非洲猪瘟病毒360-12L蛋白的原核表达及其多克隆抗体制备

Prokaryotic Expression of African Swine Fever Virus 360-12L Protein and Preparation of Its Polyclonal Antibody

【目的】获得抗原性好的非洲猪瘟病毒(African swine fever virus)12L蛋白用于ASFV血清学诊断技术研究。【方法】根据GenBank ASFV参考序列(登录号:NC_044959.2)合成360-12L基因,并插入pET-28a(+)载体,构建原核表达质粒pET-28a-12L,将经测序鉴定正确的质粒转化大肠杆菌BL21(DE3)感受态细胞进行诱导培养,筛选最佳诱导温度,同时分析其可溶性。随后通过亲和层析纯化蛋白,并对所获得的12L重组蛋白进行SDS-PAGE分析。将获得的12L重组蛋白作为免疫原免疫BALB/c雌性小鼠,以获得鼠抗ASFV 12L蛋白的多克隆抗体,通过间接ELISA方法测定所制备的多克隆抗体效价,并用间接免疫荧光试验进行验证。【结果】成功构建了ASFV 12L蛋白原核表达质粒,重组菌pET-28a-12L-BL21在37℃6 h时表达量较高,主要以包涵体形式表达,可在54.7 ku处出现明显条带。间接ELISA结果显示,制备的鼠抗ASFV 12L蛋白多克隆抗体效价>1∶512000,间接免疫荧光试验结果表明,所制备的鼠抗ASFV 12L蛋白多克隆抗体具有良好的特异性,可特异性识别12L重组蛋白。【结论】原核表达的12L重组蛋白具有良好的免疫原性,制备的鼠抗ASFV 12L蛋白的多克隆抗体具有较高的反应性和特异性,为进一步研究12L蛋白的生物学功能、ASFV血清学诊断技术和疫苗研究提供生物材料。

【Objective】The study was aimed to obtain the antigenic 12L protein of African swine fever virus(ASFV)used to serological diagnosis of ASFV.【Method】The 360-12L gene was synthesized and inserted into pET-28a(+)vector to construct prokaryotic expression plasmid pET-28a-12L.The correct plasmid identified by sequencing was transferred into E.coli BL21(DE3)competent cells for induction.The optimal induction temperature was selected and its solubility was analyzed.The recombinant protein 12L was purified by affinity chromatography and analyzed by SDS-PAGE.The recombinant protein 12L was used as immunogen to immunize female BALB/c mice to obtain mouse polyclonal antibody against ASFV 12L protein.The titer of the polyclonal antibody was determined by indirect ELISA,and its specificity was verified by indirect immunofluorescence assay.【Result】The prokaryotic expression plasmid of ASFV 12L was successfully constructed,and the recombinant strain pET-28a-12L-BL21 had a high expression level at 37℃for 6 h,mainly in the form of inclusion body,with an obvious band at 54.7 ku,which was consistent with the expectation.Indirect ELISA showed that the titer of the polyclonal antibody against ASFV 12L protein was higher than 1∶512000.Indirect immunofluorescence assay showed that the polyclonal antibody against ASFV 12L protein had good specificity and could specifically recognize recombinant protein 12L.【Conclusion】The recombinant protein 12L expressed in E.coli had good immunogenicity,and the polyclonal antibody against ASFV 12L protein prepared from mice had high reactivity and specificity,which could lay a foundation and provide biomaterials for further study on biological function of ASFV recombinant protein 12L,serological diagnosis technology of ASFV and vaccine research.