铜绿假单胞菌外膜蛋白OprH原核表达载体的构建与表达

摘要

目的：克隆铜绿假单胞菌外膜蛋白OprH基因，构建其原核表达载体并鉴定其表达。方法从铜绿假单胞菌中提取基因组DNA进行PCR扩增OprH基因；采用T-A克隆技术构建pMD19T-OprH重组质粒，并经酶切和序列测定后，获得OprH片段插入到原核表达载体pET28b中，以构建pET28b-OprH重组表达质粒，并在表达宿主菌E�Coli BL21中经IPTG诱导表达及通过SDS-PAGE电泳鉴定。结果 pET28b-OprH原核表达载体构建成功；重组表达质粒经IPTG诱导后表达外膜蛋白OprH。结论成功克隆了OprH基因并获得原核表达物，为进一步研究快速检测该菌的方法奠定了基础。%Objective To clone the gene of outer membrane protein OprH of Pseudomonas aeruginosa ( PA) , construct a prokaryotic expression vector and identify its expression. Method The DNA genome was extracted from PA, the OprH gene was amplified by primers of PCR. The recombinant plasmid pMD19T-OprH was constructed by using T-A cloning. After enzyme digestion and sequence analysis, the OprH gene was inserted into prokaryotic expression vector pET28b to construct recombinant expression plasmid pET28b-oprH, which was expressed in E. coli BL21 (DE3) cells with induction by IPTG. Then the expressed protein was analyzed by SDS-PAGE. Result Construction of the prokaryotic ex-pression vector pET28b-oprH was successful. Then the recombinant expression plasmid expressed a corresponding protein OprH after being induced by IPTG. Conclusion OprH gene was successfully cloned and the prokaryotic expression product was obtained. It provides the basis for investigating a method of rapid detection of PA.