PEDV和TGEV双基因共表达载体pVAXD-PS1-TS的构建及体外表达  

Construction and expression of PEDV and TGEV double gene co-expressing plasmid pVAXD-PS1-TS

作　　者：廖晓丹[1] 曹三杰[1] 黄小波[1] 唐莹[1] 文心田[1] 

机构地区：[1]四川农业大学动物医学院、动物传染病与基因芯片实验室、四川省动物疫病与人类健康实验室,四川雅安625014

出　　处：《中国兽医学报》2012年第5期641-644,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31072144);教育部长江学者和创新团队发展计划项目(IRT0848)

摘　　要：采用RT-PCR扩增猪传染性肠胃炎病毒(TGEV)SC-H株S基因N端主要抗原位点片段(大小约为1 916bp)和猪流行性腹泻病毒(PEDV)SC-L株S1基因(大小约为2 367bp),插入pMDl9-T simple载体,经酶切与测序鉴定后,构建重组质粒pMD19-T-TS与pMD19-T-PS1。从T载体上将PS1、TS基因切下,以亚克隆方法插入双启动子真核表达载体pVAXD,构建能同时表达TGEV S基因和PEDV S1基因的重组质粒pVAXD-PS1-TS。对重组质粒进行PCR与酶切鉴定后,以脂质体转染法转染COS7细胞,间接免疫荧光检测转染后细胞外源基因的表达情况。结果表明,重组质粒构建正确且能够在COS7细胞中得到表达,转染的细胞呈现特异性荧光。真核表达质粒pVAXD-PS1-TS的成功构建为进一步研究PEDV和TGEV的二联核酸疫苗奠定了基础。The important antigen site of S gene（1 916 bp） of the SC-H strain of TGVE and S1 gene（2 367 bp）of the SC-L strain of PEDV was amplified by RT-PCR and cloned into pMDl9-T vector,the recombinant was named pMD19-T-TS and pMD19T-PS1.which was identified by restriction enzyme and sequenced.Then the S and S1 genes were cut from the recombinant plasmid pMD19-T-TS and pMD19-T-PS1,further inserted into the expression vector pVAXD to construct S1/S eukaryotic co-expression recombinant plasmid pVAXD-PS1-TS.After identifled by restriction enzyme and PCR.the recombinant plasmids were tranfected into COS7 cells,the expressions of recombinant plasmids were confirmed by indirect immunofluorscence assay.The results showed that the eukaryotic co-expression plasmids were constructed successfully and the transfected cells displayed specific immunofluorscence.The successful construction of the pVAXD-PS1-TS provides a foundation for further research of the PEDV and TGEV DNA vaccine.

关 键 词：猪流行性腹泻病毒 猪传染性胃肠炎病毒 S基因 构建 真核表达 

分 类 号：S852.65[农业科学—基础兽医学]