title: [Cloning and prokaryotic expression of flagellum subunit gene (flaA) of Legionella pneumophila].
abstract: 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.
authors: Department of Parasiotology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
authors: Lei
authors: L
authors: Zhang
authors: Jian-Ping
authors: JP
authors: Chen
authors: Li
authors: L
authors: Zhang
authors: Tao
authors: T
authors: Wang
authors: Ming-Jie
authors: MJ
authors: Liu
authors: Yu
authors: Y
authors: Tian
mesh_terms: 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
chemical_list: D005408:Flagellin; C067138:flaA protein, bacteria