| PEDV和TGEV双基因共表达载体pVAXD-PS1-TS的构建及体外表达 |
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| 引用本文： | 廖晓丹,曹三杰,黄小波,唐莹,文心田.PEDV和TGEV双基因共表达载体pVAXD-PS1-TS的构建及体外表达[J].中国兽医学报,2012,32(5):641-644. |
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| 作者姓名： | 廖晓丹 曹三杰 黄小波 唐莹 文心田 |
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| 作者单位： | 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 |
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| 基金项目： | 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) |
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| 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。
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| 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 |
Construction and expression of PEDV and TGEV double gene co-expressing plasmid pVAXD-PS1-TS |
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| LIAO Xiao-dan,CAO San-jie,HUANG Xiao-bo,TANG Ying,WEN Xin-tian.Construction and expression of PEDV and TGEV double gene co-expressing plasmid pVAXD-PS1-TS[J].Chinese Journal of Veterinary Science,2012,32(5):641-644. |
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| Authors: | LIAO Xiao-dan CAO San-jie HUANG Xiao-bo TANG Ying WEN Xin-tian |
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| Institution: | *(Laboratory of Animal Infectious Disease and Microarray,Key Laboratory of Animal Disease and Human Health of Sichuan Province,College of Veterinary Medicine,Sichuan Agricultural University,Ya’an,Sichuan 625014,China) |
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| Abstract: | 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. |
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| Keywords: | porcine epidemic diarrhea virus transmissible gastroentefitis virus S gene construction eukaryotic expression |
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