表达HIV-1 gag重组鸡痘病毒的构建与筛选  

Construction and Selection of the Recombinant Fowlpox Expressing HIV-1 gag

作　　者：朱羿龙[1] 李昌[2] 郭焱[1] 刘存霞[2] 杜寿文[2] 王茂鹏[2] 金宁一[2] 

机构地区：[1]长春中医药大学,长春130117 [2]军事医学科学院军事兽医研究所省部共建吉林省人兽共患病预防与控制重点实验室,长春130122

出　　处：《中国生物工程杂志》2014年第1期57-63,共7页China Biotechnology

基　　金：国家科技重大专项(2012ZX10001005-006);国家自然科学基金(81001342);中国博士后科学基金(20110491803;2012T50859)资助项目

摘　　要：构建并筛选表达HIV-1 gag蛋白的重组鸡痘病毒,并对其进行鉴定。首先设计引物通过PCR技术扩增HIV-1 gag基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的鸡痘病毒穿梭载体pTKET中,获得重组质粒pTKET-HIV gag。然后将其与鸡痘病毒FPV282E4株共转染原代鸡胚成纤维细胞(CEF)进行同源重组,以增强型绿色荧光蛋白(EGFP)为筛选标记,通过噬斑筛选获得重组病毒,应用PCR,RT-PCR,Western blot方法对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组鸡痘病毒基因组中,RT-PCR,Western blot结果表明HIV-1 gag在感染细胞内成功表达且具有抗原性。连续传代20次,PCR,RT-PCR,Western blot均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,而且病毒已经纯化。结论:成功获得表达HIV-1gag的重组鸡痘病毒,为进一步免疫试验研究奠定基础。To construct and select expressing HIV-1 gag protein in recombinant fowlpox virus. Primers were designed and synthesized. HIV-1 gag was amplified by PCR and ligated with pMD 18-T Sample Vector. Theresults sequence of HIV gag was correct, and then it was inserted into pTKETwhich was made in our laboratory to construct the recombination shuttle plasmid pTKET-HIV gag. The plasmid pTKET-HIV gag and 282E4 strain fowlpox virus were co-transfected 80% confluent Chicken Embryo Fibroblast （CEF） cells to select the recombinat fowlpox virus with EGFP as the reporter gene. The recombination fowlpox virus was verified and analyzed by PCR, RT-PCR and Western blot. The gene has been integrated into recombinant fowlpox virus genome by PCR after 10 times plaques screening, and the results of RT-PCR and Western blot showed that HIV-1 gag was successfully expressed in infected ceils. The recombinant fowlpox virus which was continuously passaged 20 times also showed exogenous genes integration, transcription and expression by PCR, RT-PCR and Western blot. FPV- TK was not amplified by PCR and showed that the HIV-1 gag gene has good genetic stability in recombinant fowlpox virus which has been purified. The recombinant fowlpox virus expressing HIV gag protein was successfully generated, which provide a good foundation for future immunity experiment.

关 键 词：重组鸡痘病毒 HIV-1 GAG 强型荧光蛋白 遗传稳定性 

分 类 号：R373.9[医药卫生—病原生物学]