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猪繁殖与呼吸综合征病毒非结构蛋白Nsp4和Nsp12多克隆抗体的制备与鉴定

Preparation and identification of polyclonal antibodies to Nsp4 and Nsp12 of porcine reproductive and respiratory syndrome virus

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【作者】 田恬王海刘星王先炜姜平

【Author】 TIAN Tian;WANG Hai;LIU Xing;WANG Xian-wei;JIANG Ping;Key Laboratory of Veterinary Bacteriology,Ministry of Agriculture/Nanjing Agricultural University;

【机构】 南京农业大学农业部动物细菌学重点开放实验室

【摘要】 为了制备猪繁殖与呼吸综合征病毒(PRRSV)两种非结构蛋白Nsp4和Nsp12的多克隆抗体,将Nsp4基因和Nsp12基因分别克隆至pET-32a(+)和pGEX-6P-1,并转化至BL21(DE3),用IPTG诱导表达,经过Ni-NTA镍离子亲和层析和包涵体洗液纯化,获得纯化的重组蛋白,然后用它们分别免疫BALB/c小鼠制备抗血清。SDS-PAGE和Western-blot鉴定结果显示,Nsp4和Nsp12分别以可溶性蛋白和包涵体形式表达,大小分别为42ku和44ku,均具有良好的反应原性。Western-blot和IFA结果显示,制备的抗血清均与PRRSV发生特异性反应,ELISA抗体效价均可达1∶32 000。本研究结果为PRRSV Nsp4和Nsp12的生物学功能研究奠定了基础。

【Abstract】 In order to prepare polyclonal antibodies against nonstructural protein(Nsp)4 and Nsp12 of porcine reproductive and respiratory syndrome virus(PRRSV),PRRSV Nsp4 and Nsp12 genes were cloned into pET-32a(+)and pGEX-6P-1,respectively.The recombinant plasmids were expressed in Escherichia coli BL21(DE3)after induction with IPTG.The recombinant Nsp4 and Nsp12 proteins were purified through Ni-chelating affinity chromatography and inclusion body lotion,then they were used to immunize BALB/c mice,respectively.The recombinant Nsp4 protein was mainly soluble with a molecular weight of 42ku,and the recombinant Nsp12 protein was mainly in inclusion bodies with a molecular weight of 44ku.Western-blot showed that both had high reactionogenicity.The prepared antisera could specifically react with PRRSV in Western-blot and IFA,which laid the foundation for further functional studies for Nsp4 and Nsp12 proteins.

【基金】 国家自然科学基金重点项目(31230071);教育部科学技术研究重大项目(313031,201200971);农业部公益性行业专项(201203039);国家生猪产业技术体系专项(CARS-36)
  • 【文献出处】 中国兽医科学 ,Chinese Veterinary Science , 编辑部邮箱 ,2014年06期
  • 【分类号】S852.651
  • 【被引频次】1
  • 【下载频次】153
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