 255Ser Thr Lys Ala Glu Asn Lys Ile Gln Glu Val Phe Lys Gly Ala His260 265 270Gly Glu Ile Ser Glu His Lys Ile Lys Asn Phe Arg Lys Glu Trp Trp275 280 285Asn Glu Phe Arg Glu Lys Leu Trp Glu Ala Met Leu Ser Glu His Lys
290 295 300Asn Asn Ile Asn Asn Cys Lys Asn Ile Pro Gln Glu Glu Leu Gln Ile305 310 315 320Thr Gln Trp Ile Lys Glu Trp His Gly Glu Phe Leu Leu Glu Arg Asp325 330 335Asn Arg Ser Lys Leu Pro Lys Ser Lys Cys Lys Asn Asn Thr Leu Tyr340 345 350Glu Ala Cys Glu Lys Glu Cys Ile Asp Pro Cys Met Lys Tyr Arg Asp355 360 365Trp Ile Ile Arg Ser Lys Phe Glu Trp His Thr Leu Ser Lys Glu Tyr370 375 380Glu Thr Gln Lys Val Pro Lys Glu Asn Ala Glu Asn Tyr Leu Ile Lys385 390 395 400Ile Ser Glu Asn Lys Asn Asp Ala Lys Val Ser Leu Leu Leu Asn Asn405 410 415Cys Asp Ala Glu Tyr Ser Lys Tyr Cys Asp Cys Lys His Thr Thr Thr420 425 430Leu Val Lys Ser Val Leu Asn Gly Asn Asp Asn Thr Ile Lys Glu Lys435 440 445Arg Glu His Ile Asp Leu Asp Asp Phe Ser Lys Phe Gly Cys Asp Lys450 455 460Asn Ser Val Asp Thr Asn Thr Lys Val Trp Glu Cys Lys Asn Pro Tyr465 470 475 480Ile Leu Ser Thr Lys Asp Val Cys Val Pro Pro Arg Arg Gln Glu Leu485 490 495Cys Leu Gly Asn Ile Asp Arg Ile Tyr Asp Lys Asn Leu Leu Met Ile500 505 510Lys Glu His Ile Leu Ala Ile Ala Ile Tyr Glu Ser Arg Ile Leu Lys515 520 525Arg Lys Tyr Lys Asn Lys Asp Asp Lys Glu Val Cys Lys Ile Ile Asn530 535 540Lys Thr Phe Ala Asp Ile Arg Asp Ile Ile Gly GIy Thr Asp Tyr Trp545 550 555 560Asn Asp Leu Ser Asn Arg Lys Leu Val Gly Lys Ile Asn Thr Asn Ser565 570 575Lys Tyr Val His Arg Asn Lys Lys Asn Asp Lys Leu Phe Arg Asp Glu580 585 590Trp Trp Lys Val Ile Lys Lys Asp Val Trp Asn Val Ile Ser Trp Val595 600 605Phe Lys Asp Lys Thr Val Cys Lys Glu Asp Asp Ile Glu Asn Ile Pro610 615 620Gln Phe Phe Arg Trp Phe Ser Glu Trp Gly Asp Asp Tyr Cys Gln Asp625 630 635 640Lys Thr Lys Met Ile Glu Thr Leu Lys Val Glu Cys Lys Glu Lys Pro645 650 655Cys Glu Asp Asp Asn Cys Lys Ser Lys Cys Asn Ser Tyr Lys Glu Trp660 665 670Ile Ser Lys Lys Lys Glu Glu Tyr Asn Lys Gln Ala Lys Gln Tyr Gln675 680 685Glu Tyr Gln Lys Gly Asn Asn Tyr Lys Met Tyr Ser Glu Phe Lys Ser690 695 700Ile Lys Pro Glu Val Tyr Leu Lys Lys Tyr Ser Glu Lys Cys Ser Asn705 710 715 720Leu Asn Phe Glu Asp Glu Phe Lys Glu Glu Leu His Ser Asp Tyr Lys725 730 735Asn Lys Cys Thr Met Cys Pro Glu Val Lys Asp Val Pro Ile Ser Ile740 745 750Ile Arg Asn Asn Glu Gln Thr Ser Gln Glu Ala Val Pro Glu Glu Asn755 760 765Thr Glu Ile Ala His Arg Thr Glu Thr Pro Ser Ile Ser Glu Gly Pro770 775 780Lys Gly Asn Glu Gln Lys Glu Arg Asp Asp Asp Ser Leu Ser Lys Ile785 790 795 800Ser Val Ser Pro Glu Asn Ser Arg Pro Glu Thr Asp Ala Lys Asp Thr805 810 815Ser Asn Leu Leu Lys Leu Lys Gly Asp Val Asp Ile Ser Met Pro Lys820 825 830Ala Val Ile Gly Ser Ser Pro Asn Asp Asn Ile Asn Val Thr Glu Gln835 840 845Gly Asp Asn Ile Ser Gly Val Asn Ser Lys Pro Leu Ser Asp Asp Val850 855 860Arg Pro Asp Lys Lys Glu Leu Glu Asp Gln Asn Ser Asp Glu Ser Glu865 870 875 880Glu Thr Val Val Asn His Ile Ser Lys Ser Pro Ser Ile Asn Asn Gly885 890 895Asp Asp Ser Gly Ser Gly Ser Ala Thr Val Ser Glu Ser Ser Ser Ser900 905 910Asn Thr Gly Leu Ser Ile Asp Asp Asp Arg Asn Gly Asp Thr Phe Val915 920 925Arg Thr Gln Asp Thr Ala Asn Thr Glu Asp Val Ile Arg Lys Glu Asn930 935 940Ala Asp Lys Asp Glu Asp Glu Lys Gly Ala Asp Glu Glu Arg His Ser945 950 955 960Thr Ser Glu Ser Leu Ser Ser Pro Glu Glu Lys Met Leu Thr Asp Asn965 970 975Glu Gly Gly Asn Ser Leu Asn His Glu Glu Val Lys Glu His Thr Ser980 985 990Asn Ser Asp Asn Val Gln Gln Ser Gly Gly Ile Val Asn Met Asn Val99510001005Glu Lys Glu Leu Lys Asp Thr Leu Glu Asn Pro Ser Sar Ser Leu Asp101010151020Glu Gly Lys Ala His Glu Glu Leu Ser Glu Pro Asn Leu Ser Ser Asp1025103010351040Gln Asp Met Ser Asn Thr Pro Gly Pro Leu Asp Asn Thr Ser Glu Glu104510501055Thr Thr Glu Arg Ile Ser Asn Asn Glu Tyr Lys Val Asn Glu Arg Glu106010651070Asp Glu Arg Thr Leu Thr Lys Glu Tyr Glu Asp Ile Val Leu Lys Ser107510801085His Met Asn Arg Glu Ser Asp Asp Gly Glu Leu Tyr Asp Glu Asn Ser109010951100Asp Leu Ser Thr Val Asn Asp Glu Sar Glu Asp Ala Glu Ala Lys Met1105111011151120Lys Gly Asn Asp Thr Ser Glu Met Ser His Asn Ser Ser Gln His Ile112511301135Glu Ser Asp Gln Gln Lys Asn Asp Met Lys Thr Val Gly Asp Leu Gly114011451150Thr Thr His Val Gln Asn Glu Ile Sar Val Pro Val Thr Gly Glu Ile115511601165Asp Glu Lys Leu Arg Glu Ser Lys Glu Ser Lys Ile His Lys Ala Glu117011751180Glu Glu Arg Leu Ser His Thr Asp Ile His Lys Ile Asn Pro Glu Asp1185119011951200Arg Asn Ser Asn Thr Leu His Leu Lys Asp Ile Arg Asn Glu Glu Asn120512101215Glu Arg His Leu Thr Asn Gln Asn Ile Asn Ile Ser Gln Glu Arg Asp122012251230Leu Gln Lys His Gly Phe His Thr Met Asn Asn Leu His Gly Asp Gly123512401245Val Ser Glu Arg Ser Gln Ile Asn His Ser His His Gly Asn Arg Gln125012551260Asp Arg Gly Gly Asn Ser Gly Asn Val Leu Asn Met Arg Ser Asn Asn1265127012751280Asn Asn phe Asn Asn Ile Pro Ser Arg Tyr Asn Leu Tyr Asp Lys Lys128512901295Leu Asp Leu Asp Leu Tyr Glu Asn Arg Asn Asp Ser Thr Thr Lys Glu130013051310Leu Ile Lys Lys Leu Ala Glu Ile Asn Lys Cys Glu Asn Glu Ile Ser131513201325Val Lys Tyr Cys Asp His Met Ile His Glu Glu Ile Pro Leu Lys Thr133013351340Cys Thr Lys Glu Lys Thr Arg Asn Leu Cys Cys Ala Val Ser Asp Tyr1345135013551360Cys Met Ser Tyr Phe Thr Tyr Asp Ser Glu Glu Tyr Tyr Asn Cys Thr136513701375Lys Arg Glu Phe Asp Asp Pro Ser Tyr Thr Cys Phe Arg Lys Glu Ala138013851390Phe Ser Ser Met Ile Phe Lys Phe Leu Ile Thr Asn Lys Ile Tyr Tyr139514001405Tyr Phe Tyr Thr Tyr Lys Thr Ala Lys Val Thr Ile Lys Lys Ile Asn141014151420Phe Ser Leu Ile Phe Phe Phe Phe Phe Ser Phe142514301435(2)SEQ ID NO5資料(I)序列特征(A)長(zhǎng)度2288個(gè)堿基對(duì)(B)類型核酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型DNA(基因組)(III)假定的非(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO5CACTTTATGC TTCCGGCTCG TATGTTGTGT GGAATTGTGA GCGGATAACA ATTTCACACA 60GGAAACAGCT ATGACCATGA TTACGCCAAG CTCTAATACG ACTCACTATA GGGAAAGCTG120GTACGCCTGC AGGTCCGGTC CGGAATTCAA TAAAATATTT CCAGAAAGGA ATGTGCAAAT180TCACATATCC AATATATTCA AGGAATATAA AGAAAATAAT GTAGATATCA TATTTGGAAC240GTTGAATTAT GAATATAATA ATTTCTGTAA AGAAAAACCT GAATTAGTAT CTGCTGCCAA300GTATAATCTG AAAGCTCCAA ATGCTAAATC CCCTAGAATA TACAAATCTA AGGAGCATGA360AGAATCAAGT GTGTTTGGTT GCAAAACGAA AATCAGTAAA GTTAAAAAAA AATGGAATTG420TTATAGTAAT AATAAAGTAA CTAAACCTGA AGGTGTATGT GGACCACCAA GAAGGCAACA480ATTATGTCTT GGATATATAT TTTTGATTCG CGACGGTAAC GAGGAAGGAT TAAAAGATCA540TATTAATAAG GCAGCTAATT ATGAGGCAAT GCATTTAAAA GAGAAATATG AGAATGCTGG600TGGTGATAAA ATTTGCAATG CTATATTGGG AAGTTATGCA GATATTGGAG ATATTGTAAG660AGGTTTGGAT GTTTGGAGGG ATATAAATAC TAATAAATTA TCAGAAAAAT TCCAAAAAAT720TTTTATGGGT GGTGGTAATT CTAGGAAAAA ACAAAACGAT AATAATGAAC GTAATAAATG780GTGGGAAAAA CAAAGGAATT TAATATGGTC TAGTATGGTA AAACACATTC CAAAAGGAAA840AACATGTAAA CGTCATAATA ATTTTGAGAA AATTCCTCAA TTTTTGAGAT GGTTAAAAGA900ATGGGGTGAT GAATTTTGTG AGGAAATGGG TACGGAAGTC AAGCAATTAG AGAAAATATG960TGAAAATAAA AATTGTTCGG AAAAAAAATG TAAAAATGCA TGTAGTTCCT ATGAAAAATG 1020GATAAAGGAA CGAAAAAATG AATATAATTT GCAATCAAAG AAATTTGATA GTGATAAAAA 1080ATTAAATAAA AAAAACAATC TTTATAATAA ATTTGAGGAT TCTAAAGCTT ATTTAAGGAG 1140TGAATCAAAA CAGTGCTCAA ATATAGAATT TAATGATGAA ACATTTACAT TTCCTAATAA 1200ATATAAAGAG GCTTGTATGG TATGTGAAAA TCCTTCATCT TCGAAAGCTC TTAAACCTAT 1260AAAAACGAAT GTGTTTCCTA TAGAGGAATC AAAAAAATCT GAGTTATCAA GTTTAACAGA 1320TAAATCTAAG AATACTCCTA ATAGTTCTGG TGGGGGAAAT TATGGAGATA GACAAATATC 1380AAAAAGAGAC GATGTTCATC ATGATGGTCC TAAGGAAGTG AAATCCGGAG AAAAAGAGGT 1440ACCAAAAATA GATGCAGCTG TTAAAACAGA AAATGAATTT ACCTCTAATC GAAACGATAT 1500TGAAGGAAAG GAAAAAAGTA AAGGTGATCA TTCTTCTCCT GTTCATTCTA AAGATATAAA 1560AAATGAGGAA CCACAAAGGG TGGTGTCTGA AAATTTACCT AAAATTGAAG AGAAAATGGA 1620ATCTTCTGAT TCTATACCAA TTACTCATAT AGAAGCTGAA AAGGGTCAGT CTTCTAATTC 1680TAGCGATAAT GATCCTGCAG TAGTAAGTGG TAGAGAATCT AAAGATGTAA ATCTTCATAC 1740TTCTGAAAGG ATTAAAGAAA ATGAAGAAGG TGTGATTAAA ACAGATGATA GTTCAAAAAG 1800TATTGAAATT TCTAAAATAC CATCTGACCA AAATAATCAT AGTGATTTAT CACAGAATGC 1860AAATGAGGAC TCTAATCAAG GGAATAAGGA AACAATAAAT CCTCCTTCTA CAGAAAAAAA 1920TCTCAAAGAA ATTCATTATA AAACATCTGA TTCTGATGAT CATGGTTCTA AAATTAAAAG 1980TGAAATTGAA CCAAAGGAGT TAACGGAGGA ATCACCTCTT ACTGATAAAA AAACTGAAAG 2040TGCAGCGATT GGTGATAAAA ATCATGAATC AGTAAAAAGC GCTGATATTT TTCAATCTGA 2100GATTCATAAT TCTGATAATA GAGATAGAAT TGTTTCTGAA AGTGTAGTTC AGGATTCTTC 2160AGGAAGCTCT ATGAGTACTG AATCTATACG TACTGATAAC AAGGATTTTA AAACAAGTGA 2220GGATATTGCA CCTTCTATTA ATGGTCGGAA TTCCCGGGTC GACGAGCTCA CTAGTCGGCG 2280GCCGCTCT2288(2)SEQ ID NO6資料(I)序列特征(A)長(zhǎng)度749個(gè)氨基酸(B)類型氨基酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型蛋白質(zhì)(III)假定的非(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO6Ala Asp Asn Asn Phe Thr Gln Glu Thr Ala Met Thr Met Ile Thr Pro1 5 10 15Ser Ser Asn Thr Thr His Tyr Arg Glu Ser Trp Tyr Ala Cys Arg Ser20 25 30Gly Pro Glu Phe Asn Lys Ile Phe Pro Glu Arg Asn Val Gln Ile His35 40 45Ile Ser Asn Ile Phe Lys Glu Tyr Lys Glu Asn Asn Val Asp Ile Ile50 55 60Phe Gly Thr Leu Asn Tyr Glu Tyr Asn Asn Phe Cys Lys Glu Lys Pro65 70 75 80Glu Leu Val Ser Ala Ala Lys Tyr Asn Leu Lys Ala Pro Asn Ala Lys85 90 95Ser Pro Arg Ile Tyr Lys Ser Lys Glu His Glu Glu Ser Ser Val Phe100 105 110Gly Cys Lys Thr Lys Ile Ser Lys Val Lys Lys Lys Trp Asn Cys Tyr115 120 125Ser Asn Asn Lys Val Thr Lys Pro Glu Gly Val Cys Gly Pro Pro Arg130 135 140Arg Gln Gln Leu Cys Leu Gly Tyr Ile Phe Leu Ile Arg Asp Gly Asn145 150 155 160Glu Glu Gly Leu Lys Asp His Ile Asn Lys Ala Ala Asn Tyr Glu Ala165 170 175Met His Leu Lys Glu Lys Tyr Glu Asn Ala Gly Gly Asp Lys Ile Cys180 185 190Asn Ala Ile Leu Gly Ser Tyr Ala Asp Ile Gly Asp Ile Val Arg Gly195 200 205Leu Asp Val Trp Arg Asp Ile Asn Thr Asn Lys Leu Ser Glu Lys Phe210 215 220Gln Lys Ile Phe Met Gly Gly Gly Asn Ser Arg Lys Lys Gln Asn Asp225 230 235 240Asn Asn Glu Arg Asn Lys Trp Trp Glu Lys Gln Arg Asn Leu Ile Trp245 250 255Ser Ser Met Val Lys His Ile Pro Lys Gly Lys Thr Cys Lys Arg His260 265 270Asn Asn Phe Glu Lys Ile Pro Gln Phe Leu Arg Trp Leu Lys Glu Trp275 280 285Gly Asp Glu Phe Cys Glu Glu Met Gly Thr Glu Val Lys Gln Leu Glu290 295 300Lys Ile Cys Glu Asn Lys Asn Cys Ser Glu Lys Lys Cys Lys Asn Ala305 310 315 320Cys Ser Ser Tyr Glu Lys Trp Ile Lys Glu Arg Lys Asn Glu Tyr Asn325 330 335Leu Gln Ser Lys Lys Phe Asp Ser Asp Lys Lys Leu Asn Lys Lys Asn340 345 350Asn Leu Tyr Asn Lys Phe Glu Asp Ser Lys Ala Tyr Leu Arg Ser Glu355 360 365Ser Lys Gln Cys Ser Asn Ile Glu Phe Asn Asp Glu Thr Phe Thr Phe370 375 380Pro Asn Lys Tyr Lys Glu Ala Cys Met Val Cys Glu Asn Pro Ser Ser385 390 395 400Ser Lys Ala Leu Lys Pro Ile Lys Thr Asn Val Phe Pro Ile Glu Glu405 410 415Ser Lys Lys Ser Glu Leu Ser Ser Leu Thr Asp Lys Ser Lys Asn Thr420 425 430Pro Asn Sar Sar Gly Gly Gly Asn Tyr Gly Asp Arg Gln Ile Ser Lys435 440 445Arg Asp Asp Val His His Asp Gly Pro Lys Glu Val Lys Ser Gly Glu450 455 460Lys Glu Val Pro Lys Ile Asp Ala Ala Val Lys Thr Glu Asn Glu Phe465 470 475 480Thr Ser Asn Arg Asn Asp Ile Glu Gly Lys Glu Lys Ser Lys Gly Asp485 490 495His Ser Ser Pro Val His Ser Lys Asp Ile Lys Asn Glu Glu Pro Gln500 505 510Arg Val Val Ser Glu Asn Leu Pro Lys Ile Glu Glu Lys Met Glu Ser515 520 525Ser Asp Ser Ile Pro Ile Thr His Ile Glu Ala Glu Lys Gly Gln Ser530 535 540Ser Asn Ser Ser Asp Asn Asp Pro Ala Val Val Ser Gly Arg Glu Ser545 550 555 560Lys Asp Val Asn Leu His Thr Ser Glu Arg Ile Lys Glu Asn Glu Glu565 570 575Gly Val Ile Lys Thr Asp Asp Ser Ser Lys Ser Ile Glu Ila Ser Lys580 585 590Ile Pro Ser Asp Gln Asn Asn His Ser Asp Leu Ser Gln Asn Ala Asn595 600 605Glu Asp Ser Asn Gln Gly Asn Lys Glu Thr Ile Asn Pro Pro Ser Thr610 615 620Glu Lys Asn Leu Lys Glu Ile His Tyr Lys Thr Ser Asp Ser Asp Asp625 630 635 640His Gly Ser Lys Ile Lys Ser Glu Ile Glu Pro Lys Glu Leu Thr Glu645 650 655Glu Ser Pro Leu Thr Asp Lys Lys Thr Glu Ser Ala Ala Ile Gly Asp660 665 670Lys Asn His Glu Ser Val Lys Ser Ala Asp Ile Phe Gln Ser Glu Ile675 680 685His Asn Ser Asp Asn Arg Asp Arg Ile Val Ser Glu Ser Val Val Gln690 695700Asp Ser Ser Gly Ser Ser Met Ser Thr Glu Ser Ile Arg Thr Asp Asn705 710 715 720Lys Asp Phe Lys Thr Ser Glu Asp Ile Ala Pro Ser Ile Asn Gly Arg725 730 735Asn Ser Arg Val Asp Glu Leu Thr Ser Arg Arg Pro Leu740 745(2)SEQ ID NO7資料(I)序列特征(A)長(zhǎng)度2606個(gè)堿基對(duì)(B)類型核酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型DNA(基因組)(III)假定的非(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO7AGCTCTATTA CGACTCACTA TAGGGAAAGC TGGTACGCCT GCAGGTACCG GTCCGGAATT 60CCCGGGTCGA CGAGCTCACT AGTCGGCGGC CGCTCTAGAG GATCCAAGCT TAATAGTGTT120TATACGTCTA TTGGCTTATT TTTAAATAGC TTAAAAAGCG GACCATGTAA AAAGGATAAT180GATAATGCAG AGGATAATAT AGATTTTGGT GATGAAGGTA AAACATTTAA AGAGGCAGAT240AATTGTAAAC CATGTTCTCA ATTTACTGTT GATTGTAAAA ATTGTAATGG TGGTGATACA300AAAGGGAAGT GCAATGGCAG CAATGGCAAA AAGAATGGAA ATGATTATAT TACTGCAAGT360GATATTGAAA ATGGAGGGAA TTCTATTGGA AATATAGATA TGGTTGTTAG TGATAAGGAT420GCAAATGGAT TTAATGGTTT AGACGCTTGT GGAAGTGCAA ATATCTTTAA AGGTATTAGA480AAAGAACAAT GGAAATGTGC TAAAGTATGT GGTTTAGATG TATGTGGTCT TAAAAATGGT540AATGGTAGTA TAGATAAAGA TCAAAAACAA ATTATAATTA TTAGAGCATT GCTTAAACGT600TGGGTAGAAT ATTTTTTAGA AGATTATAAT AAAATTAATG CCAAAATTTC ACATTGTACG660AAAAAGGATA ATGAATCCAC ATGTACAAAT GATTGTCCAA ATAAATGTAC ATGTGTAGAA720GAGTGGATAA ATCAGAAAAG GACAGAATGG AAAAATATAA AAAAACATTA CAAAACACAA780AATGAAAATG GTGACAATAA CATGAAATCT TTGGTTACAG ATATTTTGGG TGCCTTGCAA840CCCCAAAGTG ATGTTAACAA AGCTATAAAA CCTTGTAGTG GTTTAACTGC GTTCGAGAGT900TTTTGTGGTC TTAATGGCGC TGATAACTCA GAAAAAAAAG AAGGTGAAGA TTACGATCTT960GTTCTATGTA TGCTTAAAAA TCTTGAAAAA CAAATTCAGG AGTGCAAAAA GAAACATGGC 1020GAAACTAGTG TCGAAAATGG TGGCAAATCA TGTACCCCCC TTGACAACAC CACCCTTGAG 1080GAGGAACCCA TAGAAGAGGA AAACCAAGTG GAAGCGCCGA ACATTTGTCC AAAACAAACA 1140GTGGAAGATA AAAAAAAAGA GGAAGAAGAA GAAACTTGTA CACCGGCATC ACCAGTACCA 1200GAAAAACCGG TACCTCATGT GGCACGTTGG CGAACATTTA CACCACCTGA GGTATTCAAG 1260ATATGGAGGG GAAGGAGAAA TAAAACTACG TGCGAAATAG TGGCAGAAAT GCTTAAAGAT 1320AAGAATGGAA GGACTACAGT AGGTGAATGT TATAGAAAAG AAACTTATTC TGAATGGACG 1380TGTGATGAAA GTAAGATTAA AATGGGACAG CATGGAGCAT GTATTCCTCC AAGAAGACAA 1440AAATTATGTT TACATTATTT AGAAAAAATA ATGACAAATA CAAATGAATT GAAATACGCA 1500TTTATTAAAT GTGCTGCAGC AGAAACTTTT TTGTTATGGC AAAACTACAA AAAAGATAAG 1560AATGGTAATG CAGAAGATCT CGATGAAAAA TTAAAAGGTG GTATTATCCC CGAAGATTTT 1620AAACGGCAAA TGTTCTATAC GTTTGCAGAT TATAGAGATA TATGTTTGGG TACGGATATA 1680TCATCAAAAA AAGATACAAG TAAAGGTGTA GGTAAAGTAA AATGCAATAT TGATGATGTT 1740TTTTATAAAA TTAGCAATAG TATTCGTTAC CGTAAAAGTT GGTGGGAAAC AAATGGTCCA 1800GTTATATGGG AAGGAATGTT ATGCGCTTTA AGTTATGATA CGAGCCTAAA TAATGTTAAT 1860CCGGAAACTC ACAAAAAACT TACCGAAGGC AATAACAACT TTGAGAAAGT CATATTTGGT 1920AGTGATAGTA GCACTACTTT GTCCAAATTT TCTGAAAGAC CTCAATTTCT AAGATGGTTG 1980ACTGAATGGG GAGAAAATTT CTGCAAAGAA CAAAAAAAGG AGTATAAGGT GTTGTTGGCA 2040AAATGTAAGG ATTGTGATGT TGATGGTGAT GGTAAATGTA ATGGAAAATG TGTTGCGTGC 2100AAAGATCAAT GTAAACAATA TCATAGTTGG ATTGGAATAT GGATAGATAA TTATAAAAAA 2160CAAAAAGGAA GATATACTGA GGTTAAAAAA ATACCTCTGT ATAAAGAAGA TAAAGACGTG 2220AAAAACTCAG ATGATGCTCG CGATTATTTA AAAACACAAT TACAAAATAT GAAATGTGTA 2280AATGGAACTA CTGATGAAAA TTGTGAGTAT AAGTGTATGC ATAAAACCTC ATCCACAAAT 2340AGTGATATGC CCGAATCGTT GGACGAAAAG CCGGAAAAGG TCAAAGACAA GTGTAATTGT 2400GTACCTAATG AATGCAATGC ATTGAGTGTA AGTGGTAGCG GTTTTCCTGA TGGTCAAGCT 2460TACGTACGCG TGCATGCGAC GTCATAGCTC TTCTATAGTG TCACCTAAAT TCAATTCACT 2520GGCCGTCGTT TTACAACGTC GTGACTGGGA AAACCTGGCG TTACCCAACT TAATCGCCTT 2580GCAGCACATC CCCCTTTCGC CAGCTG2606(2)SEQ ID NO8資料(I)序列特征(A)長(zhǎng)度921個(gè)氨基酸(B)類型氨基酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型蛋白質(zhì)(III)假定的非(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO8Lys Leu Asn Ser Val Tyr Thr Ser Ile Gly Leu Phe Leu Asn Ser Leu1 5 10 15Lys Ser Gly Pro Cys Lys Lys Asp Asn Asp Asn Ala Glu Asp Asn Ile20 25 30Asp Phe Gly Asp Glu Gly Lys Thr Phe Lys Glu Ala Asp Asn Cys Lys35 40 45Pro Cys Ser Gln Phe Thr Val Asp Cys Lys Asn Cys Asn Gly Gly Asp50 55 60Thr Lys Gly Lys Cys Asn Gly Ser Asn Gly Lys Lys Asn Gly Asn Asp65 70 75 80Tyr Ile Thr Ala Ser Asp Ile Glu Asn Gly Gly Asn Sar Ile Gly Asn85 90 95Ile Asp Met Val Val Ser Asp Lys Asp Ala Asn Gly Phe Asn Gly Leu100 105 110Asp Ala Cys Gly Ser Ala Asn Ile Phe Lys Gly Ile Arg Lys Glu Gln115 120 125Trp Lys Cys Ala Lys Val Cys Gly Leu Asp Val Cys Gly Leu Lys Asn130 135 140Gly Asn Gly Ser Ile Asp Lys Asp Gln Lys Gln Ile Ile Ile Ile Arg145 150 155 160Ala Leu Leu Lys Arg Trp Val Glu Tyr Phe Leu Glu Asp Tyr Asn Lys
165 170 175Ile Asn Ala Lys Ile Ser His Cys Thr Lys Lys Asp Asn Glu Ser Thr180 185 190Cys Thr Asn Asp Cys Pro Asn Lys Cys Thr Cys Val Glu Glu Trp Ile195 200 205Asn Gln Lys Arg Thr Glu Trp Lys Asn Ile Lys Lys His Tyr Lys Thr210 215 220Gln Asn Glu Asn Gly Asp Asn Asn Met Lys Ser Leu Val Thr Asp Ile225 230 235 240Leu Gly Ala Leu Gln Pro Gln Ser Asp Val Asn Lys Ala Ile Lys Pro245 250 255Cys Ser Gly Leu Thr Ala Phe Glu Ser Phe Cys Gly Leu Asn Gly Ala260 265 270Asp Asn Ser Glu Lys Lys Glu Gly Glu Asp Tyr Asp Leu Val Leu Cys275 280 285Met Leu Lys Asn Leu Glu Lys Gln Ile Gln Glu Cys Lys Lys Lys His290 295 300Gly Glu Thr Ser Val Glu Asn Gly Gly Lys Ser Cys Thr Pro Leu Asp305 310 315 320Asn Thr Thr Leu Glu Glu Glu Pro Ile Glu Glu Glu Asn Gln Val Glu325 330 335Ala Pro Asn Ile Cys Pro Lys Gln Thr Val Glu Asp Lys Lys Lys Glu340 345 350Glu Glu Glu Glu Thr Cys Thr Pro Ala Ser Pro Val Pro Glu Lys Pro355 360 365Val Pro His Val Ala Arg Trp Arg Thr Phe Thr Pro Pro Glu Val Phe370 375 380Lys Ile Trp Arg Gly Arg Arg Asn Lys Thr Thr Cys Glu Ile Val Ala385 390 395 400Glu Met Leu Lys Asp Lys Asn Gly Arg Thr Thr Val Gly Glu Cys Tyr405 410 415Arg Lys Glu Thr Tyr Ser Glu Trp Thr Cys Asp Glu Ser Lys Ile Lys420 425 430Met Gly Gln His Gly Ala Cys Ile Pro Pro Arg Arg Gln Lys Leu Cys435 440 445Leu His Tyr Leu Glu Lys Ile Met Thr Asn Thr Asn Glu Leu Lys Tyr450 455 460Ala Phe Ile Lys Cys Ala Ala Ala Glu Thr Phe Leu Leu Trp Gln Asn465 470 475 480Tyr Lys Lys Asp Lys Asn Gly Asn Ala Glu Asp Leu Asp Glu Lys Leu485 490 495Lys Gly Gly Ile Ile Pro Glu Asp Phe Lys Arg Gln Met Phe Tyr Thr500 505 510Phe Ala Asp Tyr Arg Asp Ile Cys Leu Gly Thr Asp Ile Ser Ser Lys515 520 525Lys Asp Thr Ser Lys Gly Val Gly Lys Val Lys Cys Asn Ile Asp Asp530 535 540Val Phe Tyr Lys Ile Ser Asn Ser Ile Arg Tyr Arg Lys Ser Trp Trp545 550 555 560Glu Thr Asn Gly Pro Val Ile Trp Glu Gly Met Leu Cys Ala Leu Ser565 570 575Tyr Asp Thr Ser Leu Asn Asn Val Asn Pro Glu Thr His Lys Lys Leu580 585 590Thr Glu Gly Asn Asn Asn Phe Glu Lys Val Ile Phe Gly Ser Asp Ser595 600 605Ser Thr Thr Leu Ser Lys Phe Ser Glu Arg Pro Gln Phe Leu Arg Trp610 615 620Leu Thr Glu Trp Gly Glu Asn Phe Cys Lys Glu Gln Lys Lys Glu Tyr625 630 635 640Lys Val Leu Leu Ala Lys Cys Lys Asp Cys Asp Val Asp Gly Asp Gly645 650 655Lys Cys Asn Gly Lys Cys Val Ala Cys Lys Asp Gln Cys Lys Gln Tyr660 665 670His Ser Trp Ile Gly Ile Trp Ile Asp Asn Tyr Lys Lys Gln Lys Gly675 680 685Arg Tyr Thr Glu Val Lys Lys Ile Pro Leu Tyr Lys Glu Asp Lys Asp690 695 700Val Lys Asn Ser Asp Asp Ala Arg Asp Tyr Leu Lys Thr Gln Leu Gln705 710 715 720Asn Met Lys Cys Val Asn Gly Thr Thr Asp Glu Asn Cys Glu Tyr Lys725 730 735Cys Met His Lys Thr Ser Ser Thr Asn Ser Asp Met Pro Glu Ser Leu740 745 750Asp Glu Lys Pro Glu Lys Val Lys Asp Lys Cys Asn Cys Val Pro Asn
755 760 765Glu Cys Asn Ala Leu Ser Val Ser Gly Sar Gly Phe Pro Asp Gly Gln770 775 780Ala Phe Gly Gly Gly Val Leu Glu Gly Thr Cys Lys Gly Leu Gly Glu785 790 795 800Pro Lys Lys Lys Ile Glu Pro Pro Gln Tyr Asp Pro Thr Asn Asp Ile805 810 815Leu Lys Ser Thr Ile Pro Val Thr Ile Val Leu Ala Leu Gly Ser Ile820 825 830Ala phe Leu Phe Met Lys Val Ile Tyr Ile Tyr Val Trp Tyr Ile Tyr835 840 845Met Leu Cys Val Gly Ala Leu Asp Thr Tyr Ile Cys Gly Cys Ile Cys850 855 860Ile Cys Ile Phe Ile Cys Val Ser Val Tyr Val Cys Val Tyr Val Tyr865 870 875 880Val Phe Leu Tyr Met Cys Val Phe Tyr Ile Tyr Phe Ile Tyr Ile Tyr885 890 895Val Phe Ile Leu Lys Met Lys Lys Met Lys Lys Met Lys Lys Met Lys900 905 910Lys Met Lys Lys Arg Lys Lys Arg Ile915 920(2)SEQ ID NO9資料(I)序列特征(A)長(zhǎng)度2101堿基對(duì)(B)類型核酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型DNA(基因組)(III)假定的非(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO9GGAACAGGGT GATAATAAAG TAGGAGCCTG TGCTCCGTAT AGACGATTAC ATTTATGTGA 60TTATAATTTG GAATCTATAG ACACAACGTC GACGACGCAT AAGTTGTTGT TAGAGGTGTG120TATGGCAGCA AAATACGAAG GAAACTCAAT AAATACACAT TATACACAAC ATCAACGAAC190TAATGAGGAT TCTGCTTCCC AATTATGTAC TGTATTAGCA CGAAGTTTTG CAGATATAGG240TGATATCGTA AGAGGAAAAG ATCTATATCT CGGTTATGAT AATAAAGAAA AAGAACAAAG300AAAAAAATTA GAACAGAAAT TGAAAGATAT TTTCAAGAAA ATACATAAGG ACGTGATGAA360GACGAATGGC GCACAAGAAC GCTACATAGA TGATGCCAAA GGAGGAGATT TTTTTCAATT420AAGAGAAGAT TGGTGGACGT CGAATCGAGA AACAGTATGG AAAGCATTAA TATGTCATGC480ACCAAAAGAA GCTAATTATT TTATAAAAAC AGCGTGTAAT GTAGGAAAAG GAACTAATGG540TCAATGCCAT TGCATTGGTG GAGATGTTCC CACATATTTC GATTATGTGC CGCAGTATCT600TCGCTGGTTC GAGGAATGGG CAGAAGACTT TTGCAGGAAA AAAAAAAAAA AACTAGAAAA660TTTGCAAAAA CAGTGTCGTG ATTACGAACA AAATTTATAT TGTAGTGGTA ATGGCTACGA720TTGCACAAAA ACTATATATA AAAAAGGTAA ACTTGTTATA GGTGAACATT GTACAAACTG780TTCTGTTTGG TGTCGTATGT ATGAAACTTG GATAGATAAC CAGAAAAAAG AATTTCTAAA840ACAAAAAAGA AAATACGAAA CAGAAATATC AGGTGGTGGT AGTGGTAAGA GTCCTAAAAG900GACAAAACGG GCTGCACGTA GTAGTAGTAG TAGTGATGAT AATGGGTATG AAAGTAAATT960TTATAAAAAA CTGAAAGAAG TTGGCTACCA AGATGTCGAT AAATTTTTAA AAATATTAAA 1020CAAAGAAGGA ATATGTCAAA AACAACCTCA AGTAGGAAAT GAAAAAGCAG ATAATGTTGA 1080TTTTACTAAT GAAAAATATG TAAAAACATT TTCTCGTACA GAAATTTGTG AACCGTGCCC 1140ATGGTGTGGA TTGGAAAAAG GTGGTCCACC ATGGAAAGTT AAAGGTGACA AAACCTGCGG 1200AAGTGCAAAA ACAAAGACAT ACGATCCTAA AAATATTACC GATATACCAG TACTCTACCC 1260TGATAAATCA CAGCAAAATA TACTAAAAAA ATATAAAAAT TTTTGTGAAA AAGGTGCACC 1320TGGTGGTGGT CAAATTAAAA AATGGCAATG TTATTATGAT GAACATAGGC CTAGTAGTAA 1380AAATAATAAT AATTGTGTAG AAGGAACATG GGACAAGTTT ACACAAGGTA AACAAACCGT 1440TAAGTCCTAT AATGTTTTTT TTTGGGATTG GGTTCATGAT ATGTTACACG ATTCTGTAGA 1500GTGGAAGACA GAACTTAGTA AGTGTATAAA TAATAACACT AATGGCAACA CATGTAGAAA 1560CAATAATAAA TGTAAAACAG ATTGTGGTTG TTTTCAAAAA TGGGTTGAAA AAAAACAACA 1620AGAATGGATG GCAATAAAAG ACCATTTTGG AAAGCAAACA GATATTGTCC AACAAAAAGG 1680TCTTATCGTA TTTAGTCCCT ATGGAGTTCT TGACCTTGTT TTGAAGGGCG GTAATCTGTT 1740GCAAAATATT AAAGATGTTC ATGGAGATAC AGATGACATA AAACACATTA AGAAACTGTT 1800GGATGAGGAA GACGCAGTAG CAGTTGTTCT TGGTGGCAAG GACAATACCA CAATTGATAA 1860ATTACTACAA CACGAAAAAG AACAAGCAGA ACAATGCAAA CAAAAGCAGG AAGAATGCGA 1920GAAAAAAGCA CAACAAGAAA GTCGTGGTCG CTCCGCCGAA ACCCGCGAAG ACGAAAGGAC 1980ACAACAACCT GCTGATAGTG CCGGCGAAGT CGAAGAAGAA GAAGACGACG ACGACTACGA 2040CGAAGACGAC GAAGATGACG ACGTAGTCCA GGACGTAGAT GTAAGTGAAA TAAGAGGTCC 2100G 2101(2)SEQ ID NO10資料(I)序列特征(A)長(zhǎng)度700個(gè)氨基酸(B)類型氨基酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型蛋白質(zhì)(III)假定的無(wú)(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO10Glu Gln Gly Asp Asn Lys Val Gly Ala Cys Ala Pro Tyr Arg Arg Leu1 5 10 15His Leu Cys Asp Tyr Asn Leu Glu Ser Ile Asp Thr Thr Ser Thr Thr20 25 30His Lys Leu Leu Leu Glu Val Cys Met Ala Ala Lys Tyr Glu Gly Asn35 40 45Ser Ile Asn Thr His Tyr Thr Gln His Gln Arg Thr Asn Glu Asp Ser50 55 60Ala Ser Gln Leu Cys Thr Val Leu Ala Arg Ser Phe Ala Asp Ile Gly65 70 75 80Asp Ile Val Arg Gly Lys Asp Leu Tyr Leu Gly Tyr Asp Asn Lys Glu85 90 95Lys Glu Gln Arg Lys Lys Leu Glu Gln Lys Leu Lys Asp Ile phe Lys100 105 110Lys Ile His Lys Asp Val Met Lys Thr Asn Gly Ala Gln Glu Arg Tyr115 120 125Ile Asp Asp Ala Lys Gly Gly Asp Phe Phe Gln Leu Arg Glu Asp Trp130 135 140Trp Thr Ser Asn Arg Glu Thr Val Trp Lys Ala Leu Ile Cys His Ala145 150 155 160Pro Lys Glu Ala Asn Tyr Phe Ile Lys Thr Ala Cys Asn Val Gly Lys165 170 175Gly Thr Asn Gly Gln Cys His Cys Ile Gly Gly Asp Val Pro Thr Tyr180 185 190Phe Asp Tyr Val Pro Gln Tyr Leu Arg Trp Phe Glu Glu Trp Ala Glu195 200 205Asp Phe Cys Arg Lys Lys Lys Lys Lys Leu Glu Asn Leu Gln Lys Gln210 215 220Cys Arg Asp Tyr Glu Gln Asn Leu Tyr Cys Ser Gly Asn Gly Tyr Asp225 230 235 240Cys Thr Lys Thr Ile Tyr Lys Lys Gly Lys Leu Val Ile Gly Glu His245 250 255Cys Thr Asn Cys Ser Val Trp Cys Arg Met Tyr Glu Thr Trp Ile Asp260 265 270Asn Gln Lys Lys Glu Phe Leu Lys Gln Lys Arg Lys Tyr Glu Thr Glu275 280 285Ile Ser Gly Gly Gly Ser Gly Lys Ser Pro Lys Arg Thr Lys Arg Ala290 295 300Ala Arg Ser Ser Ser Ser Ser Asp Asp Asn Gly Tyr Glu Ser Lys Phe305 310 315 320Tyr Lys Lys Leu Lys Glu Val Gly Tyr Gln Asp Val Asp Lys Phe Leu325 330 335Lys Ile Leu Asn Lys Glu Gly Ile Cys Gln Lys Gln Pro Gln Val Gly340 345 350Asn Glu Lys Ala Asp Asn Val Asp Phe Thr Asn Glu Lys Tyr Val Lys355 360 365Thr Phe Ser Arg Thr Glu Ile Cys Glu Pro Cys Pro Trp Cys Gly Leu370 375 380Glu Lys Gly Gly Pro Pro Trp Lys Val Lys Gly Asp Lys Thr Cys Gly385 390 395 400Ser Ala Lys Thr Lys Thr Tyr Asp Pro Lys Asn Ile Thr Asp Ile Pro405 410 415Val Leu Tyr Pro Asp Lys Ser Gln Gln Asn Ile Leu Lys Lys Tyr Lys420 425 430Asn Phe Cys Glu Lys Gly Ala Pro Gly Gly Gly Gln Ile Lys Lys Trp
435 440 445Gln Cys Tyr Tyr Asp Glu His Arg Pro Ser Ser Lys Asn Asn Asn Asn450 455 460Cys Val Glu Gly Thr Trp Asp Lys Phe Thr Gln Gly Lys Gln Thr Val465 470 475 480Lys Ser Tyr Asn Val Phe Phe Trp Asp Trp Val His Asp Met Leu His485 490 495Asp Ser Val Glu Trp Lys Thr Glu Leu Ser Lys Cys Ile Asn Asn Asn500 505 510Thr Asn Gly Asn Thr Cys Arg Asn Asn Asn Lys Cys Lys Thr Asp Cys515 520 525Gly Cys Phe Gln Lys Trp Val Glu Lys Lys Gln Gln Glu Trp MetAla530 535 540Ile Lys Asp His Phe Gly Lys Gln Thr Asp Ile Val Gln Gln Lys Gly545 550 555 560Leu Ile Val Phe Ser Pro Tyr Gly Val Leu Asp Leu Val Leu Lys Gly565 570 575Gly Asn Leu Leu Gln Asn Ile Lys Asp Val His Gly Asp Thr Asp Asp580 585 590Ile Lys His Ile Lys Lys Leu Leu Asp Glu Glu Asp Ala Val Ala Val595 600 605Val Leu Gly Gly Lys Asp Asn Thr Thr Ile Asp Lys Leu Leu Gln His610 615 620Glu Lys Glu Gln Ala Glu Gln Cys Lys Gln Lys Gln Glu Glu Cys Glu625 630 635 640Lys Lys Ala Gln Gln Glu Ser Arg Gly Arg Ser Ala Glu Thr Arg Glu645 650 655Asp Glu Arg Thr Gln Gln Pro Ala Asp Ser Ala Gly Glu Val Glu Glu660 665 670Glu Glu Asp Asp Asp Asp Tyr Asp Glu Asp Asp Glu Asp Asp Asp Val675 680 685Val Gln Asp Val Asp Val Ser Glu Ile Arg Gly Pro690 695 700(2)SEQ ID NO11資料(I)序列特征(A)長(zhǎng)度8220個(gè)堿基對(duì)(B)類型核酸
(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型DNA(基因組)(III)假定的非(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO11AAAAATGGGG CCCAAGGAGG CTGCAGGTGG GGATGATATT GAGGATGAAA GTGCCAAACA 60TATGTTTGAT AGGATAGGAA AAGATGTGTA CGATAAAGTA AAAGAGGAAG CTAAAGAACG120TGGTAAAGGC TTGCAAGGAC GTTTGTCAGA AGCAAAATTT GAGAAAAATG AAAGCGATCC180ACAAACACCA GAAGATCCAT GCGATCTTGA TCATAAATAT CATACAAATG TAACTACTAA240TGTAATTAAT CCGTGCGCTG ATAGATCTGA CGTGCGTTTT TCCGATGAAT ATGGAGGTCA300ATGTACACAT AATAGAATAA AAGATAGTCA ACAGGGTGAT AATAAAGGTG CATGTGCTCC360ATATAGGCGA TTGCATGTAT GCGATCAAAA TTTAGAACAG ATAGAGCCTA TAAAAATAAC420AAATACTCAT AATTTATTGG TAGATGTGTG TATGGCAGCA AAATTTGAAG GACAATCAAT480AACACAAGAT TATCCAAAAT ATCAAGCAAC ATATGGTGAT TCTCCTTCTC AAATATGTAC540TATGCTGGCA CGAAGTTTTG CGGACATAGG GGACATTGTC AGAGGAAGAG ATTTGTATTT600AGGTAATCCA CAAGAAATAA AACAAAGACA ACAATTAGAA AATAATTTGA AAACAATTTT660CGGGAAAATA TATGAAAAAT TGAATGGCGC AGAAGCACGC TACGGAAATG ATCCGGAATT720TTTTAAATTA CGAGAAGATT GGTGGACTGC TAATCGAGAA ACAGTATGGA AAGCCATCAC780ATGTAACGCT TGGGGTAATA CATATTTTCA TGCAACGTGC AATAGAGGAG AACGAACTAA840AGGTTACTGC CGGTGTAACG ACGACCAAGT TCCCACATAT TTTGATTATG TGCCGCAGTA900TCTTCGCTGG TTCGAGGAAT GGGCAGAAGA TTTTTGTAGG AAAAAAAATA AAAAAATAAA960AGATGTTAAA AGAAATTGTC GTGGAAAAGA TAAAGAGGAT AAGGATCGAT ATTGTAGCCG 1020TAATGGCTAC GATTGCGAAA AAACTAAACG AGCGATTGGT AAGTTGCGTT ATGGTAAGCA 1080ATGCATTAGC TGTTTGTATG CATGTAATCC TTACGTTGAT TGGATAAATA ACCAAAAAGA 1140ACAATTTGAC AAACAGAAAA AAAAATATGA TGAAGAAATA AAAAAATATG AAAATGGAGC 1200ATCAGGTGGT AGTAGGCAAA AACGGGATGC AGGTGGTACA ACTACTACTA ATTATGATGG 1260ATATGAAAAA AAATTTTATG ACGAACTTAA TAAAAGTGAA TATAGAACCG TTGATAAATT 1320TTTGGAAAAA TTAAGTAATG AAGAAATATG CACAAAAGTT AAAGACGAAG AAGGAGGAAC 1380AATTGATTTT AAAAACGTTA ATAGTGATAG TACTAGTGGT GCTAGTGGCA CTAATGTTGA 1440AAGTCAAGGA ACATTTTATC GTTCAAAATA TTGCCAACCC TGCCCTTATT GTGGAGTGAA 1500AAAGGTAAAT AATGGTGGTA GTAGTAATGA ATGGGAAGAG AAAAATAATG GCAAGTGCAA 1560GAGTGGAAAA CTTTATGAGC CTAAACCCGA CAAAGAAGGT ACTACTATTA CAATCCTTAA 1620AAGTGGTAAA GGACATGATG ATATTGAAGA AAAATTAAAC AAATTTTGTG ATGAAAAAAA 1680TGGTGATACA ATAAATAGTG GTGGTAGTGG TACGGGTGGT AGTGGTGGTG GTAACAGTGG 1740TAGACAGGAA TTGTATGAAG AATGGAAATG TTATAAAGGT GAAGATGTAG TGAAAGTTGG 1800ACACGATGAG GATGACGAGG AGGATTATGA AAATGTAAAA AATGCAGGCG GATTATGTAT 1860ATTAAAAAAC CAAAAAAAGA ATAAAGAAGA AGGTGGAAAT ACGTCTGAAA AGGAGCCTGA 1920TGAAATCCAA AAGACATTCA ATCCTTTTTT TTACTATTGG GTTGCACATA TGTTAAAAGA 1980TTCCATACAT TGGAAAAAAA AACTTCAGAG ATGTTTACAA AATGGTAACA GAATAAAATG 2040TGGAAACAAT AAATGTAATA ATGATTGTGA ATGTTTTAAA AGATGGATTA CACAAAAAAA 2100AGACGAATGG GGGAAAATAG TACAACATTT TAAAACGCAA AATATTAAAG GTAGAGGAGG 2160TAGTGACAAT ACGGCAGAAT TAATCCCATT TGATCACGAT TATGTTCTTC AATACAATTT 2220GCAAGAAGAA TTTTTGAAAG GCGATTCCGA AGACGCTTCC GAAGAAAAAT CCGAAAATAG 2280TCTGGATGCA GAGGAGGCAG AGGAACTAAA ACACCTTCGC GAAATCATTG AAAGTGAAGA 2340CAATAATCAA GAAGCATCTG TTGGTGGTGG CGTCACTGAA CAAAAAAATA TAATGGATAA 2400ATTGCTCAAC TACGAAAAAG ACGAAGCCGA TTTATGCCTA GAAATTCACG AAGATGAGGA 2460AGAGGAAAAA GAAAAAGGAG ACGGAAACGA ATGTATCGAA GAGGGCGAAA ATTTTCGTTA 2520TAATCCATGT AGTGGCGAAA GTGGTAACAA ACGATACCCC GTTCTTGCGA ACAAAGTAGC 2580GTATCAAATG CATCACAAGG CAAAGACACA ATTGGCTAGT CGTGCTGGTA GAAGTGCGTT 2640GAGAGGTGAT ATATCCTTAG CGCAATTTAA AAATGGTCGT AACGGAAGTA CATTGAAAGG 2700ACAAATTTGC AAAATTAACG AAAACTATTC CAATGATAGT CGTGGTAATA GTGGTGGACC 2760ATGTACAGGC AAAGATGGAG ATCACGGAGG TGTGCGCATG AGAATAGGAA CGGAATGGTC 2820AAATATTGAA GGAAAAAAAC AAACGTCATA CAAAAACGTC TTTTTACCTC CCCGACGAGA 2880ACACATGTGT ACATCCAATT TAGAAAATTT AGATGTTGGT AGTGTCACTA AAAATGATAA 2940GGCTAGCCAC TCATTATTGG GAGATGTTCA GCTCGCAGCA AAAACTGATG CAGCTGAGAT 3000AATAAAACGC TATAAAGATC AAAATAATAT ACAACTAACT GATCCAATAC AACAAAAAGA 3060CCAGGAGGCT ATGTGTCGAG CTGTACGTTA TAGTTTTGCC GATTTAGGAG ACATTATTCG 3120AGGAAGAGAT ATGTGGGATG AGGATAAGAG CTCAACAGAC ATGGAAACAC GTTTGATAAC 3180CGTATTTAAA AACATTAAAG AAAAACATGA TGGAATCAAA GACAACCCTA AATATACCGG 3240TGATGAAAGC AAAAAGCCCG CATATAAAAA ATTACGAGCA GATTGGTGGG AAGCAAATAG 3300ACATCAAGTG TGGAGAGCCA TGAAATGCGC AACAAAAGGC ATCATATGTC CTGGTATGCC 3360AGTTGACGAT TATATCCCCC AACGTTTACG CTGGATGACT GAATGGGCTG AATGGTATTG 3420TAAAGCGCAA TCACAGGAGT ATGACAAGTT AAAAAAAATC TGTGCAGATT GTATGAGTAA 3480GGGTGATGGA AAATGTACGC AAGGTGATGT CGATTGTGGA AAGTGCAAAG CAGCATGTGA 3540TAAATATAAA GAGGAAATAG AAAAATGGAA TGAACAATGG AGAAAAATAT CAGATAAATA 3600CAATCTATTA TACCTACAAG CAAAAACTAC TTCTACTAAT CCTGGCCGTA CTGTTCTTGG 3660TGATGACGAT CCCGACTATC AACAAATGGT AGATTTTTTG ACCCCAATAC ACAAAGCAAG 3720TATTGCCGCA CGTGTTCTTG TTAAACGTGC TGCTGGTAGT CCCACTGAGA TCGCCGCCGC 3780CGCCCCGATC ACCCCCTACA GTACTGCTGC CGGATATATA CACCAGGAAA TAGGATATGG 3840GGGGTGCCAG GAACAAACAC AATTTTGTGA AAAAAAACAT GGTGCAACAT CAACTAGTAC 3900CACGAAAGAA AACAAAGAAT ACACCTTTAA ACAACCTCCG CCGGAGTATG CTACAGCGTG 3960TGATTGCATA AATAGGTCGC AAACAGAGGA GCCGAAGAAA AAGGAAGAAA ATGTAGAGAG 4020TGCCTGCAAA ATAGTGGAGA AAATACTTGA GGGTAAGAAT GGAAGGACTA CAGTAGGTGA 4080ATGTAATCCA AAAGAGAGTT ATCCTGATTG GGATTGCAAA AACAATATTG ACATTAGTCA 4140TGATGGTGCT TGTATGCCTC CAAGGAGACA AAAACTATGT TTATATTATA TAGCACATGA 4200GAGTCAAACA GAAAATATAA AAACAGACGA TAATTTGAAA GATGCTTTTA TTAAAACTGC 4260AGCAGCAGAA ACTTTTCTTT CATGGCAATA TTATAAGAGT AAGAATGATA GTGAAGCTAA 4320AATATTAGAT AGAGGCCTTA TTCCATCCCA ATTTTTAAGA TCCATGATGT ACACGTTTGG 4380AGATTATAGA GATATATGTT TGAACACAGA TATATCTAAA AAACAAAATG ATGTAGCTAA 4440GGCAAAAGAT AAAATAGGTA AATTTTTCTC AAAAGATGGC AGCAAATCTC CTAGTGGCTT 4500ATCACGCCAA GAATGGTGGA AAACAAATGG TCCAGAGATT TGGAAAGGAA TGTTATGTGC 4560CTTAACAAAA TACGTCACAG ATACCGATAA CAAAAGAAAA ATCAAAAACG ACTACTCATA 4620CGATAAAGTC AACCAATCCC AAAATGGCAA CCCTTCCCTT GAAGAGTTTG CTGCTAAACC 4680TCAATTTCTA CGTTGGATGA TCGAATGGGG AGAAGAGTTT TGTGCTGAAC GTCAGAAGAA 4740GGAAAATATC ATAAAAGATG CATGTAATGA AATAAATTCT ACACAACAGT GTAATGATGC 4800GAAACATCGT TGTAATCAAG CATGTAGAGC ATATCAAGAA TATGTTGAAA ATAAAAAAAA 4860AGAATTTTCG GGACAAACAA ATAACTTTGT TCTAAAGGCA AATGTTCAGC CCCAAGATCC 4920AGAATATAAA GGATATGAAT ATAAAGACGG CGTACAACCG ATACAGGGGA ATGAGTATTT 4980ACTGCAAAAA TGTGATAATA ATAAATGTTC TTGCATGGAT GGAAATGTAC TTTCCGTCTC 5040TCCAAAAGAA AAACCTTTTG GAAAATATGC CCATAAATAT CCTGAGAAAT GTGATTGTTA 5100TCAAGGAAAA CATGTACCTA GCATACCACC TCCCCCCCCA CCTGTACAAC CACAACCGGA 5160AGCACCAACA GTAACAGTAG ACGTTTGCAG CATAGTAAAA ACACTATTTA AAGACACAAA 5220CAATTTTTCC GACGCTTGTG GTCTAAAATA CGGCAAAACC GCACCATCCA GTTGGAAATG 5280TATACCAAGT GACACAAAAA GTGGTGCTGG TGCCACCACC GGCAAAAGTG GTAGTGATAG 5340TGGTAGTATT TGTATCCCAC CCAGGAGGCG ACGATTATAT GTGGGGAAAC TACAGGAGTG 5400GGCTACCGCG CTCCCACAAG GTGAGGGCGC CGCGCCGTCC CACTCACGCG CCGACGACTT 5460GCGCAATGCG TTCATCCAAT CTGCTGCAAT AGAGACTTTT TTCTTATGGG ATAGATATAA 5520AGAAGAGAAA AAACCACAGG GTGATGGGTC ACAACAAGCA CTATCACAAC TAACCAGTAC 5580ATACAGTGAT GACGAGGAGG ACCCCCCCGA CAAACTGTTA CAAAATGGTA AGATACCCCC 5640CGATTTTTTG AGATTAATGT TCTATACATT AGGAGATTAT AGGGATATTT TAGTACACGG 5700TGGTAACACA AGTGACAGTG GTAACACAAA TGGTAGTAAC AACAACAATA TTGTGCTTGA 5760AGCGAGTGGT AACAAGGAGG ACATGCAAAA AATACAAGAG AAAATAGAAC AAATTCTCCC 5820AAAAAATGGT GGCACACCTC TTGTCCCAAA ATCTAGTGCC CAAACACCTG ATAAATGGTG 5880GAATGAACAC GCCGAATCTA TCTGGAAAGG TATGATATGT GCATTGACAT ATACAGAAAA 5940GAACCCTGAC ACCAGTGCAA GAGGCGACGA AAACAAAATA GAAAAGGATG ATGAAGTGTA 6000CGAGAAATTT TTTGGCAGCA CAGCCGACAA ACATGGCACA GCCTCAACCC CAACCGGCAC 6060ATACAAAACC CAATACGACT ACGAAAAAGT CAAACTTGAG GATACAAGTG GTGCCAAAAC 6120CCCCTCAGCC TCTAGTGATA CACCCCTTCT CTCCGATTTC GTGTTACGCC CCCCCTACTT 6180CCGTTACCTT GAAGAATGGG GTCAAAATTT TTGTAAAAAA AGAAAGCATA AATTGGCACA 6240AATAAAACAT GAGTGTAAAG TAGAAGAAAA TGGTGGTGGT AGTCGTCGTG GTGGTATAAC 6300AAGACAATAT AGTGGGGATG GCGAAGCGTG TAATGAGATG CTTCCAAAAA ACGATGGAAC 6360TGTTCCGGAT TTAGAAAAGC CGAGTTGTGC CAAACCTTGT AGTTCTTATA GAAAATGGAT 6420AGAAAGCAAG GGAAAAGAGT TTGAGAAACA AGAAAAGGCA TATGAACAAC AAAAAGACAA 6480ATGTGTAAAT GGAAGTAATA AGCATGATAA TGGATTTTGT GAAACACTAA CAACGTCCTC 6540TAAAGCTAAA GACTTTTTAA AAACGTTAGG ACCATGTAAA CCTAATAATG TAGAGGGTAA 6600AACAATTTTT GATGATGATA AAACCTTTAA ACATACAAAA GATTGTGATC CATGTCTTAA 6660ATTTAGTGTT AATTGTAAAA AAGATGAATG TGATAATTCT AAAGGAACCG ATTGCCGAAA 6720TAAAAATAGT ATTGATGCAA CAGATATTGA AAATGGAGTG GATTCTACTG TACTAGAAAT 6780GCGTGTCAGT GCTGATAGTA AAAGTGGATT TAATGGTGAT GGTTTAGAGA ATGCTTGTAG 6840AGGTGCTGGT ATCTTTGAAG GTATTAGAAA AGATGAATGG AAATGTCGTA ATGTATGTGG 6900TTATGTTGTA TGTAAACCGG AAAACGTTAA TGGGGAAGCA AAGGGAAAAC ACATTATACA 6960AATTAGAGCA CTGGTTAAAC GTTGGGTAGA ATATTTTTTT GAAGATTATA ATAAAATAAA 7020ACATAAAATT TCACATCGCA TAAAAAATGG TGAAATATCT CCATGTATAA AAAATTGTGT 7080AGAAAAATGG GTAGATCAGA AAAGAAAAGA ATGGAAGGAA ATTACTGAAC GTTTCAAAGA 7140TCAATATAAA AATGACAATT CAGATGATGA CAATGTGAGA AGTTTTTTGG AGACCTTGAT 7200ACCTCAAATT ACTGATGCAA ACGCTAAAAA TAAGGTTATA AAATTAAGTA AGTTCGGTAA 7260TTCTTGTGGA TGTAGTGCCA GTGCGAACGA ACAAAACAAA AATGGTGAAT ACAAGGACGC 7320TATAGATTGT ATGCTTAAAA AGCTTAAAGA TAAAATTGGC GAGTGCGAAA AGAAACACCA 7380TCAAACTAGT GATACCGAGT GTTCCGACAC ACCACAACCG CAAACCCTTG AAGACGAAAC 7440TTTGGATGAT GATATAGAAA CAGAGGAGGC GAAGAAGAAC ATGATGCCGA AAATTTGTGA 7500AAATGTGTTA AAAACAGCAC AACAAGAGGA TGAAGGCGGT TGTGTCCCAG CAGAAAATAG 7560TGAAGAACCG GCAGCAACAG ATAGTGGTAA GGAAACCCCC GAACAAACCC CCGTTCTCAA 7620ACCCGAAGAA GAAGCAGTAC CGGAACCACC ACCTCCACCC CCACAGGAAA AAGCCCCGGC 7680ACCAATACCC CAACCACAAC CACCAACCCC CCCCACACAA CTCTTGGATA ATCCCCACGT 7740TCTAACCGCC CTGGTGACCT CCACCCTCGC CTGGAGCGTT GGCATCGGTT TTGCTACATT 7800CACTTATTTT TATCTAAAGG TAAATGGAAG TATATATATG GGGATGTGGA TGTATGTGGA 7860TGTATGTGAA TGTATGTGGA TGTATGTGGA TGTATGTGGA TGTGTTTTAT GGATATGTAT 7920TTGTGATTAT GTTTGGATAT ATATATATAT ATATATATGT TTATGTATAT GTGTTTTTGG 7980ATATATATAT GTGTATGTAT ATGATTTTCT GTATATGTAT TTGTGGGTTA AGGATATATA 8040TATATGGATG TACTTGTATG TGTTTTATAT ATATATTTTA TATATATGTA TTTATATTAA 8100AAAAGAAATA TAAAAACAAA TTTATTAAAA TGAAAAAAAG AAAAATGAAA TATAAAAAAA 8160AATTTATTAA AATAAAAAAA AAAAAAAAAA AAAAGGAGAA AAATTTTTTA AAAAATAATA 8220(2)SEQ ID NO12資料(I)序列特征(A)長(zhǎng)度2710個(gè)氨基酸(B)類型氨基酸(C)鏈型單鏈(D)拓?fù)渚€型(II)分子類型蛋白質(zhì)(III)假定的非(VI)原始來(lái)源(A)生物鐮狀瘧原蟲(chóng)(XI)序列描述SEQ ID NO12Asn Val Met Val Glu Leu Ala Lys Met Gly Pro Lys Glu Ala Ala Gly1 5 10 15Gly Asp Asp Ile Glu Asp Glu Ser Ala Lys His Met Phe Asp Arg Ile20 25 30Gly Lys Asp Val Tyr Asp Lys Val Lys Glu Glu Ala Lys Glu Arg Gly35 40 45Lys Gly Leu Gln Gly Arg Leu Ser Glu Ala Lys Phe Glu Lys Asn Glu50 55 60Ser Asp Pro Gln Thr Pro Glu Asp Pro Cys Asp Leu Asp His Lys Tyr65 70 75 80His Thr Asn Val Thr Thr Asn Val Ile Asn Pro Cys Ala Asp Arg Ser85 90 95Asp Val Arg Phe Ser Asp Glu Tyr Gly Gly Gln Cys Thr His Asn Arg100 105 110Ile Lys Asp Ser Gln Gln Gly Asp Asn Lys Gly Ala Cys Ala Pro Tyr115 120 125Arg Arg Leu His Val Cys Asp Gln Asn Leu Glu Gln Ile Glu Pro Ile130 135 140Lys Ile Thr Asn Thr His Asn Leu Leu Val Asp Val Cys Met Ala Ala145 150 155 160Lys Phe Glu Gly Gln Ser Ile Thr Gln Asp Tyr Pro Lys Tyr Gln Ala165 170 175Thr Tyr Gly Asp Ser Pro Ser Gln Ile Cys Thr Met Leu Ala Arg Ser180 185 190Phe Ala Asp Ile Gly Asp Ile Val Arg Gly Arg Asp Leu Tyr Leu Gly195 200 205Asn Pro Gln Glu Ile Lys Gln Arg Gln Gln Leu Glu Asn Asn Leu Lys210 215 220Thr Ile Phe Gly Lys Ile Tyr Glu Lys Leu Asn Gly Ala Glu Ala Arg225 230 235 240Tyr Gly Asn Asp Pro Glu Phe Phe Lys Leu Arg Glu Asp Trp Trp Thr245 250 255Ala Asn Arg Glu Thr Val Trp Lys Ala Ile Thr Cys Asn Ala Trp Gly260 265 270Asn Thr Tyr Phe His Ala Thr Cys Asn Arg Gly Glu Arg Thr Lys Gly275 280 285Tyr Cys Arg Cys Asn Asp Asp Gln Val Pro Thr Tyr Phe Asp Tyr Val290 295 300Pro Gln Tyr Leu Arg Trp Phe Glu Glu Trp Ala Glu Asp Phe Cys Arg305 310 315 320Lys Lys Asn Lys Lys Ile Lys Asp Val Lys Arg Asn Cys Arg Gly Lys325 330 335Asp Lys Glu Asp Lys Asp Arg Tyr Cys Ser Arg Asn Gly Tyr Asp Cys340 345 350Glu Lys Thr Lys Arg Ala Ile Gly Lys Leu Arg Tyr Gly Lys Gln Cys355 360 365Ile Ser Cys Leu Tyr Ala Cys Asn Pro Tyr Val Asp Trp Ile Asn Asn370 375 380Gln Lys Glu Gln Phe Asp Lys Gln Lys Lys Lys Tyr Asp Glu Glu Ile385 390 395 400Lys Lys Tyr Glu Asn Gly Ala Ser Gly Gly Ser Arg Gln Lys Arg Asp405 410 415Ala Gly Gly Thr Thr Thr Thr Asn Tyr Asp Gly Tyr Glu Lys Lys Phe420 425 430Tyr Asp Glu Leu Asn Lys Ser Glu Tyr Arg Thr Val Asp Lys Phe Leu435 440 445Glu Lys Leu Ser Asn Glu Glu Ile Cys Thr Lys Val Lys Asp Glu Glu450 455 460Gly Gly Thr Ile Asp Phe Lys Asn Val Asn Ser Asp Ser Thr Ser Gly465 470 475 480Ala Ser Gly Thr Asn Val Glu Ser Gln Gly Thr Phe Tyr Arg Ser Lys485 490 495Tyr Cys Gln Pro Cys Pro Tyr Cys Gly Val Lys Lys Val Asn Asn Gly500 505 510Gly Ser Ser Asn Glu Trp Glu Glu Lys Asn Asn Gly Lys Cys Lys Ser515 520 525Gly Lys Leu Tyr Glu Pro Lys Pro Asp Lys Glu Gly Thr Thr Ile Thr530 535 540Ile Leu Lys Ser Gly Lys Gly His Asp Asp Ile Glu Glu Lys Leu Asn545 550 555 560Lys Phe Cys Asp Glu Lys Asn Gly Asp Thr Ile Asn Ser Gly Gly Ser565 570 575Gly Thr Gly Gly Ser Gly Gly Gly Asn Ser Gly Arg Gln Glu Leu Tyr580 585 590Glu Glu Trp Lys Cys Tyr Lys Gly Glu Asp Val Val Lys Val Gly His595 600 605Asp Glu Asp Asp Glu Glu Asp Tyr Glu Asn Val Lys Asn Ala Gly Gly610 615 620Leu Cys Ile Leu Lys Asn Gln Lys Lys Asn Lys Glu Glu Gly Gly Asn625 630 635 640Thr Ser Glu Lys Glu Pro Asp Glu Ile Gln Lys Thr Phe Asn Pro Phe645 650 655Phe Tyr Tyr Trp Val Ala His Met Leu Lys Asp Ser Ile His Trp Lys660 665 670Lys Lys Leu Gln Arg Cys Leu Gln Asn Gly Asn Arg Ile Lys Cys Gly675 680 685Asn Asn Lys Cys Asn Asn Asp Cys Glu Cys Phe Lys Arg Trp Ile Thr690 695 700Gln Lys Lys Asp Glu Trp Gly Lys Ile Val Gln His Phe Lys Thr Gln705 710 715 720Asn Ile Lys Gly Arg Gly Gly Ser Asp Asn Thr Ala Glu Leu Ile Pro725 730 735Phe Asp His Asp Tyr Val Leu Gln Tyr Asn Leu Gln Glu Glu Phe Leu
740 745 750Lys Gly Asp Ser Glu Asp Ala Ser Glu Glu Lys Ser Glu Asn Ser Leu755 760 765Asp Ala Glu Glu Ala Glu Glu Leu Lys His Leu Arg Glu Ile Ile Glu770 775 780Ser Glu Asp Asn Asn Gln Glu Ala Ser Val Gly Gly Gly Val Thr Glu785 790 795 800Gln Lys Asn Ile Met Asp Lys Leu Leu Asn Tyr Glu Lys Asp Glu Ala805 810 815Asp Leu Cys Leu Glu Ile His Glu Asp Glu Glu Glu Glu Lys Glu Lys820 825 830Gly Asp Gly Asn Glu Cys Ile Glu Glu Gly Glu Asn Phe Arg Tyr Asn835 840 845Pro Cys Ser Gly Glu Ser Gly Asn Lys Arg Tyr Pro Val Leu Ala Asn850 855 860Lys Val Ala Tyr Gln Met His His Lys Ala Lys Thr Gln Leu Ala Ser865 870 875 880Arg Ala Gly Arg Ser Ala Leu Arg Gly Asp Ile Ser Leu Ala Gln Phe885 890 895Lys Asn Gly Arg Asn Gly Ser Thr Leu Lys Gly Gln Ile Cys Lys Ile900 905 910Asn Glu Asn Tyr Ser Asn Asp Ser Arg Gly Asn Ser Gly Gly Pro Cys915 920 925Thr Gly Lys Asp Gly Asp His Gly Gly Val Arg Met Arg Ile Gly Thr930 935 940Glu Trp Ser Asn Ile Glu Gly Lys Lys Gln Thr Ser Tyr Lys Asn Val945 950 955 960Phe Leu Pro Pro Arg Arg Glu His Met Cys Thr Ser Asn Leu Glu Asn965 970 975Leu Asp Val Gly Ser Val Thr Lys Asn Asp Lys Ala Ser His Ser Leu980 985 990Leu Gly Asp Val Gln Leu Ala Ala Lys Thr Asp Ala Ala Glu Ile Ile995 10001005Lys Arg Tyr Lys Asp Gln Asn Asn Ile Gln Leu Thr Asp Pro Ile Gln101010151020Gln Lys Asp Gln Glu Ala Met Cys Arg Ala Val Arg Tyr Ser Phe Ala1025103010351040Asp Leu Gly Asp Ile Ile Arg Gly Arg Asp Met Trp Asp Glu Asp Lys104510501055Ser Ser Thr Asp Met Glu Thr Arg Leu Ile Thr Val Phe Lys Asn Ile106010651070Lys Glu Lys His Asp Gly Ile Lys Asp Asn Pro Lys Tyr Thr Gly Asp107510801085Glu Ser Lys Lys Pro Ala Tyr Lys Lys Leu Arg Ala Asp Trp Trp Glu109010951100Ala Asn Arg His Gln Val Trp Arg Ala Met Lys Cys Ala Thr Lys Gly1105111011151120Ile Ile Cys Pro Gly Met Pro Val Asp Asp Tyr Ile Pro Gln Arg Leu112511301135Arg Trp Met Thr Glu Trp Ala Glu Trp Tyr Cys Lys Ala Gln Ser Gln114011451150Glu Tyr Asp Lys Leu Lys Lys Ile Cys Ala Asp Cys Met Sar Lys Gly11551160 1165Asp Gly Lys Cys Thr Gln Gly Asp Val Asp Cys Gly Lys Cys Lys Ala117011751180Ala Cys Asp Lys Tyr Lys Glu Glu Ile Glu Lys Trp Asn Glu Gln Trp1185119011951200Arg Lys Ile Ser Asp Lys Tyr Asn Leu Leu Tyr Leu Gln Ala Lys Thr120512101215Thr Ser Thr Asn Pro Gly Arg Thr Val Leu Gly Asp Asp Asp Pro Asp122012251230Tyr Gln Gln Met Val Asp Phe Leu Thr Pro Ile His Lys Ala Ser Ile123512401245Ala Ala Arg Val Leu Val Lys Arg Ala Ala Gly Ser Pro Thr Glu Ile125012551260Ala Ala Ala Ala Pro Ile Thr Pro Tyr Ser Thr Ala Ala Gly Tyr Ile1265127012751280His Gln Glu Ile Gly Tyr Gly Gly Cys Gln Glu Gln Thr Gln Phe Cys128512901295Glu Lys Lys His Gly Ala Thr Ser Thr Ser Thr Thr Lys Glu Asn Lys130013051310Glu Tyr Thr Phe Lys Gln Pro Pro Pro Glu Tyr Ala Thr Ala Cys Asp131513201325Cys Ile Asn Arg Ser Gln Thr Glu Glu Pro Lys Lys Lys Glu Glu Asn
133013351340Val Glu Ser Ala Cys Lys Ile Val Glu Lys Ile Leu Glu Gly Lys Asn1345135013551360Gly Arg Thr Thr Val Gly Glu Cys Asn Pro Lys Glu Ser Tyr Pro Asp136513701375Trp Asp Cys Lys Asn Asn Ile Asp Ile Ser His Asp Gly Ala Cys Met138013851390Pro Pro Arg Arg Gln Lys Leu Cys Leu Tyr Tyr Ile Ala His Glu Ser139514001405Gln Thr Glu Asn Ile Lys Thr Asp Asp Asn Leu Lys Asp Ala Phe Ile141014151420Lys Thr Ala Ala Ala Glu Thr Phe Leu Ser Trp Gln Tyr Tyr Lys Ser1425143014351440Lys Asn Asp Ser Glu Ala Lys Ile Leu Asp Arg Gly Leu Ile Pro Ser144514501455Gln Phe Leu Arg Ser Met Met Tyr Thr Phe Gly Asp Tyr Arg Asp Ile146014651470Cys Leu Asn Thr Asp Ile Ser Lys Lys Gln Asn Asp Val Ala Lys Ala147514801485Lys Asp Lys Ile Gly Lys Phe Phe Ser Lys Asp Gly Ser Lys Ser Pro149014951500Ser Gly Leu Ser Arg Gln Glu TrP Trp Lys Thr Asn Gly Pro Glu Ile1505151015151520Trp Lys Gly Met Leu Cys Ala Leu Thr Lys Tyr Val Thr Asp Thr Asp152515301535Asn Lys Arg Lys Ile Lys Asn Asp Tyr Ser Tyr Asp Lys Val Asn Gln154015451550Ser Gln Asn Gly Asn Pro Ser Leu Glu Glu Phe Ala Ala Lys Pro Gln155515601565Phe Leu Arg Trp Met Ile Glu Trp Gly Glu Glu Phe Cys Ala Glu Arg157015751580Gln Lys Lys Glu Asn Ile Ile Lys Asp Ala Cys Asn Glu Ile Asn Ser1585159015951600Thr Gln Gln Cys Asn Asp Ala Lys His Arg Cys Asn Gln Ala Cys Arg160516101615Ala Tyr Gln Glu Tyr Val Glu Asn Lys Lys Lys Glu Phe Ser Gly Gln162016251630Thr Asn Asn Phe Val Leu Lys Ala Asn Val Gln Pro Gln Asp Pro Glu163516401645Tyr Lys Gly Tyr Glu Tyr Lys Asp Gly Val Gln Pro Ile Gln Gly Asn165016551660Glu Tyr Leu Leu Gln Lys Cys Asp Asn Asn Lys Cys Ser Cys Met Asp1665167016751680Gly Asn Val Leu Ser Val Ser Pro Lys Glu Lys Pro Phe Gly Lys Tyr168516901695Ala His Lys Tyr Pro Glu Lys Cys Asp Cys Tyr Gln Gly Lys His Val170017051710Pro Ser Ile Pro Pro Pro Pro Pro Pro Val Gln Pro Gln Pro Glu Ala171517201725Pro Thr Val Thr Val Asp Val Cys Ser Ile Val Lys Thr Leu Phe Lys173017351740Asp Thr Asn Asn Phe Ser Asp Ala Cys Gly Leu Lys Tyr Gly Lys Thr1745175017551760Ala Pro Ser Ser Trp Lys Cys Ile Pro Ser Asp Thr Lys Ser Gly Ala176517701775Gly Ala Thr Thr Gly Lys Ser Gly Ser Asp Ser Gly Ser Ile Cys Ile178017851790Pro Pro Arg Arg Arg Arg Leu Tyr Val Gly Lys Leu Gln Glu Trp Ala179518001805Thr Ala Leu Pro Gln Gly Glu Gly Ala Ala Pro Ser His Ser Arg Ala181018151820Asp Asp Leu Arg Asn Ala Phe Ile Gln Ser Ala Ala Ile Glu Thr Phe1825183018351840Phe Leu Trp Asp Arg Tyr Lys Glu Glu Lys Lys Pro Gln Gly Asp Gly184518501855Ser Gln Gln Ala Leu Ser Gln Leu Thr Ser Thr Tyr Ser Asp Asp Glu186018651870Glu Asp Pro Pro Asp Lys Leu Leu Gln Asn Gly Lys Ile Pro Pro Asp187518801885Phe Leu Arg Leu Met Phe Tyr Thr Leu Gly Asp Tyr Arg Asp Ile Leu189018951900Val His Gly Gly Asn Thr Ser Asp Ser Gly Asn Thr Asn Gly Ser Asn1905191019151920Asn Asn Asn Ile Val Leu Glu Ala Ser Gly Asn Lys Glu Asp Met Gln
192519301935Lys Ile Gln Glu Lys Ile Glu Gln Ile Leu Pro Lys Asn Gly Gly Thr194019451950Pro Leu Val Pro Lys Ser Ser Ala Gln Thr Pro Asp Lys Trp Trp Asn195519601965Glu His Ala Glu Ser Ile Trp Lys Gly Met Ile Cys Ala Leu Thr Tyr197019751980Thr Glu Lys Asn Pro Asp Thr Ser Ala Arg Gly Asp Glu Asn Lys Ile1985199019952000Glu Lys Asp Asp Glu Val Tyr Glu Lys Phe Phe Gly Ser Thr Ala Asp200520102015Lys His Gly Thr Ala Ser Thr Pro Thr Gly Thr Tyr Lys Thr Gln Tyr202020252030Asp Tyr Glu Lys Val Lys Leu Glu Asp Thr Ser Gly Ala Lys Thr Pro203520402045Ser Ala Ser Ser Asp Thr Pro Leu Leu Ser Asp Phe Val Leu Arg Pro205020552060Pro Tyr Phe Arg Tyr Leu Glu Glu Trp Gly Gln Asn Phe Cys Lys Lys2065207020752080Arg Lys His Lys Leu Ala Gln Ile Lys His Glu Cys Lys Val Glu Glu208520902095Asn Gly Gly Gly Ser Arg Arg Gly Gly Ile Thr Arg Gln Tyr Ser Gly210021052110Asp Gly Glu Ala Cys Asn Glu Met Leu Pro Lys Asn Asp Gly Thr Val211521202125Pro Asp Leu Glu Lys Pro Ser Cys Ala Lys Pro Cys Ser Ser Tyr Arg213021352140Lys Trp Ile Glu Ser Lys Gly Lys Glu Phe Glu Lys Gln Glu Lys Ala2145215021552160Tyr Glu Gln Gln Lys Asp Lys Cys Val Asn Gly Ser Asn Lys His Asp216521702175Asn Gly Phe Cys Glu Thr Leu Thr Thr Ser Ser Lys Ala Lys Asp Phe218021852190Leu Lys Thr Leu Gly Pro Cys Lys Pro Asn Asn Val Glu Gly Lys Thr219522002205Ile Phe Asp Asp Asp Lys Thr Phe Lys His Thr Lys Asp Cys Asp Pro221022152220Cys Leu Lys Phe Ser Val Asn Cys Lys Lys Asp Glu Cys Asp Asn Ser2225223022352240Lys Gly Thr Asp Cys Arg Asn Lys Asn Ser Ile Asp Ala Thr Asp Ile224522502255Glu Asn Gly Val Asp Ser Thr Val Leu Glu Met Arg Val Ser Ala Asp226022652270Ser Lys Ser Gly Phe Asn Gly Asp Gly Leu Glu Asn Ala Cys Arg Gly227522802285Ala Gly Ile Phe Glu Gly Ile Arg Lys Asp Glu Trp Lys Cys Arg Asn229022952300Val Cys Gly Tyr Val Val Cys Lys Pro Glu Asn Val Asn Gly Glu Ala2305231023152320Lys Gly Lys His Ile Ile Gln Ile Arg Ala Leu Val Lys Arg Trp Val232523302335Glu Tyr Phe Phe Glu Asp Tyr Asn Lys Ile Lys His Lys Ile Ser His234023452350Arg Ile Lys Asn Gly Glu Ile Ser Pro Cys Ile Lys Asn Cys Val Glu235523602365Lys Trp Val Asp Gln Lys Arg Lys Glu Trp Lys Glu Ile Thr Glu Arg237023752380Phe Lys Asp Gln Tyr Lys Asn Asp Asn Ser Asp Asp Asp Asn Val Arg2385239023952400Ser Phe Leu Glu Thr Leu Ile Pro Gln Ile Thr Asp Ala Asn Ala Lys240524102415Asn Lys Val Ile Lys Leu Ser Lys Phe Gly Asn Ser Cys Gly Cys Ser242024252430Ala Ser Ala Asn Glu Gln Asn Lys Asn Gly Glu Tyr Lys Asp Ala Ile243524402445Asp Cys Met Leu Lys Lys Leu Lys Asp Lys Ile Gly Glu Cys Glu Lys245024552460Lys His His Gln Thr Ser Asp Thr Glu Cys Ser Asp Thr Pro Gln Pro2465247024752480Gln Thr Leu Glu Asp Glu Thr Leu Asp Asp Asp Ile Glu Thr Glu Glu248524902495Ala Lys Lys Asn Met Met Pro Lys Ile Cys Glu Asn Val Leu Lys Thr250025052510Ala Gln Gln Glu Asp Glu Gly Gly Cys Val Pro Ala Glu Asn Ser Glu
251525202525Glu Pro Ala Ala Thr Asp Ser Gly Lys Glu Thr Pro Glu Gln Thr Pro253025352540Val Leu Lys Pro Glu Glu Glu Ala Val Pro Glu Pro Pro Pro Pro Pro2545255025552560Pro Gln Glu Lys Ala Pro Ala Pro Ile Pro Gln Pro Gln Pro Pro Thr256525702575Pro Pro Thr Gln Leu Leu Asp Asn Pro His Val Leu Thr Ala Leu Val258025852590Thr Ser Thr Leu Ala Trp Ser Val Gly Ile Gly Phe Ala Thr Phe Thr259526002605Tyr Phe Tyr Leu Lys Val Asn Gly Ser Ile Tyr Met Gly Met Trp Met261026152620Tyr Val Asp Val Cys Glu Cys Met Trp Met Tyr Val Asp Val Cys Gly2625263026352640Cys Val Leu Trp Ile Cys Ile Cys Asp Tyr Val Trp Ile Tyr Ile Tyr2645 26502655Ile Tyr Ile Cys Leu Cys Ile Cys Val Phe Gly Tyr Ile Tyr Val Tyr266026652670Val Tyr Asp Phe Leu Tyr Met Tyr Leu Trp Val Lys Asp Ile Tyr Ile267526802685Trp Met Tyr Leu Tyr Val Phe Tyr Ile Tyr Ile Leu Tyr Ile Cys Ile269026952700Tyr Ile Lys Lys Glu Ile27052710(2)SEQ ID NO13資料(I)序列特征(A)長(zhǎng)度30對(duì)堿基(B)類型核酸(C)鏈型單鏈(D)拓?fù)渚€性(II)分子類型DNA(基因組)(III)假定的非(IV)反義無(wú)(V)片段類型(VI)原始來(lái)源(XI)序列描述SEQ ID NO13
ATCGATCAGC TGGGAAGAAA TACTTCATCT(2)SEQ ID NO14資料(I)序列特征(A)長(zhǎng)度30對(duì)堿基(B)類型核酸(C)鏈型單鏈(D)拓?fù)渚€性(II)分子類型DNA(基因組)(III)假定的非(IV)反義無(wú)(V)片段類型(VI)原始來(lái)源(XI)序列描述SEQ ID NO14ATCGATGGGC CCCGAAGTTT GTTCATTATT(2)SEQ ID NO15資料(I)序列特征(A)長(zhǎng)度30對(duì)堿基(B)類型核酸(C)鏈型單鏈(D)拓?fù)渚€性(II)分子類型DNA(基因組)(III)假定的非(IV)反義無(wú)(V)片段類型(VI)原始來(lái)源(XI)序列描述SEQ ID NO15TCTCGTCAGC TGACGATCTC TAGTGCTATT(2)SEQ ID NO16資料(I)序列特征(A)長(zhǎng)度30對(duì)堿基(B)類型核酸(C)鏈型單鏈(D)拓?fù)渚€性(II)分子類型DNA(基因組)(III)假定的非(IV)反義無(wú)(V)片段類型(VI)原始來(lái)源(XI)序列描述SEQ ID NO16ACGAGTGGGC CCTGTCACAA CTTCCTGAGT
權(quán)利要求
1.一種源自鐮狀瘧原蟲(chóng)唾液酸結(jié)合蛋白(SABP)的分離的結(jié)合區(qū)多肽，其中所述的結(jié)合區(qū)多肽至少含有圖1中定義的所述的SABP中開(kāi)始于SEQ ID NO4中的大約第158位殘基的半胱氨酸富集區(qū)的一部分，或者其中含有用GAP，BESTFIT，F(xiàn)ASTA或TFASTA在使用默認(rèn)參數(shù)下確定其中至少存在SEQ ID NO4所含半胱氨酸殘基的70％的序列的一部分，所述的結(jié)合區(qū)多肽含有的氨基酸在約50個(gè)到約616個(gè)之間。
2.權(quán)利要求1的結(jié)合區(qū)多肽，其中SABP結(jié)合區(qū)多肽含大約200到大約600之間的氨基酸殘基。
3.權(quán)利要求1的結(jié)合區(qū)多肽，其中SABP結(jié)合區(qū)多肽具有大約616的氨基酸殘基。
4.權(quán)利要求1的結(jié)合區(qū)多肽，其中SABP結(jié)合區(qū)多肽含有圖1所示的開(kāi)始于SEQ ID NO4的大約第158位殘基的SABP F1序列。
5.權(quán)利要求1的結(jié)合區(qū)多肽，其中SABP結(jié)合區(qū)多肽含有圖1所示的開(kāi)始于SEQ ID NO4的大約第462位殘基的SABP F2序列。
6.權(quán)利要求1的結(jié)合區(qū)多肽，其中SABP結(jié)合區(qū)多肽是重組產(chǎn)生的。
7.一種藥物用組合物，包含藥學(xué)上可接受的載體和權(quán)利要求1的結(jié)合區(qū)多肽，其用途是在人體或動(dòng)物體內(nèi)誘導(dǎo)抗瘧原蟲(chóng)屬裂殖子的保護(hù)性免疫反應(yīng)。
8.權(quán)利要求7的藥物用組合物在人體或動(dòng)物體內(nèi)誘導(dǎo)抗瘧原蟲(chóng)屬裂殖子的保護(hù)性免疫反應(yīng)上的用途。
9.一種包含分離的編碼權(quán)利要求1的SABP結(jié)合區(qū)多肽的多核苷酸。
10.一種含有權(quán)利要求9的多核苷酸的重組細(xì)胞。
全文摘要
本發(fā)明提供了在治療和阻止由鐮狀瘧原蟲(chóng)或間日瘧原蟲(chóng)引起的瘧疾中有用的分離的多肽。特別是來(lái)自EBL家族中的蛋白質(zhì)的結(jié)合區(qū)以及鐮狀瘧原蟲(chóng)裂殖子上唾液酸結(jié)合蛋白(SABP)的多肽。也可以是來(lái)自間日瘧原蟲(chóng)裂殖子上Duffy抗原結(jié)合蛋白(DABP)的多肽。
文檔編號(hào)C12N15/09GK1414018SQ02131950
公開(kāi)日2003年4月30日 申請(qǐng)日期2002年9月5日 優(yōu)先權(quán)日1993年9月10日
發(fā)明者吉里·辛, 切坦·切特尼斯, 路易斯·H·米勒, 戴維·S·彼特森, 星?！ぬK, 托馬斯·E·威廉姆斯 申請(qǐng)人:美國(guó)國(guó)有健康與人類服務(wù)部