一步法构建塞内卡病毒感染性克隆平台
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S852.65

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Construction of Senecavirus A Infectious Clone by One-step Approach
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    摘要:

    利用同源重组技术,通过一步法快速构建A型塞内卡病毒(Senecavirus A,SVA)感染性克隆平台,从而为研究其致病机理、构建标记疫苗等奠定基础。依次扩增CMV启动子、SVA全基因组、poly(A)尾巴及丁肝核酶序列(HDVr),利用同源重组技术,将3段核酸片段整合进低拷贝载体pWSK-29。将菌落PCR鉴定正确的感染性克隆质粒(pWSK-29-SVA)转染BHK-21细胞,拯救病毒,在盲传第5代时对拯救病毒进行基因测序鉴定,并对其进行生物学特性分析。结果显示:成功构建了SVA感染性克隆质粒(pWSK-29-SVA),成功拯救了病毒,将拯救病毒命名为rSVA(rescued SVA);rSVA测序结果与亲本病毒高度同源,且二者生物学特性相似。结果表明,本试验经同源重组法一步构建pWSK-29-SVA感染性克隆平台,避免了传统感染性克隆中酶切、连接及寻找酶切位点带来的繁琐与不便,构建方案更为灵活多样,大大提高了便利性与实效性,且拯救出的病毒生物学特性与亲本毒株高度一致。本研究为进一步研究SVA致病机理、构建疫苗提供了平台。

    Abstract:

    In order to rapidly construct an infectious cloning platform of senecavirus A(SVA)by one-step approach by use of a homologous recombination technique,and subsequently to lay a foundation for studying the pathogenesis of the virus and constructing marker vaccines,CMV promoter,SVA whole genome,poly(A)tail and hepatitis delta virus ribozyme(HDVr)sequence were successively amplified,three nucleic acid fragments were integrated into low-copy vector,pWSK-29,by the homologous recombination technique. The infectious cloning plasmid(pWSK-29-SVA)identified by colony PCR was transfected into BHK-21 cells to rescue the virus,then the rescued virus was identified through gene sequencing in its fifth generation under blind passage,followed by analysis on its biological characteristics. The results showed that SVA infectious cloning plasmid(pWSK-29-SVA)was successfully constructed,and the virus was successfully rescued,which was named as rescued SVA(rSVA);the result of rSVA sequencing was highly homologous with that of its parental viruses,both of which shared similar biological characteristics. It was concluded that any possible cumbersome and inconvenience caused by enzyme digestion,connection and looking for enzyme digestion sites under traditional infectious cloning could be avoided by the platform established in the study,by which,the convenience and effectiveness were greatly improved due to its flexible and diverse construction schemes,and the biological characteristics of rSVA were highly consistent with that of its parental viruses. A platform was thereby provided in the paper for further study on the pathogenesis of SVA and construction of vaccines.

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高春柳,赵胜杰,周晓翠,魏荣,尼博.一步法构建塞内卡病毒感染性克隆平台[J].《中国动物检疫》编辑部,2021,38(9):66-72.

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  • 在线发布日期: 2021-09-03
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