中国株马传染性贫血病病毒S2基因的原核表达及其多克隆抗体制备

Expression of the S2 gene of Chinese EIAV strain in E.coli and preparation of polyclonal antibodies against the S2 protein

本实验为表达马传染性贫血病病毒(EIAV)S2蛋白和制备其抗血清,以EIAV感染性分子克隆pFDDV3-8为模板扩增S2基因,将其克隆于pMD18-T载体中,测序验证后,将S2基因亚克隆于pET-30a表达载体,构建S2基因的重组表达质粒(pET-EIAV-S2),并转化DH5α受体菌。以终浓度为0.6mmol/L的IPTG诱导,表达的重组蛋白约20ku。Westernblot分析表明,该蛋白可与EIAV的阳性血清反应,具有免疫原性。该重组蛋白通过镍离子亲和层析进行纯化后,免疫新西兰兔。ELISA及westernblot分析表明,制备的兔多克隆抗体与EIAV的S2蛋白发生特异性反应。EIAVS2蛋白的重组表达及其抗体的制备为进一步研究S2辅助蛋白在EIAV复制与致病中的作用,以及S2基因反向遗传研究奠定了基础。

The S2 gene of a Chinese EIAV strain EIAVFDOV3-8 was amplified by PCR and cloned into prokaryotic expression vector pET-30a for expression in E.coli. A 20 ku fusion protein was expressed and was shown to react with EIAV positive serum in western blot. Purified S2 protein was used to immune rabbits and higth titer of polyclonal antibody against S2 was induced. These results set fundation for further research on S2 function in EIAV replication and pathogenicity.