布鲁菌外膜蛋白OMP25的原核表达  

Prokaryocyotic Expression of Outer Membrane Protein 25 of Brucella

作　　者：刘艳琴[1] 吴树清[1] 吴杰[1] 

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018

出　　处：《黑龙江畜牧兽医》2011年第4期20-22,共3页Heilongjiang Animal Science And veterinary Medicine

基　　金："十一五"国家重大科技攻关项目(2006BAD4A16-4)

摘　　要：为了克隆布鲁菌外膜蛋白OMP25基因并构建基因的原核表达系统,试验采用聚合酶链反应(PCR)扩增得到布鲁菌基因组OMP25基因片段,T-A克隆后测定核苷酸序列,将目的基因定向插入原核表达载体pET-32a,经双酶切和DNA测序,再将构建的重组质粒转化到E.coilDH5α、Rosetta,经IPTG诱导后用SDS-PAGE检测重组蛋白pET32a-OMP25的表达情况。结果表明:经DNA测序显示OMP25基因序列和插入位点正确,成功构建了重组质粒pET32a-OMP25,经IPTG诱导表达出以包涵体形式存在的长为43 ku的OMP25融合蛋白;经Western-blot检测,OMP25融合蛋白与布鲁菌阳性血清发生特异性反应,说明重组蛋白具有良好的反应原性。To clone and construct the recombinant expression plasmid for 25 ku Outer membrane protein（OMP25） of Brucella in E.coli,The gene segments of Brucella OMP25 were amplified and obtained by polymerase chain reaction（PCR）,then sequenced after TA cloning.Subsequently the target gene was cloned into prokaryotic expression vector PET-32a and identified by restriction enzyme digestion and DNA sequencing.The recombinant plasmid was transformed into E.coli Rosetta.After induced by ITPG,the recombinant protein expressed by OMP25 gene in E.coli DH5α and Rosetta was identified by SDS-PAGE analysis.The results of DNA sequencing showed that the sequence and cloning site of the insert for OMP25 gene were exact.The recombinant expression plasmid PET-32a-OMP25 was constructed,and the protein including bodies of OMP25 43 ku fusion was successfully expressed induced by IPTG.The OMP25 gene of Brucella was cloned into PET-32a,and then recombinant plasmid pET32a-OMP25 was constructed and the recombinant OMP25 protein was successfully expressed in E.coli Rosetta.The western-blotting analysis showed that the recombinant OMP25 reacted specifically with brucella positive serum.It indicated the recombinant protein processed favorable immunogenicity.

关 键 词：布鲁菌 分离株 外膜蛋白OMP25 重组质粒 蛋白纯化 

分 类 号：S813.1[农业科学—畜牧学]