【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。更多还原

【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.更多还原