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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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)
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
porcine epidemic diarrhea virus
transmissible gastroentefitis virus
S gene
construction
eukaryotic expression
