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Published on: February 5, 2015
[Construction of a prokaryotic expression vector carrying mompS-linker-flaA fusion gene and its expression in E.coli]
Lei Zhang1, Jian-ping Chen, Li Zhang
1Department of Parasiotology, West China Medical Center, Sichuan University, Chengdu 610041, China. leigezhang@163.com
Objective:
To construct the fusion expression vector of Legionella pneumophila mompS and flaA genes linked with a flexible chain for expression in E.coli.
Methods:
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.
Results:
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.
Conclusion:
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.
