我国艾滋病病毒B'、B/C亚型流行株的表型分析、全基因组克隆及gp41主要抗原表位研究
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摘要
艾滋病病毒(Human Immunodeficiency Virus,HIV)是导致人获得性免疫缺陷综合征(Acquired Immunodeficiency Syndrome,AIDS)的病原体。截止至2002年年底，全球存活的HIV感染者已经达到4200万人。我国HIV／AIDS流行呈快速上升趋势，估计HIV感染者已经达到100万人。HIV分为HIV-1和HIV-2型，HIV-1的M群至少包含11个亚型，其中B’和B／C重组亚型是我国的主要流行株。对我国的HIV流行株进行系统的表型分析、基因组研究和表位鉴定是研究我国HIV疫苗、诊断方法和致病机理的基础，我国至今对B’和B／C重组亚型毒株缺乏这样系统的研究。本文对我国HIV-1B’和B／C亚型毒株进行了表型分析、全基因组克隆及及序列测定。对B’亚型毒株gp41的主要抗原表位进行了研究。
1．我国艾滋病病毒B’、B／C亚型流行株的表型分析
HIV为单股正链RNA病毒，具有高度变异性，其突变频率比DNA病毒高100万倍。高突变性导致HIV复杂的表型特征，HIV-1表型决定感染宿主细胞时使用辅助受体的类型，分为M嗜性、T嗜性和双嗜性。HIV-1感染的早期多为M嗜性，晚期转变成T嗜性或双嗜性，而决定病毒细胞嗜性的区域位于HIV-1gp120，其中V3环起主要作用。我们从来自河南的一份有偿献血者血液中成功分离到一株HIV-1B’亚型毒株(CNHN24)，对病毒基因突变与细胞嗜性的关系进行了研究，主要结果如下：
A．将HIV-1感染者的PBMC与健康人PBMC混合共培养，分离出HIV-1 CNHN24株，证明这是一株嗜巨噬细胞，呈快／高型复制的病毒，分析了V3环基因序列突变与细胞嗜性变化的关系；
B．将感染HIV-1 CNHN24株的PBMC与MT4细胞共培养，使毒株由嗜巨噬细胞，使用CCR5辅助受体转变为嗜T细胞，使用CXCR4辅助受体；
C．HIV-1 CNHN24株已经在人传代T淋巴细胞株MT4细胞中稳定传16代，TCID_(50)维持在10~8的高滴度；
博士学位论文
中文摘要
D.序列分析表明，在细胞嗜性改变的同时，H工v一1 CNHN24株的V3环序列发生
了明显的变化，核普酸序列的突变率达到17%;
E.经PBMC和MT4细胞培养后，培养上清中的病毒基因与细胞基因组中前病毒
DNA的V3环序列完全一致;
F.分析了病毒由M嗜性转变为T嗜性的V3环氨基酸突变特征，突变率为9%。
突变位点位于V3环的第4、11、13、14、24、27、32位，其中第11、14、
27和32位氨基酸电荷变化明显。
2.我国艾滋病病毒B’、B/C亚型流行株全基因组克隆
我国H工V流行株全基因组序列的报道较少，尚未见在国内实验室完成Hlv
全基因组克隆的报道。我们利用RACE法确定了病毒mRNA的3’端和5’端序列，
设计了两条特异性引物，以CNHN24病毒感染细胞的前病毒基因组为PCR扩增的
模板，主要完成了以下工作:
A.建立了扩增H工v一1全基因组技术平台;
B.首次在国内实验室构建了H工V一IB’亚型CNHN24株的全基因组克隆;
C.全基因组克隆测序提交GenBank，登录号为:AY180905;
D.通过V3环序列分析确定CNHN24克隆为H工V一IB’亚型，氨基酸序列比对发
现在九个位点发生氨基酸替换;
E.Gag、Pol、Vpr和Vsf基因与RL42株的一致率达到95.42%~97.08%，但gpzZo
的氨基酸一致率只有64.6%;
F，进化分析显示CNHN24株与国内RL42株的遗传距离最近。
扩增低拷贝基因的长片段链是长链PCR技术面临的一个难点，融合PCR方
法是解决这一困难的重要手段之一。根据我国HIV一IB/C亚型序列的保守区设
计引物，首先将02CNHN01株病毒全长基因分成6段相互部分重叠的片段进行PCR
扩增，然后再以融合PCR方法依次将相邻两个片段融合在一起。主要结果如下:
A.建立了H脚一IB/C亚型全长基因组DNA分子的融合PCR方法;
B.利用融合PCR方法成功克隆了H工V一IB/C亚型(OZCNHN01株)全基因组;
C.CZ一V3区序列与H工V一IC亚型国际参考株一致率为97.14%，与国内HIV一1
B/C亚型株的一致率为95.41%~96.33%;
D．系统发育分析显示，该毒株的序列与国内 HIVl 北亚型98CN009株聚在一
起，而98CN009株序列则与93IN101株聚在一起。
E．序列分析发现部分位点有HIV－IB亚型重组，确定 02CNHN01株为mV－IB／C
重组型。
3．我国艾滋病病毒B’亚型流行株PP41主要抗原表位研究
噬菌体展示技术已被证明为研究蛋白质与其配体相互关系很有效的工具。
本实验中，我们通过人抗HIV－IB’亚型PP41多克隆抗体，筛选噬菌体展示随
机12肽库，将得到的表位及两个表位的串联体与噬菌体厂＊蛋白的I～11结
构域在PoE30载体中表达，用纯化的表达蛋白检测HIV感染者血清中的特异性
抗体，证实了表位抗原和串联表位抗原用于＊ 抗体检测的可行性。主要结果
如下：
A．制备了N工＊1＊＊1表达抗原亲和层析柱：
B．从我国HIV＋’亚型感染者血清中纯化了抗PP41多克隆抗体；
C．从噬菌体展示随机12肽库中筛选到位于gP41免疫优势区588～597aa的3
个表位 ph32PI、ph33PI和 Dh39PI；
D．将Ph39PI与文献报道的一个HIV－lgP41表位串联连接；
E．将 4个表位分别插入到噬菌体厂 11蛋白的 I～11结构域中，在 PQE30载体
中实现表达；
F．纯化的表达蛋白可用于检测HIV－1感染者血清中的抗体
Human Immunodeficiency Virus (HIV) was the pathogen of Acquired Immunodeficiency Syndrome (AIDS). Up to the end 2002, survival HlV-infected individuals have amounted to 42 millions. According to the specialist estimates, the number of HIV-infected individuals and/or AIDS patients is increasing rapidly, it has been one million in China. HIV can be divided into HIV-1 and HIV-2, at least 11 subtypes in HIV-1 main group, of which HIV-1 B' and B/C are prevalent subtypes in China. Systemic phenotype analysis, study on the genome characterization and identify in HIV-1 epitopes may be the basis for the development of candidate vaccines and diagnosis methods, design of anti-HIV agents based on HIV-1 prevalent subtypes in China. Up to now, there have not been a systemic study for HIV-1 B' and B/C strains in China. IN this study, we reported the phenotypic analysis, full-length molecular cloning and major gp41 antigen epitope application of HIV-1 B' and B/C subtype strains in China.
Part I. Phenotypic analysis of HIV-1 prevalent Subtypes B' and B/C strains in China
As the single plus stranded RNA virus, HIV is of great variability. The mutation rate is one million times to DNA viruses. It resulted in difference in virus phenotypes. HIV phenotype determines its coreceptor utilization. While HIV entrying into cells, virus isolates are classified into three groups, M-tropic,T-tropic and dualtropic. Early infection with HIV-1 is dominated by M-tropic viruses,the evolution of CXCR4-tropic viruses later in the infection. As to viral factors involved in HIV-1 cell tropism, a specific region of gp!20 protein, the V3 loop, was demonstrated to be a major determinant of coreceptor usage. In this part, one HIV-1 strain was isolated from a HIV-1 infected paid blood donor in Henan province, whose viruses were identified as B' genotype. The cell tropism of the virus was identified. Major results are showed following:
A. HIV-1 CNHN24 strain was isolated by PBMC coculturing from a HIV-infected person with phytohemaggluting-stimulated donor PBMC. The phenotype was M-tropic, SI and rapid/high replication. The relationship between cell tropism change and V3 loop mutation was analyzed.
B. By coculture of PBMC and MT4 cell, HIV-1 CNHN24 strain turnovered T-tropic, CXCR4-utilization from M-tropic, CCR5-utilization.
C. HIV-1 CNHN24 isolate could infect MT4 cell successively, which had been passed 16 generations with high titer of 108 TCID50.
D. HIV-1 CNHN24 infected PBMC were cocultured with donor PBMC and/or MT4 cell, HIV-1 RNA and proviral DNA were extracted from the supernatant and PBMC and/or MT4 cell, respectively. V3 loop sequences were absolutely identities for HIV-1 RNA and proviral DNA.
E. When HIV-1 CNHN24 turnover its cell tropism, the significant changes have taken place in V3 loop sequence of HIV-1 CNHN24 strain, the mutation rate of nucleic acid sequence was 17%.
F. The mutation of V3 loop ammo acid was analyzed on T-tropic turnover from M-tropic, the mutation rate was 9%, showing amino acid mutant at positions 4,11,13,14,24,27,32, of which the changes of amino acid charge were obviously at positions 11,14,27,32.
Part II. Full-length genome cloning of subtype HIV-1 B' and B/C in China
Up to now, only a few HIV-1 full-length genome sequences of prevalent HIV-1 strains were reported, and no any reports on HIV-1 full-length genome cloning in China. We have developed 3' end and 5' end sequencing of mRMA by RACE technical, designed and synthesized two specific primers according to the sequence for amplify full-length HIV-1 genome. The PCR reaction was performed by using HIV-1 CNHN24 proviral DNA as the template.
A. With the optimization of the components of reaction, Long fragment PCR for HIV-1 full-length genome was finally set up.
B. The cloning and characterization of a virtually full-length HIV-1 B' subtype was completed first time in Chinese lab.
C. The full sequence of the cloned primary isolate CNHN24 has been submitted to GenBank database, search number is AY180905.
D. HIV-1 CNHN
1．我国艾滋病病毒B’、B／C亚型流行株的表型分析
HIV为单股正链RNA病毒，具有高度变异性，其突变频率比DNA病毒高100万倍。高突变性导致HIV复杂的表型特征，HIV-1表型决定感染宿主细胞时使用辅助受体的类型，分为M嗜性、T嗜性和双嗜性。HIV-1感染的早期多为M嗜性，晚期转变成T嗜性或双嗜性，而决定病毒细胞嗜性的区域位于HIV-1gp120，其中V3环起主要作用。我们从来自河南的一份有偿献血者血液中成功分离到一株HIV-1B’亚型毒株(CNHN24)，对病毒基因突变与细胞嗜性的关系进行了研究，主要结果如下：
A．将HIV-1感染者的PBMC与健康人PBMC混合共培养，分离出HIV-1 CNHN24株，证明这是一株嗜巨噬细胞，呈快／高型复制的病毒，分析了V3环基因序列突变与细胞嗜性变化的关系；
B．将感染HIV-1 CNHN24株的PBMC与MT4细胞共培养，使毒株由嗜巨噬细胞，使用CCR5辅助受体转变为嗜T细胞，使用CXCR4辅助受体；
C．HIV-1 CNHN24株已经在人传代T淋巴细胞株MT4细胞中稳定传16代，TCID_(50)维持在10~8的高滴度；
博士学位论文
中文摘要
D.序列分析表明，在细胞嗜性改变的同时，H工v一1 CNHN24株的V3环序列发生
了明显的变化，核普酸序列的突变率达到17%;
E.经PBMC和MT4细胞培养后，培养上清中的病毒基因与细胞基因组中前病毒
DNA的V3环序列完全一致;
F.分析了病毒由M嗜性转变为T嗜性的V3环氨基酸突变特征，突变率为9%。
突变位点位于V3环的第4、11、13、14、24、27、32位，其中第11、14、
27和32位氨基酸电荷变化明显。
2.我国艾滋病病毒B’、B/C亚型流行株全基因组克隆
我国H工V流行株全基因组序列的报道较少，尚未见在国内实验室完成Hlv
全基因组克隆的报道。我们利用RACE法确定了病毒mRNA的3’端和5’端序列，
设计了两条特异性引物，以CNHN24病毒感染细胞的前病毒基因组为PCR扩增的
模板，主要完成了以下工作:
A.建立了扩增H工v一1全基因组技术平台;
B.首次在国内实验室构建了H工V一IB’亚型CNHN24株的全基因组克隆;
C.全基因组克隆测序提交GenBank，登录号为:AY180905;
D.通过V3环序列分析确定CNHN24克隆为H工V一IB’亚型，氨基酸序列比对发
现在九个位点发生氨基酸替换;
E.Gag、Pol、Vpr和Vsf基因与RL42株的一致率达到95.42%~97.08%，但gpzZo
的氨基酸一致率只有64.6%;
F，进化分析显示CNHN24株与国内RL42株的遗传距离最近。
扩增低拷贝基因的长片段链是长链PCR技术面临的一个难点，融合PCR方
法是解决这一困难的重要手段之一。根据我国HIV一IB/C亚型序列的保守区设
计引物，首先将02CNHN01株病毒全长基因分成6段相互部分重叠的片段进行PCR
扩增，然后再以融合PCR方法依次将相邻两个片段融合在一起。主要结果如下:
A.建立了H脚一IB/C亚型全长基因组DNA分子的融合PCR方法;
B.利用融合PCR方法成功克隆了H工V一IB/C亚型(OZCNHN01株)全基因组;
C.CZ一V3区序列与H工V一IC亚型国际参考株一致率为97.14%，与国内HIV一1
B/C亚型株的一致率为95.41%~96.33%;
D．系统发育分析显示，该毒株的序列与国内 HIVl 北亚型98CN009株聚在一
起，而98CN009株序列则与93IN101株聚在一起。
E．序列分析发现部分位点有HIV－IB亚型重组，确定 02CNHN01株为mV－IB／C
重组型。
3．我国艾滋病病毒B’亚型流行株PP41主要抗原表位研究
噬菌体展示技术已被证明为研究蛋白质与其配体相互关系很有效的工具。
本实验中，我们通过人抗HIV－IB’亚型PP41多克隆抗体，筛选噬菌体展示随
机12肽库，将得到的表位及两个表位的串联体与噬菌体厂＊蛋白的I～11结
构域在PoE30载体中表达，用纯化的表达蛋白检测HIV感染者血清中的特异性
抗体，证实了表位抗原和串联表位抗原用于＊ 抗体检测的可行性。主要结果
如下：
A．制备了N工＊1＊＊1表达抗原亲和层析柱：
B．从我国HIV＋’亚型感染者血清中纯化了抗PP41多克隆抗体；
C．从噬菌体展示随机12肽库中筛选到位于gP41免疫优势区588～597aa的3
个表位 ph32PI、ph33PI和 Dh39PI；
D．将Ph39PI与文献报道的一个HIV－lgP41表位串联连接；
E．将 4个表位分别插入到噬菌体厂 11蛋白的 I～11结构域中，在 PQE30载体
中实现表达；
F．纯化的表达蛋白可用于检测HIV－1感染者血清中的抗体
Human Immunodeficiency Virus (HIV) was the pathogen of Acquired Immunodeficiency Syndrome (AIDS). Up to the end 2002, survival HlV-infected individuals have amounted to 42 millions. According to the specialist estimates, the number of HIV-infected individuals and/or AIDS patients is increasing rapidly, it has been one million in China. HIV can be divided into HIV-1 and HIV-2, at least 11 subtypes in HIV-1 main group, of which HIV-1 B' and B/C are prevalent subtypes in China. Systemic phenotype analysis, study on the genome characterization and identify in HIV-1 epitopes may be the basis for the development of candidate vaccines and diagnosis methods, design of anti-HIV agents based on HIV-1 prevalent subtypes in China. Up to now, there have not been a systemic study for HIV-1 B' and B/C strains in China. IN this study, we reported the phenotypic analysis, full-length molecular cloning and major gp41 antigen epitope application of HIV-1 B' and B/C subtype strains in China.
Part I. Phenotypic analysis of HIV-1 prevalent Subtypes B' and B/C strains in China
As the single plus stranded RNA virus, HIV is of great variability. The mutation rate is one million times to DNA viruses. It resulted in difference in virus phenotypes. HIV phenotype determines its coreceptor utilization. While HIV entrying into cells, virus isolates are classified into three groups, M-tropic,T-tropic and dualtropic. Early infection with HIV-1 is dominated by M-tropic viruses,the evolution of CXCR4-tropic viruses later in the infection. As to viral factors involved in HIV-1 cell tropism, a specific region of gp!20 protein, the V3 loop, was demonstrated to be a major determinant of coreceptor usage. In this part, one HIV-1 strain was isolated from a HIV-1 infected paid blood donor in Henan province, whose viruses were identified as B' genotype. The cell tropism of the virus was identified. Major results are showed following:
A. HIV-1 CNHN24 strain was isolated by PBMC coculturing from a HIV-infected person with phytohemaggluting-stimulated donor PBMC. The phenotype was M-tropic, SI and rapid/high replication. The relationship between cell tropism change and V3 loop mutation was analyzed.
B. By coculture of PBMC and MT4 cell, HIV-1 CNHN24 strain turnovered T-tropic, CXCR4-utilization from M-tropic, CCR5-utilization.
C. HIV-1 CNHN24 isolate could infect MT4 cell successively, which had been passed 16 generations with high titer of 108 TCID50.
D. HIV-1 CNHN24 infected PBMC were cocultured with donor PBMC and/or MT4 cell, HIV-1 RNA and proviral DNA were extracted from the supernatant and PBMC and/or MT4 cell, respectively. V3 loop sequences were absolutely identities for HIV-1 RNA and proviral DNA.
E. When HIV-1 CNHN24 turnover its cell tropism, the significant changes have taken place in V3 loop sequence of HIV-1 CNHN24 strain, the mutation rate of nucleic acid sequence was 17%.
F. The mutation of V3 loop ammo acid was analyzed on T-tropic turnover from M-tropic, the mutation rate was 9%, showing amino acid mutant at positions 4,11,13,14,24,27,32, of which the changes of amino acid charge were obviously at positions 11,14,27,32.
Part II. Full-length genome cloning of subtype HIV-1 B' and B/C in China
Up to now, only a few HIV-1 full-length genome sequences of prevalent HIV-1 strains were reported, and no any reports on HIV-1 full-length genome cloning in China. We have developed 3' end and 5' end sequencing of mRMA by RACE technical, designed and synthesized two specific primers according to the sequence for amplify full-length HIV-1 genome. The PCR reaction was performed by using HIV-1 CNHN24 proviral DNA as the template.
A. With the optimization of the components of reaction, Long fragment PCR for HIV-1 full-length genome was finally set up.
B. The cloning and characterization of a virtually full-length HIV-1 B' subtype was completed first time in Chinese lab.
C. The full sequence of the cloned primary isolate CNHN24 has been submitted to GenBank database, search number is AY180905.
D. HIV-1 CNHN
引文
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