Zhang Lei, Chen Jian-ping, Zhang Li, Wang Tao, Liu Ming-jie, Tian Yu
Department of Parasiotology, West China Medical Center, Sichuan University, Chengdu 610041, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Dec;26(12):1701-5.
To construct the fusion expression vector of Legionella pneumophila mompS and flaA genes linked with a flexible chain for expression in E.coli.
The flaA gene, an flagellum subunit gene of Legionella pneumophila, and mompS gene that encodes an major outer membrane protein of Legionella pneumophila, were amplified from the DNA of Legionella pneumophila by PCR and cloned into the prokaryotic expression vector pET32a (+) containing thioredoxin gene Trx. Following analysis of the recombinant plasmid (pET-LpSLF) with restriction endonuclease digestion, PCR and DNA sequencing, the expression of pET-LpSLF was induced with IPTG and the expressed fusion protein Trx-MOMPS-FlaA was examined with SDS-PAGE and Western blotting.
The results of restriction endonuclease digestion, PCR and DNA sequencing analysis showed that the flaA gene (1 440 bp) and the mompS gene (906 bp) were successfully amplified from Legionella pneumophila DNA, and the recombinant plasmid pET-LpSLF was constructed and expressed in E.coli as demonstrated by SDS-PAGE and Western blotting.
The fusion expression vector of mompS and flaA genes linked with a flexible chain has been successfully constructed and allows efficient expression of mompS-linker-flaA gene in E.coli, which enables further study of the immunogenicity and immunoprotection of Legionella pneumophila.
构建嗜肺军团菌mompS和flaA基因与柔性链连接的融合表达载体，以便在大肠杆菌中表达。
从嗜肺军团菌DNA中通过PCR扩增出嗜肺军团菌鞭毛亚基基因flaA和编码嗜肺军团菌主要外膜蛋白的mompS基因，并克隆到含有硫氧还蛋白基因Trx的原核表达载体pET32a(+)中。用限制性内切酶消化、PCR及DNA测序分析重组质粒（pET-LpSLF）后，用IPTG诱导pET-LpSLF表达，并用SDS-PAGE和Western印迹检测表达的融合蛋白Trx-MOMPS-FlaA。
限制性内切酶消化、PCR及DNA测序分析结果表明，成功从嗜肺军团菌DNA中扩增出flaA基因（1440 bp）和mompS基因（906 bp），构建了重组质粒pET-LpSLF，并经SDS-PAGE和Western印迹证明其在大肠杆菌中表达。
成功构建了mompS和flaA基因与柔性链连接的融合表达载体，使mompS-连接子-flaA基因在大肠杆菌中高效表达，为进一步研究嗜肺军团菌的免疫原性和免疫保护作用奠定了基础。
