HIV-1B'亚型毒株全基因组分子克隆
摘要
艾滋病又称获得性免疫缺陷综合症(acquired immunodeficiency syndrome, AIDS),引起艾滋病的病原体为人类免疫缺陷病毒(human immunodeficiency virus, HIV)。自美国1981年6月5日报告艾滋病,26年来艾滋病已在世界范围内迅速传播,给人类健康以巨大威胁。我国于1985年首次发现并报道了HIV-1感染者——一位来自境外的旅游者,随后在浙江发现因使用美国Amour公司的凝血因子VⅧ而感染HIV的感染者。研究发现,我国主要存在有B／C重组、B′、B、C、CRF01_AE等HIV-1亚型病毒流行株,但以B／C重组、B′为主。CRF_07和CRF_08 B／C重组亚型主要流行于我国西南省份地区和新疆等地的静脉注射吸毒人群中,B′亚型则是流行于华中地区河南省、湖北省及周边地区非法采供血人群中。
90年代初,因为有偿献血行为,我国中部省份河南和湖北等地出现了HIV-1 B′亚型毒株的暴发流行。本文对2005年来自湖北省武汉地区的一位AIDS患者体内的HIV-1B′病毒进行了分离,该患者因垂直传播(母婴传播)感染HIV-1,而其母亲因有偿献血感染HIV-1,发展为艾滋病后病逝。随后对分离的病毒进行基因克隆,获得了一株来自我国中部地区HIV-1 B′流行株的全基因组分子克隆。
1.通过与健康人外周血单核淋巴细胞(PBMCs)共培养,对患者体内的HIV-1 B′病毒进行分离。分别分离健康人和HIV-1感染者外周血单核淋巴细胞,将感染者PBMCs与用植物血球凝集素(PHA)刺激培养3天的健康人PBMCs共培养,在共培养体系中加入白细胞介素-2(IL-2),并且每隔3天更换50%新鲜培养液,收集培养上清测定逆转录酶活性或p24病毒抗原确定病毒的增殖。将分离的病毒株命名为05CNHB_hp。
2.利用分别转染了辅助受体CXCR4和CCR5的细胞系细胞,接种05CNHB_hp病毒培养液,观察分离病毒株的细胞嗜性。分离的病毒株能在HOS-CD4-CXCR4细胞形成明显的合胞体,而在HOS-CD4-CCR5细胞则不能观察到合胞体的形成；在HOS-CD4-CXCR4细胞培养上清中能明显检测到p24病毒抗原,而在HOS-CD4-CCR5细胞培养上清中没有检测到p24病毒抗原。实验结果表明,分离的05CNHB_hp具CXCR4辅助受体细胞嗜性。
3.将分离的HIV-1 B'05CNHB_hp病毒培养液接种于PHA刺激培养3天的健康人PBMCs,在病毒增殖高峰期收集培养细胞,提取细胞基因组DNA,设计引物扩增病毒基因组3’端近全长序列并克隆到载体PCR(?)-XL-Vector,经测序得到该病毒LTR起始序列,设计引物扩增病毒基因组剩余5′端一相对短的片段序列,克隆到同样的载体,测序后获得整个基因组的核苷酸序列。根据测序结果找到合适的酶切位点,通过限制性酶切两个克隆然后将两个克隆片段连接得到具有完整HIV-1基因组的分子克隆。
4.将测序的结果运用Bioedit软件与从HIV数据库(http://hiv-web.lanl.gov)下载的其他各亚型全长序列进行比对,并运用ClastalX1.83软件进行系统分析。从系统树分析来看,分离的病毒属于HIV-1 B′亚型。然后通过Simplot软件进行Bootscan分析,检测病毒是否存在重组现象。结果未发现重组现象。随后为了检测该病毒的耐药性突变,将病毒基因序列提交斯坦福大学的HIV耐药数据库(http://hivdb.stanford.edu/pages/ algs/HIVdb.html)分析病毒可能的基因型耐药突变。结果表明该病毒没有产生耐药突变,分离的病毒是一株能较好体现药物使用前流行的HIV-1 B′病毒,而且现有的抗艾滋病药物对这类病毒均具有较好的抑制作用。
5.病毒全基因组分子克隆转染293T细胞研究。用Lipofectamine2000转染试剂将全长克隆质粒转染到293T细胞,而后收集转染细胞培养上清测定p24病毒抗原并感染用PHA刺激培养3天的健康人PBMCs,接着检测培养上清的p24病毒抗原。通过反复的感染试验,发现构建的该克隆不具备感染活性。
本研究中提供的全基因组信息将对针对我国中部地区有效的疫苗和耐药性研究设计和发展提供参考,具有重要意义。
Acquired Immunodeficiency Syndrome (AIDS) is caused by Human Immunodeficiency Virus (HIV). It was firstly reported in American in 1981. During the last 26 years, AIDS has spread virtually every corner of the world. The first HIV-1 infection in China was reported in 1985. It was founded that there were two major HIV-1 subtypes in China:CRF07 and 08 in the IDU in Yunnan, Guangxi and Xinjiang, and subtype B'in the illegal paid blood donors (PBD) in central China, such as Henan and Hubei etc.
In the early 1990s, because of PBD, HIV-1 B'broke out circulation in central China. We isolated a strain of HIV-1 B' from a patient in Wuhan, Hubei province in 2005. We learned that the patient infected HIV-1 from his mother because of vertical infection, and his mother died of AIDS years ago. We constructed and characterized the full-length genome clone of the isolated virus HIV-1 B'.
1.Normal donor's peripheral blood mononuclear cells (PBMCs) obtained from HIV-negative blood donors were isolated from buffy coats using the Ficollhypaque method. Immediately after isolation, PBMCs were stimulated with 0.5ug/ml phytohemagglutinin (PHA). PBMCs were maintained in RPMI1640 supplemented with 10% fetal bovine serum, 2mmol/L glutamine, and antibiotics. Briefly, the isolated PBMCs from AIDS patient were co-cultivated with HIV-negative PHA-stimulated donor's PBMCs in RPMI 1640 supplemented with 10% fetal bovine serum,20U/ml IL-2,2mM glutamine, and antibiotics. Medium was changed in half every 3 days post co-culture and virus stock was used to test the RT/p24 to confirm the virus isolated and was stored at-80℃. Then we named the virus isolated 05CNHB_hp, and it replicated it's peak 11 days post co-cultivation.
2. With the cell lines transformed with CXCR4 and CCR5 respectively, inoculated with the virus 05CNHB_hp to determine its cell tropism. The virus could induce syncytium in HOS-CD4-CXCR4 cells, but it not in HOS-CD4-CCR5 cells. Also the p24 could be tested in HOS-CD4-CXCR4 but couldn't in HOS-CD4-CCR5. So, the result showed that the virus infected the cells with co-receptor CXCR4.
3.The genome DNA was isolated from PBMCs infected with 05CNHB_hp, and the primers were designed to amplify the 3'terminal near full-length part of virus genome. Then the PCR product was cloned to T vector and sequenced. Based on the nucleotide sequenced, another pair of primers was designed to amplify the rest part of genome. The PCR product was cloned to the same vector and sequenced.
4.The nucleotide sequence was aligned to the sequences of other HIV-1 subtype download from HIV Database in http://hiv-web.lanl.gov, then used the soft Clustal X1.83 to make phylogenetic tree using the neighbor-joining method and Kimura two-parameter model. From the phylogenetic tree, the virus 05CNHB_hp was confirmed to belonged to HIV-1 B'.And to investigate its possible recombination with other subtypes, we used soft Simplot to Bootscan with reference sequences HIV-1 Al, B', and C subtype. From the Bootscan, we learned that the isolated virus 05CNHB_hp didn't have any recombination with other subtypes. And no mutation associated with drug-resistance was detected when the nucleotide sequence was submitted to HIV DRUG RESISTANCE DATABASE at Stanford University, which means the drugs available domestically are effective to restrain the virus and the virus isolated could show the primaly HIV-1 B'before the use of drugs.
5.At last, we transfected the full-length genome clone to 293T cells to test its infectivity. We retrieved the supernatant to test its p24 content and inoculated in PBMCs stimulated for 3 days by PHA. The full-length genome clone 05CNHB_hp3 we constructed could express p24 in 293T cells, but unfortunately, there was no p24 in the supernatant of PBMCs inoculated with transfected cells culture supernatant. That meant the clone 05CNHB_hp we constructed haven't got its infectivity.
The full-length genome information in our research would have very important implications for future design and development of an effective vaccine, antiretroviral drugs, and of necessary assays and reagents for testing future candidate vaccine and antiretroviral drugs targeted at the HIV-1 B'strains circulating in central China.
90年代初,因为有偿献血行为,我国中部省份河南和湖北等地出现了HIV-1 B′亚型毒株的暴发流行。本文对2005年来自湖北省武汉地区的一位AIDS患者体内的HIV-1B′病毒进行了分离,该患者因垂直传播(母婴传播)感染HIV-1,而其母亲因有偿献血感染HIV-1,发展为艾滋病后病逝。随后对分离的病毒进行基因克隆,获得了一株来自我国中部地区HIV-1 B′流行株的全基因组分子克隆。
1.通过与健康人外周血单核淋巴细胞(PBMCs)共培养,对患者体内的HIV-1 B′病毒进行分离。分别分离健康人和HIV-1感染者外周血单核淋巴细胞,将感染者PBMCs与用植物血球凝集素(PHA)刺激培养3天的健康人PBMCs共培养,在共培养体系中加入白细胞介素-2(IL-2),并且每隔3天更换50%新鲜培养液,收集培养上清测定逆转录酶活性或p24病毒抗原确定病毒的增殖。将分离的病毒株命名为05CNHB_hp。
2.利用分别转染了辅助受体CXCR4和CCR5的细胞系细胞,接种05CNHB_hp病毒培养液,观察分离病毒株的细胞嗜性。分离的病毒株能在HOS-CD4-CXCR4细胞形成明显的合胞体,而在HOS-CD4-CCR5细胞则不能观察到合胞体的形成；在HOS-CD4-CXCR4细胞培养上清中能明显检测到p24病毒抗原,而在HOS-CD4-CCR5细胞培养上清中没有检测到p24病毒抗原。实验结果表明,分离的05CNHB_hp具CXCR4辅助受体细胞嗜性。
3.将分离的HIV-1 B'05CNHB_hp病毒培养液接种于PHA刺激培养3天的健康人PBMCs,在病毒增殖高峰期收集培养细胞,提取细胞基因组DNA,设计引物扩增病毒基因组3’端近全长序列并克隆到载体PCR(?)-XL-Vector,经测序得到该病毒LTR起始序列,设计引物扩增病毒基因组剩余5′端一相对短的片段序列,克隆到同样的载体,测序后获得整个基因组的核苷酸序列。根据测序结果找到合适的酶切位点,通过限制性酶切两个克隆然后将两个克隆片段连接得到具有完整HIV-1基因组的分子克隆。
4.将测序的结果运用Bioedit软件与从HIV数据库(http://hiv-web.lanl.gov)下载的其他各亚型全长序列进行比对,并运用ClastalX1.83软件进行系统分析。从系统树分析来看,分离的病毒属于HIV-1 B′亚型。然后通过Simplot软件进行Bootscan分析,检测病毒是否存在重组现象。结果未发现重组现象。随后为了检测该病毒的耐药性突变,将病毒基因序列提交斯坦福大学的HIV耐药数据库(http://hivdb.stanford.edu/pages/ algs/HIVdb.html)分析病毒可能的基因型耐药突变。结果表明该病毒没有产生耐药突变,分离的病毒是一株能较好体现药物使用前流行的HIV-1 B′病毒,而且现有的抗艾滋病药物对这类病毒均具有较好的抑制作用。
5.病毒全基因组分子克隆转染293T细胞研究。用Lipofectamine2000转染试剂将全长克隆质粒转染到293T细胞,而后收集转染细胞培养上清测定p24病毒抗原并感染用PHA刺激培养3天的健康人PBMCs,接着检测培养上清的p24病毒抗原。通过反复的感染试验,发现构建的该克隆不具备感染活性。
本研究中提供的全基因组信息将对针对我国中部地区有效的疫苗和耐药性研究设计和发展提供参考,具有重要意义。
Acquired Immunodeficiency Syndrome (AIDS) is caused by Human Immunodeficiency Virus (HIV). It was firstly reported in American in 1981. During the last 26 years, AIDS has spread virtually every corner of the world. The first HIV-1 infection in China was reported in 1985. It was founded that there were two major HIV-1 subtypes in China:CRF07 and 08 in the IDU in Yunnan, Guangxi and Xinjiang, and subtype B'in the illegal paid blood donors (PBD) in central China, such as Henan and Hubei etc.
In the early 1990s, because of PBD, HIV-1 B'broke out circulation in central China. We isolated a strain of HIV-1 B' from a patient in Wuhan, Hubei province in 2005. We learned that the patient infected HIV-1 from his mother because of vertical infection, and his mother died of AIDS years ago. We constructed and characterized the full-length genome clone of the isolated virus HIV-1 B'.
1.Normal donor's peripheral blood mononuclear cells (PBMCs) obtained from HIV-negative blood donors were isolated from buffy coats using the Ficollhypaque method. Immediately after isolation, PBMCs were stimulated with 0.5ug/ml phytohemagglutinin (PHA). PBMCs were maintained in RPMI1640 supplemented with 10% fetal bovine serum, 2mmol/L glutamine, and antibiotics. Briefly, the isolated PBMCs from AIDS patient were co-cultivated with HIV-negative PHA-stimulated donor's PBMCs in RPMI 1640 supplemented with 10% fetal bovine serum,20U/ml IL-2,2mM glutamine, and antibiotics. Medium was changed in half every 3 days post co-culture and virus stock was used to test the RT/p24 to confirm the virus isolated and was stored at-80℃. Then we named the virus isolated 05CNHB_hp, and it replicated it's peak 11 days post co-cultivation.
2. With the cell lines transformed with CXCR4 and CCR5 respectively, inoculated with the virus 05CNHB_hp to determine its cell tropism. The virus could induce syncytium in HOS-CD4-CXCR4 cells, but it not in HOS-CD4-CCR5 cells. Also the p24 could be tested in HOS-CD4-CXCR4 but couldn't in HOS-CD4-CCR5. So, the result showed that the virus infected the cells with co-receptor CXCR4.
3.The genome DNA was isolated from PBMCs infected with 05CNHB_hp, and the primers were designed to amplify the 3'terminal near full-length part of virus genome. Then the PCR product was cloned to T vector and sequenced. Based on the nucleotide sequenced, another pair of primers was designed to amplify the rest part of genome. The PCR product was cloned to the same vector and sequenced.
4.The nucleotide sequence was aligned to the sequences of other HIV-1 subtype download from HIV Database in http://hiv-web.lanl.gov, then used the soft Clustal X1.83 to make phylogenetic tree using the neighbor-joining method and Kimura two-parameter model. From the phylogenetic tree, the virus 05CNHB_hp was confirmed to belonged to HIV-1 B'.And to investigate its possible recombination with other subtypes, we used soft Simplot to Bootscan with reference sequences HIV-1 Al, B', and C subtype. From the Bootscan, we learned that the isolated virus 05CNHB_hp didn't have any recombination with other subtypes. And no mutation associated with drug-resistance was detected when the nucleotide sequence was submitted to HIV DRUG RESISTANCE DATABASE at Stanford University, which means the drugs available domestically are effective to restrain the virus and the virus isolated could show the primaly HIV-1 B'before the use of drugs.
5.At last, we transfected the full-length genome clone to 293T cells to test its infectivity. We retrieved the supernatant to test its p24 content and inoculated in PBMCs stimulated for 3 days by PHA. The full-length genome clone 05CNHB_hp3 we constructed could express p24 in 293T cells, but unfortunately, there was no p24 in the supernatant of PBMCs inoculated with transfected cells culture supernatant. That meant the clone 05CNHB_hp we constructed haven't got its infectivity.
The full-length genome information in our research would have very important implications for future design and development of an effective vaccine, antiretroviral drugs, and of necessary assays and reagents for testing future candidate vaccine and antiretroviral drugs targeted at the HIV-1 B'strains circulating in central China.
引文
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