寨卡病毒E蛋白及第三结构域的原核表达和多克隆抗体制备

Prokaryotic expression and preparation of the polyclonal antibodies of E protein and domain Ⅲ in Zika virus

目的在大肠杆菌中克隆表达和纯化寨卡病毒(Zika Virus,ZIKV)包膜糖蛋白(Envelope Protein,E)及第三结构域(Envelope DomainⅢ,EDⅢ),并制备两种免疫原的鼠多克隆抗体。方法通过Vero-E6细胞培养扩增ZIKV,提取病毒总RNA并反转录为cDNA,利用E和EDⅢ基因的cDNA序列构建原核表达载体pET32a/E和pET28a/EDⅢ,转入E.coli BL21(DE3),IPTG诱导表达,采用Ni+柱亲和层析法纯化蛋白。以纯化的重组蛋白为抗原免疫BALB/c小鼠,采集抗血清,间接ELISA法测定效价,Western Blot检测特异性。结果成功表达并纯化重组蛋白E和EDⅢ,获得的多克隆抗体效价均达到1∶409 600,Western Blot检测多克隆抗体可特异性识别重组E蛋白和EDⅢ以及天然E蛋白。结论成功制备出特异性抗寨卡病毒E蛋白和EDⅢ的鼠源多克隆抗体,为深入探索寨卡病毒致病机制、检测方法和免疫策略奠定了研究基础。

To clone,express and purify the E Protein and EDⅢ of Zika virus in E.coli and prepare two kinds of polyclonal antibodies,the virus was amplified by Vero-E6 cell culture.Total RNA was extracted by RT-PCR and reverse transcribed into cDNA.The prokaryotic expression vectors pET32 a/E and pET28 a/EDⅢ were constructed by cDNA sequence of E and EDⅢgene.Then,recombinant plasmids were transformed into E.coli BL21 and induced by IPTG,and purified by Ni+column affinity chromatography.BALB/C mice were immunized with purified recombinant proteins.Antiserum was collected and titer was determined by indirect ELISA.Western blot was used to detect the specificity.Results showed that the recombinant proteins were successfully expressed and purified.The titer of the polyclonal antibodies both reached 1:409 600.Western Blot analysis showed that the polyclonal antibodies could specifically recognize the recombinant proteins.Thus,the specific polyclonal antibody were successfully prepared,laying a foundation for further study on the pathogenesis,detection methods and immune strategies of Zika virus.