鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein

【目的】本研究选择原核表达系统表达鸭坦布苏病毒(Duck Tembusu virus,DTMUV)核衣壳蛋白(Capsid protein),并制备其多克隆抗体,为DTMUV分子机制研究奠定基础。【方法】根据DTMUV-201909株基因序列,运用一步克隆技术将Capsid基因克隆至表达载体pET-30a(+)中,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,利用IPTG进行诱导表达,通过SDS-PAGE和Western blotting鉴定重组蛋白;使用ISA206佐剂与纯化后的重组蛋白混合乳化后免疫BALB/c小鼠,以获得多克隆抗体。间接ELISA方法测定获得的多克隆抗体效价,并对多克隆抗体进行Western blotting和间接免疫荧光试验(IFA)验证。【结果】试验成功构建pET-30a-Capsid重组质粒,SDS-PAGE结果显示,表达的重组蛋白大小约为18 ku,主要以包涵体形式存在;Western blotting结果表明,该蛋白能与抗His标签鼠单克隆抗体发生特异性反应,具有良好反应原性。间接ELISA结果显示,制备的鼠抗Capisd蛋白多克隆抗体效价可达1∶256000;Western blotting和IFA结果显示,制备的多克隆抗体能特异性识别DTMUV感染细胞样品的Capsid蛋白。【结论】本研究成功制备小鼠抗Capsid蛋白多克隆抗体,为深入研究DTMUV Capsid蛋白的结构和功能提供了试验材料,为进一步阐明DTMUV的致病机制奠定基础。

【Objective】The purpose of this study was to express the Capsid protein of Duck Tembusu virus(DTMUV)by prokaryotic expression system and prepare its polyclonal antibody to lay a foundation for the study of the molecular mechanism of DTMUV.【Method】According to the gene sequence of DTMUV-201909 strain,the Capsid gene was cloned into the prokaryotic expression vector pET-30a(+)by one-step cloning technique.The recombinant plasmid was transformed into E.coli BL21(DE3)competent cells.The recombinant protein was induced by IPTG.The recombinant protein was identified by SDS-PAGE and Western blotting.The purified recombinant protein was emulsified with ISA206 adjuvant at equal volume and immunized BALB/c mice to obtain polyclonal antibodies.The titer of the polyclonal antibody obtained was measured by indirect ELISA,and the specificity of the polyclonal antibody was identified by Western blotting and indirect immunofluorescence assay(IFA).【Result】The recombinant plasmid pET-30a-Capsid was successfully obtained,SDS-PAGE showed that the molecular weight of the expressed recombinant protein was about 18 ku,mainly in the form of inclusion body.Western blotting results showed that the protein could react specifically with anti-His labeled mouse monoclonal antibody and had good reactivity.The results of indirect ELISA showed that the titer of the prepared polyclonal antibody against Capisd protein reached 1∶256000.Western blotting and IFA results showed that the polyclonal antibody could react specifically with DTMUV.【Conclusion】The mouse anti-Capsid protein polyclonal antibody was successfully prepared.It provided materials for studying the structure and function of Capsid protein,and laid a foundation for revealing the pathogenesis of DTMUV.