[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.


CONCLUSIONS
The flaA gene of Legionella pneumophila has highly expressed in prokaryotic cell in fused form with Trx.
Zhang
Lei
L
Department of Parasiotology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Chen
Jian-Ping
JP
Zhang
Li
L
Wang
Tao
T
Liu
Ming-Jie
MJ
Tian
Yu
Y
D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005408:Flagellin / Q000235:genetics*; D015870:Gene Expression; D016952:Legionella pneumophila / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D005408:Flagellin; C067138:flaA protein, bacteria