携带中国CRF07_BC HIV-1毒株env基因的嵌合病毒SHIV构建及体内外生物学活性检测

发布时间：2018-01-06 00:25

本文关键词：携带中国CRF07_BC HIV-1毒株env基因的嵌合病毒SHIV构建及体内外生物学活性检测　出处：《南开大学》2009年博士论文　论文类型：学位论文

【摘要】：艾滋病已成为影响人类健康最严重的传染性疾病之一,各国迫切需要研制安全有效的艾滋病疫苗,然而至今还未有成功的范例。疫苗的安全性、有效性以及免疫策略需要在合适的动物模型中进行评价,SHIV/恒河猴模型是应用最为广泛的动物模型之一。SHIV,即SIV/HIV嵌合病毒,是利用基因重组技术,将SIV和HIV的相应基因进行置换而构建出来的重组嵌合病毒,然而尚未有代表中国HIV流行株高致病SHIV和动物模型。构建中国HIV-1 CRF07 BC主要流行株基因CCR5嗜性的致病性SHIV/恒河猴艾滋病模型,对于设计艾滋病疫苗和免疫保护效果评价可以发挥重要作用。本研究以SHIV-89.6P的克隆株SHIV-KB9为主要骨架,将从AIDS患者临床样本中制备gp120和部分gp41(从N末端的50到780个氨基酸)的KpnI-BamHI基因片段替换SHIV-KB9的相应区域,得到76个携带中国主要流行亚型CRF07 BC不同毒株env基因的SHIV cDNA嵌合克隆。将这些重组病毒cDNA转染293T细胞,收集病毒上清,分别感染TZM细胞系、人和猴的PBMC细胞,对所有克隆的生物学活性进行细胞水平鉴定。通过SIV p27衣壳蛋白滴定,筛选到4株具有感染活性的SHIV cDNA克隆,分别为SHIV-XJN0363A4,SHIV-XJN036388,SHIV-XJN0421A17和SHIV-XJDC6431。通过对这4株SHIV病毒在恒河猴淋巴细胞中感染能力的比较,筛选活性最高的SHIV-XJDC6431进行一系列细胞水平生物活性的比较。透射电镜观察到SHIV-XJDC6431 cDNA克隆在293T转染细胞系中包装出病毒颗粒;利用表达不同辅助受体的细胞系确定SHIV-XJDC6431具有CCR5嗜性;SIVp27衣壳蛋白滴定描绘出其在人、恒河猴PBMC中的复制动力学曲线,复制能力低于SHW-KB9,但高于本室构建的SHIV-XJ01270和SHIV-CN97001;被感染过的恒河猴PBMC出现明显的细胞病变;DNA PCR检测到病毒已成功整合到人、恒河猴PBMC的基因组中;RT-PCR检测到病毒在人、恒河猴PBMC培养上清中gag-pol RNA转录本和(或)RNA基因组的形成。随后,将1000TCID_(50)的SHIV-XJDC6431病毒静脉注射两只中国恒河猴443号和444号,两只恒河猴感染特征基本一致。感染后20天左右出现病毒载量,峰值出现时间较晚,仅达到10~(5.12)pg/ml和10~(5.47)pg/ml。检测结果表明SHIV-XJDC6431可在第一代恒河猴443、444中有效复制,两只动物均表现出潜伏期稳定的低水平病毒血症过程。在444感染后第65天进行了一次猴体传代实验,发现经过体内适应的病毒在第二代恒河猴体内(445号)表现出较高的感染活性和复制能力,感染445后第15天可检测到病毒载量,且峰值出现在19天,可达到10_(6.98)pg/ml。通过病毒载量和外周血CD4~+T淋巴细胞计数分析,发现第一代动物表现出的感染活性较弱,与SHIV-KB9相比病毒载量峰值较低并且检测到载量的时间点滞后。总之,本研究成功构建了四株在细胞水平具有感染活性的携带中国流行亚型HIV-1 env基因片断的SHIV病毒株,其中一株SHIV-XJDC6431在动物体内已验证具有生物学活性。同时,经过猴体传代试验能够提高其感染、复制能力,并期望经过后期多次动物体内传代实验,能够筛选到高致病性的携带中国流行亚型env基因的SHIV病毒,为构建评价AIDS/HIV疫苗的动物模型提供物质基础。
[Abstract]:AIDS has become one of the most severe infectious diseases that affect human health, the urgent need for countries to develop a safe and effective vaccine against HIV, however there has not been successful so far. The vaccine's safety and effectiveness and immunization strategies need to be evaluated in an appropriate animal model, SHIV/ Ganges RIver monkey model is the most widely used animal model of.SHIV application of SIV/HIV chimeric virus, using gene recombination technology, recombinant chimeric virus corresponding gene SIV and HIV were constructed and replacement, but has not yet China representative HIV strains of highly pathogenic SHIV and animal model.
Construction of HIV-1 CRF07 BC China strains pathogenic SHIV/ gene CCR5 from Ganges RIver simian AIDS model, can play an important role in the evaluation of design AIDS vaccine and immune protection. In this study, the SHIV-89.6P clone of SHIV-KB9 as the main skeleton, from AIDS patients in clinical samples prepared by gp120 and gp41 (from 50 to the N terminal 780 amino acids) the corresponding region of the KpnI-BamHI gene fragment by SHIV instead of SHIV-KB9 and cDNA chimeric clone 76 carrying China major epidemic subtypes CRF07 BC strains env gene. The recombinant virus cDNA was transfected into 293T cells, the viral supernatant was collected, were infected TZM cells, human and monkey PBMC cells cell level, identification of the biological activity of all the clones. The SIV p27 capsid protein titration, screened 4 strains with SHIV cDNA clone infection activity, respectively, SHIV-XJN0363A4, SHIV-XJN036388, SHIV-XJN0421A17 and SHIV-XJDC6431. compared the biological activity of these 4 SHIV strains in Ganges RIver monkey lymphocytes by screening the SHIV-XJDC6431 with the highest activity.
Were observed by transmission electron microscopy SHIV-XJDC6431 cDNA clone packing of virus particles in 293T transfected cell lines; using the auxiliary receptor expression in different cell lines to determine SHIV-XJDC6431 with CCR5 tropism; SIVp27 capsid protein titration depict the people in Ganges RIver, replication kinetics curve of monkey PBMC in the replication capacity of less than SHW-KB9, but higher than the construction of SHIV-XJ01270 SHIV-CN97001; Ganges RIver monkey was infected PBMC cells appear obvious lesions; DNA PCR detected the virus has been successfully integrated into the genome of PBMC, Ganges RIver monkey; RT-PCR virus was detected in Ganges RIver, the monkey PBMC culture supernatant of gag-pol RNA transcripts and (or) the formation of RNA genome.
Subsequently, 1000TCID_ (50) SHIV-XJDC6431 intravenous injection of China virus two monkeys in Ganges RIver No. 443 and No. 444, two Ganges RIver monkey infected features are basically the same. 20 days after infection occurs around the viral load, the peak time of late, only reached 10~ (5.12) pg/ml and 10~ (5.47) PG /ml. detection results show that the SHIV-XJDC6431 can in the first generation of Ganges RIver in the 443444 monkey copy, two animal showed low levels of viremia during incubation period stable. A monkey body passage experiment was carried out in sixty-fifth days after infection 444, found that after in vivo adaptation of the virus in the second generation of Ganges RIver rhesus monkeys (445) showed higher activity of infection and replication capabilities 445, fifteenth days after infection can be detected viral load, and the peak appeared on the 19 day, can reach 10_ (6.98) pg/ml. by viral CD4~+T lymphocyte count in peripheral blood and analysis, found the first generation animal show sense The staining activity is weak, the peak value of the viral load is low compared with SHIV-KB9 and the time point of detecting the load is lagging.
In conclusion, this study successfully constructed four strains at the cellular level with infection activity to carry China subtypes HIV-1 env gene fragment of SHIV virus strains, one strain in SHIV-XJDC6431 animal model has been verified with biological activity. At the same time, after the monkey passage experiment can improve the ability of infection, replication, and expected after several animal in vivo passage experiment, screened to carry China subtypes of env gene of SHIV virus is highly pathogenic, and provide the material basis for establishing the animal model evaluation of AIDS/HIV vaccine.

【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R512.91;R392

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