牛病毒性腹泻黏膜病毒E2基因的原核表达与免疫原性分析

摘要

[Objective]To prokaryotic express the bovine viral diarrhea-muscosal disease viruses E2 gene encoding protein.[Method]Bovine viral diarrhea-muscosal disease viruses E2 gene were amplified by PCR and linked into the pET-32a prokaryotic expression vector,prokaryotic expression recombinant plasmid pET32a-E2 was constructed,which was then trandformed into E.coli(Rosetta)cells for protein expression.E2 protein of recombinant strains was induced to express by 1 mmol/L IPTG and SDS-PAGE electrophoresis,target protein was purified by Ni-NTA affinity chromatography column and the immunogenicity was identified by Western blot analysis.[Result]The recombinant plasmid pET32a-E2 was confirmed by PCR,restriction enzyme.It had high-level expression in E.coli.SDS-PAGE showed that recombinant protein with molecular weight of 58 kDa,with concerntration of 0.521 mg/mL.Western blot showed that recombinant protien can reacts with positive serum,indicating good immunogenicity.[Conclusion]E2 protein is expressed in successfully,which lays foundation for establishing BVDV detection method in future.%[目的]原核表达牛病毒性腹泻黏膜病毒(BVDV)E2基因编码蛋白.[方法]采用PCR方法从BVDV中扩增E2基因片段,与原核表达载体pET-32a连接,构建重组表达质粒pET-32a-E2,转化E.coli(Rosetta)感受态细胞,重组菌用1 mmol/L IPTG诱导表达E2蛋白,进行SDS-PAGE电泳,并用Ni-NTA亲和层析柱纯化目的蛋白,经Western blot分析鉴定免疫原性.[结果]重组质粒pET-32a-E2经PCR及酶切鉴定证明构建正确,重组质粒能够在大肠杆菌中大量表达,表达产物的分子质量大小约为58 kDa,纯化后E2重组蛋白浓度0.521 mg/mL,Western blot分析表明,其能被BVDV阳性血清识别,具有很好的免疫原性.[结论]E2蛋白成功表达,为后续建立BVDV检测方法奠定了基础.