中国中部地区人类免疫缺陷病毒流行株HIV-1 B’的全长基因组序列比较分析及其侵染性克隆的构建
发布时间：2018-11-03 12:43
【摘要】：我们前期的工作证明,中国主要存在两种HIV-1亚型流行株,一种是流行于河南有偿献血人群(PBD)中的B`亚型,另一种是流行于云南、广西和新疆的静脉吸毒人群(IDU)中的CRF07和08 CB`重组亚型。本研究是我们前期工作的延伸。我们对2002年来自河南的不同地区的3个有偿献血者体内的HIV-1全长基因组进行了分离和系统地分析,获得了国际上第一株来自中国河南地区有偿献血人群中的HIV-1的全长基因组,证明流行于河南的B`亚型毒株没有流行于中国其他地区或者世界上任何其它地区的HIV-1毒株发生重组。之后,我们又进一步获得了3个HIV-1 B`毒株的全长基因组,并在此基础上成功构建了中国第一株侵染性全长分子克降,同时也是世界上首株HIV-1 B`侵染性克隆。对3个有偿献血者体内的HIV-1全长基因组的系统分析发现,这3个克隆彼此之间极其相似,并与分离自中国云南和河南的HIV-1 B`毒株关系紧密。对3个全长基因组序列的重组分析进一步很好的证实了我们先前的结论,即河南省的HIV-1 B`毒株没有与流行于中国的其他亚型毒株发生重组的迹象。3个有偿献血者尽管来自河南的不同地区但是体内的HIV-1全长基因组序列及其相似这一特性表明,河南省得HIV-1很可能来源于一个共同的单一的毒株,在传入河南后才扩敞到其他地区。3个克隆中的两个具有所有已知的病毒的结构和功能蛋白的完整的开放阅读框,没有在蛋白酶和逆转录酶基因上发现耐药突变,与样品取自中国开始全国范围内的免费抗逆转录病毒治疗之前的时间相一致。大多数有效中和抗体识别的抗原决定簇也很保守。完成全长基因组序列分析后,利用获得的来自3个PBD的HIV-1 B`基因组片断,我们用常规克隆技术构建了6个HIV-1 B`亚型侵染性全长分子克隆。其中2个克隆来自单一病人,另外4个是用来自不同的病人的HIV-1 B`基因组片断组合成的嵌合病毒。6个克隆都为R5嗜性,能有效感染TZM-b1细胞并激活报告基因的表达。病毒在体外细胞中的复制动力学研究初步表明6个病毒都能够在CEM×174-5.25M7细胞中有效复制,5个克隆能够在人外周血单核细胞中复制,其中02HNsq4-11A和02HNsq4-sc11-smx2较其它克隆显示更好的体外细胞中的复制能力。我们相信,本研究中提供的全长基因组信息将对未来设计和发展针对对中国中部地区的有效的疫苗和抗逆转录病毒药物意义重大。构建的侵染性克隆也将成为研究中国中部地区HIV-1生物特性和测试针对此地区的HIV-1的疫苗和药物的重要工具。
[Abstract]:Our previous work has proved that there are mainly two HIV-1 subtypes in China, one is the B subtype prevalent in Henan paid blood donation population, and the other is prevalent in Yunnan. CRF07 and 08 CB `Recombinant subtypes in (IDU) of intravenous Drug users in Guangxi and Xinjiang. This study is an extension of our earlier work. We isolated and systematically analyzed the full-length HIV-1 genome of three paid blood donors from different regions of Henan Province in 2002. The first full-length HIV-1 genome from paid blood donors from Henan region of China was obtained. The results showed that the B subtype B strain was not recombined in other regions of China or in any other region of the world. After that, we further obtained the full-length genome of three HIV-1 B` strains, and successfully constructed the first infective full-length molecular clone of HIV-1 B` in China, and was the first HIV-1 B` infective clone in the world at the same time. A systematic analysis of the full-length HIV-1 genomes of three paid blood donors showed that the three clones were very similar to each other and were closely related to the HIV-1 B` strains isolated from Yunnan and Henan provinces of China. The recombination analysis of three full-length genomic sequences further confirms our previous conclusion. That is, the HIV-1 B strain in Henan Province showed no sign of recombination with other subtype strains prevalent in China. Three paid blood donors, although from different regions in Henan Province, showed the full-length HIV-1 genome sequence and its phase in the body. This property suggests that, The HIV-1 from Henan Province is probably derived from a common single strain, which was introduced to Henan and only opened to other regions. Two of the three clones have a complete open reading frame with all known structural and functional proteins of the virus. No drug-resistant mutations were found in protease and reverse transcriptase genes, consistent with the time before samples were taken from China to begin free antiretroviral therapy nationwide. The antigenic determinants recognized by most effective neutralizing antibodies are also conservative. After the complete sequence analysis of the full-length genome, we constructed six full-length molecular clones of HIV-1 B` subtype by conventional cloning technique, using the HIV-1 B` genome fragments from three PBD. Two of the clones were from a single patient, the other four were chimeric viruses composed of HIV-1 B` genome fragments from different patients. The six clones were R5 eosinophilic and could effectively infect TZM-b1 cells and activate the expression of reporter genes. The study on the replication kinetics of the virus in vitro showed that all 6 viruses could replicate effectively in CEM 脳 174-5.25M7 cells, and 5 clones could replicate in human peripheral blood monocytes. 02HNsq4-11A and 02HNsq4-sc11-smx2 showed better replication ability in vitro than other clones. We believe that the full length genome information provided in this study will be of great significance for the future design and development of effective vaccines and antiretroviral drugs for central China. The constructed infectious clones will also be an important tool for studying the biological characteristics of HIV-1 and testing vaccines and drugs for HIV-1 in central China.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R373
，
本文编号：2307825
[Abstract]:Our previous work has proved that there are mainly two HIV-1 subtypes in China, one is the B subtype prevalent in Henan paid blood donation population, and the other is prevalent in Yunnan. CRF07 and 08 CB `Recombinant subtypes in (IDU) of intravenous Drug users in Guangxi and Xinjiang. This study is an extension of our earlier work. We isolated and systematically analyzed the full-length HIV-1 genome of three paid blood donors from different regions of Henan Province in 2002. The first full-length HIV-1 genome from paid blood donors from Henan region of China was obtained. The results showed that the B subtype B strain was not recombined in other regions of China or in any other region of the world. After that, we further obtained the full-length genome of three HIV-1 B` strains, and successfully constructed the first infective full-length molecular clone of HIV-1 B` in China, and was the first HIV-1 B` infective clone in the world at the same time. A systematic analysis of the full-length HIV-1 genomes of three paid blood donors showed that the three clones were very similar to each other and were closely related to the HIV-1 B` strains isolated from Yunnan and Henan provinces of China. The recombination analysis of three full-length genomic sequences further confirms our previous conclusion. That is, the HIV-1 B strain in Henan Province showed no sign of recombination with other subtype strains prevalent in China. Three paid blood donors, although from different regions in Henan Province, showed the full-length HIV-1 genome sequence and its phase in the body. This property suggests that, The HIV-1 from Henan Province is probably derived from a common single strain, which was introduced to Henan and only opened to other regions. Two of the three clones have a complete open reading frame with all known structural and functional proteins of the virus. No drug-resistant mutations were found in protease and reverse transcriptase genes, consistent with the time before samples were taken from China to begin free antiretroviral therapy nationwide. The antigenic determinants recognized by most effective neutralizing antibodies are also conservative. After the complete sequence analysis of the full-length genome, we constructed six full-length molecular clones of HIV-1 B` subtype by conventional cloning technique, using the HIV-1 B` genome fragments from three PBD. Two of the clones were from a single patient, the other four were chimeric viruses composed of HIV-1 B` genome fragments from different patients. The six clones were R5 eosinophilic and could effectively infect TZM-b1 cells and activate the expression of reporter genes. The study on the replication kinetics of the virus in vitro showed that all 6 viruses could replicate effectively in CEM 脳 174-5.25M7 cells, and 5 clones could replicate in human peripheral blood monocytes. 02HNsq4-11A and 02HNsq4-sc11-smx2 showed better replication ability in vitro than other clones. We believe that the full length genome information provided in this study will be of great significance for the future design and development of effective vaccines and antiretroviral drugs for central China. The constructed infectious clones will also be an important tool for studying the biological characteristics of HIV-1 and testing vaccines and drugs for HIV-1 in central China.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R373
，
本文编号：2307825
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