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  

Construction and expression of adenovirus-based coxsackievirus B3 VP1 recombinant adenovirus vector vaccine rAd/VP22-L-VP1

作　　者：李剑[1] 刘贵霞[1] 米立国[1] 蓝佳明[1] 张永红[1] 金玉怀[1] 

机构地区：[1]河北医科大学病原生物学教研室,河北省石家庄市050017

出　　处：《中国组织工程研究》2012年第7期1280-1284,共5页Chinese Journal of Tissue Engineering Research

基　　金：河北省科技支撑计划(09276418D-5);河北省自然科学基金(C2009001087)~~

摘　　要：背景:VP22是单纯疱疹病毒1型(Herpes simplex virus type1,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-1VP22和CVB3VP1,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6.77×107pfu/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.

关 键 词：柯萨奇病毒B3 腺病毒载体 基因疫苗 单纯疱疹病毒1型VP22 佐剂 组织工程 

分 类 号：R318[医药卫生—生物医学工程]