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.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
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
