非洲猪瘟病毒DP71L蛋白原核表达及其多克隆抗体制备

Prokaryotic expression of African swine fever virus DP71L protein and preparation of polyclonal antibody

为深入研究非洲猪瘟病毒(ASFV)DP71L蛋白的生物学功能,根据非洲猪瘟病毒ASFVCN/GS/2018DP71L基因序列设计特异性引物,通过扩增DP71L基因并克隆到原核表达载体pET28a(+)后转入大肠杆菌BL21(DE3)诱导表达,通过SDS-PAGE、Western-blot进行鉴定,并以纯化出的重组DP71L蛋白免疫BALB/c小鼠,制备多克隆抗体,间接ELISA测定多克隆抗体效价,Western-blot鉴定多克隆抗体的特异性。结果显示,重组DP71L蛋白以可溶物和包涵体两种形式表达;间接ELISA测得多克隆抗体血清效价为1∶64000;Western-blot结果显示,该抗体能特异性识别DP71L蛋白。结论:成功地表达了DP71L重组蛋白,制备了多克隆抗体,为深入研究DP71L蛋白生物学功能奠定了基础。

To study the biological function of DP71L protein of African swine fever virus(ASFV),the specific primers of ASFV CN/GS/2018 DP71L were designed according to the gene sequence of ASFV CN/GS/2018 DP71L.The DP71L gene was amplified and cloned into the prokaryotic expression vector pET28a,and then transformed into Escherichia coli BL21(DE3)to induce expression.BALB/c mice were immunized with the purified recombinant DP71L protein to prepare polyclonal antibody.The serum titer of the polyclonal antibody was determined by indirect ELISA,and the specificity of the polyclonal antibody was identified by Western-blot.The results showed that the recombinant DP71L protein was expressed in soluble form and inclusion body form.The titer of polyclonal antibody was 1∶64000 measured by indirect ELISA.Westernblot results showed that the antibody specifically recognized the DP71L protein.The results showed that the recombinant protein DP71L was successfully expressed and the polyclonal antibody was prepared,which laid a foundation for further study on the biological function of DP71L protein.