猪丁型冠状病毒S基因的原核表达及多克隆抗体的制备

Prokaryotic Expression of Porcine Deltacoronavirus S Gene and Preparation of Polyclonal Antibody

纤突(Spike,S)蛋白是猪丁型冠状病毒(Porcine deltacoronavirus,PDCoV)诱导机体产生抗体的主要抗原,为制备其重组蛋白和多克隆抗体,本实验对分离株CHN/20GXNN-1/2020的S基因进行序列分析,选择抗原指数较高的b区S1b,克隆至表达载体pET-32a进行原核表达,重组蛋白S1b主要以包涵体形式表达,大小约为34.1 kDa。利用Ni-NTA琼脂糖树脂纯化的重组蛋白免疫新西兰大白兔,制备多克隆抗体。通过间接ELISA测定多克隆抗体的效价高于1∶128000,Western blot和IFA均证明其可以与病毒S蛋白特异性结合。本研究制备的多克隆抗体特异性良好,为进一步研发PDCoV的检测方法和研究S蛋白的功能奠定了基础。

Spike(S)protein is the main antigen for porcine deltacoronavirus(PDCoV)to induce the body to produce antibodies.In order to prepare its recombinant protein and polyclonal antibody,the S gene of the isolated strain CHN/20GXNN-1/2020 was sequenced.The b region S1b with high antigen index was selected and cloned into the expression vector pET-32a for prokaryotic expression.The recombinant protein S1b was mainly expressed in the form of inclusion bodies,and the size was about 34.1 kDa.The recombinant protein purified by Ni-NTA agarose resin was applied to immunize New Zealand white rabbits to prepare polyclonal antibodies.The titer of the polyclonal antibody was higher than1∶128000 by indirect ELISA.Western blot and IFA showed that it could specifically bind to the S protein of the virus.The polyclonal antibody prepared in this study had good specificity,which laid a foundation for further development of PDCoV detection methods and study of the function of S protein.