[Cloning the lvgA gene of Legionella pneumophila and detecting its expression in Escherichia coli].
In order to clone lvgA gene (Legionella virulence gene) of Legionella pneumophila and detect its expression in prokaryotic cell, we amplified the lvgA gene from the total cell DNA of Legionella pneumophila with PCR,and then inserted it into the coloning vector pUC18. The recombinant plasmid pUlvgA was obtained. After the recombinant plasmid pUlvgA was identified with restriction enzyme analysis, polymerase chain reaction and nucleotide sequencing analysis, the lvgA gene was subcloned into the prokaryotic expression vector pGEX-4T-1. The prokaryotic expression recombinant plasmid pGlvgA was constructed. After IPTG induction, the E. coli JM109 containing the recombinant plasmid pGlvgA expressed fusion protein. The expression of lvgA was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The results indicated that the lvgA gene of 627 bp long was amplified, the recombinant plasmids pUlvgA and pGlvgA were constructed successfully, and the GST-LvgA fusion protein of approximately 36 KDa in size was expressed in prokaryotic cell efficiently as expected.
Liu
Mingjie
M
Department of Parasiotology and Lab of Morphology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Chen
Jianping
J
Liao
Tao
T
Lu
Dianxiang
D
Chen
Xian
X
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D016952:Legionella pneumophila / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D037521:Virulence Factors / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; C513417:LvgA protein, Legionella pneumophila; D011994:Recombinant Proteins; D037521:Virulence Factors