[Cloning and prokaryotic expression of flagellum subunit gene (flaA) of Legionella pneumophila].

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-beta-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.