立氏立克次体外膜蛋白B基因片段的克隆与表达

目的立氏立克次体(Rickettsiarickettsii)外膜蛋白B(OmpB)基因(ompB)片段的克隆与表达。方法采用PCR方法,从立氏立克次体基因组中扩增其ompB基因片段,将该基因片段与原核表达载体pQE30连接,构建重组原核表达质粒pQE30/ompB;将pQE30/ompB转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果获得长为1188bp的ompB基因片段,SDS-PAGE分析发现pQE30/ompB转化菌表达了一44kDa蛋白,该蛋白与立氏立克次体免疫兔血清在免疫印迹分析中呈阳性反应,与其它立克次体免疫血清反应呈阴性,用该重组蛋白免疫血清做间接免疫荧光,检测立氏立克次体结果为阳性,而检测贝氏柯克斯体、恙虫病立克次体和普氏立克次体的结果为阴性。结论pQE30/ompB转化的大肠杆菌表达了ompB基因片段,所产生的重组蛋白具有立氏立克次体抗原特异性和良好的免疫反应性。

Cloning and expression of the gene fragment encoding Orienlia tsulsugamushi the outer mem brane protein B of Rickettsia rickettsii in E.coli

A gene fragment encoding the outer membrane protein B(OmpB) of Rickettsia rickettsii was amplified from the genomic DNA of R.rickettsii by PCR, and was cloned into the prokaryotic expression vector pQE30 to construct the recombinant plasmid pQE30/ompB.E.coli cells were transformed with this recombinant plasmid and the transformants were induced to express recombinant protein with IPTG. It was found that a ompB gene fragment with length of 1188 bp was obtained, and the recombinant protein of approximately 44 kDa as demonstrated by SDS PAGE could react with sera from rabbits immunized with R.rickettsii,but did not react with immune sera to other reckettsia agents as demonstrated by immunoblotting assay.The R.rickettsii cells were recognized by the immune serum against the recombinant protein, as shown in the indirect immunofluororescence assay,but against Coxiella burnetis,were not recongized by the immune sera Orienlia tsulsugamushi and R.prowazeckii. These results suggest that E coli cells transformed with plasmid pQE30/ompB can express a 44 kDa recombinant protein and this protein possesses high specificity and good immunogenicity of R.rickettsii.