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  

Prokaryotic Expression and Inmunogenicity of E2 Gene of BVDV

作　　者：王宇婷[1] 马莉莉[1] 李晓月[1] 王士霞[1] 毕莹[1] 倪宏波[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《安徽农业科学》2017年第12期122-123,129,共3页Journal of Anhui Agricultural Sciences

基　　金：黑龙江省农垦总局"十二五"重点科技攻关项目(HNK125B-11-10A;HNK125B-11-02)

摘　　要：[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-32aE2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。[ Objective ] To prokaryotic express the bovine viral diarrhea-muscosal disease viruses E2 gene encoding protein. [ Method J 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 immuno- genicity. [ Conclusion]E2 protein is expressed in successfully,which lays foundation for establishing BVDV detection method in future.

关 键 词：牛病毒性腹泻黏膜病毒 E2基因 原核表达 

分 类 号：S852.4[农业科学—基础兽医学]