猪流行性腹泻病毒CH/GX/2015/750A株S2蛋白的原核表达及抗原性分析

本研究旨在了解猪流行性腹泻病毒（PEDV）S2蛋白的抗原性，为下一步诊断试剂盒及亚单位疫苗的研究奠定基础。试验通过反转录PCR的方法扩增PEDV CH/GX/2015/750A株S2基因部分片段（S2A），将其克隆后插入原核表达载体pET-32a（+）中，构建原核表达质粒pET32a-S2A。将原核表达质粒转化入大肠杆菌BL21感受态细胞中，IPTG诱导表达重组蛋白，Ni柱亲和层析法纯化重组蛋白，Western blotting检测重组蛋白S2A的反应原性。纯化复性的重组蛋白S2A免疫昆明小鼠制备多克隆抗体，用间接ELISA法检测获得的多克隆抗体效价，间接免疫荧光法验证制备的多克隆抗体的特异性。结果显示，重组S2A蛋白在IPTG终浓度为0.2 mmol/L时，37 ℃诱导表达3 h可获得最高表达量，该重组蛋白主要以包涵体的形式存在；Western blotting结果显示，纯化复性后的重组蛋白S2A能够与PEDV阳性血清发生特异性结合，具有良好的反应原性。制备的多克隆抗体效价可达1：32 000，间接免疫荧光结果表明，制备的多克隆抗体能够特异性识别和结合PEDV。结果表明，PEDV S2A蛋白具有良好的抗原性，可作为诊断试剂盒或亚单位疫苗的候选抗原。

Prokaryotic Expression and Antigenicity Analysis of S2 Protein of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A Strain

The purpose of this study was to understand the antigenicity of porcine epidemic diarrhea virus (PEDV) S2 protein and lay a foundation for the further study of diagnostic kit and subunit vaccine.In this study,the partial fragment of S2 gene (S2A) of PEDV CH/GX/2015/750A strain was amplified by RT-PCR and inserted into prokaryotic expression vector pET-32a(+) to construct prokaryotic expression plasmid pET32a-S2A.The prokaryotic expression plasmid pET32a-S2A was transformed into E.coli BL21(DE3).The recombinant protein S2A was induced by IPTG and purified by affinity chromatography on Ni column.The activity of S2A protein was detected by Western blotting.The purified recombinant protein S2A was used to immunize Kunming mice to prepare polyclonal antibody.The titer of the polyclonal antibody was detected by indirect ELISA,and the specificity of the polyclonal antibody was verified by indirect immunofluorescence.The results showed that the optimal induction condition of recombinant S2A protein was that when the final concentration of IPTG was 0.2 mmol/L,the induction temperature was 37 ℃,and the induction time was 3 h.The recombinant protein mainly existed in the form of inclusion body.Western blotting results showed that the purified and renatured recombinant protein S2A could specifically bind to PEDV positive serum and had good react ogenicity.The titer of the polyclonal antibody was 1:32 000.IFA results showed that the polyclonal antibody could specifically recognize and bind PEDV virus.The results of this study showed that PEDV S2A protein had good antigenicity and could be used as a candidate antigen of diagnostic kit or subunit vaccine.