嗜肺军团菌flaA基因的克隆及原核融合表达

Cloning and Prokaryotic Expression of Flagellum Subunit Gene (flaA) of Legionella pneumophila

目的构建嗜肺军团菌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,为进一步研究鞭毛亚单位蛋白的致病作用和免疫保护性提供前提条件。方法以嗜肺军团菌1型DNA为模板,PCR扩增获得嗜肺军团菌flaA基因,与带有硫氧还蛋白基因(Trx)的高效原核表达质粒pET32a(+)定向重组,构建重组质粒,转化大肠杆菌BL21(DE3),并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-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1435bp的flaA基因,成功构建了重组质粒pET-flaA,SDS-PAGE及Western blot分析显示重组质粒pET-flaA在大肠杆菌中得到了高效融合表达。结论成功构建嗜肺军团菌flaA基因重组质粒,并在原核系统中得到了高效表达。

Objective To construct the fused expression vector of flaA gene of Legionella pneumophila and realize the flaA gene expressing in E. coli so as to set the basis for future research on the disease-causing role and immune protective response of flaA. Methods In this study, the flaA gene, an flagellum subunit gene （flaA） of Legionella pneumophila, was obtained from DNA of Legionella pneumophila by polymerase chain reaction （PCR）, and was harbored into prokaryote expression vector, pET32a （＋）containing thioredoxin gene Trx. The recombinant plasmid （pET-flaA） was analyzed with restriction-endonuclease digestion, PCR and DNA sequencing analysis, and transferred into E. coli strain BL21 （DE3） for transformation. The expression of fusion protein Trx- flaA was induced with isopropy-β-D-thiogalactoside （IPTG） and examined with SDS-PAGE and Western blot techniques. Results The restriction endonuclease digestion,PCR amplification and DNA sequencing analysis showed that the flaA gene of 1435 bp was amplified from Legionella pneumophila DNA, and the recombinant plasmid pET-flaA was constructed successfully. The Trx-flaA protein was expressing in E. coli and could be detected with SDS-PAGE and Western blot techniques. Conclusion The flaA gene of Legionella pneumophila has highly expressed in prokaryotic cell in fused form with Trx.