猪流行性腹泻病毒S基因片段的克隆及原核表达

为研究猪流行性腹泻病毒(porcine epidemic diarrhea virus PEDV)S基因片段的原核表达产物是否具有抗原性,分析S基因抗原位点后,用PCR技术扩增S蛋白主要抗原区的核酸序列,经克隆后将目的片段连接到原核表达载体pET-28a(+)中,成功构建了重组质粒pET-28a-PEDV-Sp,其重组菌于37℃、0.5 mmol/L IPTG诱导表达4 h后进行SDS-PAGE分析,在分子质量约为29 kDa处出现一新蛋白带,与预期相符。质谱鉴定表明,已成功表达了目的蛋白。纯化后的重组蛋白免疫兔制备多克隆抗体,抗体效价检测结果显示该蛋白具有良好的抗原性。该研究为猪流行性腹泻基因工程疫苗的研制奠定基础。

Cloning and Prokaryotic Expression of the S Gene Segment of Porcine Epidemic Diarrhea Virus

In order to study the antigenicity of prokaryotic expression of the S gene segment of porcine epidemic diarrhea virus (PEDV), its antigen site was analyzed, and the segment was amplified with PCR. Then we constructed the recombinant with the vector pET-28a(+) named pET-28a-PEDV-Sp, which was induced to express in E.coli BL21(DE3) by 0.5 mmol/L IPTG at 37℃. Futhermore, the expressed product was analysed by SDS-PAGE, and the result indicated that there was a new 29 kDa-electrophoretic band corresponding to the presumptive interested protein. In addition, the result of mass spectrometry also showed that the recombinant protein was expressed successfully. The purified protein was then used for preparation of polyclonal antibody. The result of ELISA showed the protein had good antigenicity. This work lays a foundation for the development of genetic engineering vaccine of PEDV.