[Legionella pneumophila lvgA and Hsp60 gene splicing and the fusion gene expression in E.coli].
OBJECTIVE
To fuse Legionella virulence gene (lvgA) with heat shock protein 60 gene (Hsp60) by PCR and detect the fusion gene expression in E.coli.


METHODS
The fragments of lvgA and Hsp60 genes having matching sequences at their ends to be fused were amplified from the genomic DNA of Legionella pneumophila by PCR, and the PCR products were mixed, denatured, reannealed, so that the strands with matching sequences at their 3' ends overlapped to serve as primers for each other. Extension of this overlap by DNA polymerase produced recombinant products. After amplification with outer primers, sufficient product of the fusion gene was harvested, which was inserted into the prokaryotic expression vector pGEX-4T-1 to construct the prokaryotic expression recombinant plasmid. After identification with restriction enzyme analysis, polymerase chain reaction and nucleotide sequence analysis, the E.coli BL21 containing the recombinant plasmid pGlvgA/Hsp60 was induced with IPTG and the expression of lvgA/Hsp60 was detected by SDS-PAGE and Western blot analysis.


RESULTS
The lvgA/Hsp60 fusion gene of 2,292 bp was amplified and the recombinant plasmid pGlvgA/Hsp60 was constructed successfully. A 117-kD GST-lvgA-Hsp60 fusion protein was detected in the E.coli containing the recombinant plasmid.


CONCLUSIONS
The recombinant plasmid for Legionella pneumophila lvgA/Hsp60 fusion gene is constructed successfully and this fusion protein can be expressed in prokaryotic cells efficiently, which make possible the immunological characterization of this fusion gene.
Liu
Ming-Jie
MJ
Department of Parasitology, West China Medical Center, Sichuan University, Chengdu 610041, China. lmj912@sohu.com
Chen
Jian-ping
JP
Wang
Tao
T
Liao
Tao
T
Chen
Xian
X
Lu
Dian-xiang
DX
Tian
Yu
Y
D017398:Alternative Splicing; D018834:Chaperonin 60 / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D050939:Gene Fusion; D005822:Genetic Vectors; D006801:Humans; D007877:Legionnaires' Disease / Q000276:immunology / Q000517:prevention & control; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D019444:Vaccines, DNA / Q000096:biosynthesis / Q000276:immunology; D037521:Virulence Factors / Q000096:biosynthesis / Q000235:genetics*
D018834:Chaperonin 60; C513417:LvgA protein, Legionella pneumophila; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D037521:Virulence Factors