嗜肺军团菌主要外膜蛋白与鞭毛亚单位蛋白双基因融合表达载体的构建及其诱导表达

目的构建嗜肺军团菌主要外膜蛋白S基因(mompS)与鞭毛亚单位蛋白基因(flaA)的融合表达载体,并在原核系统表达。方法以嗜肺军团菌1型DNA为模板,PCR分别扩增获得嗜肺军团菌mompS基因和flaA基因,与带有硫氧还蛋白(Trx)基因的高效原核表达质粒pET32a(+)定向重组,构建mompS与flaA基因完全融合的重组质粒,经限制性核酸内切酶酶切鉴定、PCR和核酸序列分析后,以IPTG诱导表达Trx-MOMPS-FlaA融合蛋白,用SDS-PAGE及Western blot进行鉴定。结果限制性核酸内切酶酶切鉴定、PCR和核酸序列分析表明,扩增出了嗜肺军团菌904bp的mompS及1432bp的flaA基因,成功构建了重组质粒pET-LpSF,SDS-PAGE及Western blot分析显示重组质粒pET-LpSF在原核系统中得到了表达。结论成功构建了嗜肺军团菌mompS-flaA双基因的原核融合表达载体,并在大肠杆菌中得到了表达,为进一步研究嗜肺军团菌核酸双价疫苗提供研究基础。

Construction fusion vector of mompS and flaA gene of Legionella pneumophila and its induced expression in E. coli

To construct the fused expression vector of mompS-flaA gene of Legionella pneumophila and to realize the expression of mompS-flaA gene of Legionella pneumophila in E.coli. The flaA gene, an flagellum subunit gene(flaA) of Legionella pneumophila, and mompS gene, an major outer membrane protein gene of Legionella pneumophila, were obtained from DNA of Legionella pneumophila by polymerase chain reaction (PCR), and was cloned into prokaryotic expression vector, pET32a(+)containing thioredoxin Trx gene. The recombinant plasmid (pET-LpSF) was analyzed with restriction-endonuclease digestion, PCR and DNA sequencing analysis and the expression of pET-LpSF was induced with isopropy-β-D-thiogalactoside (IPTG).The fusion protein Trx-MOMPS-FlaA was examined with SDS-PAGE and Western blot techniques. It was found that PCR and DNA sequencing analysis showed that the flaA gene of 1432bp and the mompS gene of 904 bp were amplified from Legionella pneumophila DNA, and the recombinant plasmid pET-LpSF was constructed and detected its expression in prokaryotic cell successfully with SDS-PAGE and Western blot techniques. If is concluded that the fused expression vector of mompS-flaA gene of Legionella pneumophila was successfully constructed and induced in E.coli, which provide the basis for future research on the double valence DNA vaccine of Legionella pneumophila.