猪瘟流行毒株E2基因主要抗原区的原核高效表达

Study on pronucleus expression of key antigen region domain E2 gene of Classical Swine Fever Virus prevailing strain in E.coli

【目的】构建猪瘟病毒(Classical Swine Fever Virus,CSFV)E2基因主要抗原区原核表达质粒载体,高效表达E2蛋白,为进一步研究E2亚单位疫苗提供物质基础。【方法】用RT-nested PCR技术扩增CSFV流行毒株SX-YL株E2基因主要抗原区,克隆于表达载体pET32a中,成功构建表达质粒pET32a-E2,将pET32a-E2转入BL21(DE3)受体菌并用IPTG诱导表达E2蛋白,对E2蛋白进行SDS-PAGE电泳、Western-blot分析和可溶性鉴定。【结果】CSFVE2基因得到了高效表达,表达蛋白量占菌体蛋白量的33.2%,表达蛋白E2主要以包涵体形式存在;免疫印迹分析结果表明,表达蛋白E2能识别天然猪瘟多克隆抗体。【结论】E2蛋白表达量高并具有生物学活性。

[Objective] The study is to construct plasmid vector of key antigen region domain CSFV E2 gene and express E2 protein highly efficient, to offer substantial foundation for studding E2 subunit vaccine. [Method] Clonied key antigen region domain E2 gene of strain SXYL by RT-nested PCR,connected it with pET32a to fabricate expressing plasmid pET32a-E2 successfully,translated pET32a-E2 to BL21（DE3） and induced it express E2 protein by IPTG,identified E2 protein by SDS-PAGE,Western-blot and ultrasonic. [Result] Recombined E. coli could express E2 protein highly efficient, the quantity of expression is 33.2%, most E2 protein is inclusion bodies, the E2 protein could identify natural polyclonal antibody of CSFV. [Conclusion] The quantity of expression protein is high and it has biological activity.