鸭坦布苏病毒E蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression of E protein of Duck Tembusu virus and preparation of polyclonal antibodies

目的用RT-PCR技术扩增鸭坦布苏病毒(DTMUV)AH-F10株E基因,并克隆至pET32a(+)载体,构建重组表达质粒pET32a-E,表达E蛋白。方法重组表达质粒转化感受态细胞BL21(DE3),经IPTG诱导后,获得含6个His标签的融合蛋白,大小约54kDa。表达的蛋白以包涵体形式存在。对目的蛋白进行纯化,用纯化的E蛋白免疫Balb/c小鼠,制备多克隆抗体血清。结果SDS-PAGE和Western blot试验结果表明E基因在大肠埃希菌中成功表达,并能与抗DTMUV多克隆抗体产生特异性反应,具有良好的反应原性。间接免疫荧光试验表明免疫小鼠后获得的多克隆抗体能与DTMUV反应。结论本研究为DTMUV新型疫苗和诊断试剂盒的进一步研究奠定了基础。

Objective To construct,express and purify the envelope protein of Duck Tembusu virus（DTMUV）.Methods The E gene of Duck Tembusu virus AH-F10 strain was amplified by RT-PCR and inserted into the multiple cloning sites of pET32a（＋）vector.The recombinant plasmid was transformed into BL21（DE3）.The expression of E protein was induced with IPTG.The 6His-E protein was expressed in a form of inclusion body with a molecular weight of 54 kDa.Six Balb/c mice were injected with the purified protein for preparation of E protein specific polyclonal antibodies.Results SDS-PAGE and Western blot analysis showed that the E protein was successfully expressed in BL21.Indirect immunofluorescence assay（IFA）showed that the polyclonal antibody from the inoculated mice was able to react with DTMUV.Conclusion The study laid a foundation for further study on new vaccines and diagnosis kits of DTMUV.