中國(guó)中部地區(qū)人類免疫缺陷病毒流行株HIV-1 B’的全長(zhǎng)基因組序列比較分析及其侵染性克隆的構(gòu)建
發(fā)布時(shí)間：2018-11-03 12:43
【摘要】：我們前期的工作證明,中國(guó)主要存在兩種HIV-1亞型流行株,一種是流行于河南有償獻(xiàn)血人群(PBD)中的B`亞型,另一種是流行于云南、廣西和新疆的靜脈吸毒人群(IDU)中的CRF07和08 CB`重組亞型。本研究是我們前期工作的延伸。我們對(duì)2002年來自河南的不同地區(qū)的3個(gè)有償獻(xiàn)血者體內(nèi)的HIV-1全長(zhǎng)基因組進(jìn)行了分離和系統(tǒng)地分析,獲得了國(guó)際上第一株來自中國(guó)河南地區(qū)有償獻(xiàn)血人群中的HIV-1的全長(zhǎng)基因組,證明流行于河南的B`亞型毒株沒有流行于中國(guó)其他地區(qū)或者世界上任何其它地區(qū)的HIV-1毒株發(fā)生重組。之后,我們又進(jìn)一步獲得了3個(gè)HIV-1 B`毒株的全長(zhǎng)基因組,并在此基礎(chǔ)上成功構(gòu)建了中國(guó)第一株侵染性全長(zhǎng)分子克降,同時(shí)也是世界上首株HIV-1 B`侵染性克隆。對(duì)3個(gè)有償獻(xiàn)血者體內(nèi)的HIV-1全長(zhǎng)基因組的系統(tǒng)分析發(fā)現(xiàn),這3個(gè)克隆彼此之間極其相似,并與分離自中國(guó)云南和河南的HIV-1 B`毒株關(guān)系緊密。對(duì)3個(gè)全長(zhǎng)基因組序列的重組分析進(jìn)一步很好的證實(shí)了我們先前的結(jié)論,即河南省的HIV-1 B`毒株沒有與流行于中國(guó)的其他亞型毒株發(fā)生重組的跡象。3個(gè)有償獻(xiàn)血者盡管來自河南的不同地區(qū)但是體內(nèi)的HIV-1全長(zhǎng)基因組序列及其相似這一特性表明,河南省得HIV-1很可能來源于一個(gè)共同的單一的毒株,在傳入河南后才擴(kuò)敞到其他地區(qū)。3個(gè)克隆中的兩個(gè)具有所有已知的病毒的結(jié)構(gòu)和功能蛋白的完整的開放閱讀框,沒有在蛋白酶和逆轉(zhuǎn)錄酶基因上發(fā)現(xiàn)耐藥突變,與樣品取自中國(guó)開始全國(guó)范圍內(nèi)的免費(fèi)抗逆轉(zhuǎn)錄病毒治療之前的時(shí)間相一致。大多數(shù)有效中和抗體識(shí)別的抗原決定簇也很保守。完成全長(zhǎng)基因組序列分析后,利用獲得的來自3個(gè)PBD的HIV-1 B`基因組片斷,我們用常規(guī)克隆技術(shù)構(gòu)建了6個(gè)HIV-1 B`亞型侵染性全長(zhǎng)分子克隆。其中2個(gè)克隆來自單一病人,另外4個(gè)是用來自不同的病人的HIV-1 B`基因組片斷組合成的嵌合病毒。6個(gè)克隆都為R5嗜性,能有效感染TZM-b1細(xì)胞并激活報(bào)告基因的表達(dá)。病毒在體外細(xì)胞中的復(fù)制動(dòng)力學(xué)研究初步表明6個(gè)病毒都能夠在CEM×174-5.25M7細(xì)胞中有效復(fù)制,5個(gè)克隆能夠在人外周血單核細(xì)胞中復(fù)制,其中02HNsq4-11A和02HNsq4-sc11-smx2較其它克隆顯示更好的體外細(xì)胞中的復(fù)制能力。我們相信,本研究中提供的全長(zhǎng)基因組信息將對(duì)未來設(shè)計(jì)和發(fā)展針對(duì)對(duì)中國(guó)中部地區(qū)的有效的疫苗和抗逆轉(zhuǎn)錄病毒藥物意義重大。構(gòu)建的侵染性克隆也將成為研究中國(guó)中部地區(qū)HIV-1生物特性和測(cè)試針對(duì)此地區(qū)的HIV-1的疫苗和藥物的重要工具。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R373
，
本文編號(hào)：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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R373
，
本文編號(hào)：2307825
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