表达猴免疫缺陷病毒Env蛋白的重组鸡痘病毒的构建和筛选

Construction and Selection of the Recombinant Fowlpox Virus Expressing Env Protein of Simian Immunodeficiency Virus

目的:构建并筛选表达猴免疫缺陷病毒(SIV)Env蛋白的重组鸡痘病毒(FPV),并对其进行鉴定。方法:设计引物,通过PCR技术扩增SIV env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FPV穿梭载体pTKET中,获得重组质粒pTKET-SIV env,然后将其与FPV282E4株共转染原代鸡胚成纤维细胞进行同源重组,以增强型绿色荧光蛋白为筛选标记,通过噬斑筛选获得重组病毒,应用PCR、RT-PCR、Western印迹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FPV基因组中,RT-PCR、Western印迹结果表明SIV Env蛋白在感染细胞内表达且具有抗原性;连续传代20次,PCR、RT-PCR、Western印迹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,且病毒已经纯化。结论:获得表达SIV Env蛋白的重组FPV,为进一步免疫试验研究奠定了基础。

Objective： To construct and select expressing Env protein of simian immunodeficiency virus（SIV） in recombiant fowlpox virus（FPV）. Methods： Primers were designed and synthesized. SIV env gene was amplified by PCR and ligated with pMDI8-T simple vector. The results sequences of SIV env was correct, and then it was inserted into pTKET which was made in our laboratory to construct the recombination shuttle plasmid pTKET-SIV env. The plasmid pTKET-SIV env and FPV282E4 strain were co-transfected 80% confluent chicken embryo fibroblast cells to select the recombination FPV with EGFP as the reporter gene. The recombination FPV was verified and analyzed by PCR, RT-PCR and Western blot. Results： The gene has been integrated into recombinant FPV genome by PCR after 10 times plaques screening, and the results of RT-PCR and Western blot showed that SIV env was successfully expressed in infected cell. The recombinant FPV 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 SIV env gene has good genetic stability in recombinant FPV which has been purified. Conclusion： The recombinant FPV was successfully gained which could express SIV Env protein, and to provide basis for next immunity test.