專(zhuān)利名稱(chēng):Hiv-1亞型分離株調(diào)節(jié)/附加基因及其修飾物和衍生物的制作方法
背景技術(shù):
本發(fā)明涉及篩選HIV-1 C亞型(分化體)分離株調(diào)節(jié)/附加基因的方法,和被選的HIV-1 C亞型分離株調(diào)節(jié)/附加基因及其修飾物和衍生物用于預(yù)防性和治療性疫苗中以產(chǎn)生蛋白質(zhì)和多肽而達(dá)到預(yù)防HIV感染或疾病的目的。
獲得性免疫缺陷綜合癥(AIDS)是由人類(lèi)免疫缺陷病毒(HIV)引起的。全世界超過(guò)三千四百萬(wàn)的人口攜帶HIV/AIDS,且95%以上的感染者生活在發(fā)展中國(guó)家(UNAIDS,1999)。據(jù)估計(jì),二千四百五十萬(wàn)感染人口居住在撒哈拉以南的非洲國(guó)家,南非現(xiàn)在已經(jīng)成為世界上HIV-1流行病蔓延最快的地區(qū)之一。到2000年末,南非的政府?huà)D產(chǎn)診所里超過(guò)24%的孕婦呈HIV陽(yáng)性(衛(wèi)生部,2001)。從長(zhǎng)遠(yuǎn)角度看,預(yù)防性疫苗被認(rèn)為是控制這種流行病的唯一可行的手段。
HIV表現(xiàn)出顯著的遺傳多樣性,這給疫苗的研制造成很大困難。變異的分子基礎(chǔ)在于病毒逆轉(zhuǎn)錄酶,它不僅會(huì)在每一輪復(fù)制中引入錯(cuò)誤,還會(huì)促使病毒RNA之間發(fā)生重組。根據(jù)對(duì)序列的系統(tǒng)發(fā)育學(xué)分析,將HIV劃分為下列幾種類(lèi)型M(主要類(lèi)型)、O(異常型)和N(非M、非O型),其中M類(lèi)型包括A-H和K亞型。近來(lái),重組體病毒已被更為頻繁地鑒別,且許多已經(jīng)嚴(yán)重傳播和形成蔓延(循環(huán)重組體形式或CRF),例如西非的A/G亞型重組體,泰國(guó)的CRF A/E重組體(Robertson等人,2000)。
亞型C在南非地區(qū),包括博茨瓦納、津巴布韋、贊比亞、馬拉維、莫桑比克和南非,占有主導(dǎo)地位。而且,在坦桑尼亞的南部地區(qū)檢測(cè)到感染C亞型的人數(shù)在增加。這種亞型還主要分布于埃塞俄比亞和印度,并且正在中國(guó)變得日益重要。
研制疫苗可能遇到的另一個(gè)障礙是,HIV病毒的生物學(xué)特性隨病情而發(fā)生變化。HIV需要兩個(gè)受體來(lái)感染細(xì)胞,CD4和共受體,其中CCR5和CXCR4是HIV-1株使用的主要共受體。最常見(jiàn)的傳播表型是非合胞體誘導(dǎo)株(NSI),巨噬細(xì)胞嗜性病毒,該病毒利用共受體CCR5作為入口(R5病毒)。粘膜中的朗格漢斯細(xì)胞選擇入口處的R5變體并將它們轉(zhuǎn)移到淋巴結(jié),在那里R5變體被復(fù)制和擴(kuò)增。當(dāng)感染加重時(shí),病毒進(jìn)化,在T細(xì)胞系中的復(fù)制和生長(zhǎng)能力增強(qiáng)。這些合胞體誘導(dǎo)株(SI)T-嗜性病毒聯(lián)合使用CXCR4和CCR5,或優(yōu)選使用CCR5,在有些情況下也使用其他次要的共受體(Conner等人,1997;Richman和Bozzette,1994)。但是,HIV-1 C亞型病毒似乎并不常見(jiàn),表現(xiàn)在它們不容易發(fā)生這種表型轉(zhuǎn)換,因?yàn)镽5病毒在患有嚴(yán)重的AIDS病人體內(nèi)同樣占主導(dǎo)地位(Bjorndal等人,1999;Peeters等人,1999;Tscherning等人,1998;Scarlatti等人,1997)。
HIV疫苗的目的是誘導(dǎo)CD8+細(xì)胞毒T淋巴細(xì)胞(CTL)免疫反應(yīng)和中和抗體反應(yīng)。許多目前使用的疫苗手段主要是針對(duì)誘導(dǎo)CTL反應(yīng)。人們認(rèn)為CTL反應(yīng)可能更加重要,因?yàn)樗c感染后病毒復(fù)制的最初控制和發(fā)病期復(fù)制的控制有關(guān),且與病情的發(fā)展呈負(fù)相關(guān)(Koup等人,1994;Ogg等人,1999;Schmitz等人,1999)。CTL保護(hù)人體不受感染的重要性表現(xiàn)在,CTL存在于高度暴露的血清陰性個(gè)體中,例如肯尼亞的某些性服務(wù)者(Rowland-Jones等人,1998)。
對(duì)基因多樣性的了解與以誘導(dǎo)引起細(xì)胞毒素T淋巴細(xì)胞(CTL)反應(yīng)為目的的疫苗設(shè)計(jì)密切相關(guān)。病毒之間存在許多共同的CTL表位(HIV分子免疫數(shù)據(jù)庫(kù),1998)。而且,研究表明,存在可交叉反應(yīng)的CTL反應(yīng)以B亞型為基礎(chǔ)的疫苗接種的個(gè)體能夠裂解用多種類(lèi)型的分離株感染的自體目標(biāo)(Ferrari等人,1997);來(lái)自非B亞型感染個(gè)體的CTL能夠裂解B亞型誘導(dǎo)的目標(biāo)(Betts等人,1997;Durali等人,1998)。對(duì)比HIV-1序列數(shù)據(jù)庫(kù)中的CTL表位發(fā)現(xiàn),同一個(gè)亞型中的細(xì)胞毒素T表位比各亞型之間的細(xì)胞毒素T表位更保守,且如果攻擊病毒是與疫苗株相同的亞型,發(fā)生CTL反應(yīng)的機(jī)會(huì)將會(huì)更大。
HIV的調(diào)節(jié)基因?qū)τ诋a(chǎn)生免疫反應(yīng)非常重要。Tat、Rev和Nef在感染循環(huán)的初期被表達(dá),從而在感染初期為細(xì)胞毒素T淋巴細(xì)胞(CTL)提供靶子,這可能導(dǎo)致在病毒傳播之前感染病毒的細(xì)胞被破壞(Klotman等人,1991;Addo等人,2001)。而且,具有說(shuō)服力的數(shù)據(jù)表明,Tat蛋白在感染HIV的無(wú)癥狀的人體中產(chǎn)生有效的免疫反應(yīng)(Calarota等人,1999;Calarota等人,2001)。近來(lái)報(bào)道,Tat蛋白是第一種從感染的短尾猿的CTL中逃逸的蛋白之一(Allen等人,2000)。這表明tat有免疫壓力,且對(duì)Tat蛋白有早期反應(yīng)。
用于疫苗設(shè)計(jì)的病毒株必須通過(guò)遺傳型分析證實(shí)是典型的循環(huán)型菌株,而不是罕見(jiàn)或異常株。而且,重要的是,疫苗株還具有近來(lái)傳播病毒的表型,所述病毒是NSI,其使用的共受體是CCR5。
定義在下面的說(shuō)明書(shū)中,特定的術(shù)語(yǔ)具有以下含義“野生型”是指自然產(chǎn)生的病毒分離株的HIV密碼子變體;“密碼子優(yōu)化”是指利用人類(lèi)密碼子變體取代HIV密碼子變體而重新合成基因;“截取”是指將前十個(gè)氨基酸從Nef蛋白中截去,從而使其功能滅活但仍保持免疫原性;“改排”是指Tat蛋白的重排,從而使其功能滅活但仍保持免疫原性。
發(fā)明概述根據(jù)本發(fā)明的第一方面,提供一種分子,該分子具有(I)如圖1所示的核苷酸序列(SEQ I.D.No.1);(II)圖1所示的核苷酸序列(SEQ I.D.No.1)所相應(yīng)的RNA序列;(III)一種與圖1所示的核苷酸序列(SEQ I.D.No.1)相比具有至少97%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,其顯示基本相似的免疫原性;(IV)一種與SEQ I.D.No.1給出的核苷酸序列同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其為(I)-(IV)中任一序列的修飾物或衍生物。
所述修飾序列優(yōu)選如圖9和10任一所示的序列,圖中所示的序列是Du422和Du151的共有序列(SEQ I.D.Nos.9和10)。
根據(jù)本發(fā)明的另一方面,提供一種分子,該分子具有(I)如圖3所示的核苷酸序列(SEQ I.D.No.3);(II)圖3所示的核苷酸序列(SEQ I.D.No.3)所相應(yīng)的RNA序列;(III)一種與圖3所示的核苷酸序列(SEQ I.D.No.3)相比具有至少97%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,其顯示基本相似的免疫原性;(IV)一種與圖3所示的核苷酸序列(SEQ I.D.No.3)同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其為(I)-(IV)中任一序列的修飾物或衍生物。
所述修飾序列優(yōu)選如圖9和10任一所示的序列,圖中所示的序列是Du422和Du151的共有序列(SEQ I.D.Nos.9和10)。
根據(jù)本發(fā)明的第三方面,提供一種分子,該分子具有(I)如圖5所示的核苷酸序列(SEQ I.D.No.5);(II)圖5所示的核苷酸序列(SEQ I.D.No.5)所相應(yīng)的RNA序列;(III)一種與圖5所示的核苷酸序列(SEQ I.D.No.5)相比具有至少98%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,其顯示基本相似的免疫原性;(IV)一種與圖5所示的核苷酸序列(SEQ I.D.No.5)同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其為(I)-(IV)中任一序列的修飾物或衍生物。
所述修飾序列優(yōu)選如圖12和13(SEQ I.D.Nos.11和13)任一所示的序列。
根據(jù)本發(fā)明的第四方面,提供一種分子,該分子具有(I)如圖7所示的核苷酸序列(SEQ I.D.No.7);(II)圖7所示的核苷酸序列(SEQ I.D.No.7)所相應(yīng)的RNA序列;(III)一種與圖7所示的核苷酸序列(SEQ I.D.No.7)相比具有至少96%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,其顯示基本相似的免疫原性;
(IV)一種與圖7所示的核苷酸序列(SEQ I.D.No.7)同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其為(I)-(IV)中任一序列的修飾物或衍生物。
所述修飾序列優(yōu)選具有與圖12和13(SEQ I.D.Nos.12和13)任一所示的分離株Du151的nef基因相似或相同的修飾。
根據(jù)本發(fā)明的第五方面,提供一種分子,該分子具有(I)如圖15所示的核苷酸序列(SEQ I.D.No.15);(II)圖15所示的核苷酸序列(SEQ I.D.No.15)所相應(yīng)的RNA序列;(III)一種與圖15所示的核苷酸序列(SEQ I.D.No.15)相比具有至少90%或者更高的DNA相似性的序列、或該序列所相應(yīng)的RNA序列,其顯示基本相似的免疫原性;(IV)一種與圖15所示的核苷酸序列(SEQ I.D.No.15)同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其為(I)-(IV)中任一序列的修飾物或衍生物。
根據(jù)本發(fā)明的第六方面,提供一種分子,該分子具有(I)如圖17所示的核苷酸序列(SEQ I.D.No.17);(II)圖17所示的核苷酸序列(SEQ I.D.No.17)所相應(yīng)的RNA序列;(III)一種與圖17所示的核苷酸序列(SEQ I.D.No.17)相比具有至少90%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,其顯示基本相似的免疫原性;(IV)一個(gè)與圖17所示的核苷酸序列(SEQ I.D.No.17)或與該核苷酸序列相應(yīng)的RNA序列同源的序列;或者(V)一種序列,其為(I)-(IV)中任一序列的修飾物或衍生物。
根據(jù)本發(fā)明的第七方面,提供一種多肽,該多肽具有(I)如圖2所示的氨基酸序列(SEQ I.D.No.2);(II)一種與圖2所示的序列相比具有至少95%相似性的序列,且其具有基本相似的免疫原性;或者(III)一種序列,其為圖2所示的氨基酸序列(SEQ I.D.No.2)的修飾物或衍生物。
所述修飾序列優(yōu)選如圖11所示,它是Du422和Du151的共有序列(SEQI.D.No.11)。
根據(jù)本發(fā)明的第八方面,提供一種多肽,該多肽具有(I)如圖4所示的氨基酸序列(SEQ I.D.No.4);(II)一種與圖4所示的序列相比具有至少95%相似性的序列,且其具有基本相似的免疫原性;或者(III)一種序列,其為圖4所示的氨基酸序列(SEQ I.D.No.4)的修飾物或衍生物。
所述修飾序列優(yōu)選如圖11所示,它是Du422和Du151的共有序列(SEQI.D.No.11)。
根據(jù)本發(fā)明的第九方面,提供一種多肽,該多肽具有(I)如圖6所示的氨基酸序列(SEQ I.D.No.6);(II)一種與圖6所示的序列相比具有至少92%相似性的序列,且其具有基本相似的免疫原性;或者(III)一種序列,其為圖6所示的氨基酸序列(SEQ I.D.No.6)的修飾物或衍生物。
所述修飾序列優(yōu)選如圖14所示(SEQ I.D.No.14)。
根據(jù)本發(fā)明的第十方面,提供一種多肽,該多肽具有(I)如圖8所示的氨基酸序列(SEQ I.D.No.8);(II)一種與圖8所示的序列相比具有至少95%相似性的序列,且其具有基本相似的免疫原性;或者(III)一種序列,其為圖8所示的氨基酸序列(SEQ I.D.No.8)的修飾物或衍生物。
所述修飾序列優(yōu)選具有與圖14(SEQ I.D.No.14)所示的分離株Du151的nef基因相似或相同的修飾。
根據(jù)本發(fā)明的第十一方面,提供一種多肽,該多肽具有(I)如圖16所示的氨基酸序列(SEQ I.D.No.16);(II)一種與圖16所示的序列相比具有至少90%相似性的序列,且其具有基本相似的免疫原性;或者
(III)一種序列,其為圖16所示的氨基酸序列(SEQ I.D.No.16)的修飾物或衍生物。
根據(jù)本發(fā)明的第十二方面,HIV-1 C亞型的tat基因的共有氨基酸序列如下MEPVDPNLEPWNHPGSQPKTACNKCYCKHCSYHCLVCFQTKGLGISYGRKKRRQRRSAPP60SSEDHQNLISKQPLPQTRGDPTGSEESKKKVESKTETDPFD101(SEQ ID NO18)根據(jù)本發(fā)明的第十三方面,HIV-1 C亞型的部分nef基因的共有氨基酸序列如下MGGKWSKSSIVGWPAVRERIRRTEPAAEGVGAASQDLDKHGALTSSNTAHNNADCAWLQA60QEEEEEVGFPVRPQVPLRPMTYKGAFDLSFFLKEKGGLEGLIYSKKRQEILDLWVYHTQG120FFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANEGENNCLIHPMSQHGMEDEDRE180VLKWKFDSSLARRHMARELHPEYYKDC207(SEQ ID NO19)根據(jù)本發(fā)明的第十四方面,HIV-1 C亞型的部分rev基因的共有氨基酸序列如下MAGRSGDSDEALIQAVRIIKILYQSNPYPKPEGTRQARKNRRRRWRARQRQIHSISERIL60STCLGRPAEPVPLQLPPIERLHIDCSESSGTSGTQQSQQTTEGVGSP107(SEQ ID NO20)根據(jù)本發(fā)明的第十五方面,提供了上述至少一種序列在生產(chǎn)用于治療或預(yù)防HIV感染的疫苗中的應(yīng)用。優(yōu)選在疫苗中使用至少兩種序列。
根據(jù)本發(fā)明的第十六方面,提供了一種含有至少兩種上述序列的疫苗。
根據(jù)本發(fā)明的第十七方面,提供了一種疫苗,該疫苗含有至少部分的gag基因序列、逆轉(zhuǎn)錄酶(pol)基因序列、改排tat基因序列和截取的nef基因序列,這些基因序列結(jié)合在一起形成框內(nèi)多基因,以grttnC表示(SEQI.D.No30)。
該疫苗可用于治療或預(yù)防HIV。
圖1(SEQ I.D.No 1)表示分離株Du422的測(cè)序tat基因的核酸序列(cDNA);圖2(SEQ I.D.No 2)表示分離株Du422的測(cè)序tat基因的氨基酸序列,它是從核酸序列衍生得到的;圖3(SEQ I.D.No 3)表示分離株Du151的測(cè)序tat基因的核酸序列(cDNA);圖4(SEQ I.D.No 4)表示分離株Du151的測(cè)序tat基因的氨基酸序列,它是從核酸序列衍生得到的;圖5(SEQ I.D.No 5)表示分離株Du151的測(cè)序nef基因的核酸序列(cDNA);圖6(SEQ I.D.No 6)表示分離株Du151的測(cè)序nef基因的氨基酸序列,它是從核酸序列衍生得到的;圖7(SEQ I.D.No 7)表示分離株Du422的測(cè)序rev基因的核酸序列,它是從核酸序列衍生得到的;圖8(SEQ I.D.No 8)表示分離株Du422的測(cè)序rev基因的氨基酸序列,它是從核酸序列衍生得到的;圖9(SEQ I.D.No 9)表示分離株Du422和Du151的共有序列的野生型、改排和測(cè)序tat基因的核酸序列(DNA);圖10(SEQ I.D.No 10)表示分離株Du422和Du151的共有序列的密碼子優(yōu)化、改排和測(cè)序tat基因的核酸序列(DNA),它用于增強(qiáng)表達(dá);圖11(SEQ I.D.No 11)表示分離株Du422和Du151的共有序列的改排和測(cè)序Tat蛋白的氨基酸序列;圖12(SEQ I.D.No 12)表示分離株Du151的野生型、截取和測(cè)序nef基因的核酸序列(DNA);圖13(SEQ I.D.No 13)表示分離株Du151的密碼子優(yōu)化、截取和測(cè)序nef基因的核酸序列(DNA),它用于增強(qiáng)表達(dá);圖14(SEQ I.D.No 14)表示分離株Du151的截取和測(cè)序Nef蛋白的氨基酸序列;圖15(SEQ I.D.No 15)表示由分離株Du422和Du151的改排tat(SEQI.D.No 10)-截取Nef(SEQ I.D.No 11)基因組成的野生型多基因的核酸序列(DNA);圖16(SEQ I.D.No 16)表示分離株Du422和Du151的測(cè)序的改排Tat(SEQ I.D.No 10)-截取Nef(SEQ I.D.No 12)多蛋白的氨基酸序列;圖17(SEQ I.D.No 17)表示由分離株Du422和Du151的改排tat-截取Nef基因組成的測(cè)序多基因的核酸序列(DNA),該序列被修飾以反映人類(lèi)密碼子在表達(dá)增加中的應(yīng)用;圖18示意性地表示HIV-1的基因組,并且圖示了由逆轉(zhuǎn)錄酶產(chǎn)生的測(cè)序重疊片斷的位置,重疊片斷是聚合酶鏈反應(yīng)之后產(chǎn)生的,其目的是生成南非共有序列;圖19表示基于各種分離株的tat基因序列的各種HIV-1 C亞型分離株的核酸序列的系統(tǒng)發(fā)育樹(shù),包括大量的共有序列和本發(fā)明的南非共有序列以及本發(fā)明的篩選分離株Du422和Du151;圖20表示基于各種分離株的nef基因序列的各種HIV-1 C亞型分離株的核酸序列的系統(tǒng)發(fā)育樹(shù),包括大量的共有序列和本發(fā)明的南非共有序列以及本發(fā)明的篩選分離株Du151;圖21表示基于各種分離株的rev基因序列的各種HIV-1 C亞型分離株的核酸序列的系統(tǒng)發(fā)育樹(shù),包括大量的共有序列和本發(fā)明的南非共有序列以及本發(fā)明的篩選分離株Du422;圖22表示多種分離株的每一種的Tat蛋白序列與根據(jù)本發(fā)明制備的tat基因的南非共有序列如何不同;圖23表示多種分離株的每一種的Nef蛋白序列與根據(jù)本發(fā)明制備的nef基因的南非共有序列如何不同;圖24表示多種分離株的每一種的Rev蛋白序列與根據(jù)本發(fā)明制備的rev基因的南非共有序列如何不同;圖25表示基于各種分離株的Tat蛋白序列的各種HIV-1 C亞型分離株的氨基酸序列的系統(tǒng)發(fā)育樹(shù),包括大量的共有序列和本發(fā)明的南非共有序列以及本發(fā)明的篩選分離株Du422和Du151;圖26表示基于各種分離株的Nef蛋白序列的各種HIV-1 C亞型分離株的氨基酸序列的系統(tǒng)發(fā)育樹(shù),它包括大量的共有序列和本發(fā)明的南非共有序列以及本發(fā)明的篩選分離株Du422和Du151;圖27表示基于各種分離株的Tat蛋白序列的各種HIV-1 C亞型分離株的氨基酸序列的系統(tǒng)發(fā)育樹(shù),它包括大量的共有序列和本發(fā)明的南非共有序列以及本發(fā)明的篩選分離株Du422和Du151;圖28表示各種分離株的測(cè)序的Tat蛋白彼此之間相對(duì)于篩選的Du422和Du151Tat克隆體以及相對(duì)于Tat蛋白的南非共有序列的氨基酸序列同源性百分比,它是基于分離株的Tat蛋白的兩兩對(duì)比;圖29表示各種分離株的測(cè)序的Nef蛋白彼此之間相對(duì)于篩選的Du151Nef克隆體以及相對(duì)于Nef蛋白的南非共有序列的氨基酸序列同源性百分比,它是基于分離株的Nef蛋白的兩兩對(duì)比;圖30表示各種分離株的測(cè)序的Rev蛋白彼此之間相對(duì)于篩選的Du422Rev克隆體以及相對(duì)于Rev蛋白的南非共有序列的氨基酸序列同源性百分比,它是基于分離株的Rev蛋白的兩兩對(duì)比;圖31示意性地表示改排Tat蛋白,其包括用于保持CTL表位的重疊片斷;圖32a表示由gag(Du422)、RT(Du151)、改排tat(SEQ I.D.No.10)和截取nef(SEQ I.D.No.13)(SEQ I.D.No.29)組成的GrttnC的核酸序列;圖32b表示由gag(Du422)、RT(Du151)、改排tat和截取nef(SEQ I.D.No.16)(SEQ I.D.No.30)組成的GrttnC的氨基酸序列;圖33表示pTHgrttnC的質(zhì)粒圖,表達(dá)GrttnC用于預(yù)防或治療HIV感染的DNA疫苗載體;圖34表示形成部分grttnC的Du422gag核苷酸序列(SEQ I.D.No.31);圖35表示形成部分grttnC的Du422Gag氨基酸序列(SEQ I.D.No.32);圖36表示形成部分grttnC的Du151逆轉(zhuǎn)錄酶(RT)核苷酸序列(SEQI.D.No.33);以及圖37表示形成部分grttnC的Du151逆轉(zhuǎn)錄酶(RT)氨基酸序列(SEQI.D.No.34)。
發(fā)明詳述本發(fā)明涉及HIV-1 C亞型分離株調(diào)節(jié)和附加基因的篩選,以及這些基因及其修飾物和衍生物在制備抵抗HIV-1 C亞型的預(yù)防性和治療性藥物組合物和制劑,特別是疫苗中的應(yīng)用。因此所述組合物能夠用于預(yù)防性地防止感染或者治療性地預(yù)防或改善疾病。在研制HIV疫苗時(shí)要考慮許多因素,本發(fā)明的一個(gè)方面涉及篩選合適的HIV分離株附加和調(diào)節(jié)基因的方法,所述基因可用于研制疫苗。
申請(qǐng)人設(shè)想,根據(jù)上述方法研制的疫苗可用于抵抗一種或多種HIV亞型和HIV-1 C亞型。
提出了一種鑒定用于研制抗HIV-1 C亞型的疫苗的合適菌株的方法。收集來(lái)自急性感染個(gè)體的病毒株。對(duì)所述分離株的tat、rev和nef區(qū)進(jìn)行測(cè)序,并且比較了從這些分離株上得到的tat、rev和nef基因的氨基酸序列。通過(guò)篩選在每個(gè)位點(diǎn)最常出現(xiàn)的氨基酸,形成了一種共有序列,即南非共有序列。HIV-1 C亞型的tat、rev和nef基因中任一種的共有序列還構(gòu)成了本發(fā)明的一個(gè)方面。通過(guò)將菌株與共有序列對(duì)比并利用表型手段表征,篩選適合疫苗開(kāi)發(fā)的菌株。這些分離株也構(gòu)成了本發(fā)明的一個(gè)方面。
為了篩選使用CCR5共受體的NSI菌株,用一群確定的性服務(wù)者來(lái)鑒定合適的菌株。通過(guò)比較菌株和已經(jīng)形成的共有序列,從急性感染的個(gè)體體內(nèi)鑒定合適的菌株。對(duì)來(lái)自12個(gè)急性感染的人體的病毒株的tat、rev和/或nef區(qū)進(jìn)行測(cè)序并進(jìn)行表型表征。將該序列與來(lái)自另一地區(qū)的、具有超過(guò)500個(gè)CD4細(xì)胞的15個(gè)無(wú)癥狀的人體的病毒株以及來(lái)自Gauteng的TB傳染病醫(yī)院的9個(gè)AIDS患者和2個(gè)患有AIDS的兒童的11個(gè)病毒株進(jìn)行對(duì)比。還包括其他存放在Los Alamos數(shù)據(jù)庫(kù)中的已公開(kāi)的C亞型序列(http//ww.hjv-web.lanl.gov/)。
篩選了2個(gè)標(biāo)記為Du422和Du151的潛在疫苗株。這樣篩選是基于tat、rev和nef三個(gè)基因區(qū)的共有序列的氨基酸同源性、CCR5嗜性以及在組織培養(yǎng)物中生長(zhǎng)和復(fù)制的能力。三個(gè)分離株及其修飾物和衍生物的三個(gè)基因區(qū)的核酸序列和氨基酸序列也構(gòu)成了本發(fā)明的幾個(gè)方面。
分離和篩選病毒株以設(shè)計(jì)疫苗使用下面的標(biāo)準(zhǔn)來(lái)篩選合適的菌株,以引入南非HIV-1疫苗中該菌株是循環(huán)型菌株的遺傳性代表;
該菌株不是奇異株;該菌株在HIV-1 C亞型的tat、rev和nef基因方面盡可能地與根據(jù)本發(fā)明得到的共有氨基酸序列相似;該菌株具有一個(gè)R5表型,即與選擇性傳播有關(guān)的表型;以及所述疫苗能夠在組織培養(yǎng)物中生長(zhǎng)。
按照下述步驟篩選病毒株,用于設(shè)計(jì)疫苗。用南非夸祖魯那塔的一群確定的性服務(wù)者來(lái)鑒定用于HIV疫苗的合適菌株。來(lái)自12個(gè)急性感染者的病毒株的tat、rev和nef區(qū)被測(cè)序、分離并采用表型手段表征。將該序列與來(lái)自南非Gauteng地區(qū)和西開(kāi)普的開(kāi)普敦地區(qū)的無(wú)癥狀A(yù)IDS感染(nef區(qū))個(gè)體的相似收集以及其他被公開(kāi)的C亞型序列進(jìn)行了對(duì)比。
患者來(lái)自南非的4個(gè)地區(qū)的HIV感染者被征集在一起。獲得來(lái)自夸祖魯那塔的近期感染的性服務(wù)者的血樣(n=12)。將近期感染定義為早先血清反應(yīng)陰性而在之前一年內(nèi)變?yōu)檠宸磻?yīng)陽(yáng)性的人。還收集了來(lái)自開(kāi)普敦的門(mén)診診所的患者(n=2)、來(lái)自約翰內(nèi)斯堡的產(chǎn)前檢查診所的婦女(n=6)和來(lái)自約翰內(nèi)斯堡外的一個(gè)金礦上的STD診所的男性患者(n=7)的血樣。后面兩組在臨床上是穩(wěn)定的,被劃分為無(wú)癥狀感染。另外,為了比較nef基因,獲得了來(lái)自Gauteng的AIDS患者的11個(gè)分離株、來(lái)自TB傳染病醫(yī)院的患者的9個(gè)分離株和來(lái)自感染AIDS的兒童的2個(gè)分離株。將血樣收集在EDTA中,用于確定CD4 T細(xì)胞計(jì)數(shù)和進(jìn)行病毒基因分析。對(duì)于近期感染,為測(cè)定血漿病毒量,進(jìn)行了支鏈(bDNA)分析(Chiron),并分離病毒。使用免疫檢測(cè)法(ELISA)測(cè)定HIV-1的血清狀態(tài)。得到了針對(duì)性服務(wù)者的CD4 T細(xì)胞計(jì)數(shù)和病毒載量結(jié)果,有關(guān)血清轉(zhuǎn)化日的臨床狀態(tài)信息、CD4和共受體使用數(shù)據(jù)如表1所示。
病毒分離采用標(biāo)準(zhǔn)的共培養(yǎng)技術(shù),利用有絲分裂素活化的供體外周血單核細(xì)胞(PBMC),從PBMC中分離HIV。2×106個(gè)的患者PBMC與2×106個(gè)供體PBMC共培養(yǎng)于具有2mL RPMI1640、20%的FCS、抗生素和5%的IL-2(Boehringer)的12孔培養(yǎng)平板中。每周向培養(yǎng)物中補(bǔ)充兩次含有IL-2的新鮮介質(zhì),每次使用5×105/mL的供體PBMC。使用商業(yè)化的p24抗原分析法(Coulter)每周一次地監(jiān)控病毒的生長(zhǎng)。抗原陽(yáng)性培養(yǎng)物增大,再培養(yǎng)2周,得到40mL含有病毒的上清液,將其存放在-70℃待用。從商業(yè)化的性服務(wù)者體內(nèi)得到的病毒的分離結(jié)果如表1所示。
病毒表型使用含病毒上清液,評(píng)估MT-2和共受體轉(zhuǎn)染細(xì)胞系上的病毒分離株的生物表型。為了進(jìn)行MT-2分析,使用5×104個(gè)MT-2細(xì)胞在PRMI、10%FCS和抗生素中培養(yǎng)500μL上清液。為了形成syncitia,每天監(jiān)控培養(yǎng)物為期6天以上。表達(dá)CCR5或CXCR4共受體的U87.CD4細(xì)胞在含有10%FCS、抗生素、500μg/mL G418和1μg/mL的嘌呤霉素的DMEM中生長(zhǎng)。表達(dá)少數(shù)共受體的GHOST細(xì)胞在含有10%FCS、500μg/mL G418、1μg/mL的嘌呤霉素和100μg/mL的潮霉素的DMEM中生長(zhǎng)。通過(guò)胰蛋白酶化每周兩次將細(xì)胞系傳代。在12孔的培養(yǎng)平板上進(jìn)行共受體分析;在每個(gè)孔里固定5×104個(gè)細(xì)胞,使其附著過(guò)夜。第二天加入含病毒的500μL上清液,培養(yǎng)過(guò)夜,使病毒附著并感染,第三天洗三次。為了形成syncitia和產(chǎn)生p24抗原,在第4、8和12天監(jiān)控培養(yǎng)物。有syncitia和p24抗原產(chǎn)生濃度增長(zhǎng)跡象的培養(yǎng)物被認(rèn)為對(duì)于病毒生長(zhǎng)是有利的。來(lái)自商業(yè)化的性服務(wù)者的病毒的共受體處理結(jié)果如表1所示。
表1用于篩選候選疫苗的急性感染人群
*來(lái)自98年11月的數(shù)據(jù)測(cè)序從血漿分離RNA,用逆轉(zhuǎn)錄酶從RNA擴(kuò)增基因片斷,接著通過(guò)聚合酶鏈反應(yīng)(PCR)生成擴(kuò)增的DNA片斷。PCR引物的位置如下,(用HIV-1HXBr序列編碼)tat向外向前引物(5’GGC CGC AGA GGG AAC CATAC3’(SEQ ID NO21)5584-5703bp),或者rev向外逆引物(5’GCC CTG TCTTAT TCT TCT AGG3’(SEQ ID NO22)8753-8774bp)。余下的用于巢式PCR的引物是tat向外逆引物(5’CCT CAA TAT CCC CAT CAC TCT C3’(SEQ IDNO23)6226-6248bp)、tat向內(nèi)向前引物(5’TGC CAG CAT AGC AGA ATAGG3’(SEQ ID NO24)5785-5804bp)和tat向內(nèi)逆引物(5’CTA TCA ATG CTCCTA CTC CTA ATC3’(SEQ ID NO25)6078-6101bp),以及rev向外向前引物(5’GAT AGT AGG AGG CTT GAT AGG3’(SEQ ID NO26)8285-8302bp)、rev向內(nèi)向前引物(5’GGT GTA CTC GCT ATA GTG3’(SEQ IDNO27)8321-8339bp)和rev向內(nèi)逆引物(5’CCT TCA GCT ACT GCT ATTGC3’(SEQ ID NO28)8689-8698bp)。
用QIAQUICK PCR純化設(shè)備(Qiagen,德國(guó))對(duì)擴(kuò)增的DNA片斷進(jìn)行純化。然后,使用上游PCR引物作為測(cè)序引物對(duì)DNA片斷測(cè)序。采用Sanger雙脫氧終止劑法,用附著在雙脫氧核苷酸的熒光染料進(jìn)行測(cè)序。通過(guò)電泳,用ABI377測(cè)序儀測(cè)定序列。表示進(jìn)行上述測(cè)序的tat、rev和nef基因的HIV-1前病毒基因組的示意圖見(jiàn)圖18。
遺傳型特征為了篩選疫苗分離株,對(duì)包括tat(101個(gè)密碼子,306個(gè)堿基)、rev(107個(gè)相鄰密碼子,324個(gè)堿基)和nef(207個(gè)密碼子,618個(gè)堿基)的三個(gè)HIV基因進(jìn)行了測(cè)試(圖18)。圖18表示5’長(zhǎng)終端重覆區(qū)域,結(jié)構(gòu)和功能基因(gag、pol和env)以及調(diào)節(jié)和附加蛋白(vif、tat、rev、nef、vpr和vpu)。gag開(kāi)放式閱讀框表示編碼p17基質(zhì)蛋白、p24核蛋白以及p7和p6核衣殼蛋白的區(qū)域。pol開(kāi)放式閱讀框表示蛋白酶(PR)p15、逆轉(zhuǎn)錄酶(RT)p66和核糖核酸酶H整合酶p51。env開(kāi)放式閱讀框表示編碼gp120和gp41的區(qū)域。
在所有的38個(gè)分離株中,有12個(gè)來(lái)自德班群(DU)、24個(gè)來(lái)自約翰內(nèi)斯堡(GG、RB、COT和SW)、2個(gè)來(lái)自開(kāi)普敦(CT)。其中有17個(gè)的tat基因被測(cè)序,有17個(gè)的rev基因被測(cè)序,有32個(gè)的nef基因被測(cè)序。被測(cè)序的分離株如表2所示。
表2分離株和被測(cè)序的基因區(qū)域列表
將來(lái)自德班(Du)、約翰內(nèi)斯堡(GG、RB、SW和COT)和開(kāi)普敦(CT)的核酸序列與大量可得的已公開(kāi)的C亞型序列(來(lái)自L(fǎng)os Alamos HIV序列數(shù)據(jù)庫(kù))進(jìn)行系統(tǒng)發(fā)育學(xué)對(duì)比,后者包括來(lái)自其他南非國(guó)家的序列和來(lái)自L(fǎng)os Alamos HIV序列數(shù)據(jù)庫(kù)的所有C亞型共有序列。對(duì)比的目的是保證被選的疫苗分離株與南非序列相比,不是系統(tǒng)發(fā)育學(xué)上的異常株,對(duì)比結(jié)果如圖19、圖20和圖21所示。圖19-21表示南非序列是不同的,印度序列通常會(huì)形成與非洲序列不同的分離簇。南非序列并非獨(dú)一無(wú)二的,一般來(lái)說(shuō),它們相互之間是有聯(lián)系的,就像它們與來(lái)自南非的其他序列有關(guān)一樣。這表明印度序列與南非C亞型序列不同,在南非沒(méi)有無(wú)性傳染病,但是南非病毒反映出南非地區(qū)的C亞型病毒具有多樣性。
共有序列的測(cè)定氨基酸序列來(lái)自如表2所示的序列,這些序列被用來(lái)測(cè)定南非共有序列。選擇在每個(gè)位置出現(xiàn)最頻繁的氨基酸作為該位置處的共有氨基酸。這樣,沿著每個(gè)被測(cè)序基因(tat、rev和nef基因)的線(xiàn)性長(zhǎng)度測(cè)定共有序列。使用DNAMAN程序(DNAMAN2輸出)進(jìn)行排列,產(chǎn)生共有序列。這樣會(huì)在每個(gè)基因區(qū)域產(chǎn)生共有序列。氨基酸序列和產(chǎn)生的共有序列的排列如圖22、23和24所示。氨基酸相似性如圖25-27所示。
最終選用哪個(gè)分離株是根據(jù)一個(gè)特定分離株的tat、rev和nef基因序列與上面得到的南非共有序列的相似性,以及表1所示的具有好的復(fù)制動(dòng)力學(xué)的R5分離株的可獲得性來(lái)確定的。
疫苗分離株的篩選基于上述考慮和方法,從急性感染的人群篩選兩個(gè)菌株作為疫苗株。第一個(gè)菌株是用于tat和nef基因的Du151,第二個(gè)菌株是用于tat和rev基因的Du422。篩選這三個(gè)菌株的原因如下1.當(dāng)獲得樣品時(shí),Du151已被感染長(zhǎng)達(dá)6周,CD4計(jì)數(shù)達(dá)到367個(gè)細(xì)胞每μL血液,病毒載量大于500000個(gè)拷貝每mL血漿??紤]到高的病毒載量和從感染開(kāi)始的記錄時(shí)間,患者可能還處于免疫系統(tǒng)控制HIV復(fù)制之前的病毒血癥初期。
2.當(dāng)獲得樣品時(shí),Du422已被感染長(zhǎng)達(dá)4個(gè)月,CD4計(jì)數(shù)達(dá)到397個(gè)細(xì)胞每μL血漿,病毒載量達(dá)到17118個(gè)拷貝每mL血漿。與Du151相比,該患者體內(nèi)的病毒復(fù)制已經(jīng)得到了一定程度的控制。
這兩個(gè)分離株能夠在細(xì)胞培養(yǎng)物中生長(zhǎng),而且對(duì)它們的整個(gè)基因組進(jìn)行了序列分析。
根據(jù)圖28所示的Du151和Du422Tat蛋白序列以及其他分離株的氨基酸兩兩對(duì)比分析,所示的Du151和Du422tat序列與圖19和22所示的南非共有序列非常相似。它們與共有序列具有89.4%(Du151)和91.6%(Du422)的氨基酸序列一致性。因此Du151和Du422用于生成野生型(非密碼子優(yōu)化)或人化(密碼子優(yōu)化)的、再合成的改排Tat。它們是在與南非共有序列略微相關(guān)的病毒株中篩選出來(lái)的,原因是它們能夠在組織培養(yǎng)物中生長(zhǎng),兩分離株的整個(gè)基因組都已被測(cè)序和表征。
Nef基因表現(xiàn)出最大的序列不同性。根據(jù)如圖29所示的與SA共有序列的Nef氨基酸兩兩同一的分?jǐn)?shù)(93.4%)的分析結(jié)果,選用Du151分離株作為nef基因源。當(dāng)與其他近期血清轉(zhuǎn)換體相比時(shí),與任一種Du151分離株序列的所有兩兩同一分?jǐn)?shù)都在80.2%以上,如圖29所示。該結(jié)果中的其他貢獻(xiàn)性因素是,這是為env和pol基因源篩選的相同分離株,且其在生物體內(nèi)具有優(yōu)異的生長(zhǎng)性能。
Rev基因是三個(gè)之中最保守的。根據(jù)與SA共有序列的氨基酸兩兩同一分?jǐn)?shù)(95.2%),篩選Du422 rev基因。另外,當(dāng)與其他近期血清轉(zhuǎn)換體相比時(shí),與Du422分離株序列的所有兩兩同一的分?jǐn)?shù)都在83%以上,如圖29所示。這些兩兩分?jǐn)?shù)使得Du422與該序列庫(kù)中的最佳分?jǐn)?shù)相似,該兩兩分?jǐn)?shù)將與R5病毒的相似水平與好的細(xì)胞培養(yǎng)物復(fù)制動(dòng)力學(xué)結(jié)合起來(lái)。
基因的再合成多蛋白基因Tat-nef是通過(guò)合成寡核苷酸片斷制備的,GeneArt(geneart股份有限公司,雷根斯堡(Regensburg))將所述寡核苷酸片斷連接起來(lái)組成整個(gè)基因。合成了密碼子優(yōu)化和非密碼子優(yōu)化的基因,并將其克隆到pPCR-Script(Stratagene)載體。通過(guò)將插入物的兩股測(cè)序,并將其與原始序列對(duì)比,從而鑒定插入物。Du422/Du151和單獨(dú)的Du151的tat和nef基因序列以及Tat-nef多蛋白基因序列的修飾物如SEQ I.D.Nos.9-17所示。
將Tat蛋白分成三個(gè)重疊片斷并改排(如圖31所示),使蛋白失活從而更加安全,但不會(huì)失去潛在的CTL表位。Nef蛋白被截去10個(gè)氨基酸,去掉了允許Nef蛋白離開(kāi)細(xì)胞的N終端十四烷基化部位(SEQ I.D.No.12)。除了使蛋白更安全外,還希望當(dāng)?shù)鞍紫萋涞郊?xì)胞中時(shí)會(huì)產(chǎn)生更有效的CTL反應(yīng)。
疫苗的研制本發(fā)明的疫苗可以使用多種不同的載體用多種不同的方法制備。所述疫苗包括來(lái)自多種不同載體的病毒的膠囊式RNA或轉(zhuǎn)錄的DNA序列。這些疫苗可以含有至少部分的來(lái)自Du422分離株的tat和rev基因以及至少部分的來(lái)自本發(fā)明的Du151分離株或其衍生物或修飾物的tat和nef基因。
用于DNA疫苗中的基因被再合成以反映人密碼子的用途。Tat Du422基因被分為三個(gè)具有重疊端的片斷,使得沒(méi)有潛在的CTL表位被丟失和重新改排以提高Tat蛋白的安全性。鑒于安全原因,Du151nef基因的前10個(gè)氨基酸被截去,以除去十四烷基化部位。將改排tat和截短的nef合成在同一個(gè)閱讀框中,以構(gòu)成Tat-Nef多蛋白。合成人型和非人型Tat-nef多蛋白用于其他替代性疫苗。相似的密碼子優(yōu)化的rev基因可以由DNA疫苗表達(dá)。
其他疫苗含有從來(lái)自Du422和Du151分離株的tat基因的RNA轉(zhuǎn)錄的DNA、從來(lái)自Du151分離株的nef基因的RNA轉(zhuǎn)錄的DNA和從來(lái)自Du422分離株的rev基因的RNA轉(zhuǎn)錄的DNA。這些基因也可以被表達(dá)為寡聚包膜糖蛋白復(fù)合物(Progenics,USA),其公開(kāi)見(jiàn)J Virol 2000 Jan;74(2)627-43(Binley,J.L.等人),或在腺相關(guān)病毒(AAV)(Target Genetics)、委內(nèi)瑞拉馬腦炎病毒(美國(guó)專(zhuān)利申請(qǐng)USSN 60/216,995,引為本文參考)、變性牛痘病毒安卡拉(MVA)(Amara等人,2002)、BCG以及其他正在開(kāi)普敦大學(xué)研發(fā)的疫苗中表達(dá)。
含有框內(nèi)多基因、GrttnC(圖32a和32b;SEQ I.D.Nos.29和30)和重組tat-截取nef(SEQ I.D.No.17)的疫苗構(gòu)造已被研制,并將被引入多個(gè)候選疫苗中,包括DNA疫苗、用pTH DNA疫苗載體(Hanke等人,2000)的pTHgrttnC(圖33),以及MVA疫苗(Amara等人,2002),所述GrttnC包括密碼子優(yōu)化的Du422gag(圖34和35;SEQ I.D.Nos.31和32)和Du151RT(逆轉(zhuǎn)錄酶)(圖36和37;SEQ I.D.Nos.33和34)(WO 02/04494,其內(nèi)容引為本文參考)。從Du422和Du151分離出來(lái)的gag和pol基因的核苷酸和氨基酸序列分別如SEQ I.D.Nos.35-38所示。
本發(fā)明并不僅限于所描述的具體實(shí)施方式
。
保藏下述材料已經(jīng)保藏在歐洲細(xì)胞培養(yǎng)物中心(ECACC),它是應(yīng)用微生物學(xué)與研究中心,位于英國(guó)威爾特郡SP4OJG索爾茲伯里。
材料ECACC保藏號(hào)保藏日HIV-1病毒分離株Du151保藏號(hào)000727242000年7月27日HIV-1病毒分離株Du422臨時(shí)保藏號(hào)000727262000年7月27日臨時(shí)保藏號(hào)010321142001年3月22日保藏是根據(jù)用于專(zhuān)利程序微生物保藏的國(guó)際公約布達(dá)佩斯條約的有關(guān)規(guī)定及其細(xì)則而做出的。
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序列表PA134027-mrcuct2.ST25SEQUENCE LISTING<110> South African Medical Research Council and University of Cape Town<120> HIV-1 Subtype Isolate Regulatory/Accessory Genes,and Modifications andDerivatives Thereof<130> PA134027/PCT<140> ZA 2001/8978<141> 2001-10-31<160> 38<170> PatentIn version 3.1<210> 1<211> 306<212> PRT<213> Human immunodeficiency virus type 1<400> 1Ala Thr Gly Gly Ala Gly Cys Cys Ala Ala Thr Ala Gly Ala Thr Cys1 5 10 15Cys Thr Ala Ala Cys Cys Thr Ala Gly Ala Gly Cys Cys Cys Thr Gly20 25 30Gly Ala Ala Cys Cys Ala Thr Cys Cys Ala Gly Gly Ala Ala Gly Thr35 40 45Cys Ala Gly Cys Cys Thr Ala Ala Thr Ala Cys Thr Cys Cys Thr Thr50 55 60Gly Thr Ala Ala Thr Ala Ala Cys Thr Gly Cys Thr Ala Thr Thr Gly65 70 75 80Thr Ala Ala Ala Cys Ala Cys Thr Gly Thr Ala Gly Cys Thr Ala Cys85 90 95Cys Ala Thr Thr Gly Thr Cys Thr Ala Gly Thr Thr Thr Gly Cys Thr100 105 110Thr Thr Cys Ala Gly Ala Cys Ala Ala Ala Ala Gly Gly Cys Thr Thr115 120 125
PA134027-mrcuct2.ST25Ala Gly Gly Cys Ala Thr Thr Thr Cys Cys Thr Ala Thr Gly Gly Cys130 135 140Ala Gly Gly Ala Ala Gly Ala Ala Gly Cys Gly Gly Ala Gly Ala Cys145 150 155 160Ala Gly Cys Gly Ala Cys Gly Ala Ala Gly Cys Ala Cys Thr Cys Cys165 170 175Thr Cys Cys Ala Ala Gly Cys Ala Gly Thr Gly Ala Ala Gly Ala Thr180 185 190Cys Ala Thr Cys Ala Ala Ala Ala Thr Cys Cys Thr Ala Thr Ala Thr195 200 205Cys Ala Ala Ala Gly Cys Ala Ala Cys Cys Cys Thr Thr Ala Thr Cys210 215 220Cys Cys Gly Ala Cys Ala Gly Gly Cys Thr Cys Gly Gly Ala Ala Gly245 250 255Ala Ala Thr Cys Gly Ala Ala Gly Ala Ala Gly Ala Ala Gly Gly Thr260 265 270Gly Gly Ala Gly Ala Gly Cys Ala Ala Gly Ala Cys Ala Ala Ala Gly275 280 285Ala Cys Ala Gly Ala Thr Cys Cys Ala Thr Thr Cys Gly Ala Thr Thr290 295 300Ala Gly305<210> 2<211> 101<212> PRT<213> Human immunodeficiency virus type 1<400> 2Met Glu Pro Ile Asp Pro Asn Leu Glu Pro Trp Asn His Pro Gly Ser1 5 10 15Gln Pro Asn Thr Pro Cys Asn Asn Cys Tyr Cys Lys His Cys Ser Tyr20 25 30His Cys Leu Val Cys Phe Gln Thr Lys Gly Leu Gly Ile Ser Tyr Gly35 40 45Arg Lys Lys Arg Arg Gln Arg Arg Ser Thr Pro Pro Ser Ser Glu Asp
PA134027-mrcuct2.ST2550 55 60His Gln Asn Pro Ile Ser Lys Gln Pro Leu Ser Gln Thr Arg Gly Asp65 70 75 80Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val Glu Ser Lys Thr Lys85 90 95Thr Asp Pro Phe Asp100<210> 3<211> 306<212> DNA<213> Human immunodeficiency virus type 1<400> 3atggagccaa tagatcctaa cctagagccc tggaaccatc caggaagtca acctaacact60ccttgtacta aatgctattg taaatactgc agctatcatt gtctagtttg ctttcagaca 120aaaggcttag gcatttccta tggcaggaag aagcggagac agcgacgaag cactcctcca 180agcagtgagg atcatcaaaa tcttatatca gagcagccct taccccaagc ccgaggggtc 240ccgacaggct cggaagaatc gaagaagaag gtggagagca agacaaaaac agatccattc 300gattag 306<210> 4<211> 101<212> PRT<213> Human immunodeficiency virus type 1<400> 4Met Glu Pro Ile Asp Pro Asn Leu Glu Pro Trp Asn His Pro Gly Ser1 5 10 15Gln Pro Asn Thr Pro Cys Thr Lys Cys Tyr Cys Lys Tyr Cys Ser Tyr20 25 30His Cys Leu Val Cys Phe Gln Thr Lys Gly Leu Gly Ile Ser Tyr Gly35 40 45Arg Lys Lys Arg Arg Gln Arg Arg Ser Thr Pro Pro Ser Ser Glu Asp50 55 60His Gln Asn Leu Ile Ser Glu Gln Pro Leu Pro Gln Ala Arg Gly Val65 70 75 80Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val Glu Ser Lys Thr Lys85 90 95
PA134027-mrcuct2.ST25Thr Asp Pro Phe Asp100<210> 5<211> 618<212> DNA<213> Human immunodeficiency virus type 1<400> 5atggggggca agtggtcaaa aagcagcata gtgggatggc ctgctgtaag agaaagaata60agaagaactg agccagcagc agagggagta ggaccagcat ctcaagactt agataaacat 120ggagcgctta caagcagcaa cacagcccac aataatcctg actgtgcctg gctacaagca 180caagaggagg aagaagacgt aggttttcca gtcagacctc aggtgcctct aagaccaatg 240acttataagg cagcattcga tctcagcttc tttttaaaag aaaagggggg actggaaggg 300ttaattcact ctaagagaag acaagacatt cttgatttgt gggtctatca cacacaaggc 360tacttccctg attggcaaaa ctacacgccg ggaccaggag tcagataccc actgaccttt 420ggatggtgct tcaagctagt gccagttgat ccaagggaag tagaagaggc caacaaagga 480gaaaacaact gtttgctaca ccctatgagc cagcatggaa tggaggatgc agacagagaa 540gtattaagat gggtgtttga cagcagtcta gcacgcagac acctggcccg cgagaaacat 600ccggagtatt acaaagac 618<210> 6<211> 207<212> PRT<213> Human immunodeficiency virus type 1<400> 6Met Gly Gly Lys Trp Ser Lys Ser Ser Ile Val Gly Trp Pro Ala Val1 5 10 15Arg Glu Arg Ile Arg Arg Thr Glu Pro Ala Ala Glu Gly Val Gly Ala20 25 30Ala Ser Gln Asp Leu Asp Lys His Gly Ala Leu Thr Ser Ser Asn Thr35 40 45Ala His Asn Asn Pro Asp Cys Ala Trp Leu Gln Ala Gln Glu Glu Glu50 55 60Pro Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met65 70 75 80Thr Tyr Lys Ala Ala Phe Asp Leu Ser Phe Phe Leu Lys Glu Lys Gly85 90 95Gly Leu Glu Gly Leu Ile Tyr Ser Lys Lys Arg Gln Asp Ile Leu Asp
PA134027-mrcuct2.ST25100 105 110Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr115 120 125Thr Pro Gly Pro Gly Val Arg Leu Pro Leu Thr Phe Gly Trp Cys Phe130 135 140Lys Leu Val Pro Val Asp Pro Glu Glu Val Glu Glu Ala Asn Lys Gly145 150 155 160Glu Asn Asn Cys Leu Leu His Pro Leu Ser Gln His Gly Met Glu Asp165 170 175Ala Asp Arg Glu Val Leu Lys Trp Val Phe Asp Ser Ser Leu Ala Arg180 185 190Arg His Leu Ala Arg Glu Lys His Pro Glu Tyr Tyr Lys Asp Cys195 200 205<210> 7<211> 324<212> DNA<213> Human immunodeficiency virus type 1<400> 7atggcaggaa gaagcggaga cagcgacgaa gcactcctcc aagcagtgaa gatcatcaaa60atcctatatc aaagcaaccc ttatcccaaa cccgagggga cccgacaggc tcggaagaat 120cgaagaagaa ggtggagagc aagacaaaga cagatccatt cgattagtga gcggattctt 180agcacttgcc tgggacgatc tgcggagcct gtgcctcttc agctaccacc aattgagaga 240cttcatattg actgcagcga gagcagcgga acttctggga cgcagcagtc tcaggggact 300gcagagaggg tgggaagtcc ttaa 324<210> 8<211> 107<212> PRT<213> Human immunodeficiency virus type 1<400> 8Met Ala Gly Arg Ser Gly Asp Ser Asp Glu Ala Leu Leu Gln Ala Val1 5 10 15Lys Ile Ile Lys Ile Leu Tyr Gln Ser Asn Pro Tyr Pro Lys Pro Glu20 25 30Gly Thr Arg Gln Ala Arg Lys Asn Arg Arg Arg Arg Trp Arg Ala Arg35 40 45
PA134027-mrcuct2.ST25Gln Arg Gln Ile His Ser Ile Ser Glu Arg Ile Leu Ser Thr Cys Leu50 55 60Gly Arg Ser Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Ile Glu Arg65 70 75 80Leu His Ile Asp Cys Ser Glu Ser Ser Gly Thr Ser Gly Thr Gln Gln85 90 95Ser Gln Gly Thr Ala Glu Arg Val Gly Ser Pro100 105<210> 9<211> 392<212> DNA<213> Human immunodeficiency virus type 1<400> 9ggatccgcgg ccgcaagctt gccaccatgg taggcatttc ccatggcagg aagaagcgga60gacagcgacg aagcactcct ccaagcagtg aggatcatca aaatcctata tcaaagcagc 120ccttacccca aacccgaggg gacccgacag gctcggaaga atcgaagaag aaggtggaga 180gcaagacaaa aacagatcca ttcgattgta aatactgcag ctatcattgt ctagtttgct 240ttcagacaaa aggcttaggt attagctatg gaaggaagaa acggatggag ccaatagatc 300ctaacctaga gccctggaac catccaggaa gtcaacctaa cactccttgt aataaatgct 360attgtaagta ctgttcatat cattgcctag tt 392<210> 10<211> 392<212> DNA<213> Human immunodeficiency virus type 1<400> 10ggatccgcgg ccgcaagctt gccaccatgg tgggcatcag ctacggccgc aagaagcgcc60gccagcgccg cagcaccccg cccagcagcg aggaccacca gaaccccatc agcaagcagc 120ccctgcccca gacccgcggc gaccccaccg gcagcgagga gagcaagaag aaggtggaga 180gcaagaccaa gaccgacccc ttcgactgca agtactgcag ctaccactgt ctggtgtgct 240tccagaccaa gggcctgggc atctcctacg ggcgcaagaa acggatggag cccatcgacc 300ccaacctgga gccctggaac caccccggca gccagcccaa caccccctgc aacaagtgct 360actgcaaata ctgctcctac cactgcctcg tg 392<210> 11<211> 122<212> PRT<213> Human immunodeficiency virus type 1
PA134027-mrcuct2.ST25<400> 11Met Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Ser1 5 10 15Thr Pro Pro Ser Ser Glu Asp His Gln Asn Pro Ile Ser Lys Gln Pro20 25 30Leu Pro Gln Thr Arg Gly Asp Pro Thr Gly Ser Glu Glu Ser Lys Lys35 40 45Lys Val Glu Ser Lys Thr Lys Thr Asp Pro Phe Asp Cys Lys Tyr Cys50 55 60Ser Tyr His Cys Leu Val Cys Phe Gln Thr Lys Gly Leu Gly Ile Ser65 70 75 80Tyr Gly Arg Lys Lys Arg Met Glu Pro Ile Asp Pro Asn Leu Glu Pro85 90 95Trp Asn His Pro Gly Ser Gln Pro Asn Thr Pro Cys Asn Lys Cys Tyr100 105 110Cys Lys Tyr Cys Ser Tyr His Cys Leu Val115 120<210> 12<211> 617<212> DNA<213> Human immunodeficiency virus type 1<400> 12gtgggatggc ctgctgtaag agaaagaata agaagaactg agccagcagc agagggagta60ggaccagcat ctcaagactt agataaacat ggagcgctta caagcagcaa cacagcccac 120aataatcctg actgtgcctg gctacaagca caagaggagg aagaagacgt aggttttcca 180gtcagacctc aggtgcctct aagaccaatg acttataagg cagcattcga tctcagcttt 240tttttaaaag aaaagggggg actggaaggg ttaattcact ctaagagaag acaagacatt 300cttgatttgt gggtctatca cacacaaggc tactcccctg attggcaaaa ctacacgccg 360ggaccaggag tcagataccc actgaccttt ggatggtgct tcaagctagt gccagttgat 420ccaagggaag tagaagaggc caacaaagga gaaaacaact gtttgctaca ccctatgagc 480cagcatggaa tggaggatgc agacagagaa gtattaagat gggtgtttga cagcagtcta 540gcacgcagac acctggcccg cgagaaacat ccggagtatt acaaagacta ggaattctct 600agagcggccg cgtcgac 617<210> 13<211> 617
PA134027-mrcuct2.ST25<212> DNA<213> Human immunodeficiency virus type 1<400> 13gtgggctggc ccgccgtgcg cgagcgcatc cgccgcaccg agcccgccgc cgagggcgtg60ggccccgcca gccaggacct ggacaagcac ggcgccctga ccagcagcaa caccgcccac 120aacaaccccg actgcgcctg gctgcaggcc caggaggagg aggaggacgt gggcttcccc 180gtgcgccccc aggtgcccct gcgccccatg acctacaagg ccgccttcga cctgagcttc 240ttcctgaagg agaagggcgg cctggagggc ctgatccaca gcaagcgccg ccaggacatc 300ctggacctgt gggtgtacca cacccagggc tacttccccg actggcagaa ctacaccccc 360ggccccggcg tgcgctaccc cctgaccttc ggctggtgct tcaagctggt gcccgtggac 420ccccgcgagg tggaggaggc caacaagggc gagaacaact gcctgctgca ccccatgagc 480cagcacggca tggaggacgc cgaccgcgag gtgctgcgct gggtgttcga cagcagcctg 540gcccgccgcc acctggcccg cgagaagcac cccgagtact acaaggactg agaattctct 600agagcggccg cgtcgac 617<210> 14<211> 196<212> PRT<213> Human immunodeficiency virus type 1<400> 14Val Gly Trp Pro Ala Val Arg Glu Arg Ile Arg Arg Thr Glu Pro Ala1 5 10 15Ala Glu Gly Val Gly Pro Ala Ser Gln Asp Leu Asp Lys His Gly Ala20 25 30Leu Thr Ser Ser Asn Thr Ala His Asn Asn Pro Asp Cys Ala Trp Leu35 40 45Gln Ala Gln Glu Glu Glu Glu Asp Val Gly Phe Pro Val Arg Pro Gln50 55 60Val Pro Leu Arg Pro Met Thr Tyr Lys Ala Ala Phe Asp Leu Ser Phe65 70 75 80Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His Ser Lys Arg85 90 95Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe100 105 110Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu115 120 125Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Asp Pro Arg Glu Val
PA134027-mrcuct2.ST25130 135 140Glu Glu Ala Asn Lys Gly Glu Asn Asn Cys Leu Leu His Pro Met Ser145 150 155 160Gln His Gly Met Glu Asp Ala Asp Arg Glu Val Leu Arg Trp Val Phe165 170 175Asp Ser Ser Leu Ala Arg Arg His Leu Ala Arg Glu Lys His Pro Glu180 185 190Tyr Tyr Lys Asp195<210> 15<211> 1009<212> DNA<213> Human immunodeficiency virus type 1<400> 15ggatccgcgg ccgcaagctt gccaccatgg taggcatttc ctatggcagg aagaagcgga60gacagcgacg aagcactcct ccaagcagtg aggatcatca aaatcctata tcaaagcagc 120ccttacccca aacccgaggg gacccgacag gctcggaaga atcgaagaag aaggtggaga 180gcaagacaaa aacagatcca ttcgattgta aatactgcag ctatcattgt ctagtttgct 240ttcagacaaa aggcttaggc atttcctatg gcaggaagaa gcggatggag ccaatagatc 300ctaacctaga gccctggaac catccaggaa gtcaacctaa cactccttgt aataaatgct 360attgtaaata ctgcagctat cattgtctag ttgtgggatg gcctgctgta agagaaagaa 420taagaagaac tgagccagca gcagagggag taggaccagc atctcaagac ttagataaac 480atggagcgct tacaagcagc aacacagccc acaataatcc tgactgtgcc tggctacaag 540cacaagagga ggaagaagac gtaggttttc cagtcagacc tcaggtgcct ctaagaccaa 600tgacttataa ggcagcattc gatctcagct tctttttaaa agaaaagggg ggactggaag 660ggttaattca ctctaagaga agacaagaca ttcttgattt gtgggtctat cacacacaag 720gctacttccc tgattggcaa aactacacgc cgggaccagg agtcagatac ccactgacct 780ttggatggtg cttcaagcta gtgccagttg atccaaggga agtagaagag gccaacaaag 840gagaaaacaa ctgtttgcta caccctatga gccagcatgg aatggaggat gcagacagag 900aagtattaag atgggtgttt gacagcagtc tagcacgcag acacctggcc cgcgagaaac 960atccggagta ttacaaagac taggaattct ctagagcggc cgcgtcgac 1009<210> 15<211> 318<212> PRT<213> Human immunodeficiency virus type 1
PA134027-mrcuct2.ST25<400> 16Met Val Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Ser1 5 10 15Thr Pro Pro Ser Ser Glu Asp His Gln Asn Pro Ile Ser Lys Gln Pro20 25 30Leu Pro Gln Thr Arg Gly Asp Pro Thr Gly Ser Glu Glu Ser Lys Lys35 40 45Lys Val Glu Ser Lys Thr Lys Thr Asp Pro Phe Asp Cys Lys Tyr Cys50 55 60Ser Tyr His Cys Leu Val Cys Phe Gln Thr Lys Gly Leu Gly Ile Ser65 70 75 80Tyr Gly Arg Lys Lys Arg Met Glu Pro Ile Asp Pro Asn Leu Glu Pro85 90 95Trp Asn His Pro Gly Ser Gln Pro Asn Thr Pro Cys Asn Lys Cys Tyr100 105 110Cys Lys Tyr Cys Ser Tyr His Cys Leu Val Val Gly Trp Pro Ala Val115 120 125Arg Glu Arg Ile Arg Arg Thr Glu Pro Ala Ala Glu Gly Val Gly Pro130 135 140Ala Sar Gln Asp Leu Asp Lys His Gly Ala Leu Thr Ser Ser Asn Thr145 150 155 160Ala His Asn Asn Pro Asp Cys Ala Trp Leu Gln Ala Gln Glu Glu Glu165 170 175Glu Asp Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met180 185 190Thr Tyr Lys Ala Ala Phe Asp Leu Ser Phe Phe Leu Lys Glu Lys Gly195 200 205Gly Leu Glu Gly Leu Ile His Ser Lys Arg Arg Gln Asp Ile Leu Asp2l0 215 220Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr225 230 235 240Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe245 250 255Lys Leu Val Pro Val Asp Pro Arg Glu Val Glu Glu Ala Asn Lys Gly260 265 270Glu Asn Asn Cys Leu Leu His Pro Met Ser Gln His Gly Met Glu Asp275 280 285Ala Asp Arg Glu Val Leu Arg Trp Val Phe Asp Ser Ser Leu Ala Arg
PA134027-mrcuct2.ST25290 295 300Arg His Leu Ala Arg Glu Lys His Pro Glu Tyr Tyr Lys Asp305 310 315<210> 17<211> 1009<212> DNA<213> Human immunodeficiency virus type 1<400> 17ggatccgcgg ccgcaagctt gccaccatgg tgggcatcag ctacggccgc aagaagcgcc60gccagcgccg cagcaccccg cccagcagcg aggaccacca gaaccccatc agcaagcagc 120ccctgcccca gacccgcggc gaccccaccg gcagcgagga gagcaagaag aaggtggaga 180gcaagaccaa gaccgacccc ttcgactgca agtactgcag ctaccactgt ctggtgtgct 240tccagaccaa gggcctgggc atctcctacg ggcgcaagaa acggatggag cccatcgacc 300ccaacctgga gccctggaac caccccggca gccagcccaa caccccctgc aacaagtgct 360actgcaaata ctgctcctac cactgcctcg tggtgggctg gcccgccgtg cgcgagcgca 420tccgccgcac cgagcccgcc gccgagggcg tgggccccgc cagccaggac ctggacaagc 480acggcgccct gaccagcagc aacaccgccc acaacaaccc cgactgcgcc tggctgcagg 540cccaggagga ggaggaggac gtgggcttcc ccgtgcgccc ccaggtgccc ctgcgcccca 600tgacctacaa ggccgccttc gacctgagct tcttcctgaa ggagaagggc ggcctggagg 660gcctgatcca cagcaagcgc cgccaggaca tcctggacct gtgggtgtac cacacccagg 720gctacttccc cgactggcag aactacaccc ccggccccgg cgtgcgctac cccctgacct 780tcggctggtg cttcaagctg gtgcccgtgg acccccgcga ggtggaggag gccaacaagg 840gcgagaacaa ctgcctgctg caccccatga gccagcacgg catggaggac gccgaccgcg 900aggtgctgcg ctgggtgttc gacagcagcc tggcccgccg ccacctggcc cgcgagaagc 960accccgagta ctacaaggac tgagaattct ctagagcggc cgcgtcgac 1009<210> 18<211> 101<212> PRT<213> Human immunodeficiency virus type 1<400> 18Met Glu Pro Val Asp Pro Asn Leu Glu Pro Trp Asn His Pro Gly Ser1 5 10 15Gln Pro Lys Thr Ala Cys Asn Lys Cys Tyr Cys Lys His Cys Ser Tyr20 25 30His Cys Leu Val Cys Phe Gln Thr Lys Gly Leu Gly Ile Ser Tyr Gly35 40 45
PA134027-mrcuct2.ST25Arg Lys Lys Arg Arg Gln Arg Arg Ser Ala Pro Pro Ser Ser Glu Asp50 55 60His Gln Asn Leu Ile Ser Lys Gln Pro Leu Pro Gln Thr Arg Gly Asp65 70 75 80Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val Glu Ser Lys Thr Glu85 90 95Thr Asp Pro Phe Asp100<210> 19<211> 207<212> PRT<213> Human immunodeficiency virus type 1<400> 19Met Gly Gly Lys Trp Ser Lys Ser Ser Ile Val Gly Trp Pro Ala Val1 5 10 15Arg Glu Arg Ile Arg Arg Thr Glu Pro Ala Ala Glu Gly Val Gly Ala20 25 30Ala Ser Gln Asp Leu Asp Lys His Gly Ala Leu Thr Ser Ser Asn Thr35 40 45Ala His Asn Asn Ala Asp Cys Ala Trp Leu Gln Ala Gln Glu Glu Glu50 55 60Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met65 70 75 80Thr Tyr Lys Gly Ala Phe Asp Leu Ser Phe Phe Leu Lys Glu Lys Gly85 90 95Gly Leu Glu Gly Leu Ile Tyr Ser Lys Lys Arg Gln Glu Ile Leu Asp100 105 110Leu Trp Val Tyr His Thr Gln Gly Phe Phe Pro Asp Trp Gln Asn Tyr115 120 125Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe130 135 140Lys Leu Val Pro Val Asp Pro Arg Glu Val Glu Glu Ala Asn Glu Gly145 150 155 160Glu Asn Asn Cys Leu Leu His Pro Met Ser Gln His Gly Met Glu Asp165 170 175Glu Asp Arg Glu Val Leu Lys Trp Lys Phe Asp Ser Ser Leu Ala Arg
PA134027-mrcuct2.ST25180 185 190Arg His Met Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys195 200 205<210> 20<211> 107<212> PRT<213> Human immunodeficiency virus type 1<400> 20Met Ala Gly Arg Ser Gly Asp Ser Asp Glu Ala Leu Leu Gln Ala Val1 5 10 15Arg Ile Ile Lys Ile Leu Tyr Gln Ser Asn Pro Tyr Pro Lys Pro Glu20 25 30Gly Thr Arg Gln Ala Arg Lys Asn Arg Arg Arg Arg Trp Arg Ala Arg35 40 45Gln Arg Gln Ile His Ser Ile Ser Glu Arg Ile Leu Ser Thr Cys Leu50 55 60Gly Arg Pro Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Ile Glu Arg65 70 75 80Leu His Ile Asp Cys Ser Glu Ser Ser Gly Thr Ser Gly Thr Gln Gln85 90 95Ser Gln Gln Thr Thr Glu Gly Val Gly Ser Pro100 105<210> 21<21l> 20<212> DNA<213> Human immunodeficiency virus type 1<400> 21ggccgcagag ggaaccatac20<210> 22<211> 21<212> DNA<213> Human immunodeficiency virus type 1<400> 22gccctgtctt attcttctag g 21
PA134027-mrcuct2.ST25<210> 23<211> 21<212> DNA<213> Human immunodeficiency virus type 1<400> 23cctcaatatc cccatcactc t 21<210> 24<211> 20<212> DNA<213> Human immunodeficiency virus type 1<400> 24tgccagcata gcagaatagg20<210> 25<211> 24<212> DNA<213> Human immunodeficiency virus type 1<400> 25ctatcaatgc tcctactcct aatc 24<210> 26<211> 21<212> DNA<213> Human immunodeficiency virus type 1<400> 26gatagtagga ggcttgatag g 21<210> 27<211> 18<212> DNA<213> Human immunodeficiency virus type 1<400> 27ggtgtactcg ctatagtg 18<210> 28<211> 20<212> DNA
PA134027-mrcuct2.ST25<213> Human immunodeficiency virus type 1<400> 28ccttcagcta ctgctattgc20<210> 29<211> 3687<212> DNA<213> Human immunodeficiency virus type 1<400> 29aagcttgcca ccatggctgc tcgcgcatct atcctcagag gcgaaaagtt ggataagtgg60gaaaaaatca gactcaggcc aggaggtaaa aaacactaca tgctgaagca tatcgtgtgg 120gcatctaggg agttggagag atttgcactg aaccccggac tgctggaaac ctcagagggc 180tgtaagcaaa tcatgaaaca gctccaacca gccttgcaga ccggaacaga agagctgaag 240tccctttaca ataccgtggc aaccctctat tgcgtccacg agaagatcga ggtgagagac 300acaaaggagg ccctggacaa aatcgaggag gagcagaata agtgccagca gaagacccag 360caggcaaagg ctgctgacgg aaaggtctct cagaactatc ctatcgttca gaaccttcag 420gggcagatgg tgcaccaagc aatcagccct agaaccctga acgcatgggt gaaggtgatc 480gaggagaaag ccttttctcc cgaggttatc cccatgttta ccgccctgag cgaaggcgcc 540actcctcaag acctgaacac tatgctgaac acagtgggag gacaccaggc cgctatgcag 600atgttgaagg ataccatcaa cgaggaggca gccgaatggg accgcctcca ccccgtgcac 660gccggaccta tcgcccccgg acaaatgaga gaacctcgcg gaagtgatat tgccggtact 720accagcaccc ttcaagagca gattgcttgg atgaccagca acccacccat cccagtgggc 780gatatttaca aaaggtggat tattctgggg ctgaacaaaa ttgtgagaat gtactccccc 840gtctccatcc tcgacatccg ccaaggaccc aaggagcctt ttagggatta cgtggacaga 900ttcttcaaaa cccttagagc tgagcaagcc actcaggagg ttaagaactg gatgacagat 960actctgctcg tgcaaaacgc taaccccgat tgcaaaacca tcttgagagc tctcggtcca 1020ggtgccaccc ttgaggaaat gatgacagca tgtcaaggcg tgggaggacc tgggcacaag 1080gccagagttc tcgctgaggc catgagccag acaaactcag gcaatatcat gatgcagagg 1140agtaacttta agggtcccag gagaatcgtc aagtgcttca attgtggcaa ggagggtcac 1200attgccagga actgccgcgc ccccaggaag aaaggctgct ggaagtgtgg caaagagggc 1260caccagatga aggattgcac cgagcgccaa gcaaacttcc tgggaaagat ttggcccagt 1320cataagggcc gccctggcga attctgcggc aagaaggcca tcggcaccgt gctggtgggc 1380cccacccccg tgaacatcat cggccggaac atgctgaccc agctgggctg caccctgaac 1440ttccccatca gccccatcga gaccgtgccc gtgaagctga agcccggcat ggacggcccc 1500aaggtgaagc agtggcccct gaccgaggtg aagatcaagg ccctgaccgt catctgcgag 1560gagatggaga aggagggcaa gatcaccaag atcggccccg agaaccccta caacaccccc 1620atcttcgcca tcaagaagga ggacagcacc aagtggcgga agctggtgga cttccgggag 1680
PA134027-mrcuct2.ST25ctgaacaagc ggacccagga cttctgggag gtgcagctgg gcatccccca ccccgccggc 1740ctgaagaaga agaagagcgt gaccgtgctg gacgtgggcg acgcctactt cagcgtgccc 1800ctggacgagg gcttccggaa gtacaccgcc ttcaccatcc ccagcatcaa caacgagacc 1860cccggcatcc ggtaccagta caacgtgctg ccccagggct ggaagggcag ccccgccatc 1920ttccaggcca gcatgaccaa gatcctggag cccttccggg ccaagaaccc cgagatcgtg 1980atctaccagt acatggccgc cctgtacgtg ggcagcgacc tggagatcgg ccagcaccgg 2040gccaagatcg aggagctgcg ggagcacctg ctgaagtggg gcttcaccac ccccgacaag 2100aagcaccaga aggagccccc cttcctgtgg atgggctacg agctgcaccc cgacaagtgg 2160accgtgcagc ccatccagct gcccgagaag gacagctgga ccgtgaacga catccagaag 2220ctggtgggca agctgaactg gaccagccag atctaccccg gcatcaaggt gcggcagctg 2280tgcaagctgc tgcggggcac caaggccctg accgacatcg tgcccctgac cgaggaggcc 2340gagctggagc tggccgagaa ccgggagatc ctgaaggagc ccgtgcacgg cgtgtactac 2400gaccccagca aggacctgat cgccgagatc cagaagcagg gcgacgacca gtggacctac 2460cagatctacc aggagccctt caagaacctg aaaaccggca agtacgccaa gcggcggacc 2520acccacacca acgacgtgaa gcagctgacc gaggccgtgc agaagatcag cctggagagc 2580atcgtgacct ggggcaagac ccccaagttc cggctgccca tccagaagga gacctgggag 2640atctggtgga ccgactactg gcaggccacc tggatccccg agtgggagtt cgtgaacagc 2700ggccgcaagc ttgccaccat ggtgggcatc agctacggcc gcaagaagcg ccgccagcgc 2760cgcagcaccc cgcccagcag cgaggaccac cagaacccca tcagcaagca gcccctgccc 2820cagacccgcg gcgaccccac cggcagcgag gagagcaaga agaaggtgga gagcaagacc 2880aagaccgacc ccttcgactg caagtactgc agctaccact gtctggtgtg cttccagacc 2940aagggcctgg gcatctccta cgggcgcaag aaacggatgg agcccatcga ccccaacctg 3000gagccctgga accaccccgg cagccagccc aacaccccct gcaacaagtg ctactgcaaa 3060tactgctcct accactgcct cgtggtgggc tggcccgccg tgcgcgagcg catccgccgc 3120accgagcccg ccgccgaggg cgtgggcccc gccagccagg acctggacaa gcacggcgcc 3180ctgaccagca gcaacaccgc ccacaacaac cccgactgcg cctggctgca ggcccaggag 3240gaggaggagg acgtgggctt ccccgtgcgc ccccaggtgc ccctgcgccc catgacctac 3300aaggccgcct tcgacctgag cttcttcctg aaggagaagg gcggcctgga gggcctgatc 3360cacagcaagc gccgccagga catcctggac ctgtgggtgt accacaccca gggctacttc 3420cccgactggc agaactacac ccccggcccc ggcgtgcgct accccctgac cttcggctgg 3480tgcttcaagc tggtgcccgt ggacccccgc gaggtggagg aggccaacaa gggcgagaac 3540aactgcctgc tgcaccccat gagccagcac ggcatggagg acgccgaccg cgaggtgctg 3600cgctgggtgt tcgacagcag cctggcccgc cgccacctgg cccgcgagaa gcaccccgag 3660tactacaagg actgagaatt ctctaga 3687<210> 30<211> 1228<212> PRT
PA134027-mrcuct2.ST25<213> Human immunodeficiency virus type 1<400> 30Lys Leu Ala Thr Met Ala Ala Arg Ala Ser Ile Leu Arg Gly Glu Lys1 5 10 15Leu Asp Lys Trp Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys His20 25 30Tyr Met Leu Lys His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe35 40 45Ala Leu Asn Pro Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile50 55 60Met Lys Gln Leu Gln Pro Ala Leu Gln Thr Gly Thr Glu Glu Leu Lys65 70 75 80Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr Cys Val His Glu Lys Ile85 90 95Glu Val Arg Asp Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln100 105 110Asn Lys Cys Gln Gln Lys Thr Gln Gln Ala Lys Ala Ala Asp Gly Lys115 120 125Val Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val130 135 140His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile145 150 155 160Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu165 170 175Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val180 185 190Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu195 200 205Glu Ala Ala Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile210 215 220Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr225 230 235 240Thr Ser Thr Leu Gln Glu Gln Ile Ala Trp Met Thr Ser Asn Pro Pro245 250 255Ile Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn260 265 270
PA134027-mrcuct2.ST25Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln275 280 285Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr290 295 300Leu Arg Ala Glu Gln Ala Thr Gln Glu Val Lys Asn Trp Met Thr Asp305 310 315 320Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg325 330 335Ala Leu Gly Pro Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln340 345 350Gly Val Gly Gly Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met355 360 365Ser Gln Thr Asn Ser Gly Asn Ile Met Met Gln Arg Ser Asn Phe Lys370 375 380Gly Pro Arg Arg Ile Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His385 390 395 400Ile Ala Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys405 410 415Gly Lys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn420 425 430Phe Leu Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly Glu Phe435 440 445Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Val Gly Pro Thr Pro Val450 455 460Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Leu Gly Cys Thr Leu Asn465 470 475 480Phe Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly485 490 495Met Asp Gly Pro Lys Val Lys Gln Trp Pro Leu Thr Glu Val Lys Ile500 505 510Lys Ala Leu Thr Ala Ile Cys Glu Glu Met Glu Lys Glu Gly Lys Ile515 520 525Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr Pro Ile Phe Ala Ile530 535 540Lys Lys Glu Asp Ser Thr Lys Trp Arg Lys Leu Val Asp Phe Arg Glu545 550 555 560Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu Val Gln Leu Gly Ile Pro565 570 575
PA134027-mrcuct2.ST25His Pro Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp Val580 585 590Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp Glu Gly Phe Arg Lys Tyr595 600 605Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg610 615 620Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile625 630 635 640Phe Gln Ala Ser Met Thr Lys Ile Leu Glu Pro Phe Arg Ala Lys Asn645 650 655Pro Glu Ile Val Ile Tyr Gln Tyr Met Ala Ala Leu Tyr Val Gly Ser660 665 670Asp Leu Glu Ile Gly Gln His Arg Ala Lys Ile Glu Glu Leu Arg Glu675 680 685His Leu Leu Lys Trp Gly Phe Thr Thr Pro Asp Lys Lys His Gln Lys690 695 700Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu His Pro Asp Lys Trp705 710 715 720Thr Val Gln Pro Ile Gln Leu Pro Glu Lys Asp Ser Trp Thr Val Asn725 730 735Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Thr Ser Gln Ile Tyr740 745 750Pro Gly Ile Lys Val Arg Gln Leu Cys Lys Leu Leu Arg Gly Thr Lys755 760 765Ala Leu Thr Asp Ile Val Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu770 775 780Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro Val His Gly Val Tyr Tyr785 790 795 800Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln Lys Gln Gly Asp Asp805 810 815Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr820 825 830Gly Lys Tyr Ala Lys Arg Arg Thr Thr His Thr Asn Asp Val Lys Gln835 840 845Leu Thr Glu Ala Val Gln Lys Ile Ser Leu Glu Ser Ile Val Thr Trp850 855 860Gly Lys Thr Pro Lys Phe Arg Leu Pro Ile Gln Lys Glu Thr Trp Glu865 870 875 880
PA134027-mrcuct2.ST25Ile Trp Trp Thr Asp Tyr Trp Gln Ala Thr Trp Ile Pro Glu Trp Glu885 890 895Phe Val Asn Ser Gly Arg Lys Leu Ala Thr Met Val Gly Ile Ser Tyr900 905 910Gly Arg Lys Lys Arg Arg Gln Arg Arg Ser Thr Pro Pro Ser Ser Glu9l5 920 925Asp His Gln Asn Pro Ile Ser Lys Gln Pro Leu Pro Gln Thr Arg Gly930 935 940Asp Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val Glu Ser Lys Thr945 950 955 960Lys Thr Asp Pro Phe Asp Cys Lys Tyr Cys Ser Tyr His Cys Leu Val965 970 975Cys Phe Gln Thr Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg980 985 990Met Glu Pro Ile Asp Pro Asn Leu Glu Pro Trp Asn His Pro Gly Ser995 10001005Gln Pro Asn Thr Pro Cys Asn Lys Cys Tyr Cys Lys Tyr Cys Ser101010151020Tyr His Cys Leu Val Val Gly Trp Pro Ala Val Arg Glu Arg Ile102510301035Arg Arg Thr Glu Pro Ala Ala Glu Gly Val Gly Pro Ala Ser Gln104010451050Asp Leu Asp Lys His Gly Ala Leu Thr Ser Ser Asn Thr Ala His105510601065Asn Asn Pro Asp Cys Ala Trp Leu Gln Ala Gln Glu Glu Glu Glu107010751080Asp Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met108510901095Thr Tyr Lys Ala Ala Phe Asp Leu Ser Phe Phe Leu Lys Glu Lys110011051110Gly Gly Leu Glu Gly Leu Ile His Ser Lys Arg Arg Gln Asp Ile111511201125Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp113011351140Gln Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe114511501155Gly Trp Cys Phe Lys Leu Val Pro Val Asp Pro Arg Glu Val Glu
PA134027-mrcuct2.ST25116011651170Glu Ala Asn Lys Gly Glu Asn Asn Cys Leu Leu His Pro Met Ser117511801185Gln His Gly Met Glu Asp Ala Asp Arg Glu Val Leu Arg Trp Val119011951200Phe Asp Ser Ser Leu Ala Arg Arg His Leu Ala Arg Glu Lys His120512101215Pro Glu Tyr Tyr Lys Asp Glu Phe Ser Arg12201225<210> 31<211> 1326<212> DNA<213> Human immunodeficiency virus type 1<400> 31atggctgctc gcgcatctat cctcagaggc gaaaagttgg ataagtggga aaaaatcaga60ctcaggccag gaggtaaaaa acactacatg ctgaagcata tcgtgtgggc atctagggag 120ttggagagat ttgcactgaa ccccggactg ctggaaacct cagagggctg taagcaaatc 180atgaaacagc tccaaccagc cttgcagacc ggaacagaag agctgaagtc cctttacaat 240accgtggcaa ccctctattg cgtccacgag aagatcgagg tgagagacac aaaggaggcc 300ctggacaaaa tcgaggagga gcagaataag tgccagcaga agacccagca ggcaaaggct 360gctgacggaa aggtctctca gaactatcct atcgttcaga accttcaggg gcagatggtg 420caccaagcaa tcagccctag aaccctgaac gcatgggtga aggtgatcga ggagaaagcc 480ttttctcctg aggttatccc catgtttacc gccctgagcg aaggcgccac tcctcaagac 540ctgaacacta tgctgaacac agtgggagga caccaggccg ctatgcagat gttgaaggat 600accatcaacg aggaggcagc cgaatgggac cgcctccacc ccgtgcacgc cggacctatc 660gcccccggac aaatgagaga acctcgcgga agtgatattg ccggtactac cagcaccctt 720caagagcaga ttgcttggat gaccagcaac ccacccatcc cagtgggcga tatttacaaa 780aggtggatta ttctggggct gaacaaaatt gtgagaatgt actcccccgt ctccatcctc 840gacatccgcc aaggacccaa ggagcctttt agggattacg tggacagatt cttcaaaacc 900cttagagctg agcaagccac tcaggaggtt aagaactgga tgacagatac tctgctcgtg 960caaaacgcta accccgattg caaaaccatc ttgagagctc tcggtccagg tgccaccctt 1020gaggaaatga tgacagcatg tcaaggcgtg ggaggacctg ggcacaaggc cagagttccc 1080gctgaggcca tgagccagac aaactcaggc aatatcatga tgcagaggag taactttaag 1140ggtcccagga gaatcgtcaa gtgcttcaat tgtggcaagg agggtcacat tgccaggaac 1200tgccgcgccc ccaggaagaa aggctgctgg aagtgtggca aagagggcca ccagatgaag 1260gattgcaccg agcgccaagc aaacttcctg ggaaagattt ggcccagtca taagggccgc 1320cctggc 1326
PA134027-mrcuct2.ST25<210> 32<211> 442<212> PRT<213> Human immunodeficiency virus type 1<400> 32Met Ala Ala Arg Ala Ser Ile Leu Arg Gly Glu Lys Leu Asp Lys Trp1 5 10 15Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys His Tyr Met Leu Lys20 25 30His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro35 40 45Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Met Lys Gln Leu50 55 60Gln Pro Ala Leu Gln Thr Gly Thr Glu Glu Leu Lys Ser Leu Tyr Asn65 70 75 80Thr Val Ala Thr Leu Tyr Cys Val His Glu Lys Ile Glu Val Arg Asp85 90 95Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Cys Gln100 105 110Gln Lys Thr Gln Gln Ala Lys Ala Ala Asp Gly Lys Val Ser Gln Asn115 120 125Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile130 135 140Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala145 150 155 160Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Ala165 170 175Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln180 185 190Ala Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala Glu195 200 205Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln210 215 220Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu225 230 235 240Gln Glu Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly
PA134027-mrcuct2.ST25245 250 255Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg260 265 270Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu275 280 285Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu290 295 300Gln Ala Thr Gln Glu Val Lys Asn Trp Met Thr Asp Thr Leu Leu Val305 310 315 320Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg Ala Leu Gly Pro325 330 335Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly340 345 350Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Thr Asn355 360 365Ser Gly Asn Ile Met Met Gln Arg Ser Asn Phe Lys Gly Pro Arg Arg370 375 380Ile Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile Ala Arg Asn385 390 395 400Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly405 410 415His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu Gly Lys420 425 430Ile Trp Pro Ser His Lys Gly Arg Pro Gly435 440<210> 33<211> 1397<212> DNA<213> Human immunodeficiency virus type 1<400> 33gggaaagatt tggcccagtc ataagggccg ccctggcgaa ttctgcggca agaaggccat60cggcaccgtg ctggtgggcc ccacccccgt gaacatcatc ggccggaaca tgctgaccca 120gctgggctgc accctgaact tccccatcag ccccatcgag accgtgcccg tgaagctgaa 180gcccggcatg gacggcccca aggtgaagca gtggcccctg accgaggtga agatcaaggc 240cctgaccgcc atctgcgagg agatggagaa ggagggcaag atcaccaaga tcggccccga 300gaacccctac aacaccccca tcttcgccat caagaaggag gacagcatca agtggcggaa 360gctggtggac ttccgggagc tgaacaagcg gacccaggac ttctgggagg tgcagctggg 420
PA134027-mrcuct2.ST25tatcccccac cccgccggcc tgaagaagaa gaagagcgtg accgtgctgg acgtgggcga 480cgcctacttc agcgtgcccc tggacgaggg cttccggaag tacaccgcct tcaccatccc 540cagcatcaac aacgagaccc ccggcatccg gtaccagtac aacgtgctgc cccagggctg 600gaagggcagc cccgccatct tccaggccag catgaccaag atcctggagc ccttccgggc 660caagaacccc gagatcgtga tctaccagta catggccgcc ctgtacgtgg gcagcgacct 720ggagatcggc cagcaccggg ccaagatcga ggagctgcgg gagcacctgc tgaagtgggg 780cttcaccacc cccgacaaga agcaccagaa ggagcccccc ttcctgtgga tgggctacga 840gctgcacccc gacaagtgga ccgtgcagcc catccagctg cccgagaagg acagctggac 900cgtgaacgac atccagaagc tggtgggcaa gctgaactgg accagccaga tctaccccgg 960catcaaggtg cggcagctgt gcaagctgct gcggggcacc aaggccctga ccgacatcgt 1020gcccctgacc gaggaggccg agctggagct ggccgagaac cgggagatcc tgaaggagcc 1080cgtgcacggc gtgtactacg accccagcaa ggacctgatc gccgagatcc agaagcaggg 1140cgacgaccag tggacctacc agatctacca ggagcccttc aagaacctga aaaccggcaa 1200gtacgccaag cggcggacca cccacaccaa cgacgtgaag cagctgaccg aggccgtgca 1260gaagatcagc ctggagagca tcgtgacctg gggcaagacc cccaagttcc ggctgcccat 1320ccagaaggag acctgggaga tctggtggac cgactactgg caggccacct ggatccccga 1380gtgggagttc gtgaaca 1397<210> 34<211> 451<212> PRT<213> Human immunodeficiency virus type 1<400> 34Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Val Gly Pro Thr Pro Val1 5 10 15Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Leu Gly Cys Thr Leu Asn20 25 30Phe Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly35 40 45Met Asp Gly Pro Lys Val Lys Gln Trp Pro Leu Thr Glu Val Lys Ile50 55 60Lys Ala Leu Thr Ala Ile Cys Glu Glu Met Glu Lys Glu Gly Lys Ile65 70 75 80Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr Pro Ile Phe Ala Ile85 90 95Lys Lys Glu Asp Ser Thr Lys Trp Arg Lys Leu Val Asp Phe Arg Glu100 105 110
PA134027-mrcuct2.ST25Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu Val Gln Leu Gly Ile Pro115 120 125His Pro Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp Val130 135 140Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp Glu Gly Phe Arg Lys Tyr145 150 155 160Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg165 170 175Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile180 185 190Phe Gln Ala Ser Met Thr Lys Ile Leu Glu Pro Phe Arg Ala Lys Asn195 200 205Pro Glu Ile Val Ile Tyr Gln Tyr Met Ala Ala Leu Tyr Val Gly Ser210 215 220Asp Leu Glu Ile Gly Gln His Arg Ala Lys Ile Glu Glu Leu Arg Glu225 230 235 240His Leu Leu Lys Trp Gly Phe Thr Thr Pro Asp Lys Lys His Gln Lys245 250 255Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu His Pro Asp Lys Trp260 265 270Thr Val Gln Pro Ile Gln Leu Pro Glu Lys Asp Ser Trp Thr Val Asn275 280 285Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Thr Ser Gln Ile Tyr290 295 300Pro Gly Ile Lys Val Arg Gln Leu Cys Lys Leu Leu Arg Gly Thr Lys305 310 315 320Ala Leu Thr Asp Ile Val Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu325 330 335Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro Val His Gly Val Tyr Tyr340 345 350Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln Lys Gln Gly Asp Asp355 360 365Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr370 375 380Gly Lys Tyr Ala Lys Arg Arg Thr Thr His Thr Asn Asp Val Lys Gln385 390 395 400Leu Thr Glu Ala Val Gln Lys Ile Ser Leu Glu Ser Ile Val Thr Trp405 410 415
PA134027-mrcuct2.ST25Gly Lys Thr Pro Lys Phe Arg Leu Pro Ile Gln Lys Glu Thr Trp Glu420 425 430Ile Trp Trp Thr Asp Tyr Trp Gln Ala Thr Trp Ile Pro Glu Trp Glu435 440 445Phe Val Asn450<210> 35<211> 1479<212> DNA<213> Human immunodeficiency virus type 1<400> 35atgggtgcga gagcgtcaat attaagaggg gaaaaattag ataaatggga aaaaattagg60ttaaggccag ggggaaagaa acattatatg ttaaaacaca tagtatgggc aagcagggag 120ctggaaagat ttgcacttaa ccctggcctt ttagaaacat cagaaggatg taaacaaata 180atgaaacagc tacaaccagc tctccagaca ggaacagagg aacttaaatc attatacaac 240acagtagcaa ctctctattg tgtacatgaa aagatagaag tacgagacac caaggaagcc 300ttagataaga tagaggaaga acaaaacaaa tgtcagcaaa aaacgcagca ggcaaaagcg 360gctgacggga aagtcagtca aaattatcct atagtgcaga atctccaagg gcaaatggta 420catcaagcca tatcacctag aaccttgaat gcatgggtaa aagtaataga agaaaaggct 480tttagcccag aggtaatacc catgtttaca gcattatcag aaggagccac cccacaagat 540ttaaacacca tgttaaatac agtgggggga catcaagcag ccatgcaaat gttaaaagat 600actattaatg aagaggctgc agaatgggat agagtacatc cagtccatgc ggggcctatt 660gcaccaggcc agatgagaga accaagggga agtgacatag caggaactac tagtaccctt 720caggaacaaa tagcatggat gacaagtaac ccacctattc cagtgggaga catctataaa 780agatggataa ttctggggtt aaataaaata gtgagaatgt atagcccggt cagcattttg 840gacataagac aagggccaaa ggaacccttt cgagactatg tagatcggtt ctttaaaact 900ttaagagctg aacaagctac acaagaagta aaaaattgga tgacagacac cttgttagtc 960caaaatgcga acccagattg taagaccatt ttgagagcat taggaccagg ggctacatta 1020gaagaaatga tgacagcatg tcaaggggtg ggaggacctg gtcacaaagc aagagtattg 1080gctgaggcaa tgagtcaagc aaacagtgga aacataatga tgcagagaag caattttaaa 1140ggccctagaa gaattgttaa atgttttaac tgtggcaagg aagggcacat agccagaaat 1200tgcagagccc ctaggaaaaa aggctgttgg aaatgtggaa aggaaggaca ccaaatgaaa 1260gactgtactg aaaggcaggc taatttttta gggaaaattt ggccttccca caaggggagg 1320ccagggaatt tccttcagaa cagaccagag ccaacagccc caccagcaga gagcttcagg 1380ttcgaagaga caacccccgc tccgaaacag gagccgatag aaagggaacc cttaacttcc 1440ctcaaatcac tctttggcag cgaccccttg tctcaataa 1479
PA134027-mrcuct2.ST25<210> 36<211> 492<212> PRT<213> Human immunodeficiency virus type 1<400> 36Met Gly Ala Arg Ala Ser Ile Leu Arg Gly Glu Lys Leu Asp Lys Trp1 5 10 15Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys His Tyn Met Leu Lys20 25 30His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro35 40 45Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Met Lys Gln Leu50 55 60Gln Pro Ala Leu Gln Thr Gly Thr Glu Glu Leu Lys Ser Leu Tyr Asn65 70 75 80Thr Val Ala Thr Leu Tyr Cys Val His Glu Lys Ile Glu Val Arg Asp85 90 95Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Cys Gln100 105 110Gln Lys Thr Gln Gln Ala Lys Ala Ala Asp Gly Lys Val Ser Gln Asn115 120 125Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile130 135 140Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala145 150 155 160Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Ala165 170 175Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln180 185 190Ala Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala Glu195 200 205Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln210 215 220Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu225 230 235 240Gln Glu Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly245 250 255
PA134027-mrcuct2.ST25Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg260 265 270Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu275 280 285Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu290 295 300Gln Ala Thr Gln Glu Val Lys Asn Trp Met Thr Asp Thr Leu Leu Val305 310 315 320Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg Ala Leu Gly Pro325 330 335Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly340 345 350Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Ala Asn355 360 365Ser Gly Asn Ile Met Met Gln Arg Ser Asn Phe Lys Gly Pro Arg Arg370 375 380Ile Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile Ala Arg Asn385 390 395 400Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly405 410 415His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu Gly Lys420 425 430Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe Leu Gln Asn Arg435 440 445Pro Glu Pro Thr Ala Pro Pro Ala Glu Ser Phe Arg Phe Glu Glu Thr450 455 460Thr Pro Ala Pro Lys Gln Glu Pro Ile Glu Arg Glu Pro Leu Thr Ser465 470 475 480Leu Lys Ser Leu Phe Gly Ser Asp Pro Leu Ser Gln485 490<210> 37<211> 2997<212> DNA<213> Human immunodeficiency virus type 1<400> 37tttagggaaa atttggcctt cccacaaggg gaggccaggg aatttccttc agaacagacc60
PA134027-mrcuct2.ST25agagccaaca gccccaccag cagagagctt caggttcgaa gaaacaaccc ccgctccgaa 120acaggagccg agagaaaggg aacccttaac ttccctcaaa tcactctttg gcagcgaccc 180cttgtctcaa taaaaatagg gggccagaca agggaggctc tcttagacac aggagcagat 240gatacagtat tagaagacat aaatttgcca ggaaaatgga aaccaaaaat gataggagga 300attggaggtt ttatcaaagt aagacagtat gatcaaatac ttatagaaat ttgtggaaaa 360aaggctatag gtacagtatt agtagggcct acacctgtca acataattgg cagaaacatg 420ttgactcagc ttggatgcac actaaacttt ccaatcagtc ccattgaaac tgtaccagta 480aaactgaagc caggaatgga tggcccaaag gttaaacaat ggccgttaac agaagagaaa 540ataaaagcat taacagcaat ttgtgaagaa atggaaaagg aaggaaaaat tacaaaaatt 600gggcctgaaa atccatataa cactccaata tttgccataa aaaagaaaga cagcactaag 660tggagaaaat tagtagattt cagggaactc aataaaagaa ctcaagactt ttgggaggtt 720caattaggaa taccacaccc agcagggtta aaaaagaaaa aatcagtgac agtactggat 780gtgggagatg catatttttc agttccttta gatgaaggct tcaggaaata tactgcattc 840accataccta gtataaacaa tgaaacacca gggattagat atcaatataa tgtgcttcca 900caaggatgga aagggtcacc agcaatattc cagggtagca tgacaaaaat cttagagccc 960tttagagctc aaaatccaga aatagtcatc tatcaatata tggatgactt gtatgtagga1020tctgacttag aaatagggca acatagagca aaaatagaag agttaagaga acatctatta1080aagtggggat ttaccacacc agacaaaaaa catcagaaag aacccccatt tctttggatg1140gggtatgaac tccatcctga caaatggaca gtacagccta tacagctgcc agaaaaggat1200agctggactg tcaatgatat acagaagtta gtgggaaaat taaactgggc aagtcagatt1260tacccaggga ttaaagtaag gcaactttgt aagctcctta gggggaccaa agcactaaca1320gacatagtac cactaactga agaagcagaa ttagaattgg cagagaacag ggaaattcta1380aaagaaccag tgcatggagt atattatgac ccatcaaaag acttgatagc tgaaatacag1440aaacaggggg atgaccaatg gacatatcaa atttaccaag aaccattcaa aaacctgaag1500acaggaaagt atgcaaaaag gaggactacc cacactaatg atgtaaaaca gttaacagag1560gcagtgcaaa aaatatcctt ggaaagcata gtaatatggg gaaagactcc taaatttaga1620ctacccatcc aaaaagaaac atgggaaata tggtggacag actattggca agccacatgg1680attcctgagt gggagtttgt taatacccct cccctagtaa aactatggta ccagctagaa1740aaagaaccca tagcaggagc agaaactttc tatgtagatg gagcagctaa tagggaaact1800aaaataggaa aagcggggta tgttactgac agaggaaggc agaaaattgt aactctaagt1860gaaacaacaa atcagaagac tgaattacaa gcaattcagc tagctttgca agattcagaa1920tcagaagtaa acataataac agactcacag tacgcattag gaatcattca agcacaacca1980gataggagtg aatcagagtt ggtcaatcaa ataatagaac aattaataaa aaaggaaagg2040gtctatctgt catgggtacc agcacacaac ggacttgcag gaaatgaaca tgtagataaa2100ttagtaagta ggggaatcag gaaagtgctg gttctagatg gaatagataa ggctcatgaa2160gagcatgaaa agtatcacag caattggaga gcaatggcta gtgagtttaa tctgccaccc2220gtagtagcaa gagaaatagt agccagctgt gataaatgtc agctaaaagg ggaagccata2280catggacaag tagattgtag tccggggata tggcaattag attgtacaca tttagaagga2340
PA134027-mrcuct2.ST25aaaatcatcc tggtagcagt ccatgtagcc agtggctaca tagaagcaga ggttatccca 2400gcagaaacag gacaagaaac agcatactat atactaaaat tagcaggaag atggccagtc 2460aaagtaatac atacagacaa tggcagtaat ttcaccagtg ctgcagttaa ggcagcctgt 2520tggtgggcag gtatccaaca ggaatttggg attccctaca atccccaaag tcagggagta 2580gtagaatcca tgaataaaga attaaagaaa atcatagggc aggtaagaga tcaagctgag 2640caccttaaga cagcagtaca aatggcagta ttcattcaca attttaaaag aaaagggggg 2700attggggggt acagtgcagg ggaaagaata atagacataa tagcaacaga catacaaact 2760aaagaattac aaaaacaaat tataaaaatt caaaattttc gggtttatta cagagacagc 2820agagatccta tttggaaagg accagccaag ctactctgga aaggtgaagg ggcagtagta 2880atacaagaca acagtgacat aaaggtagta ccaaggagga aagtaaaaat cattagggac 2940tatggaaaac agatggcagg tgctgattgt gtggcaggta gacaggatga agattag 2997<210> 38<211> 998<212> PRT<213> Human immunodeficiency virus type 1<400> 38Phe Arg Glu Asn Leu Ala Phe Pro Gln Gly Glu Ala Arg Glu Phe Pro1 5 10 15Ser Glu Gln Thr Arg Ala Asn Ser Pro Thr Ser Arg Glu Leu Gln Val20 25 30Arg Arg Asn Asn Pro Arg Ser Glu Thr Gly Ala Glu Arg Lys Gly Thr35 40 45Leu Asn Phe Pro Gln Ile Thr Leu Trp Gln Arg Pro Leu Val Ser Ile50 55 60Lys Ile Gly Gly Gln Thr Arg Glu Ala Leu Leu Asp Thr Gly Ala Asp65 70 75 80Asp Thr Val Leu Glu Asp Ile Asn Leu Pro Gly Lys Trp Lys Pro Lys85 90 95Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln100 105 110Ile Leu Ile Glu Ile Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Val115 120 125Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Leu130 135 140Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Val Pro Val145 150 155 160
PA134027-mrcuct2.ST25Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Lys Gln Trp Pro Leu165 170 175Thr Glu Glu Lys Ile Lys Ala Leu Thr Ala Ile Cys Glu Glu Mer Glu180 185 190Lys Glu Gly Lys Ile Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr195 200 205Pro Ile Phe Ala Ile Lys Lys Lys Asp Ser Thr Lys Trp Arg Lys Leu210 215 220Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu Val225 230 235 240Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys Lys Lys Lys Ser Val245 250 255Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp Glu260 265 270Gly Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu275 280 285Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys290 295 300Gly Ser Pro Ala Ile Phe Gln Gly Ser Met Thr Lys Ile Leu Glu Pro305 310 315 320Phe Arg Ala Gln Asn Pro Glu Ile Val Ile Tyr Gln Tyr Mer Asp Asp325 330 335Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln His Arg Ala Lys Ile340 345 350Glu Glu Leu Arg Glu His Leu Leu Lys Trp Gly Phe Thr Thr Pro Asp355 360 365Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu370 375 380His Pro Asp Lys Trp Thr Val Gln Pro Ile Gln Leu Pro Glu Lys Asp385 390 395 400Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp405 410 415Ala Ser Gln Ile Tyr Pro Gly Ile Lys Val Arg Gln Leu Cys Lys Leu420 425 430Leu Arg Gly Thr Lys Ala Leu Thr Asp Ile Val Pro Leu Thr Glu Glu435 440 445Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro Val450 455 460
PA134027-mrcuct2.ST25His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln465 470 475 480Lys Gln Gly Asp Asp Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe485 490 495Lys Asn Leu Lys Thr Gly Lys Tyr Ala Lys Arg Arg Thr Thr His Thr500 505 510Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln Lys Ile Ser Leu Glu515 520 525Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe Arg Leu Pro Ile Gln530 535 540Lys Glu Thr Trp Glu Ile Trp Trp Thr Asp Tyr Trp Gln Ala Thr Trp545 550 555 560Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro Leu Val Lys Leu Trp565 570 575Tyr Gln Leu Glu Lys Glu Pro Ile Ala Gly Ala Glu Thr Phe Tyr Val580 585 590Asp Gly Ala Ala Asn Arg Glu Thr Lys Ile Gly Lys Ala Gly Tyr Val595 600 605Thr Asp Arg Gly Arg Gln Lys Ile Val Thr Leu Ser Glu Thr Thr Asn610 615 620Gln Lys Thr Glu Leu Gln Ala Ile Gln Leu Ala Leu Gln Asp Ser Glu625 630 635 640Ser Glu Val Asn Ile Ile Thr Asp Ser Gln Tyr Ala Leu Gly Ile Ile645 650 655Gln Ala Gln Pro Asp Arg Ser Glu Ser Glu Leu Val Asn Gln Ile Ile660 665 670Glu Gln Leu Ile Lys Lys Glu Arg Val Tyr Leu Ser Trp Val Pro Ala675 680 685His Asn Gly Leu Ala Gly Asn Glu His Val Asp Lys Leu Val Ser Arg690 695 700Gly Ile Arg Lys Val Leu Val Leu Asp Gly Ile Asp Lys Ala His Glu705 710 715 720Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala Met Ala Ser Glu Phe725 730 735Asn Leu Pro Pro Val Val Ala Arg Glu Ile Val Ala Ser Cys Asp Lys740 745 750Cys Gln Leu Lys Gly Glu Ala Ile His Gly Gln Val Asp Cys Ser ProPage 32
PA134027-mrcuct2.ST25755 760 765Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu Gly Lys Ile Ile Leu770 775 780Val Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro785 790 795 800Ala Glu Thr Gly Gln Glu Thr Ala Tyr Tyr Ile Leu Lys Leu Ala Gly805 810 815Arg Trp Pro Val Lys Val Ile His Thr Asp Asn Gly Ser Asn Phe Thr820 825 830Ser Ala Ala Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Gln Gln Glu835 840 845Phe Gly Ile Pro Tyr Asr Pro Gln Ser Gln Gly Val Val Glu Ser Met850 855 860Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu865 870 875 880His Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile His Asn Phe Lys885 890 895Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Ile Asp900 905 910Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Ile915 920 925Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Ile930 935 940Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val945 950 955 960Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro Arg Arg Lys Val Lys965 970 975Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala Gly Ala Asp Cys Val Ala980 985 990Gly Arg Gln Asp Glu Asp99權(quán)利要求
1.一種分子,該分子包含(I)如圖1所示的核苷酸序列(SEQ I.D.No.1);(II)圖1所示的核苷酸序列(SEQ I.D.No.1)所相應(yīng)的RNA序列;(III)一種與圖1所示的核苷酸序列(SEQ I.D.No.1)相比至少具有97%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,且其顯示基本相似的免疫原性;(IV)一種與SEQ I.D.No.1給出的核苷酸序列同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其是(I)-(IV)中任一序列的修飾物或衍生物。
2.如權(quán)利要求1的分子,其中修飾序列是圖9和10任一所示的序列(SEQI.D.Nos.9和10)。
3.一種分子,該分子包含(I)如圖3所示的核苷酸序列(SEQ I.D.No.3);(II)圖3所示的核苷酸序列(SEQ I.D.No.3)所相應(yīng)的RNA序列;(III)一種與圖3所示的核苷酸序列(SEQ I.D.No.3)相比至少具有97%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,且其顯示基本相似的免疫原性;(IV)一種與圖3所示的核苷酸序列(SEQ I.D.No.3)同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其是(I)-(IV)中任一序列的修飾物或衍生物。
4.如權(quán)利要求3的分子,其中修飾序列是圖9和10任一所示的序列(SEQI.D.Nos.9和10)。
5.一種分子,該分子包含(I)如圖5所示的核苷酸序列(SEQ I.D.No.5);(II)圖5所示的核苷酸序列(SEQ I.D.No.5)所相應(yīng)的RNA序列;(III)一種與圖5所示的核苷酸序列(SEQ I.D.No.5)相比至少具有98%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,且其顯示基本相似的免疫原性;(IV)一種與圖5所示的核苷酸序列(SEQ I.D.No.5)同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其是(I)-(IV)中任一序列的修飾物或衍生物。
6.如權(quán)利要求5的分子,其中修飾序列是圖12和13任一所示的序列(SEQI.D.Nos.11和13)。
7.一種分子,該分子包含(I)如圖7所示的核苷酸序列(SEQ I.D.No.7);(II)圖7所示的核苷酸序列(SEQ I.D.No.7)所相應(yīng)的RNA序列;(III)一種與圖7所示的核苷酸序列(SEQ I.D.No.7)相比至少具有96%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,且其顯示基本相似的免疫原性;(IV)一種與圖7所示的核苷酸序列(SEQ I.D.No.7)同源的序列、或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其是(I)-(IV)中任一序列的修飾物或衍生物。
8.如權(quán)利要求7的分子,其中修飾序列是圖12和13任一所示的序列(SEQI.D.Nos.12和13)。
9.一種分子,該分子包含(I)如圖15所示的核苷酸序列(SEQ I.D.No.15);(II)圖15所示的核苷酸序列(SEQ I.D.No.15)所相應(yīng)的RNA序列;(III)一種與圖15所示的核苷酸序列(SEQ I.D.No.15)相比至少具有90%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,且其顯示基本相似的免疫原性;(IV)一種與圖15所示的核苷酸序列(SEQ I.D.No.15)同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其是(I)-(IV)中任一序列的修飾物或衍生物。
10.一種分子,該分子包含(I)如圖17所示的核苷酸序列(SEQ I.D.No.17);(II)圖17所示的核苷酸序列(SEQ I.D.No.17)所相應(yīng)的RNA序列;(III)一種與圖17所示的核苷酸序列(SEQ I.D.No.17)相比至少具有90%DNA相似性的序列、或該序列所相應(yīng)的RNA序列,且其顯示基本相似的免疫原性;(IV)一種與圖17所示的核苷酸序列(SEQ I.D.No.17)同源的序列,或該序列所相應(yīng)的RNA序列;或者(V)一種序列,其是(I)-(IV)中任一序列的修飾物或衍生物。
11.一種多肽,該多肽包含(I)如圖2所示的氨基酸序列(SEQ I.D.No.2);(II)一種與圖2的序列相比具有至少95%相似性的序列,且其具有基本相似的免疫原性;或者(III)一種序列,其是圖2所示的氨基酸序列(SEQ I.D.No.2)的修飾物或衍生物。
12.如權(quán)利要求11的多肽,其中修飾序列是圖11所示的序列(SEQ I.D.No.11)。
13.一種多肽,該多肽包含(I)如圖4所示的氨基酸序列(SEQ I.D.No.4);(II)一種與圖4的序列相比具有至少95%相似性的序列,且其具有基本相似的免疫原性;或者(III)一種序列,其是圖4所示的氨基酸序列(SEQ I.D.No.4)的修飾物或衍生物。
14.如權(quán)利要求13的多肽,其中修飾序列是圖11所示的序列(SEQ I.D.No11)。
15.一種多肽,該多肽包含(I)如圖6所示的氨基酸序列(SEQ I.D.No.6);(II)一種與圖6的序列相比具有至少92%相似性的序列,且其具有基本相似的免疫原性;或者(III)一種序列,其是圖6所示的氨基酸序列(SEQ I.D.No.6)的修飾物或衍生物。
16.如權(quán)利要求15的多肽,其中修飾序列是圖14所示的序列(SEQ I.D.No.14)。
17.一種多肽,該多肽包含(I)如圖8所示的氨基酸序列(SEQ I.D.No.8);(II)一種與圖8的序列相比具有至少95%相似性的序列,且其具有基本相似的免疫原性;或者(III)一種序列,其是圖8所示的氨基酸序列(SEQ I.D.No.8)的修飾物或衍生物。
18.如權(quán)利要求17的多肽,其中修飾序列是圖14所示的序列(SEQ I.D.No.14)。
19.一種多肽,該多肽包含(I)如圖16所示的核苷酸序列(SEQ I.D.No.16);(II)一種與圖16的序列相比具有至少90%相似性的序列,且其具有基本相似的免疫原性;或者(III)一種序列,其是圖16所示的氨基酸序列(SEQ I.D.No.16)的修飾物或衍生物。
20.HIV-1C亞型的tat基因的共有氨基酸序列如下MEPVDPNLEPWNHPGSQPKTACNKCYCKHCSYHCLVCFQTKGLGISYGRKKRRQRRSAPP60SSEDHQNLISKQPLPQTRGDPTGSEESKKKVESKTETDPFD101(SEQ ID NO18)
21.HIV-1C亞型的部分nef基因的共有氨基酸序列如下MGGKWSKSSIVGWPAVRERIRRTEPAAEGVGAASQDLDKHGALTSSNTAHNNADCAWLQA60QEEEEEVGFPVRPQVPLRPMTYKGAFDLSFFLKEKGGLEGLIYSKKRQEILDLWVYHTQG120FFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANEGENNCLIHPMSQHGMEDEDRE180VLKWKFDSSLARRHMARELHPEYYKDC207(SEQ ID NO19)
22.HIV-1C亞型的部分rev基因的共有氨基酸序列如下MAGRSGDSDEALIQAVRIIKILYQSNPYPKPEGTRQARKNRRRRWRARQRQIHSISERIL60STCLGRPAEPVPLQLPPIERLHIDCSESSGTSGTQQSQQTTEGVGSP107(SEQ ID NO20)
23.權(quán)利要求1和2所述序列的至少一種、權(quán)利要求3和4所述序列的至少一種、權(quán)利要求5和6所述序列的至少一種以及權(quán)利要求7和8所述序列的至少一種在生產(chǎn)用于治療或預(yù)防HIV感染的疫苗中的應(yīng)用。
24.權(quán)利要求1至22所述的序列中至少兩種序列在生產(chǎn)用于治療或預(yù)防HIV感染的疫苗中的應(yīng)用。
25.包含權(quán)利要求1和2所述序列的至少一種、權(quán)利要求3和4所述序列的至少一種、權(quán)利要求5和6所述序列的至少一種以及權(quán)利要求7和8所述序列的至少一種的疫苗。
26.包含權(quán)利要求1至22所述序列中至少兩種序列的疫苗。
27.包含至少部分的gag基因序列、逆轉(zhuǎn)錄酶(pol)基因序列、改排tat基因序列和截取的nef基因序列的疫苗,這些基因序列結(jié)合在一起形成框內(nèi)多基因,以grttnC表示(SEQ I.D.No30)。
28.權(quán)利要求25至27任一所述的用于治療或預(yù)防HIV的疫苗。
29.權(quán)利要求25至28任一所述的基本上如這里所述或舉例說(shuō)明的疫苗。
全文摘要
本發(fā)明描述HIV-1亞型分離調(diào)節(jié)/附加基因及其修飾物和衍生物。所描述的基因有tat、nef和rev基因。并公開(kāi)了共有氨基酸系列。本發(fā)明還涉及包括兩個(gè)或多個(gè)核苷酸序列的疫苗以及來(lái)自HIV-1的pol和/或gag基因的核苷酸序列。
文檔編號(hào)C12N15/49GK1606625SQ02825711
公開(kāi)日2005年4月13日 申請(qǐng)日期2002年10月31日 優(yōu)先權(quán)日2001年10月31日
發(fā)明者卡羅琳·威廉森, 喬安妮·海迪·范哈梅倫, 克萊夫·莫里斯·格雷, 威廉·博恩, 薩利姆·阿卜杜勒·卡里姆 申請(qǐng)人:南非醫(yī)學(xué)研究會(huì), 開(kāi)普敦大學(xué)