马疱疹病毒1型gD基因主要中和抗原区原核表达载体的构建和表达

摘要

According to equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) published on GenBank,a pair of primers were designed to amplify equine herpesvitus type 1 and 4 gD gene.The amplified EHV-1 gD gene passage was cloned into pET-30a expression vector,which were screened to obtain recombinant plasmid pET-30a-gD.The appraisal indicated that prokaryotic vector pET-30a-gD was successfully constracted,which were transformed into E.coli BL-21 and the induction expression was conducted by IPTG to optimize the culture conditions such as cultrure time,IPTG induction density.The bacterial solution was collected to make SDS-PAE test.The result showed that there was the highest effective mixed expression in pET-30a-gD,whose protein molecular weight was about 29 kD.The Western-blot analysis showed that there was pecular reaction between compromizing protein obtained and EHV-1/EHV-4 positive serum,which has a good immunogenicity.%根据GenBank上已发表的马疱疹病毒1型(EHV-1)和马疱疹病毒4型(EHV-4) gD基因序列,设计一对特异性引物.将扩增的EHV-1gD基因片段克隆至pET-30a表达载体,筛选获得重组质粒pET-30a-gD.经鉴定证明pET-30a-gD原核载体构建成功.将其转化入BL-21大肠杆菌中用IPTG进行诱导表达,并对培养时间、IPTG诱导浓度等培养条件进行优化,收集菌液进行SDS-PAGE检测,结果显示pET-30a-gD获得了高效融合表达,其表达的蛋白分子量约为29 kD.Western blot分析结果表明,显示获得的融合蛋白与EHV-1/EHV-4阳性血清发生特异性反应,具有良好的免疫原性.