CN1419456A - 用于hiv预防或治疗性免疫的疫苗 - Google Patents
用于hiv预防或治疗性免疫的疫苗 Download PDFInfo
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- CN1419456A CN1419456A CN01807156A CN01807156A CN1419456A CN 1419456 A CN1419456 A CN 1419456A CN 01807156 A CN01807156 A CN 01807156A CN 01807156 A CN01807156 A CN 01807156A CN 1419456 A CN1419456 A CN 1419456A
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明提供:a)HIV Tat蛋白或多核苷酸;或b)HIV Nef蛋白或多核苷酸;或c)与HIV Nef蛋白或多核苷酸连接的HIV Tat蛋白或多核苷酸(Nef-Tat);和HIV gp120蛋白或多核苷酸在生产人类HIV预防或治疗性免疫疫苗中的应用。
Description
本发明涉及HIV蛋白在医药中的新用途以及包含所述HIV蛋白的疫苗组合物。本发明尤其涉及HIV Tat和HIV gp120蛋白的联合应用。此外,本发明涉及HIV Nef和HIV gp120蛋白的联合应用。
HIV-1是获得性免疫缺陷综合征(AIDS)的主要原因,获得性免疫缺陷综合征被认为是世界主要健康问题之一。尽管世界范围内进行了深入研究以便获得疫苗,但是这方面的努力迄今仍然没有获得成功。
HIV包膜糖蛋白gp120是用于粘附宿主细胞的病毒蛋白。这种粘附通过与称为CD4的辅助T细胞和巨噬细胞的两种表面分子和两种趋化因子受体CCR-4或CXCR-5之一结合而介导产生。gp120蛋白首先表达为较大的前体分子(gp160),然后翻译后切割产生gp120和gp41。gp120蛋白通过与gp41分子(它插入病毒膜中)连接而保留在病毒体表面。
gp120蛋白是中和抗体的主要靶,但遗憾的是gp120蛋白最具免疫原性的区段(V3环)也是该蛋白变异性最大的部分。因此,认为前体gp120(或其前体gp160)用作激发中和抗体的疫苗抗原对于广泛的保护性疫苗仅具有有限的作用。gp120确实还含有细胞毒性T淋巴细胞(CTL)识别性表位。CTL效应细胞能够消除病毒感染细胞,因此构成第二种主要抗病毒免疫机制。与中和抗体的目标区段相反,某些CTL表位似乎在不同HIV病毒株中相对保守。为此,认为gp120和gp160是疫苗有效抗原性组分,目的在于激发细胞介导的免疫应答(特别是CTL)。
已经有关于HIV-1非包膜蛋白的介绍,包括例如内部结构蛋白,例如gag和pol基因产物,以及其它非结构蛋白,例如Rev、Nef、Vif和Tat(Greene等,New England J.Med,324,5,308以及下列文献等等(1991);Bryant等(Ed.Pizzo),Pediatr.Infect.Dis.J.,11,5,390以下下列文献等等(1992)。
HIV Tat和Nef蛋白是早期蛋白,即它们在感染早期及缺乏结构蛋白的情况下表达。
在会议论文集中(C.David Pauza,Immunization with Tat toxoidattenuates SHIV89.6PD infection in rhesus macaques,12th Cent Gardesmeeting,Mames-La-Coquette,26.10.1999),介绍了各种实验,其中单独用Tat类毒素或者联用包膜糖蛋白gp160疫苗组合(一剂重组痘苗病毒和一剂重组蛋白)免疫恒河猴。然而,所观测到的结果表明,存在包膜糖蛋白并不优于单独用Tat进行的实验。
但是,我们发现在保护恒河猴免受嵌合人-猿猴免疫缺陷病毒(SHIV)致病性攻击时,包含Tat-和/或Nef-的免疫原(尤其是Nef-Tat融合蛋白)与gp120协同作用。迄今,认为恒河猴SHIV感染是人AIDS最相关的动物模型。所以,我们应用该临床前模型评价了单独或者组合包含gp120抗原和含Nef-及Tat-的抗原的疫苗保护效力。分析两种病毒感染和致病性标记,即恒河猴外周血CD4阳性细胞百分率和血浆游离SHIV RNA基因组浓度,表明所述两种抗原协同作用。单独用gp120或NefTat+SIV Nef免疫与只用佐剂免疫相比,没有任何差异。相反,联合给予gp120和NefTat+SIV Net抗原导致上述具体实验组所有动物的两种上述参数明显改善。
因此,本发明提供HIV Tat和/或Nef蛋白和HIV gp120一起在生产用于人HIV预防或治疗性免疫的疫苗中的新用途。
如上所述,相对于只用NefTat+SIV Nef或gp120观测到的结果,一起给予NefTat蛋白、SIV Nef蛋白和gp120蛋白的免疫应答增强。这种增强作用或协同作用作为应用上述组合蛋白免疫的结果可以表现为病毒负荷下降。另一方面,增强作用本身还表现为CD4+维持水平高于没有用HIV NefTat、SIV Nef和HIV gp120免疫的维持水平。这种协同作用的原因是gp120与Tat或者gp120与Nef、或者gp120与Nef和Tat二者组合所致。
加入其它HIV蛋白还可进一步增强gp120与Tat和/或Nef之间观测到的协同作用。所述其它蛋白也可以与含gp120、Tat和/或Nef的疫苗各个组分协同作用,而不需要存在所有原抗原组合。所述其它蛋白可以是HIV调节蛋白,例如Rev、Vif、Vpu和Vpr。它们也可以是得自HIV gag或pol基因的结构蛋白。
HIV gag基因编码前体蛋白p55,p55能够自动装配成未成熟病毒样颗粒(VLP)。然后,前体蛋白p55蛋白酶解为主要结构蛋白p24(壳体蛋白)和p18(基质蛋白),以及若干较小蛋白。可以认为前体蛋白p55和其主要衍生物p24及p18均为合适疫苗抗原,它们可以进一步增强gp120与Tat和/或Nef之间观测到的协同作用。前体蛋白p55和壳体蛋白p24可用作VLP或单体蛋白。
本发明疫苗中HIV Tat蛋白可以任选与HIV Nef蛋白连接,例如作为融合蛋白。
本发明中的HIV Tat蛋白、HIV Nef蛋白或NefTat融合蛋白可以具有C末端组氨酸尾,优选包含5-10个组氨酸残基。存在的组氨酸(或者“His”)尾有助于纯化。
在一个优选实施方案中,所述蛋白表达有组氨酸尾,包含5-10个、优选6个组氨酸残基。它们的优点是有助于纯化。已有报道,Nef(Macreadie I.G.等,1993,Yeast 9(6)565-573)和Tat(Braddock M等,1989,Cell 58(2)269-79)在酵母(酿酒酵母(Saccharomyces cerevisiae))中独立表达。Nef蛋白和Gag蛋白p55和p18是豆蔻酸化蛋白。以前的WO99/16884介绍Nef和Tat在毕赤酵母表达系统(Nef-His和Tat-His构建体)中独立表达,以及表达融合构建物Nef-Tat-His。
典型的Nef-His(Seq.ID.No.8和9)、Tat-His(Seq.ID.No.10和11)和Nef-Tat-His融合蛋白(Seq.ID.No.12和13)的DNA和氨基酸序列见图1。
本发明HIV蛋白可以以其天然构象使用,或者对于疫苗应用更优选可以被修饰。所述修饰可能因为涉及纯化方法的技术原因需要进行修饰,或者可用于使Tat或Nef蛋白的一种或多种功能特性生物失活。所以,本发明包括HIV蛋白衍生物,HIV蛋白衍生物可以为例如突变蛋白。术语“突变”本文用以指使用广为人知的定点诱变技术或任何其它常规方法缺失、添加或置换了一个或多个氨基酸的分子。
举例来说,可以使Tat突变蛋白突变,使其生物失活,同时还保留其免疫原性表位。D.Clements(Tulane University)构建的一种可能突变tat基因(源自BH10分子克隆)在活性位点区(Lys41→Ala)和RGD基元(Arg78→Lys和Asp80→Glu)中存在突变(Virology 235:48-64,1997)。
图1(Seq.ID.No.22和23)图示突变Tat以及Nef-Tat突变体-His(Seq.ID.No.24和25)。
本发明疫苗中HIV Tat或Nef蛋白可以在纯化过程中通过化学方法修饰,使得所述蛋白为稳定单体蛋白。一种防止诸如Tat或Nef的蛋白氧化聚集的方法是利用化学修饰所述蛋白巯基。第一步是用还原剂如DTT、β-巯基乙醇或谷胱甘肽处理还原二硫键。第二步是使产生的巯基与烷化剂反应而使其封闭(例如可用碘乙酰胺使所述蛋白羧酰胺化/脲基甲基化)。根据细胞结合测定以及对人外周血单核细胞的淋巴细胞增殖的抑制作用评价,所述化学修饰不会改变Tat或Nef的功能特性。
可用以下实施例概述的方法纯化HIV Tat蛋白和HIV gp120蛋白。
本发明疫苗包含免疫保护量或者免疫治疗量的Tat和/或Nef或者NefTat及gp120抗原,而且可用常规技术制备。
有关疫苗制剂的全面介绍参见New Trends and Developments inVaccines,Voller等主编,University Park Press,Baltimore,Maryland,U.S.A.1978。例如Fullerton的美国专利4,235,877介绍了脂质体中的包囊化。例如Likhite的美国专利4,372,945和Armor等的美国专利4,474,757公开了蛋白质与大分子的缀合。
疫苗制剂中蛋白量根据在典型接种者体内诱导免疫保护应答而没有显著副作用的剂量进行选择。所述蛋白量随所使用的具体免疫原而不同。一般来说,预期每剂包含1-1000μg各蛋白,优选2-200μg、最优选4-40μg的Tat或Nef或NefTat,而优选1-150μg、最优选2-25μg gp120。具体疫苗的最佳量可通过包括观测患者体内的抗体滴度和其它反应的标准研究而确定。一个疫苗剂量具体实例包含20μgNefTat和5或20μg gp120。初次免疫接种后,患者可于约4周内接受一次加强免疫,以及可以接受后续的第二次加强免疫。
在本发明疫苗制剂中本发明蛋白优选佐剂化。关于佐剂的全面介绍见Vaccine Design-the Subunit and Adjuvant Approach,Powell和Newman主编,Plenum Press,New York,1995。
合适的佐剂包括铝盐,诸如氢氧化铝凝胶(alum)或磷酸铝,但也可以是钙盐、铁盐或锌盐,或者可以是酰化酪氨酸或酰化糖的不溶性悬浮液,阳离子或阴离子衍化的多糖或聚磷腈。
本发明疫苗制剂中,优选所述佐剂组合物优先诱导Th1型应答。然后,应当知道,不排除其它应答,包括其它体液免疫应答。
通过所述抗原与免疫系统细胞相互作用,从而对所述抗原产生免疫应答。产生的免疫应答可主要分为两大类:体液免疫应答或细胞介导的免疫应答(传统上其特征分别在于保护性抗体机制和效应细胞机制)。这两类免疫应答称为Th1型反应(细胞介导的应答)以及Th2型免疫应答(体液免疫应答)。
非常典型的Th1型免疫应答的特征可能在于产生抗原特异性单倍型限制性细胞毒性T淋巴细胞以及天然杀伤细胞反应。在小鼠中,通常Th1型应答的特征在于产生IgG2a亚型抗体,而在人类中这些抗体相当于IgG1型抗体。Th2型免疫应答的特征在于产生各种各样的免疫球蛋白同种型,在小鼠包括IgG1、IgA和IgM。
可以认为产生这两种类型免疫应答的驱动力是细胞因子,细胞因子是为数众多的已鉴定的蛋白信使,其作用是辅助免疫系统细胞,控制最终免疫应答为Th1或Th2型应答。因此,高水平Th1型细胞因子往往促进诱导对给定抗原的细胞介导的免疫应答,而高水平Th2型细胞因子往往促进诱导对抗原的体液免疫应答。
重要的是记住,Th1型免疫应答和Th2型免疫应答的区别并不是绝对的。实际上,有个别人支持将免疫应答描述为主要是Th1型或主要是Th2型的免疫应答。然而,按照Mosmann和Coffman描述小鼠CD4+ve T细胞克隆时采用的细胞因子家族分类来考虑细胞因子家族常常是便利的(Mosmann,T.R.和Coffman,R.L.(1998)Th1和Th2细胞:不同淋巴因子分泌谱诱导致不同功能特性.Annual Review ofImmunology,7,第145-173页)。传统上,Th1型反应与T淋巴细胞产生INF-γ和IL-2细胞因子有关。其它常常与诱导Th1型免疫应答直接相关的细胞因子如IL-12并不是T细胞产生的。相反,Th2型反应与IL-4、IL-5、IL-6、IL-10和肿瘤坏死因子β(TNF-β)分泌有关。
已知某些疫苗佐剂特别适于刺激Th1型或Th2型细胞因子反应。传统上,在免疫接种或感染后免疫应答的Th1:Th2平衡的最佳指标包括用抗原再刺激后直接测量体外T淋巴细胞产生的Th1或Th2细胞因子,和/或测量抗原特异性抗体反应的IgG1:IgG2a比例。
因此,Th1型佐剂是在体外用抗原再刺激时刺激分离的T细胞产生高水平Th1型细胞因子而且诱导产生与Th1型同种型有关的抗原特异性免疫球蛋白反应的佐剂。
适用于本发明、可配制产生佐剂的优选Th1-型免疫刺激剂包括但不限于下列佐剂。
单磷酰脂质A、尤其是3-de-O-酰化单磷酰脂质A(3D-MPL)是优选用于本发明的Th1-型免疫刺激剂。3D-MPL是Ribi Immunochem,Montana生产的众所周知的佐剂。化学上它常常以具有4、5或6个酰化链的3-de-O-酰化单磷酰脂质A的混合物提供。3-de-O-酰化单磷酰脂质A的混合物可用GB 2122204B中介绍的方法纯化制得,所述专利文献还公开了二磷酰脂质A及其3-O-脱酰化变异体的制备。其它纯化的合成脂多糖已有介绍(US 6,005,099和EP 0729 473 B1;Hilgers等,1986,Int.Arch.Allrgy.Immunol.,79(4):392-6;Hilgers等,1987,Immunology,60(1):141-6;以及EP 0 549 074 B1)。优选型3D-MPL为0.2μm以下直径的颗粒型制剂,其生产方法公开于EP 0689 454。
皂苷也是本发明的优选Th1免疫刺激剂。皂苷是周知的佐剂,参见:Lacaille-Dubois,M和Wagner H.(1996,皂苷生物及药理活性综述,Phytomedicine第2卷,第363-386页)。例如Quil A(从南美洲的树Quilaja Saponaria Molina获得)及其各部分介绍见US 5,057,540和“皂苷用作疫苗佐剂”,Kensil,C.R.,Crit Rev Ther Drug Carrier Syst,1996,12(1-2):1-55;以及EP 0 362 279 B1。已有介绍溶血性皂苷QS21和QS17(Quil A的HPLC纯化部分)为有效的系统佐剂,其生产方法公开于美国专利号5,057,540和EP 0 362 279 B1。此外,上述参考文献还介绍QS7(Quil-A的非溶血性部分)用作系统疫苗的有效佐剂。QS21的应用详见Kensil等(1991,J.Immunology第146卷,431-437)。QS21和聚山梨醇酯或环糊精联合应用也是已知的(WO 99/10008)。包含Quil A部分如QS21和QS7的颗粒性佐剂系统见WO 96/33739和WO 96/11711。
另一种优选免疫刺激剂为包含未甲基化CpG二核苷酸(“CpG”)的免疫刺激性寡核苷酸。CpG是存在于DNA中的胞嘧啶-鸟苷二核苷酸基元的缩写。CpG为通过系统和粘膜途径给予的佐剂,这是本领域已知的(WO 96/02555,EP 468520,Davis等,J.Immunol,1998,160(2):870-876;McCluskie和Davis,J.Immunol.,1998,161(9):4463-6)。曾经观测到BCG的DNA部分可以发挥抗肿瘤效应。进一步研究表明,根据BCG基因序列获得的合成寡核苷酸能够诱导免疫刺激作用(体外及体内均具有这种作用)。上述研究的作者得出结论,某些回文序列,包括中央CG基元,具有所述免疫刺激作用。CG基元在免疫刺激中的关键作用后来由Krieg,Nature 374,第546页,1995公开阐明。详细分析表明,CG基元必定存在于某一序列环境,而且所述序列在细菌DNA中是普遍性的,而在脊椎动物DNA中是罕见性的。免疫刺激性序列常常是:嘌呤、嘌呤、C、G、嘧啶、嘧啶;其中CG基元是非甲基化的,但是已知其它未甲基化CpG序列也是免疫刺激性的而且可用于本发明。
在某些6个核苷酸组合中存在回文序列。数个所述基元无论是重复的一个基元还是不同基元的组合,均可存在于同一寡核苷酸中。存在一种或多种包含所述免疫刺激性序列的寡核苷酸可以激活各种免疫细胞亚群,包括天然杀伤细胞(产生γ干扰素而且具有细胞溶解活性)和巨噬细胞(Wooldrige等,第89卷(第8期),1977)。目前,还证实其它不具有所述共有序列的含未甲基化CpG的序列具有免疫调节性。
配制成疫苗时,CpG的一般给予形式为:与游离抗原一起的游离溶液(WO 96/02555;McCluskie及Davis,同上),或者与抗原共价结合(WO 98/16247),或者用诸如氢氧化铝的载体配制((肝炎表面抗原)Davis等,同上;Brazolot-Millan等,Proc.Natl.Acad.Sci.,USA,1998,95(26),15553-8)。
如上所述的免疫刺激剂可以与载体配制在一起,例如脂质体、水包油乳剂,和或金属盐,包括铝盐(例如氢氧化铝)。例如3D-MPL可以用氢氧化铝(EP 0 689 454)或水包油乳剂(WO 95/17210)配制;QS21可优选用含胆固醇的脂质体(WO 96/33739)、水包油乳剂(WO95/17210)或alum(WO 98/15287)配制;CpG可用alum(Davis等,同上;Brazolot-Millan,同上)或者用其它阳离子载体配制。
此外,优选免疫刺激剂组合应用,尤其是单磷酰脂质A和皂苷衍生物组合(WO 94/00153;WO 95/17210;WO 96/33739;WO 98/56414;WO 99/12565;WO 99/11241),更优选WO 94/00153公开的QS21和3D-MPL组合。另一方面,CpG+皂苷如QS21组合也构成可用于本发明的有效佐剂。
因此,合适的佐剂系统包括例如单磷酰脂质A、优选3D-MPL与铝盐组合。增强系统包括单磷酰脂质A和皂苷衍生物组合,特别是在WO 94/00153中公开的QS21和3D-MPL的组合,或者在WO96/33739中公开的QS21在含胆固醇的脂质体(DQ)中猝灭的反应原性较小的组合物。
WO 95/17210介绍了特别有效的佐剂制剂,它包括QS21、3D-MPL和维生素E的水包油乳剂,是可用于本发明的另一种优选制剂。
另一种优选制剂仅含有CpG寡核苷酸或者含有CpG寡核苷酸和铝盐。
在本发明另一方面中,所述疫苗可包含编码Tat、Nef和gp120多肽中的一种或多种多肽的DNA,使得原位产生所述多肽。所述DNA可以存在于本领域技术人员已知的众多传递系统中的任何一种传递系统中,包括核酸表达系统(如质粒DNA)、细菌和病毒表达系统。众多基因传递技术是本领域周知的,例如Rolland,Crit.Rev.Therap.Drug Carrier Systems 15:143-198,1998和其中引用的参考文献描述的传递技术。合适的核酸表达系统包含在患者体内表达所必需的DNA序列(例如合适的启动子和终止信号)。当表达系统为重组的活微生物时,例如病毒或细菌,目的基因可以插入活的重组病毒或细菌基因组中。用这种活的载体接种和体内感染导致体内表达所述抗原并诱导免疫应答。用于该目的的病毒和细菌例如:痘病毒(如痘苗病毒、禽痘病毒、金丝雀痘病毒、修饰的痘病毒如Modified VirusAnkara(MVA))、甲病毒属(新培斯病毒、塞姆利基森林病毒、委内瑞拉马脑炎病毒)、黄病毒(黄热病病毒、登革病毒、日本脑炎病毒)、腺病毒、腺伴随病毒、小核糖核酸病毒(脊髓灰质炎病毒、鼻病毒)、疱疹病毒(水痘-带状疱疹病毒等)、李斯特氏菌属(Listeria)、沙门氏菌属(Salmonella)、志贺氏菌属(Shigella)、奈瑟氏菌属(Neisseria)、BCG。这些病毒和细菌可以是有毒力的,或者以各种方式减毒以获得活疫苗。这样的活疫苗也构成本发明的一部分。
因此,本发明优选疫苗的Nef、Tat和gp120组分可以以编码所需蛋白的多核苷酸形式提供。
而且,可用组合的蛋白及DNA型制剂按照本发明进行免疫接种。认为初免-加强免疫诱导宽范围免疫应答是有效的。佐剂化蛋白疫苗主要诱导抗体和T辅助性免疫应答,而质粒或活载体传递的DNA诱导强细胞毒性T淋巴细胞(CTL)应答。因此,蛋白和DNA免疫结合可产生各种各样的免疫应答。这在HIV情况下特别有关,因为认为中和抗体和CTL对免疫防御HIV均是重要的。
按照本发明,gp120、Nef和Tat单独或组合免疫方案可以包括序贯(“初免-加强”)或同时给予蛋白抗原和编码上述蛋白的DNA。所述DNA可以以质粒DNA或重组活载体形式传递,例如痘病毒载体或者任何其它合适活载体,例如本文上述活载体。蛋白抗原可以注射一次或多次,然后给予一次或多次DNA,或者首先给予DNA一次或多次,然后进行一次或多次蛋白免疫。
本发明初免-加强免疫的具体实例包括用重组活载体形式(例如修饰痘病毒载体,如Modified Virus Ankara(MVA),或者甲病毒,如委内瑞拉马脑炎病毒)的DNA初免,然后用蛋白、优选佐剂化蛋白加强免疫。
所以,本发明进一步提供包含以下组分的药盒:
a)包含gp120、Nef和Tat蛋白中的一种或多种蛋白和药学上可接受的赋形剂的组合物;和
b)包含gp120、Nef和Tat编码多核苷酸中的一种或多种多核苷酸和药学上可接受的赋形剂的组合物;前提是(a)或(b)中的至少一种包含gp120和Nef和/或Tat和/或Nef-Tat。
组合物a)和b)可以单独以任何顺序给予或者一起给予。优选a)包含全部三种gp120、Nef和Tat蛋白。优选b)包含全部三种gp120、Nef和Tat DNA。最优选Nef和Tat为NefTat融合蛋白形式。
本发明再一方面提供生产本文上述疫苗制剂的方法,其中所述方法包括混合本发明组合蛋白。所述蛋白组合物可以与合适佐剂、任选载体混合。
可用于本发明制剂的特别优选的佐剂和/或载体组合如下:
i)3D-MPL+DQ中的QS21
ii)Alum+3D-MPL
iii)Alum+DQ中的QS21+3D-MPL
iv)Alum+CpG
v)3D-MPL+DQ中的QS21+水包油乳剂
vi)CpG
下面的实施例和附图阐明本发明。实施例:总体介绍
从Bru/Lai分离株(Cell 40:9-17,1985)选择Nef基因用于以下实验的构建物,因为该基因属于与共有Nef最密切相关的基因。
用于Bru/Lai Nef基因的原料是在哺乳动物表达载体pcDNA3(pcDNA3/Nef)上克隆的1170bp DNA片段。
Tat基因源自BH10分子克隆。该基因作为被称作pCV1的HTLVIII cDNA克隆为人们所接受,在Science,229,第69-73页,1985中对其进行了描述。
可在毕赤酵母或者任何其它宿主中表达Nef和Tat基因。实施例1.在巴斯德毕赤酵母中表达HIV-1 nef和tat序列
在甲基营养酵母巴斯德毕赤酵母中,在诱导型醇氧化酶(AOX1)启动子的控制下,表达Nef蛋白、Tat蛋白和Nef-Tat融合蛋白。
为了表达这些HIV-1基因,使用修饰型整合载体PHIL-D2(INVITROGEN)。该载体以这样一种方式修饰:异源蛋白的表达紧接在AOXl基因的天然ATG密码子之后,并产生具有一个甘氨酸和六个组氨酸残基尾的重组蛋白。通过在PHIL-D2载体的相邻AsuII和EcoRI位点之间克隆寡核苷酸接头,构建此PHIL-D2-MOD载体(参见图2)。除了His尾之外,此接头还携带NcoI、SpeI和XbaI限制位点,在所述限制位点之间插入nef、tat和nef-tat融合物。1.1构建整合型载体pRIT14597(编码Nef-His蛋白)、pRIT14598(编码Tat-His蛋白)和pRIT14599(编码融合物Nef-Tat-His)。用引物01和02经PCR由pcDNA3/Nef质粒扩增nef基因。
获得的PCR片段和PHIL-D2-MOD整合型载体都用NcoI和SpeI限制,在琼脂糖凝胶上纯化,连接产生整合型质粒pRIT14597(见图2)。
在所述PCR片段的5’末端引入一个NcoI限制位点,而在3’末端用引物04引入一个SpeI位点。获得的PCR片段和PHIL-D2-MOD载体都用NcoI和SpeI限制,在琼脂糖凝胶上纯化,连接产生整合型质粒pRIT14598。
为构建pRIT14599,在PHIL-D2-MOD载体的EcoRI平端(T4聚合酶)和NcoI位点之间连接相当于nef-tat-His编码序列的910bp DNA片段。通过XbaI平端(T4聚合酶)和NcoI消化pRIT14596获得nef-tat-His编码片段。1.2巴斯德毕赤酵母菌株GS115(his4)的转化。
为获得表达Nef-His、Tat-His和融合蛋白Nef-Tat-His的巴斯德毕赤酵母菌株,用携带相应表达盒加上HIS4基因的线性NotI片段转化菌株GS115,以互补宿主基因组中的his4。用NotI-线性片段转化GS115有利于在AOXI基因座重组。
通过定量斑点印迹分析选择多拷贝整合克隆,并确定整合、插入(Mut+表型)或置换(Muts表型)的类型。
从每次转化中选择显示高水平产生重组蛋白的一种转化体:
产生重组Nef-His蛋白(一种豆蔻酸化的215个氨基酸的蛋白)的菌株Y1738(Mut+表型),所述重组Nef-His蛋白组成如下:
°豆蔻酸
°蛋氨酸,利用PHIL-D2-MOD载体的NcoI克隆位点产生
°205个氨基酸的Nef蛋白(从氨基酸2开始,延伸至氨基酸206)
°通过克隆步骤产生的一个苏氨酸和一个丝氨酸(在PHIL-D2-MOD载体的SpeI位点克隆)。
°一个甘氨酸和六个组氨酸。
产生Tat-His蛋白(一种95个氨基酸的蛋白)的菌株Y1739(Mut+表型),所述Tat-His蛋白组成如下:
°蛋氨酸,利用NcoI克隆位点产生
°85个氨基酸的Tat蛋白(从氨基酸2开始,延伸至氨基酸86)
°通过克隆步骤引入的一个苏氨酸和一个丝氨酸
°一个甘氨酸和六个组氨酸
产生重组Nef-Tat-His融合蛋白(一种豆蔻酸化的302个氨基酸的蛋白)的菌株Y1737(Muts表型),所述Nef-Tat-His融合蛋白组成如下:
°豆蔻酸
°蛋氨酸,利用NcoI克隆位点产生
°205个氨基酸的Nef蛋白(从氨基酸2开始,延伸至氨基酸206)
°通过克隆步骤产生的一个苏氨酸和一个丝氨酸
°85个氨基酸的Tat蛋白(从氨基酸2开始,延伸至氨基酸86)
°通过克隆步骤引入的一个苏氨酸和一个丝氨酸
°一个甘氨酸和六个组氨酸实施例2.
在巴斯德毕赤酵母中表达HIV-1 Tat-突变体
还表达了突变重组Tat蛋白。突变Tat蛋白在保持其免疫原性表位的同时,必须是生物学上无活性的。
选择由D.Clements(Tulane大学)构建的双突变tat基因用于这些构成物。
该tat基因(源自BH10分子克隆)在活性位点区(Lys41→Ala)和在RGD基元(Arg78→Lys和Asp80→Glu)中具有突变(Virology235:48-64,1997)。
所述突变tat基因被认为是在CMV表达质粒(pCMVLys41/KGE)中于EcoRI和HindIII位点之间亚克隆的cDNA片段。2.1构建整合型载体pRIT14912(编码Tat突变体-His蛋白)和pRIT14913(编码融合物Nef-Tat突变体-His)。
用引物05和04经PCR(参见1.1部分pRIT14598的构建)由pCMVLys41/KGE质粒扩增tat突变基因。
在所述PCR片段的5’末端引入一个NcoI限制位点,而在3’末端用引物04引入一个SpeI位点。获得的PCR片段和PHIL-D2-MOD载体都用NcoI和SpeI限制,在琼脂糖凝胶上纯化,连接产生整合型质粒pRIT14912。
为了构建pRIT14913,用引物03和04经PCR由pCMVLys41/KGE质粒扩增tat突变基因。
获得的PCR片段和质粒pRIT14597(表达Nef-His蛋白)都用SpeI限制酶消化,在琼脂糖凝胶上纯化,连接产生整合型质粒pRIT14913。2.2巴斯德毕赤酵母菌株GS115的转化。
通过使用先前在1.2部分中描述的整合和重组菌株选择策略,获得表达Tat突变体-His蛋白和融合物Nef-Tat突变体-His的
巴斯德毕 赤酵母菌株。
选择出两种产生Tat突变体-His蛋白(一种95个氨基酸的蛋白)的重组菌株:Y1775(Mut+表型)和Y1776(Muts表型)。
选择出一种表达Nef-Tat突变体-His融合蛋白(一种302个氨基酸的蛋白)的重组菌株:Y1774 Mut+表型)。实施例3:发酵产生重组蛋白TAT-HIS的巴斯德毕赤酵母典型过程见下表。
发酵包括产生高细胞密度培养物的生长期(根据适当曲线添加甘油型培养基)以及诱导期(添加甲醇和盐/微量元素溶液)。发酵中,生长期跟踪取样检测其620nm的吸光度。诱导期中通过泵加入甲醇,通过气相色谱(对培养物样品)和用质谱仪在线气体分析监测甲醇浓度。发酵后,于2-8℃以5020g离心30’回收细胞,细胞浆状物保藏于-20℃。为了进行后续工作,解冻细胞浆状物,以150 OD(620nm)重悬浮于缓冲液(Na2HPO4 pH7 50mM、PMSF 5%、异丙醇4mM),通过DynoMill(空间0.6L,3000rpm,6L/H,珠粒直径0.40-0.70mm)破碎细胞4次。
为了评价表达情况,诱导期取出样品,破碎细胞,应用SDS-PAGE或蛋白质印迹方法进行分析。考马斯蓝染色SDS-凝胶时,明确鉴定重组Tat-his为深带,约72-96H诱导后出现最大密度。
发酵用培养基:固体预培养:(YNB+葡萄糖+琼脂)葡萄糖: 10g/l Na2MoO4.2H2O: 0.0002g/l Acide folique: 0.000064g/lKH2PO4: 1g/l MnSO4.H2O: 0.0004g/l 肌醇: 0.064g/lMgSO4.7H2O 0.5g/l H3BO3: 0.0005g/l 维生素B6: 0.008g/lCaCl2.2H2O: 0.1g/l KI: 0.0001g/l 硫胺素: 0.008g/lNaCl: 0.1g/l CoC12.6H2O: 0.00009g/l 烟酸: 0.000032g/lFeCl3.6H2O: 0.0002g/l 核黄素: 0.000016g/l Panthoténate Ca:0.008g/lCuSO4.5H2O: 0.00004g/l 生物素: 0.000064g/l 对氨基苯甲酸: 0.000016g/lZnSO4.7H2O: 0.0004g/l (NH4)2SO4: 5g/l 琼脂: 18g/l液体预培养:(YNB+甘油)甘油: 2%(v/v) Na2MoO4.2H2O:0.0002g/l Acide folique: 0.000064g/lKH2PO4: 1g/l MnSO4.H2O: 0.0004g/l 肌醇: 0.064g/lMgSO4.7H2O 0.5g/l H3BO3: 0.0005g/l 维生素B6: 0.008g/lCaCl2.2H2O: 0.1g/l KI: 0.0001g/l 硫胺素: 0.008g/lNaCl: 0.1g/l CoCl2.6H2O: 0.00009g/l 烟酸: 0.000032g/lFeCl3.6H2O: 0.0002g/l 核黄素: 0.000016g/l Panthoténate Ca:0.008g/lCuSO4.5H2O: 0.00004g/l 生物素: 0.000064g/l 对氨基苯甲酸: 0.000016g/lZnSO4.7H2O: 0.0004g/l (NH4)2SO4: 5g/l初始发酵罐装料:(FSC006AA)(NH4)2SO4: 6.4g/lKH2PO4: 9g/l Na2MoO4.2H2O: 2.04mg/lMgSO4.7H2O 4.7g/l MnSO4.H2O: 4.08mg/lCaCl2.2H2O: 0.94g/l H3BO3: 5.1mg/lFeCl3.6H2O: 10mg/l KI: 1.022mg/lHCl: 1.67mg/l CoCl2.6H2O: 0.91mg/lCuSO4.5H2O: 0.408mg/l NaCl: 0.06g/lZnSO4.7H2O: 4.08mg/l 生物素: 0.534mg/l生长期用加料液:(FFB005AA)甘油 38.7%v/v Na2MoO4.2H2O: 5.7mg/lMgSO4.7H2O 13g/l CuSO4.5H2O: 1.13mg/lCaCl2.2H2O: 2.6g/l CoCl2.6H2O: 2.5mg/lFeCl3.6H2O: 27.8mg/l H3BO3: 14.2mg/lZnSO4.7H2O: 11.3mg/l 生物素: 1.5mg/lMnSO4.H2O: 11.3mg/l KI: 2.84mg/lKH2PO4: 24.93g/l NaCl: 0.167g/l诱导期用盐和微量元素加料液(FSE021AB):KH2PO4: 45g/l Na2MoO4.2H2O: 10.2mg/lMgSO4.7H2O 23.5g/l MnSO4.H2O: 20.4mg/lCaCl2.2H2O: 4.70g/l H3BO3: 25.5mg/lNaCl: 0.3g/l KI: 5.11mg/lHCl: 8.3ml/l CoCl2.6H2O: 4.55mg/lCuSO4.5H2O: 2.04mg/l FeCl3.6H2O: 50.0mg/lZnSO4.7H2O: 20.4mg/l 生物素: 2.70mg/l实施例4:纯化Nef-Tat-His融合蛋白(巴斯德毕赤酵母)
解冻工作种子瓶 | |
↓ | |
固体预培养30℃,14-16H | 合成培养基:YNB+葡萄糖+琼脂 |
↓ | |
在两个2L锥形瓶中液体预培养30℃,200rpm | 合成培养基:2×400ml YNB+甘油当OD>1(620nm)时终止 |
↓ | |
接种到20L发酵罐 | 5L初始培养基(FSC006AA)3ml消泡剂SAG471(Witco)设定条件:温度:30℃超压:0.3barg气流:20Nl/min溶解氧:调控>40%pH:用氢氧化铵调节于5 |
↓ | |
补料分批发酵:生长期持续约40H | 加料:甘油型培养基FFB005AA最终OD值:200-500 OD(620nm) |
补料分批发酵:诱导期持续至多97H | 加料:甲醇和盐/微量元素溶液(FSE021AB) |
↓ |
离心 | 5020g/30min/2-8℃ |
↓ | |
回收细胞浆状物,保藏于-20℃ | |
↓ | |
解冻细胞,以OD150(620nm)重悬浮于缓冲液 | 缓冲液:Na2HPO4 pH7 50mM,PMSF5%,异丙醇4mM |
↓ | |
通过Dyno-mill破碎细胞4次 | Dyno-mill:(空间0.6L,3000rpm,6L/H,珠粒直径0.40-0.70mm) |
↓ | |
转移用于提取/纯化 |
由146克重组巴斯德毕赤酵母细胞(湿重)或2L Dyno-mill匀浆OD55进行纯化流程。在室温下进行层析步骤。在步骤之间,Nef-Tat阳性流分在冷室(+4℃)中保存过夜;更长时间时,将样品于-20℃冷冻保存。146克巴斯德毕赤酵母细胞
↓
匀浆
缓冲液:2L 50mM PO4 pH7.0
最终OD:50
↓Dyno-mill破碎(4次)
↓
离心 JA10转子/9500rpm/30min/室温
↓Dyno-mill沉淀
↓洗涤(1小时-4℃)
缓冲液:+2L 10mM PO4pH7.5-150
mM-NaCl 0.5%empigen
↓
离心 JA10转子/9500rpm/30min/室温
↓
沉淀
↓溶解(O/N-4℃)
缓冲液:+660ml 10mM PO4 pH7.5-
150mM NaCl-4.0M GuHCl
↓
还原 +0.2M 2-巯基乙磺酸钠盐(加入粉末)/在
(4小时-室温-暗处) 温育前调节pH至7.5(用0.5M NaOH
溶液)
↓
脲基甲基化 +0.25M碘乙酰胺(加入粉末)/在温育前
(半小时-室温-暗处) 调节pH至7.5(用0.5M NaOH溶液)
↓在Ni++-NTA-琼脂糖(Qiagen-
平衡缓冲液:10mM PO4 pH7.5-15030ml树脂)上固定化金属离子 mM NaCl-4.0M GuHCl亲和层析
洗涤缓冲液:
1)平衡缓冲液
2)10mM PO4 pH7.5-150mM NaCl-
6M尿素
3)10mM PO4 pH7.5-150mM NaCl-
6M尿素-25aM咪唑
洗脱缓冲液:10mM PO4 pH7.5-150mM
NaCl-6M尿素-0.5M咪唑
↓稀释 离子强度降至18mS/cm2
稀释缓冲液:10mM PO4 pH7.5-6M
尿素
↓SP Sepharose FF(Pharmacia-
平衡缓冲液:10mM PO4 pH7.5-150mM30ml树脂)阳离子交换层析 NaCl-6.0M尿素
洗涤缓冲液:
1)平衡缓冲液
2)10mM PO4 pH7.5-250mM NaCl-
6M尿素
洗脱缓冲液:10mM硼酸盐pH9.0-2
M NaCl-6M尿素
↓
浓缩 至5mg/ml
10kDa Omega膜(Filtron)
↓Superdex200 XK 16/60凝胶过
洗脱缓冲液:10mM PO4 pH7.5-150mM滤层析(Pharmacia-120ml树 NaCl-6M尿素
脂) 5ml样品/注射→注射5次
↓
透析(O/N-4℃)
缓冲液:10mM PO4 pH6.8-150mM
NaCl-0.5M精氨酸*
↓
过滤除菌 Millex GV 0.22μm
*比率:0.5M精氨酸:1600μg/ml蛋白浓度。纯度
图3通过Daiichi银染色和图4通过考马斯蓝G250显示由SDS-PAGE估计的纯度水平。
Superdex200步骤后: >95%
透析和过滤除菌步骤后: >95%回收
由146克重组巴斯德毕赤酵母细胞(=2L Dyno-mill匀浆OD 55)纯化得到51mg Nef-Tat-his蛋白。实施例5:纯化巴斯德毕赤酵母中的氧化型Nef-Tat-His融合蛋白
由73克重组巴斯德毕赤酵母细胞(湿重)或1L Dyno-mill匀浆OD50进行纯化流程。在室温下进行层析步骤。在步骤之间,Nef-Tat阳性流分在冷室(+4℃)中保存过夜;更长时间时,将样品于-20℃冷冻保存。
73克巴斯德毕赤
酵母细胞
↓
匀浆
缓冲液:1L 50mM PO4 pH7.0-Pefabloc
5mM
最终OD:50
↓Dyno-mill破碎(4次)
↓
离心 JA10转子/9500rpm/30min/室温
↓Dyno-mill沉淀
↓洗涤(2小时-4℃)
缓冲液:+1L 10mM PO4 pH7.5-150
mM-NaCl 0.5%Empigen
↓
离心 JA10转子/9500rpm/30min/室温
↓
沉淀
↓溶解(O/N-4℃)
缓冲液:+330ml 10mM PO4 pH7.5-
↓ 150mM NaCl-4.0M GuHCl在Ni++-NTA-琼脂糖(Qiagen-
平衡缓冲液:10mM PO4 pH7.5-15015ml树脂)上固定化金属离子 mMNaCl-4.0M GuHCl
亲和层析
洗涤缓冲液:
1)平衡缓冲液
2)10mM PO4 pH7.5-150mM NaCl-
6M尿素
3)10mM PO4 pH7.5-150mM NaCl-
6M尿素-25mM咪唑
洗脱缓冲液:10mM PO4 pH7.5-150mM
NaCl-6M尿素-0.5M咪唑
↓
稀释 离子强度降至18mS/cm2
稀释缓冲液:10mM PO4 pH7.5-6M
尿素
↓SP Sepharose FF(Pharmacia-
平衡缓冲液:10mM PO4 pH7.5-150mM7ml树脂)阳离子交换层析 NaCl-6.0M尿素
洗涤缓冲液:
1)平衡缓冲液
2)10mM PO4 pH7.5-250mM NaCl-
6M尿素
洗脱缓冲液:10mM硼酸盐pH9.0-2
M NaCl-6M尿素
↓
浓缩 至0.8mg/ml
10kDa Omega膜(Filtron)
↓
透析(O/N-4℃)
缓冲液:10mM PO4 pH6.8-150mM
NaCl-0.5M精氨酸
↓
过滤除菌 Millex GV 0.22μm→
图6显示由SDS-PAGE估计的纯度水平(Daiichi银染色、考马斯蓝 G250、蛋白质印迹法):透析和过滤除菌步骤后: >95%→
回收(蛋白质比色测定评价:DOC TCA BCA)
由73克重组巴斯德毕赤酵母细胞(湿重)或1L Dyno-mill匀浆OD50纯化得到2.8mg氧化型Nef-Tat-his蛋白。实施例6:纯化还原型TAT-HIS蛋白(巴斯德毕赤酵母)
由160克重组巴斯德毕赤酵母细胞(湿重)或2L Dyno-mill匀浆OD66进行纯化流程。在室温下进行层析步骤。在步骤之间,Tat阳性流分在冷室(+4℃)中保存过夜;更长时间时,将样品于-20℃冷冻保存。
160克巴斯德毕赤
酵母细胞
↓
匀浆
缓冲液:+2L 50mM PO4 pH7.0-4mM
PMSF
最终OD:66
↓Dyno-mill破碎(4次)
↓
离心 JA10转子/9500rpm/30min/室温
↓Dyno-mill沉淀
↓洗涤(1小时-4℃)
缓冲液:+2L 10mM PO4 pH7.5-150
mM NaCl-1%Empigen
↓
离心 JA10转子/9500rpm/30min/室温
↓
沉淀
↓溶解(O/N-4℃)
缓冲液:+660ml 10mM PO4 pH7.5-
150mM NaCl-4.0M GuHCl
↓
离心 JA10转子/9500rpm/30min/室温
↓
还原 +0.2M 2-巯基乙磺酸钠盐(加入粉末)/在(4小时-室温-暗处) 温育前调节pH至7.5(用1M NaOH溶
液)
↓
脲基甲基化 +0.25M碘乙酰胺(加入粉末)/在温育前
(半小时-室温-暗处) 调节pH至7.5(用1M NaOH溶液)
↓在Ni++-NTA-琼脂糖(Qiagen-
平衡缓冲液:10mM PO4 pH7.5-15060ml树脂)上固定化金属离子 mM NaCl-4.0M GuHCl
亲和层析
洗涤缓冲液:
1)平衡缓冲液
2)10mM PO4 pH7.5-150mM NaCl-
6M尿素
3)10mM PO4 pH7.5-150mM NaCl-
6M尿素-35mM咪唑
洗脱缓冲液:10mM PO4pH7.5-150mM
NaCl-6M尿素-0.5M咪唑
↓
稀释 离子强度降至12mS/cm
稀释缓冲液:20mM硼酸盐pH8.5-6M
尿素
↓SP Sepharose FF(Pharmacia-
平衡缓冲液:20mM硼酸盐pH8.5-30ml树脂)阳离子交换层析 150mM NaCl-6.0M尿素
洗涤缓冲液:平衡缓冲液
洗脱缓冲液:20mM硼酸盐pH8.5-
400mM NaCl-6.0M尿素
↓
浓缩 至1.5mg/ml
10kDa Omega膜(Filtron)
↓
透析(O/N-4℃)
缓冲液:10mM PO4 pH6.8-150mM
NaCl-0.5M精氨酸
↓
过滤除菌 Millex GV 0.22μm→
图7显示由SDS-PAGE估计的纯度水平(Daiichi银染色、考马斯蓝 G250、蛋白质印迹法):
透析和过滤除菌步骤后: >95%→
回收(蛋白质比色测定评价:DOC TCA BCA)
由160克重组巴斯德毕赤酵母细胞(湿重)或2L Dyno-mill匀浆OD66纯化得到48mg还原型Tat-his蛋白。实施例7:纯化氧化型Tat-his蛋白(巴斯德毕赤酵母)
由74克重组巴斯德毕赤酵母细胞(湿重)或1L Dyno-mill匀浆OD60进行纯化流程。在室温下进行层析步骤。在步骤之间,Tat阳性流分在冷室(+4℃)中保存过夜;更长时间时,将样品于-20℃冷冻保存。
74克巴斯德毕赤
酵母细胞
↓
匀浆
缓冲液:+1L 50mM PO4 pH7.0-5mM
Pefabloc
最终OD:60
↓Dyno-mill破碎(4次)
↓
离心 JA10转子/9500rpm/30min/室温
↓Dyno-mill沉淀
↓洗涤(1小时-4℃)
缓冲液:+1L 10mM PO4 pH7.5-150
mM-NaCl-1%Empigen
↓离心 JA10转子/9500rpm/30min/室温
↓沉淀
↓
溶解(O/N-4℃)
缓冲液:+330ml 10mM PO4 pH7.5-
150mM NaCl-4.0M GuHCl
↓
离心 JA10转子/9500rpm/30min/室温
↓在Ni++-NTA-琼脂糖(Qiagen-30
平衡缓冲液:10mM PO4 pH7.5-150ml树脂)上固定化金属离子亲 mM NaCl-4.0M GuHCl
和层析
洗涤缓冲液:
1)平衡缓冲液
2)10mM PO4 pH7.5-150mM NaCl-
6M尿素
3)10mM PO4 pH7.5-150mM NaCl-
6M尿素-35mM咪唑
洗脱缓冲液:10mM PO4 pH7.5-150mM
NaCl-6M尿素-0.5M咪唑
↓
稀释 离子强度降至12mS/cm
稀释缓冲液:20mM硼酸盐pH8.5-
6M尿素
↓SP Sepharose FF(Pharmacia-
平衡缓冲液:20mM硼酸盐pH8.5-15ml树脂)阳离子交换层析 150mM NaCl-6.0M尿素
洗涤缓冲液:
1)平衡缓冲液
2)20mM硼酸盐pH8.5-400mM
NaCl-6.0M尿素
洗脱缓冲液:20mM哌嗪pH11.0-2M
NaCl-6M尿素
↓
浓缩 至1.5mg/ml
10kDa Omega膜(Filtron)
↓
透析(O/N-4℃) 缓冲液:10mM PO4 pH6.8-150mM
NaCl-0.5M精氨酸
↓
过滤除菌 Millex GV 0.22μm→
图8显示由SDS-PAGE估计的纯度水平(Daiichi银染色、考马斯蓝 G250、蛋白质印迹法):
透析和过滤除菌步骤后: >95%→
回收(蛋白质比色测定评价:DOC TCA BCA)
由74克重组巴斯德毕赤酵母细胞(湿重)或1L Dyno-mill匀浆OD60纯化得到19mg氧化型Tat-his蛋白。实施例8:纯化SIV还原型NEF-HIS蛋白(巴斯德毕赤酵母)
由340克重组巴斯德毕赤酵母细胞(湿重)或4L Dyno-mill匀浆OD 100进行纯化流程。在室温下进行层析步骤。在步骤之间,Nef阳性流分在冷室(+4℃)中保存过夜;更长时间时,将样品于-20℃冷冻保存。
340克巴斯德毕赤
酵母细胞
↓
匀浆
缓冲液:4L 50mM PO4 pH7.0-PMSF 4
mM
最终OD:100
↓Dyno-mill破碎(4次)
↓
离心 JA10转子/9500rpm/60min/室温
↓Dyno-mill沉淀
↓溶解(O/N-4℃)
缓冲液:+2.6L 10mM PO4 pH7.5-150
mM NaCl-4.0M GuHCl
↓
离心 JA10转子/9500rpm/30min/室温
↓
还原 +0.2M 2-巯基乙磺酸钠盐(加入粉末)/在(4小时-室温-暗处) 温育前调节pH至7.5(用1M NaOH溶液)
↓
脲基甲基化 +0.25M碘乙酰胺(加入粉末)/在温育前(半小时-室温-暗处) 调节pH至7.5(用1M NaOH溶液)
↓在Ni++-NTA-琼脂糖(Qiagen- 平衡缓冲液:10mM PO4 pH7.5-15040ml树脂)上固定化金属离子 mM NaCl-4.0M GuHCl
亲和层析
洗涤缓冲液:
1)平衡缓冲液
2)10mM PO4 pH7.5-150mM NaCl-
6M尿素-25mM咪唑
洗脱缓冲液:10mM PO4 pH7.5-150mM
NaCl-6M尿素-0.5M咪唑
↓
浓缩 至3mg/ml
10kDa Omega膜(Filtron)
↓Superdex 200凝胶过滤层析
洗脱缓冲液:10mM PO4 pH7.5-150mM(Pharmacia-120ml树脂) NaCl-6M尿素
↓
浓缩 至1.5mg/ml
10kDa Omega膜(Filtron)
↓
透析(O/N-4℃)
缓冲液:10mM PO4 pH6.8-150mM
NaCl-Empigen 0.3%
↓
过滤除菌 Millex GV 0.22μm→
图9显示由SDS-PAGE估计的纯度水平(Daiichi银染色、考马斯蓝 G250、蛋白质印迹法):
透析和过滤除菌步骤后: >95%→
回收(蛋白质比色测定评价:DOC TCA BCA)由340克重组巴斯德毕赤酵母细胞(湿重)或4L Dyno-mill匀浆OD 100纯化得到20mg SIV还原型Nef-his蛋白。实施例9:纯化HIV还原型NEF-HIS蛋白(巴斯德毕赤酵母)
由160克重组巴斯德毕赤酵母细胞(湿重)或3L Dyno-mill匀浆OD 50进行纯化流程。在室温下进行层析步骤。在步骤之间,Nef阳性流分在冷室(+4℃)中保存过夜;更长时间时,将样品于-20℃冷冻保存。160克巴斯德毕赤酵母细胞
↓
匀浆
缓冲液:3L 50mM PO4 pH7.0-Pefabloc
5mM最终OD:50
↓Dyno-mill破碎(4次)
↓冷冻/解冻
↓离心 JA10转子/9500rpm/60min/室温
↓Dyno-mill沉淀
↓溶解(O/N-4℃)
缓冲液:+1L 10mM PO4 pH7.5-150
mM NaCl-4.0M GuHCl
↓离心 JA10转子/9500rpm/60min/室温
↓
还原 +0.1M 2-巯基乙磺酸钠盐(加入粉末)/在
(3小时-室温-暗处) 温育前调节pH至7.5(用1M NaOH溶
液)
↓
脲基甲基化 +0.15M碘乙酰胺(加入粉末)/在温育前
(半小时-室温-暗处) 调节pH至7.5(用1M NaOH溶液)
↓在Ni++-NTA-琼脂糖(Qiagen-
平衡缓冲液:10mM PO4 pH7.5-15010ml树脂)上固定化金属离子 mM NaCl-4.0M GuHCl
亲和层析
洗涤缓冲液:
1)平衡缓冲液
2)10mM PO4 pH7.5-150mM NaCl-
6M尿素
3)10mM PO4 pH7.5-150mM NaCl-
6M尿素-25mM咪唑
洗脱缓冲液:10mM柠檬酸盐pH6.0-
150mM NaCl-6M尿素-0.5M咪唑
↓
浓缩 至3mg/ml
10kDa Omega膜(Filtron)
↓Superdex 200凝胶过滤层析
洗脱缓冲液:10mM PO4 pH7.5-150mM(Pharmacia-120ml树脂) NaCl-6M尿素
↓
透析(O/N-4℃)
缓冲液:10mM PO4 pH6.8-150mM
NaCl-0.5M精氨酸
↓
过滤除菌 Millex GV 0.22μm→
图10显示由SDS-PAGE估计的纯度水平(Daiicbi银染色、考马斯 蓝G250、蛋白质印迹法):
透析和过滤除菌步骤后: >95%→
回收(蛋白质比色测定评价:DOC TCA BCA)
由160克重组巴斯德毕赤酵母细胞(湿重)或3L Dyno-mill匀浆OD 50纯化得到20mg HIV还原型Nef-his蛋白。实施例10:在巴斯德毕赤酵母中表达SIV nef序列
为了评价在致病性SHIV攻击模型中的Nef和Tat抗原,我们表达了猕猴中的猿猴免疫缺陷病毒(SIV)Nef蛋白SIVmac239(AidsResearch and Human Retroviruses,6:1221-1231,1990)。在Nef编码区中,SIVmac239在92aa之后具有一个符合读框的终止密码子,预期为仅10kD的截短产物。Nef读框的其余部分为可读框,预计它编码其完全可读形式的263aa蛋白(30kD)。
我们使用的SIVmac239 nef基因起始材料为相当于克隆在LX5N质粒上的完整编码序列的DNA片段(得自Dr R.C.Desrosiers,Southborough,MA,USA)。
使这种SIV nef基因在成熟前终止密码子处突变(9353位核苷酸G取代原T核苷酸),以便表达全长SIVmac239 Nef蛋白。
为了在巴斯德毕赤酵母中表达所述SIV nef基因,使用PHIL-D2-MOD载体(前面用于表达HIV-1 nef和tat序列)。在诱导型醇氧化酶(AOX1)启动子控制下表达所述重组蛋白,该蛋白的C末端延长为有助于纯化的组氨酸亲和尾。10.1构建整合型载体pRIT14908
为了构建pRIT 14908,用引物SNEF1和SNEF2经PCR由pLX5N/SIV-NEF质粒扩增SIV nef基因。
扩增的SIV nef DNA区段从核苷酸9077开始,于核苷酸9865终止(Aids Research and Human Retroviruses,6:1221-1231,1990)。
在所述PCR片段的5’末端导入一个NcoI限制位点(带有nef基因的ATG密码子),而在3’末端导入一个SpeI位点。获得的PCR片段和整合型载体PHIL-D2-MOD都用NcoI和SpeI限制。因为一个NcoI限制位点位于SIV nef扩增序列上(位置9286),所以获得分别约200bp和约600bp的两个片段,在琼脂糖凝胶上纯化,与PHIL-D2-MOD载体连接。自动测序证实nef扩增区段后,收集获得的重组质粒,命名为pRIT14908。10.2巴斯德毕赤酵母菌株GS115(his4)的转化。
为获得表达SIV nef-His的
巴斯德毕赤酵母菌株,用仅携带表达盒和HIS4基因的线性NotI片段转化菌株GS115(图11)。
因为两个末端与巴斯德毕赤酵母具有的AOXI基因同源,所以所述线性NotI DNA片段有利于在AOXI基因座重组。
通过定量斑点印迹分析选择多拷贝整合克隆。
选择出一个显示重组蛋白产量最高的转化体,称为Y1772。
菌株Y1772产生重组SIV Nef-His蛋白(一种272个氨基酸的蛋白),所述重组SIV Nef-His蛋白组成如下:
°豆蔻酸
°蛋氨酸,利用PHIL-D2-MOD载体的NcoI克隆位点产生
°262个氨基酸的Nef蛋白(从氨基酸2开始,延伸至氨基酸263,见图12)
°通过克隆步骤产生的一个苏氨酸和一个丝氨酸(在PHIL-D2-MOD载体的SpeI位点克隆(图11))
°一个甘氨酸和六个组氨酸
核酸和蛋白序列见图12。10.3 Y1772菌株表达产物的表征 表达水平
在含有1%甲醇作为碳源的培养基中诱导16小时后,估测重组Nef-His蛋白丰度为10%总蛋白(图13,3-4泳道)。溶解度
离心产生Nef-His蛋白的重组菌株Y1772的诱导培养物。细胞沉淀重悬浮于破碎缓冲液中,用0.5mm玻璃珠破碎细胞,离心细胞提取物。在考马斯蓝染色的SDS-PAGE10%上比较不溶性沉淀包含的蛋白(P)和溶解上清液包含的蛋白(S)。
如图13所示,菌株Y1772主要重组蛋白(泳道3-4)在不溶性部分。
具有满意重组蛋白表达水平的菌株Y1772用于产生及纯化SIVNef-His蛋白。实施例11:在CHO中表达GP120
建立了产生gP120糖蛋白的稳定CHO-K1细胞系。重组gP120糖蛋白为HIV-1分离株W61D的重组截短型,120包膜蛋白。gP120糖蛋白分泌入细胞培养基中,然后可从培养基中纯化所述蛋白。构建gp120转染质粒pRIT13968
获得的HIV-1分离株W61D的包膜DNA编码序列(包括tat和rev的5’外显子)(Dr.Tersmette,CCB,Amsterdam)为含基因组gp160包膜编码序列的质粒W61D(Nco-XhoI)。该质粒称为pRIT13965。
为了构建gp120表达盒,必须利用引物寡核苷酸序列(DIR 131)和PCR技术使一个终止密码子插入pRIT13965中的gp160编码序列的氨基酸glu515密码子。引物DIR 131含有3个终止密码子(在所有可读框中)和一个SalI限制位点。
然后,用得自pRIT13965的gp160质粒亚克隆pW61d env(pRIT13966)的N-末端BamH1-DraI片段(170bp)和通过PCR由pRIT13965产生的DraI-SalI片段(510bp)重新构建完整的gp120包膜序列。将两种片段凝胶纯化,一起连接入大肠杆菌质粒pUC18中,首先用SalI(klenow处理)切割,然后用BamH1切割。获得质粒pRIT13967。对含有gp120编码盒的XmaI-SalI片段(1580bp)的基因序列进行测序,发现与预测序列相同。首先用BclI(klenow处理)切割,然后用XmaI切割,使质粒RIT13967连接入CHO GS表达载体pEE14(Celltech Ltd.,UK)。获得的质粒称为pRIT13968。制备主细胞库
应用经典磷酸钙沉淀/甘油冲击法使gp120构建物(pRIT13968)转染入CHO细胞中。2天后,CHOK1细胞用选择性生长培养基(GMEM+磺基亚胺蛋氨酸(MSX)25μM+谷氨酸盐+天冬酰胺+10%胎牛血清)培养。进一步在175m2培养瓶中扩增3个选定的转染克隆,很少细胞的培养瓶保藏于-80℃。选择C-env 23,9进一步扩大培养。
制备小型细胞预备库,冷冻20个安瓿。为了制备预备库和MCB,在GMEM培养基中培养细胞,培养基中加入7.5%胎牛血清并含有50μM MSX。测试上述细胞培养物的无菌和支原体情况,证实为阴性。
利用从预备主细胞库获得的细胞制备主细胞库CHOKl env 23.9(12代)。简而言之,将2个安瓿的预备主种子细胞接种在添加7.5%渗析胎牛血清的培养基中。将细胞分入4个培养瓶中,于37℃培养。细胞贴壁后,培养基改为添加50μM MSX的新鲜培养基。细胞汇合时,用胰酶消化收集细胞,以1/8分瓶比例在T-培养瓶-滚瓶-细胞工厂装置中传代培养。通过胰酶消化和离心从细胞工厂装置收集细胞。细胞沉淀重悬浮于添加DMSO作为冷冻保藏剂的培养基中。预先标记安瓿,高压灭菌,加热密封(250个瓶)。检查小瓶有无渗漏,于-70℃保藏过夜,然后保藏于液氮中。细胞培养和制备粗品收获物
快速解冻2瓶主细胞库。合并细胞,接种入2个装有添加7.5%渗析胎牛血清(FBS)的合适培养基的T-培养瓶(37±1℃)。当细胞达到汇合时(13代),胰酶消化收集细胞,合并细胞,在10个上述T-培养瓶中扩大培养。胰酶消化汇合细胞(14代),连续在2个细胞工厂装置(每个装置6000cm2;15代)、然后在10个细胞工厂装置(16代)中扩大培养。所述生长培养基中添加7.5%渗析胎牛血清(FBS)和1%MSX。当细胞达到汇合时,弃去生长培养基,代之以仅含有1%渗析胎牛血清而无MSX的“生产培养基”。每2天(间隔48小时)收集上清液,直至32天。立即通过1.2-0.22μm过滤装置澄清收获的培养液,纯化前保藏于-20℃。实施例12:从细胞培养液纯化HIV GP 120(W61D CHO)
所有纯化步骤在2-8℃的冷室中进行。在此温度下调节缓冲液pH,通过0.2μm滤膜过滤。测试其致热原含量(LAL测定)。连续监测柱洗出液的280nm光密度、pH和电导率。(i)澄清培养液
收获的澄清细胞培养液(CCF)过滤除菌,加入Tris缓冲液pH8.0至30mM终浓度。纯化前CCF冷冻保藏于-20℃。(ii)疏水作用层析
解冻后,在澄清培养液中加入硫酸铵至1M。使溶液通过以30mMTris缓冲液-pH8.0-1M硫酸铵平衡的TSK/TOYOPEARL-BUTYL650M(TOSOHAAS)柱过夜。在上述条件下,抗原与凝胶基质结合。用梯度逐步降低的硫酸铵洗涤柱。抗原于30mM Tris缓冲液-pH8.0-0.25M硫酸铵时洗出。(iii)阴离子交换层析
在降低溶液电导率5-6mS/cm后,将合并的gP120流分加样至以Tris盐缓冲液-pH8.0平衡的Q-sepharose Fast Flow(Pharmacia)柱。以阴性模式即gP120不结合凝胶操作柱,同时滞留绝大部分杂质。(iv)浓缩及超滤性渗滤
为了提高蛋白浓度,将gP120合并液加至截留50kDa的FILTRON膜“Omega Screen Channel”。浓缩完成后,用包含氯化钙0.3mM的5mM磷酸缓冲液pH7.0通过渗滤交换所述缓冲液。如果不立即进行进一步处理,将,120合并液冷冻保藏于-20℃。解冻后,使所述溶液通过0.2μM膜以便去除不溶性物质。(v)羟基磷灰石层析
将gP120 UF合并液加样至以5mM磷酸缓冲液+氯化钙0.3mM,pH7.0平衡的macro-Prep Ceramic Hydroxyapatite II型(Biorad)柱。用相同缓冲液洗涤柱。抗原通过柱,而杂质与柱结合。(vi)阳离子交换层析
将gP120合并液加样至以醋酸缓冲液20mM,pH5.0平衡的CM/TOYOPEARL-650 S(TOSOHAAS)柱。用相同缓冲液、然后用醋酸盐20mM,pH5.0和NaCl 10mM洗涤柱。之后用含80mM NaCl的相同缓冲液洗脱抗原。(vii)超滤
为了增强纯化过程的病毒清除能力,再进行一个超滤步骤。使gP120合并液通过截留150kDa的FILTRON膜“Omega ScreenChannel”超滤。该孔径膜不会阻留抗原。上述过程后,在截留50kDa的相同类型膜(Filtron)上浓缩稀释抗原。(viii)大小排阻凝胶层析
将gP120合并液加样至SUPERDEX 200(PHARMACIA)柱,以便交换所述缓冲液以及消除残余杂质。用磷酸缓冲盐溶液(PBS)洗脱柱。(ix)过滤除菌和保藏
使流分通过0.2μM PVDF膜(Millipore)过滤除菌。过滤除菌后,纯化物冷冻保藏于-20℃,直至进行配制。以下流程表概述纯化流程。
SDS-PAGE分析估测纯化物的纯度水平(银染色/考马斯蓝/蛋白质印迹法)≥95%。
产率约2.5mg/L CCF(根据Lowry测定)-总纯化产率约25%(根据Elisa测定)。
纯化物质于37℃稳定1周(根据WB分析)。
从培养液纯化gp120
标记√表示去除病毒的关键步骤。
澄清培养液
↓
疏水作用层析
(BUTYL-TOYOPEARL 650M)
↓
阴离子交换层析
(阴性模式)
(Q-SEPHAROSE)
↓
50KD超滤
(浓度及缓冲液交换)
↓
(保藏于-20℃)
↓
羟基磷灰石层析
(阴性模式)
(MACROPREP CERAMIC HYDROXYAPATITE II)
↓
阳离子交换层析
(CM-TOYOPEARL 650 S)
↓
150KD超滤 √
(OMEGA膜/FILTRON)
↓
50KD超滤
(浓缩)
↓
大小排阻层析 √
(SUPERDEX 200)
过滤除菌
↓
纯化物
保藏于-20℃实施例13:疫苗制备
按照本发明制备的疫苗包括一种或多种编码抗原的DNA重组体的表达产物。此外,所述制剂包含在油/水乳剂中的3脱氧酰化单磷酰脂质A 3D-MPL和QS21的混合物、或者含未甲基化CpG二核苷酸基元的寡核苷酸以及氢氧化铝载体。
3D-MPL:是革兰氏阴性菌明尼苏达沙门氏菌(Salmonellaminnesota)的化学解毒形式脂多糖(LPS)。
在Smith Kline Beecham Biologicals进行的实验表明,与各种载体组合的3D-MPL强有力地增强体液免疫和Th1型细胞免疫。
QS21:是一种由Quillaja Saponaria Molina树的树皮粗提物纯化的皂苷,它具有很强的佐剂活性:它诱导对若干抗原的抗原特异性淋巴细胞增殖和CTL。
在Smith Kline Beecham Biologicals进行的实验证明,3D-MPL和QS21组合在诱导体液免疫应答和Th1型细胞免疫应答中存在明显的协同作用。
油/水乳剂包含2种油(维生素E和鲨烯)和含乳化剂吐温80的PBS。所述乳剂包含5%鲨烯、5%维生素E、2%吐温80,平均颗粒大小为180nm(参见WO 95/17210)。
在Smith Kline Beecham Biologicals进行的实验证实,O/W乳剂与3D-MPL/QS21联合使用进一步增强其免疫刺激特性。制备油/水乳剂(2倍浓缩物)
将吐温80溶于磷酸缓冲盐溶液(PBS)中,得到在PBS中的2%溶液。为获得100ml两倍浓缩的乳剂,涡旋混合5克DLα-生育酚和5ml鲨烯,以充分混合。加入90ml的PBS/吐温溶液并充分混合。然后将得到的乳剂通过注射器,最后将其用M110S微流设备微流化。得到的油滴大小约为180nm。制备水包油制剂
以10倍浓缩的PBS pH6.8和水稀释抗原(单独或组合的100μggp120、20μg NefTat和20μg SIV Nef),然后以5分钟间隔连续加入水包油乳剂、3D-MPL(50μg)、QS21(50μg)和1μg/ml硫柳汞(作为防腐剂)。乳剂体积等于总体积的50%(对于500μl剂量为250μl)。
所有的温育都在室温搅拌下进行。
CpG寡核苷酸(CpG)是含有一个或多个CpG序列基元的合成未甲基化寡核苷酸。与主要诱导混合型TH1/TH2应答的水包油制剂相比,CpG是非常有效的TH1型免疫诱导剂。CpG比水包油制剂诱导更低水平的抗体,但是诱导良好水平的细胞介导的免疫应答。预计CpG诱导较低的局部反应原性。
制备CpG寡核苷酸溶液:使CpG干粉溶解于水获得5mg/ml CpG溶液。制备CpG制剂
使所述3种抗原对氯化钠150mM透析,消除抑制gp120吸附在氢氧化铝上的磷酸根离子。
在吸附在氢氧化铝上之前,将用水稀释的抗原(100μg gp120、20μg NefTat和20μg SIV Nef)与CpG溶液(500μg CpG)温育30分钟,有利于NefTat和Nef抗原的His尾与寡核苷酸之间的潜在相互作用(与游离CpG相比,当与所述抗原结合时所述CpG的免疫刺激作用更强)。然后,间隔5分钟连续加入氢氧化铝(500μg)、10倍浓缩氯化钠和1μg/ml硫柳汞(作为防腐剂)。
所有温育在室温搅拌下进行。实施例14:对恒河猴进行的免疫接种以及SHIV攻击实验第一项研究
用下列疫苗组合物在第0、1和3个月肌肉内免疫各组(4只/组)恒河猴:
第1组:佐剂2+gp120
第2组:佐剂2+gp120+NefTat+SIV Nef
第3组:佐剂2 +NefTat*+SIV Nef
第4组:佐剂6+gp120+NefTat+SIV Nef
第5组:佐剂2 +NefTat+SIV Nef
第6组:佐剂2
佐剂2包含鲨烯/维生素E/吐温80/3D-MPL/QS21
佐剂6包含alum和CpG。
Tat*表示突变Tat,其中Lys41→Ala,在RGD基元中Arg78→Lys,Asp80→Glu(Virology 235:48-64,1997)。
最后一次免疫后1个月,用致病性SHIV(病毒株89.6p)攻击所有动物。从用病毒攻击周(16周)开始,在指定时间点周期性取血样,通过FACS分析测定外周血单核细胞中的CD4阳性细胞百分率(图14)以及通过bDNA检测测定血浆RNA病毒基因组浓度(图15)。结果
所有动物用SHIV89.6P攻击后均成为感染动物。
在第1、3、5、6组所有动物中(只有第1、6组(对照组)各有1只动物例外),病毒株攻击后CD4阳性细胞均下降。第2组所有动物CD4阳性细胞略微下降,随着时间回复至基线水平。在第4组动物中观测到相似趋势(图14)。
病毒负荷数据几乎与CD4数据相反。3/4的第2组动物(以及在1只保持CD4阳性细胞的对照动物)中,病毒负荷下降到检测水平以下,第4只动物仅显示临界水平的病毒负荷量。大多数其它动物保持高水平或中间水平病毒负荷量(图15)。
令人惊奇的是,在整个研究中,通过ELISA检测的抗Tat和抗Nef抗体滴度第3组(突变Tat)比第5组(非突变Tat的等同组)高2-3倍。
68周时(攻击后56周),接受完全抗原组合的两组(第2、4组)所有动物仍存活,而其它组大多数动物均因为AIDS样症状不得不进行安乐死。各组存活动物为:
第1组:2/4
第2组:4/4
第3组:0/4
第4组:4/4
第5组:0/4
第6组:1/4结论
gp120和NefTat(在存在SIV Nef的情况下)组合防止CD4阳性细胞丧失,降低致病性SHIV89.6P感染动物体内的病毒负荷量,延迟或者防止出现AIDS样疾病症状,而单独的gp120或NefTat/SIV Nef不对SHIV攻击的病理结果产生保护作用。
佐剂2为包含鲨烯、维生素E和吐温80以及3D-MPL和QS21的水包油乳剂,它似乎对最终研究结果的作用比alum/CpG佐剂更强。第二项研究
进行第二个恒河猴SHIV攻击研究的目的是确证候选疫苗gp120/NefTat+佐剂的效果以及比较不同Tat型抗原。该研究由一个不同的实验室进行。
研究设计如下。
在第0、4、12周肌肉内注射免疫各组(6只/组)恒河猴,在第16周用标准剂量的致病性SHIV89.6p攻击。
第1组是重复第一项研究的第2组。
第1组:佐剂2+gp120+NefTat+SIV Nef
第2组:佐剂2+gp120+Tat(氧化型)
第3组:佐剂2+gp120+Tat(还原型)
第4组:佐剂2
随访/最终指标仍为CD4阳性细胞百分率、RT-PCR检测的病毒负荷量、发病率和死亡率。结果
除了第2组的1只动物外,所有动物均在用SHIV89.6p攻击后变成感染动物。
第4对照组和笫3组所有动物以及第2组(1只动物例外)所有动物攻击后的CD4阳性细胞显著降低。第1组只有1只动物显示CD4阳性细胞显著降低。与第一项研究的动物不同,第二项实验的恒河猴在病毒攻击后1个月,不同水平的CD4阳性细胞稳定(图16)。稳定性一般低于初始的CD4阳性细胞百分率,但是决不使CD4阳性细胞完全丧失。可以说明这一点是:用于第二项研究的恒河猴种群对SHIV诱导疾病的易感性降低。尽管如此,gp120/NefTat/SIV Nef疫苗和两种gp120/Tat疫苗的有益作用得到证实。在免疫接种动物中,CD4阳性细胞百分率20以上的动物数为5,没有1只佐剂组对照动物保持高于所述水平。
分析血浆RNA病毒负荷量证实了研究动物的易感性相对较低(图17)。6只对照动物中只有2只保持高病毒负荷量,而其它动物中病毒从血浆中消失。因此,用病毒负荷量参数难以证实疫苗的作用。结论
分析CD4阳性细胞表明,疫苗gp120/NefTat+佐剂(存在SIV Nef)防止大多数免疫接种动物CD4阳性细胞下降。这进一步证实了第一个SHIV研究获得的结果。由于研究动物缺乏易感性,所以不能用病毒负荷量参数证实疫苗作用。总而言之,根据SHIV模型证据,gp120和Tat及Nef HIV抗原组合可以对HIV感染的病理结果产生保护作用。
单独的Tat抗原与gp120组合也可以对CD4阳性细胞下降提供一定保护作用。其作用不如gp120/NefTat/SIV Nef抗原组合明显,但是证实gp120和Tat能够介导抗SHIV诱导性疾病表现的一定保护作用。
用与第一个研究动物来源完全无关的不同来源的恒河猴进行第二个SHIV攻击研究。CD4阳性细胞百分率和血浆病毒负荷量两个参数均提示,第二个研究的动物对SHIV诱导性疾病的易感性更低,而且动物之间的变异性明显更大。尽管如此,用包含gp120/NefTat和SIV Nef的实验疫苗仍然可见gp120/NefTat/SIV Nef疫苗对保持CD4阳性细胞的有益作用。说明所述疫苗作用不仅在独立研究中获得重现,而且在不相关猴群中得到证实。
序列表<110>SmithKline Beecham Biologicals S.A.<120>用于HIV预防或治疗性免疫的疫苗<130>B45209<160>31<170>FastSEQ for Windows Version 3.0<210>1<211>28<212>DNA<213>人工序列<220><223>引物<220><221>misc feature<222>11,15,19,23,27<223>n=A,T,C或G<400>1atcgtccatg nggtnggcna agntggnt 28<210>2<211>23<212>DNA<213>人工序列<220><223>引物<400>2cggctactag tgcagttctt gaa 23<210>3<211>29<212>DNA<213>人工序列<220><223>引物<220><221>misc feature<222>12,16,20,24,28<223>n=A,T,C或G<400>3atcgtactag tngagnccan gtangatnc 29<210>4<211>24<212>DNA<213>人工序列<220><223>引物<400>4cggctactag tttccttcgg gcct 24<210>5<211>23<212>DNA<213>人工序列<220><223>引物<400>5atcgtccatg gagccagtag atc 23<210>6<211>24<212>DNA<213>人工序列<220><223>引物<400>6atcgtccatg ggtggagcta tttt 24<210>7<211>23<212>DNA<213>人工序列<220><223>引物<400>7cggctactag tgcgagtttc ctt 23<210>8<211>648<212>DNA<213>人<400>8atgggtggca agtggtcaaa aagtagtgtg gttggatggc ctactgtaag ggaaagaatg 60agacgagctg agccagcagc agatggggtg ggagcagcat ctcgagacct ggaaaaacat 120ggagcaatca caagtagcaa tacagcagct accaatgctg cttgtgcctg gctagaagca 180caagaggagg aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact 240tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta 300attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac 360ttccctgatt ggcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga 420tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa taaaggagag 480aacaccagct tgttacaccc tgtgagcctg catggaatgg atgaccctga gagagaagtg 540ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga gctgcatccg 600gagtacttca agaactgcac tagtggccac catcaccatc accattaa 648<210>9<211>215<212>PRT<213>人<400>9Met Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val1 5 10 15Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala
20 25 30Ala Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr
35 40 45Ala Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu
50 55 60Glu Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr65 70 75 80Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly
85 90 95Leu Glu Gly Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu
100 105 110Trp Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr
115 120 125Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys
130 135 140Leu Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu145 150 155 160Asn Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro
165 170 175Glu Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His
180 185 190His Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser
195 200 205Gly His His His His His His
210 215<210>10<211>288<212>DNA<213>人<400>10atggagccag tagatcctag actagagccc tggaagcatc caggaagtca gcctaaaact 60gcttgtacca attgctattg taaaaagtgt tgctttcatt gccaagtttg tttcataaca 120aaagccttag gcatctccta tggcaggaag aagcggagac agcgacgaag acctcctcaa 180ggcagtcaga ctcatcaagt ttctctatca aagcaaccca cctcccaatc ccgaggggac 240ccgacaggcc cgaaggaaac tagtggccac catcaccatc accattaa 288<210>11<211>95<212>PRT<213>人<400>11Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser1 5 10 15Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30His Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly
35 40 45Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr
50 55 60His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp65 70 75 80Pro Thr Gly Pro Lys Glu Thr Ser Gly His His His His His His
85 90 95<210>12<211>909<212>DNA<213>人<400>12atgggtggca agtggtcaaa aagtagtgtg gttggatggc ctactgtaag ggaaagaatg 60agacgagctg agccagcagc agatggggtg ggagcagcat ctcgagacct ggaaaaacat 120ggagcaatca caagtagcaa tacagcagct accaatgctg cttgtgcctg gctagaagca 180caagaggagg aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact 240tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta 300attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac 360ttccctgatt ggcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga 420tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa taaaggagag 480aacaccagct tgttacaccc tgtgagcctg catggaatgg atgaccctga gagagaagtg 540ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga gctgcatccg 600gagtacttca agaactgcac tagtgagcca gtagatccta gactagagcc ctggaagcat 660ccaggaagtc agcctaaaac tgcttgtacc aattgctatt gtaaaaagtg ttgctttcat 720tgccaagttt gtttcataac aaaagcctta ggcatctcct atggcaggaa gaagcggaga 780cagcgacgaa gacctcctca aggcagtcag actcatcaag tttctctatc aaagcaaccc 840acctcccaat cccgagggga cccgacaggc ccgaaggaaa ctagtggcca ccatcaccat 900caccattaa 909<210>13<211>302<212>PRT<213>人<400>13Met Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val1 5 10 15Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala
20 25 30Ala Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr
35 40 45Ala Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu
50 55 60Glu Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr65 70 75 80Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly
85 90 95Leu Glu Gly Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu
100 105 110Trp Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr
115 120 125Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys
130 135 140Leu Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu145 150 155 160Asn Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro
165 170 175Glu Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His
180 185 190His Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser
195 200 205Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln
210 215 220Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His225 230 235 240Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg
245 250 255Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr His
260 265 270Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp Pro
275 280 285Thr Gly Pro Lys Glu Thr Ser Gly His His His His His His
290 295 300<210>14<211>1029<212>DNA<213>人<400>14atggatccaa aaactttagc cctttcttta ttagcagctg gcgtactagc aggttgtagc 60agccattcat caaatatggc gaatacccaa atgaaatcag acaaaatcat tattgctcac 120cgtggtgcta gcggttattt accagagcat acgttagaat ctaaagcact tgcttttgca 180caacaggctg attatttaga gcaagattta gcaatgacta aggatggtcg tttagtggtt 240attcacgatc actttttaga tggcttgact gatgttgcga aaaaattccc acatcgtcat 300cgtaaagatg gccgttacta tgtcatcgac tttaccttaa aagaaattca aagtttagaa 360atgacagaaa actttgaaac catgggtggc aagtggtcaa aaagtagtgt ggttggatgg 420cctactgtaa gggaaagaat gagacgagct gagccagcag cagatggggt gggagcagca 480tctcgagacc tggaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct 540gcttgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag 600gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa 660aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg 720atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg gccaggggtc 780agatatccac tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta 840gaagaggcca ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatggaatg 900gatgaccctg agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac 960gtggcccgag agctgcatcc ggagtacttc aagaactgca ctagtggcca ccatcaccat 1020caccattaa 1029<210>15<211>324<212>PRT<213>人<400>15Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys Ser Asp1 5 10 15Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro Glu His
20 25 30Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp Tyr Leu
35 40 45Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val Ile His
50 55 60Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe Pro His65 70 75 80Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr Leu Lys
85 90 95Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met Gly Gly
100 105 110Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val Arg Glu Arg
115 120 125Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala Ala Ser Arg
130 135 140Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr145 150 155 160Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu Val Gly
165 170 175Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Ala
180 185 190Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly
195 200 205Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp Ile Tyr
210 215 220His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro225 230 235 240Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu Val Pro
245 250 255Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu Asn Thr Ser
260 265 270Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu Arg Glu
275 280 285Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His His Val Ala
290 295 300Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser Gly His His305 310 315 320His His His His<210>16<211>1290<212>DNA<213>人<400>16atggatccaa aaactttagc cctttcttta ttagcagctg gcgtactagc aggttgtagc 60agccattcat caaatatggc gaatacccaa atgaaatcag acaaaatcat tattgctcac 120cgtggtgcta gcggttattt accagagcat acgttagaat ctaaagcact tgcgtttgca 180caacaggctg attatttaga gcaagattta gcaatgacta aggatggtcg tttagtggtt 240attcacgatc actttttaga tggcttgact gatgttgcga aaaaattccc acatcgtcat 300cgtaaagatg gccgttacta tgtcatcgac tttaccttaa aagaaattca aagtttagaa 360atgacagaaa actttgaaac catgggtggc aagtggtcaa aaagtagtgt ggttggatgg 420cctactgtaa gggaaagaat gagacgagct gagccagcag cagatggggt gggagcagca 480tctcgagacc tggaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct 540gcttgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag 600gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa 660aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg 720atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg gccaggggtc 780agatatccac tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta 840gaagaggcca ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatggaatg 900gatgaccctg agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac 960gtggcccgag agctgcatcc ggagtacttc aagaactgca ctagtgagcc agtagatcct 1020agactagagc cctggaagca tccaggaagt cagcctaaaa ctgcttgtac caattgctat 1080tgtaaaaagt gttgctttca ttgccaagtt tgtttcataa caaaagcctt aggcatctcc 1140tatggcagga agaagcggag acagcgacga agacctcctc aaggcagtca gactcatcaa 1200gtttctctat caaagcaacc cacctcccaa tcccgagggg acccgacagg cccgaaggaa 1260actagtggcc accatcacca tcaccattaa 1290<210>17<211>411<212>PRT<213>人<400>17Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys Ser Asp1 5 10 15Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro Glu His
20 25 30Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp Tyr Leu
35 40 45Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val Ile His
50 55 60Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe Pro His65 70 75 80Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr Leu Lys
85 90 95Glu Ile Gln Ser Leu Glu Met Thr Glu Ash Phe Glu Thr Met Gly Gly
100 105 110Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val Arg Glu Arg
115 120 125Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala Ala Ser Arg
130 135 140Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr145 150 155 160Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu Val Gly
165 170 175Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Ala
180 185 190Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly
195 200 205Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp Ile Tyr
210 215 220His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro225 230 235 240Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu Val Pro
245 250 255Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu Asn Thr Ser
260 265 270Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu Arg Glu
275 280 285Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His His Val Ala
290 295 300Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser Glu Pro Val305 310 315 320Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro Lys Thr
325 330 335Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val
340 345 350Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg
355 360 365Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr His Gln Val Ser
370 375 380Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro385 390 395 400Lys Glu Thr Ser Gly His His His His His His
405 410<210>18<211>981<212>DNA<213>人<400>18atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300caaagtttag aaatgacaga aaactttgaa accatgggtg gcaagtggtc aaaaagtagt 360gtggttggat ggcctactgt aagggaaaga atgagacgag ctgagccagc agcagatggg 420gtgggagcag catctcgaga cctggaaaaa catggagcaa tcacaagtag caatacagca 480gctaccaatg ctgcttgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca 540gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac 600tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc 660cttgatctgt ggatctacca cacacaaggc tacttccctg attggcagaa ctacacacca 720gggccagggg tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag 780ccagataagg tagaagaggc caataaagga gagaacacca gcttgttaca ccctgtgagc 840ctgcatggaa tggatgaccc tgagagagaa gtgttagagt ggaggtttga cagccgccta 900gcatttcatc acgtggcccg agagctgcat ccggagtact tcaagaactg cactagtggc 960caccatcacc atcaccatta a 981<210>19<211>326<212>PRT<213>人<400>19Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys1 5 10 15Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe65 70 75 80Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val Arg
115 120 125Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala Ala
130 135 140Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala145 150 155 160Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu
165 170 175Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr
180 185 190Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
195 200 205Glu Gly Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp
210 215 220Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro225 230 235 240Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu
245 250 255Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu Asn
260 265 270Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu
275 280 285Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His His
290 295 300Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser Gly305 310 315 320His His His His His His
325<210>20<211>1242<212>DNA<213>人<400>20atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300caaagtttag aaatgacaga aaactttgaa accatgggtg gcaagtggtc aaaaagtagt 360gtggttggat ggcctactgt aagggaaaga atgagacgag ctgagccagc agcagatggg 420gtgggagcag catctcgaga cctggaaaaa catggagcaa tcacaagtag caatacagca 480gctaccaatg ctgcttgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca 540gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac 600tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc 660cttgatctgt ggatctacca cacacaaggc tacttccctg attggcagaa ctacacacca 720gggccagggg tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag 780ccagataagg tagaagaggc caataaagga gagaacacca gcttgttaca ccctgtgagc 840ctgcatggaa tggatgaccc tgagagagaa gtgttagagt ggaggtttga cagccgccta 900gcatttcatc acgtggcccg agagctgcat ccggagtact tcaagaactg cactagtgag 960ccagtagatc ctagactaga gccctggaag catccaggaa gtcagcctaa aactgcttgt 1020accaattgct attgtaaaaa gtgttgcttt cattgccaag tttgtttcat aacaaaagcc 1080ttaggcatct cctatggcag gaagaagcgg agacagcgac gaagacctcc tcaaggcagt 1140cagactcatc aagtttctct atcaaagcaa cccacctccc aatcccgagg ggacccgaca 1200ggcccgaagg aaactagtgg ccaccatcac catcaccatt aa 1242<210>21<211>413<212>PRT<213>人<400>21Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys1 5 10 15Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe65 70 75 80Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val Arg
115 120 125Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala Ala
130 135 140Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala145 150 155 160Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu
165 170 175Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr
180 185 190Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
195 200 205Glu Gly Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp
210 215 220Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro225 230 235 240Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu
245 250 255Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu Asn
260 265 270Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu
275 280 285Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His His
290 295 300Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser Glu305 310 315 320Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro
325 330 335Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys
340 345 350Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys
355 360 365Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr His Gln
370 375 380Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp Pro Thr385 390 395 400Gly Pro Lys Glu Thr Ser Gly His His His His His His
405 410<210>22<211>288<212>DNA<213>人<400>22atggagccag tagatcctag actagagccc tggaagcatc caggaagtca gcctaaaact 60gcttgtacca attgctattg taaaaagtgt tgctttcatt gccaagtttg tttcataaca 120gctgccttag gcatctccta tggcaggaag aagcggagac agcgacgaag acctcctcaa 180ggcagtcaga ctcatcaagt ttctctatca aagcaaccca cctcccaatc caaaggggag 240ccgacaggcc cgaaggaaac tagtggccac catcaccatc accattaa 288<210>23<211>95<212>PRT<213>人<400>23Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser1 5 10 15Gln Pro Lys Thr Ala Cys Thr Ash Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30His Cys Gln Val Cys Phe Ile Thr Ala Ala Leu Gly Ile Ser Tyr Gly
35 40 45Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr
50 55 60His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Lys Gly Glu65 70 75 80Pro Thr Gly Pro Lys Glu Thr Ser Gly His His His His His His
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195 200 205Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln
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260 265 270Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Lys Gly Glu Pro
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35 40 45Gly Leu Ser Ser Leu Ser Cys Glu Gly Gln Lys Tyr Asn Gln Gly Gln
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245 250 255Ala Asp Lys Lys Glu Thr Arg Thr Ser Gly His His His His His His
260 265 270
Claims (19)
1.a)HIV Tat蛋白或多核苷酸;或者
b)HIV Nef蛋白或多核苷酸;或者
c)与HIV Nef蛋白或多核苷酸结合的HIV Tat蛋白或多核苷酸(Nef-Tat);
和HIV gp120蛋白或多核苷酸在生产用于人类HIV预防或治疗性免疫的疫苗中的应用。
2.权利要求1要求保护的用途,其中所述Tat、Nef或Nef-Tat与gp120在治疗或预防HIV中协同作用。
3.权利要求1或2要求保护的用途,其中使用所述疫苗可降低HIV感染病人体内的HIV病毒负荷量。
4.权利要求1或2要求保护的用途,其中使用所述疫苗使得CD4+维持水平高于没有用HIV Tat、Nef或Nef-Tat和HIV gp120免疫的水平。
5.权利要求1-4中任一项要求保护的用途,其中所述疫苗还包含选自gag、rev、vif、vpr、vpu的抗原。
6.权利要求1-5中任一项要求保护的用途,其中所述Tat蛋白为突变蛋白。
7.权利要求1-6中任一项要求保护的用途,其中所述Tat、Nef或Nef-Tat蛋白是还原型蛋白。
8.权利要求1-7中任一项要求保护的用途,其中所述Tat、Nef或Nef-Tat蛋白为脲基甲基化蛋白。
9.权利要求1-6中任一项要求保护的用途,其中所述Tat、Nef或Nef-Tat蛋白为氧化型蛋白。
10.权利要求1-9中任一项要求保护的用途,它还包括佐剂。
11.权利要求10要求保护的用途,其中所述佐剂为TH1诱导型佐剂。
12.权利要求10或11要求保护的用途,其中所述佐剂包括单磷酰脂质A或其衍生物,例如3-de-O-酰化单磷酰脂质A。
13.权利要求10-12中任一项要求保护的用途,它还包括皂苷佐剂。
14.权利要求10-13中任一项要求保护的用途,它还包括水包油乳剂。
15.权利要求10或11要求保护的用途,其中所述佐剂包括含CpG基元的寡核苷酸。
16.权利要求15要求保护的用途,它还包括铝盐。
17.a)HIV Tat蛋白或多核苷酸;或者
b)HIV Nef蛋白或多核苷酸;.或者
c)与HIV Nef蛋白或多核苷酸结合的HIV Tat蛋白或多核
苷酸;
和HIV gp120蛋白或多核苷酸在生产适合人类HIV预防或治疗性免疫的疫苗中的应用,所述疫苗适用于初免-加强传递。
18.一种免疫人类HIV的方法,该方法包括给予所述人类对象包含HIV Tat或HIV Nef或HIV NefTat和HIV gp120蛋白、或者编码它们的多核苷酸的疫苗。
19.一种用于人类的疫苗组合物,该疫苗组合物包含HIV Tat或HIV Nef或HIV Nef-Tat和HIV gp120蛋白、或者编码它们的多核苷酸。
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GB0009336A GB0009336D0 (en) | 2000-04-14 | 2000-04-14 | Novel use |
GB0013806A GB0013806D0 (en) | 2000-06-06 | 2000-06-06 | Novel use |
GB0013806.5 | 2000-06-06 | ||
EPPCT/EP00/05998 | 2000-06-28 | ||
PCT/EP2000/005998 WO2001000232A2 (en) | 1999-06-29 | 2000-06-28 | Use of cpg as an adjuvant for hiv vaccine |
WOPCT/EP00/05998 | 2000-06-28 |
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BG (1) | BG106964A (zh) |
BR (1) | BR0107972A (zh) |
CA (1) | CA2398611A1 (zh) |
CZ (1) | CZ20022643A3 (zh) |
DZ (1) | DZ3286A1 (zh) |
EA (1) | EA200200724A1 (zh) |
HK (1) | HK1051317A1 (zh) |
HU (1) | HUP0204250A3 (zh) |
IL (1) | IL150756A0 (zh) |
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NO (1) | NO20023616L (zh) |
NZ (1) | NZ520327A (zh) |
OA (1) | OA12168A (zh) |
PL (1) | PL211762B1 (zh) |
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WO (1) | WO2001054719A2 (zh) |
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2002
- 2002-07-30 NO NO20023616A patent/NO20023616L/no unknown
- 2002-07-30 BG BG106964A patent/BG106964A/bg unknown
-
2003
- 2003-03-20 HK HK03102061.7A patent/HK1051317A1/zh unknown
-
2005
- 2005-04-29 US US11/119,212 patent/US20050266025A1/en not_active Abandoned
-
2008
- 2008-05-08 US US12/117,205 patent/US20090104229A1/en not_active Abandoned
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1930184B (zh) * | 2004-03-11 | 2012-11-28 | 国家高等卫生院 | 新型tat复合物,及包含其的疫苗 |
CN104001155A (zh) * | 2014-06-12 | 2014-08-27 | 中山大学 | 一种Tat蛋白及其制备方法和应用 |
CN104001155B (zh) * | 2014-06-12 | 2016-04-13 | 中山大学 | 一种Tat蛋白及其制备方法和应用 |
Also Published As
Publication number | Publication date |
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PL357210A1 (en) | 2004-07-26 |
AP2002002592A0 (en) | 2002-09-30 |
NZ520327A (en) | 2004-06-25 |
US20090104229A1 (en) | 2009-04-23 |
IL150756A0 (en) | 2003-02-12 |
US20030158134A1 (en) | 2003-08-21 |
HK1051317A1 (zh) | 2003-08-01 |
US20050266025A1 (en) | 2005-12-01 |
KR20070073987A (ko) | 2007-07-10 |
BR0107972A (pt) | 2002-11-05 |
AU783005B2 (en) | 2005-09-15 |
AU5791001A (en) | 2001-08-07 |
KR100808348B1 (ko) | 2008-02-27 |
NO20023616D0 (no) | 2002-07-30 |
EA200200724A1 (ru) | 2003-02-27 |
CZ20022643A3 (cs) | 2003-02-12 |
NO20023616L (no) | 2002-09-17 |
BG106964A (bg) | 2004-01-30 |
SK11122002A3 (sk) | 2003-01-09 |
KR20020073569A (ko) | 2002-09-27 |
PL211762B1 (pl) | 2012-06-29 |
CN1326873C (zh) | 2007-07-18 |
HUP0204250A3 (en) | 2005-06-28 |
WO2001054719A2 (en) | 2001-08-02 |
EP1251870A2 (en) | 2002-10-30 |
JP2003529559A (ja) | 2003-10-07 |
WO2001054719A3 (en) | 2001-12-20 |
MXPA02007413A (es) | 2004-07-30 |
DZ3286A1 (fr) | 2001-08-02 |
HUP0204250A1 (hu) | 2003-03-28 |
CA2398611A1 (en) | 2001-08-02 |
OA12168A (en) | 2006-05-08 |
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