A型塞内卡病毒VP1蛋白的原核表达及其多克隆抗体的制备

A型塞内卡病毒(SVA)是一种新发传染病病原。研究旨在对SVA的衣壳蛋白VP1进行原核表达,并制备其多克隆抗体。以SVA广西分离株SVA-GX01(GenBank登录号:MK039162)RNA为模版,通过RT-PCR扩增结构蛋白VP1基因,构建重组质粒pET-32a-VP1并转化表达菌株大肠杆菌BL21(DE3)进行诱导表达。在非变性条件下,将纯化的重组蛋白免疫新西兰大白兔制备多克隆抗体。纯化的多克隆抗体进行间接ELISA测定效价,同时进行Western blot与间接免疫荧光分析。结果表明,重组蛋白pET-32a-VP1在大肠杆菌BL21(DE3)以可溶性与包涵体两种形式表达,分子量约为49 kDa。纯化后多克隆抗体效价高达1∶64000。Western blot与间接免疫荧光(IFA)分析显示,多克隆抗体能跟SVA抗原特异性结合。重组SVA-VP1蛋白及其多克隆抗体的成功制备为猪塞内卡病毒血清学检测方法的建立提供了良好的生物材料。

Prokaryotic Expression of the VP1 Protein of Senecavirus A and Preparation of Its Polyclonal Antibodies

Senecavirus A is a new infectious agent.The aim of this study was to express the VP1 protein of Senecavirus A(SVA)and prepare its polyclonal antibodies.The structural protein of SVA VP1 gene was amplifi ed by RT-PCR using the SVA RNA of SVA strain as the template.The amplicon was cloned into the prokaryotic expression vector pET-32a,resulting a recombinant plasmid pET-32a-SVA-VP1.The plasmid was transformed into E.coli BL21(DE3)competent cells which were then induced.The purifi ed VP1 protein was used to inject the New Zealand White rabbits to prepare its polyclonal antibodies.The polyclonal antibodies against VP1 in BHK-21 cells infected with SVA was analyzed by Western blot and indirect immunofl uorescence assay.The results showed that the recombinant SVA VP1 protein was expressed as inclusion bodies and soluble solid in E.coli BL21(DE3)cells.This polyclonal antibodies effectiveness to be determined up to 1:64000.Results of western blot and indirect immunofl uorescence assay showed that the polyclonal antibodies could interact specifi cally with VP1 of SVA.Its polyclonal antibodies provided valuable biomaterials for the establishment of serological detection method for SVA.