粪肠球菌心内膜炎抗原efaA蛋白的原核表达及纯化  

Prokaryotic expression and purification of the efaA protein from Enterococus faecalis antigens

作　　者：强华[1] 吴海珍[1] 羽晓瑜[1] 朱苹[1] 

机构地区：[1]福建医科大学病原生物学系,福州350004

出　　处：《中国人兽共患病学报》2010年第2期168-170,共3页Chinese Journal of Zoonoses

基　　金：福建省教育厅科技项目(JA07094);福建省自然科学基金(C0510008)联合资助

摘　　要：目的原核表达纯化粪肠球菌心内膜炎抗原efaA蛋白,为粪肠球菌心内膜炎的致病机制研究及临床血清学诊断奠定基础。方法从粪肠球菌中扩增efaA基因,相应酶切后,克隆到原核表达载体pET30a中,构建pET30a-efaA重组质粒。经BamhI、XhoI酶切及测序鉴定,将pET30a-efaA质粒转化入BL21(DE3)。以IPTG诱导BL21(DE3)表达efaA融合蛋白,亲和层析纯化重组蛋白,SDS-PAGE、WesternBlot分析鉴定。结果PCR体外扩增efaA基因产物约943bp,重组表达质粒pET30a-efaA在大肠杆菌BL21(DE3)中表达,通过亲和层析获得纯化重组蛋白。SDS-PAGE、Western免疫印迹显示蛋白表达带的分子量约为34kd。结论粪肠球菌心内膜炎抗原efaA蛋白在大肠杆菌BL21(DE3)中成功表达并纯化。To prokaryotie express prokaryotically and to purify the efaA protein from Enterococus faecalis so as to provide the basis for the further study on the pathogenesis and clinical sero-diagnosis of endocarditis caused by E. faecalis, efaA gene of E. faecalis was amplified by PCR, the PCR-amplified product was digested with restriction enzymes and cloned into prokaryotic vector pET32a to construct the recombinant plasmid pET30a/efaA. This recombinant plasmid was confirmed by double enzyme digestion with BamhI and Xhol and then subjected to sequencing, and transformed to E. coli BL21 （DE3）. Expression of the fusion protein was induced by IPTG, and analyzed by SDS-PAGE and Western blotting. The recombinant fusion protein was purified by His-binding affinity chromatography. It was shown that efaA gene of 943 bp in size was amplified from Enterococus faecalis and the recombinant plasmid pET30a,/ efaA was successfully constructed and expressed in E. coli BL21. The purified product was found to be 34 kDa in molecular weight as demonstrated by SDS-PAGE and Western blotting. It is evident that the efaA protein of E. faecalis can be successfully expressed and purified.

关 键 词：肠球菌 粪肠球菌心内膜炎抗原 efaA 原核表达 

分 类 号：R379.1[医药卫生—病原生物学]