TGEV双基因嵌合表达质粒pVAX-S-N的构建及体外表达  

Construction and expression of TGEV chimeric plasmid pVAX-S-N

作　　者：杨恒[1] 曹三杰[1] 黄小波[1] 文心田[1] 秦学远[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重点实验室,四川雅安625014

出　　处：《中国兽医学报》2009年第3期254-257,共4页Chinese Journal of Veterinary Science

基　　金：教育部长江学者和创新团队发展计划资助项目(IR-T0555)

摘　　要：采用RT-PCR扩增猪传染性肠胃炎病毒(TGEV)SC-H株S基因5′端主要抗原位点片段和N基因,插入pMD19-T载体,经酶切与测序鉴定后,构建重组质粒19T-N与19T-S。从T载体上将S、N基因切下,以亚克隆方法插入真核表达载体pVAX1,构建嵌合表达S、N基因的重组质粒pVAX-S-N。对重组质粒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pVAX-S-N的成功构建为进一步研究TGEV核酸疫苗奠定了物质基础。The important antigen site of S and N genes of the SC-H strain of TGVE was amplified by RT-PCR and cloned into pMD-19T vector,the recombinant was named 19T-S and 19T N, which was identified by restriction enzyme and sequenced. Then the S and N genes were cut from the recombinant plasmid 19T-S and 19T-N,further in- serted into the expression vector pVAX1 got S/N chimeric eukaryotic expression plasmid pVAX-S-N. After identified by restriction enzyme and PCR, the recombinant plasmids were tranfected into COS7 cells, the expressions of re combinant plasmids were confirmed by indirect immunofluorscence assay. The results showed that the eukaryotic expression plasmids were constructed successfully and the transfected cells displayed specific immunofluorscence. The successful construction of the pVAX S-N provides foundation for advance research of the TGEV DNA vaccine.

关 键 词：猪传染性胃肠炎病毒 S基因 N基因 构建 真核表达 

分 类 号：S852.65[农业科学—基础兽医学]