猪乙型脑炎病毒E蛋白结构域Ⅲ原核表达和抗原性分析

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

为分析猪乙型脑炎病毒(JEV)E蛋白Ⅲ结构域的抗原性,本研究克隆了JEV疫苗株SA14-14-2的E蛋白结构域Ⅲ,并通过pET-28a载体进行融合表达和纯化。用纯化后蛋白作为免疫原,免疫8周龄BALB/c小鼠,通过SDS-PAGE、Western blotting、间接ELISA及间接免疫荧光方法(IFA)检测小鼠及猪抗体滴度,验证E蛋白结构域Ⅲ的抗原性。SDS-PAGE结果表明融合蛋白以包涵体形式表达;Western blotting、间接ELISA检测结果表明表达产物具有良好的抗原性;纯化的蛋白免疫BALB/c小鼠ELISA方法检测特异性抗体滴度可达1×105;猪JEV阳性血清ELISA抗体滴度可达5.1×104;IFA结果表明JEV EⅢ蛋白产生的抗血清能很好的识别乙型脑炎病毒抗原。以上结果表明,表达、纯化的重组JEV EⅢ蛋白具有良好的抗原性。本试验结果为建立以E蛋白结构域为抗原的诊断方法提供了重要依据。

In order to analyze the antigenicity of porcine Japanese encephalitis virus（JEV）E protein domain Ⅲ,which was expressed by pET-28 avector with His-tag and purified through NiNTA,the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲof E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE,Western blotting,indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting,ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10^5 anti-JEV E Ⅲ protein specific antibody titers by ELISA,and the porcine immunized with the porcine JEV generated 5.1×10^4 anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.