柯萨奇病毒B3VP1重组腺病毒载体疫苗rAd/VP22-L-VP1的构建及表达

摘要

背景:VP22 是单纯疱疹病毒1 型(Herpes simplex virus type 1,HSV-1)UL49 基因编码的碱性蛋白质,具有蛋白转导结构域(protein transduction domain,PTD),能够把与之融合的蛋白或与之结合的DNA 等大分子跨膜送递到邻近细胞,在基因靶向预防中表现出优势.目的:构建表达单纯疱疹病毒1 型VP22 与柯萨奇病毒B3 主要中和抗原VP1 融合蛋白的重组腺病毒载体疫苗,观察外源基因在HEK293 细胞中的良好表达.方法:PCR法扩增目的基因HSV-1 VP22 和CVB3 VP1,经Linker 连接,将VP22-L-VP1 插入腺病毒穿梭载体pAdTrack-CMV,构建重组穿梭质粒AdTrack-CMV/VP22-L-VP1.再将此载体与腺病毒骨架载体pAdEasy-1 在大肠杆菌BJ5183 中进行同源重组,生成重组腺病毒质粒pAd/VP22-L-VP1,脂质体介导pAd/VP22-L-VP1 转染HEK293 细胞包装重组腺病毒rAd/VP22-L-VP1.HEK293 细胞上进行病毒扩增和滴定并检测外源基因的表达.结果与结论:构建的重组腺病毒载体pAd/VP22-L-VP1 经过第4 轮扩增,其滴度达到6.77×107 pfu/mL,体外感染293 细胞可见VP22 和VP1 融合蛋白的表达.说明实验成功构建并包装重组腺病毒rAd/VP22-L-VP1.%BACKGROUND: Encoded by the UL49 gene of herpes simplex virus type 1 (HSV-1), VP22 is an alkaline protein. With the protein transduction domain (PTD), VP22 can mediate a transmembrane transduction of VP22-linked protein or DNA from the cells in which it is synthesized endogenously to adjacent cells, which shows advantage in gene targeting prevention. OBJECTIVE: To construct a recombinant adenovirus based vaccine in order to express Vp22 of HSV-1 and the VP1, the main neutralizing antigen of coxsackievirus B3 (CV B3), and to observe the expression of exogenous gene in HEK293 cells. METHODS: The DNA fragments of HSV-1 VP22 and CVB3 VP1 were amplified by PCR and linked by linker to produce VP22-L-VP1. The VP22-L-VP1 coding sequence was cloned to the pAdTrack-CMV plasmid to construct AdTrack-CMV/VP22-L-VP1. Then AdTrack-CMV/VP22-L-VP1 was transformed into Ecoli.Bj5183 carrying backbone plasmid pAdEasy-1 to produce the recombinant adenovirus plasmid pAd/VP22-L-VP1. HEK293 cells were transfected with pAd/VP22-L-VP1 to produce recombinant adenovirus rAd/VP22-L-VP1. The virus amplification and titration was preformed on HEK293 cells and the exogenous gene expression was detected by Western blot.RESULTS AND CONCLUSION: The titer of recombinant adenovirus rAd/VP22-L-VP1 of passage 4 was 6.77×107 pfu/ml. And the expression of VP22-L-VP1 was verified on HEK293 cells by Western blotting. Recombinant adenovirus rAd/VP22-L-VP1 was generated successfully, which laid a foundation for further study on CVB3VP1 gene vaccine.