布鲁氏菌猪种标准株外膜蛋白OMP25的原核表达

Expression of 25kDa outer membrane protein of Brucell in E.coli

目的克隆布鲁氏菌外膜蛋白Omp25基因并构建基因的原核表达系统。方法用聚合酶链反应技术扩增得到布鲁氏菌基因组,得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pGEX-4T-1,经双酶切和DNA测序测定,将构建的重组质粒转化大肠杆菌(E.coliHB101,TOP10),经IPTG诱导后,用SDS-PAGE检测重组蛋白rOmp25表达情况。结果经DNA测序显示Omp25DNA序列和插入位点正确,成功构建了重组质粒pGEX-4T-1-Omp25,经IPTG诱导,特异性表达出以包涵体形式存在48kDa的Omp25融合蛋白。结论制备、克隆了布鲁氏菌外膜蛋白Omp25基因片段,并在大肠杆菌中表达出Omp25融合蛋白。

Objective To clone and construct the recombinant expression plasmid for 25kDa Outer membrane protein （OMP25） of BruceUa in E. coli. Methods The OMP25 gene of BruceUa was amplified from genomic DNA and cloned into PMD - 18 vector. It was then cloned into prokaryotic espression vector pGEX - 4T - 1 and identified by enzyme digestion and DNA sequencing. The recombinant plasmid was then transformed into E. coli HB101 and TOP10.The expression of OMP25 gene was induced by ITPG and the recombinant protein was identified bv SDS - PAGE analysis. Results The results of DNA sequencing showed that the sequence and cloning site of the inserted OMP25 gene were correct, the recombinant expression plasmid pGEX - 4T - 1 - Omp25 was constructed. Conclusion The OMP25 gene of Brucella are cloned and the recombinant OMP25 protein are successfully expressed in E. coli HB101.