[Construction and expression of eucaryotic recombinant plasmid of Legionella pneumophila mip gene].

OBJECTIVE: To construct recombinant plasmid of Legionella pneumophila mip gene and detect its expression in NIH3T3 cells. METHODS: mip gene of Legionella pneumophila was amplified by PCR. The amplified DNA was ligated to pcDNA3.1(+) vector. The recombinant plasmid was named pcDNA3.1-mip. NIH3T3 cell was transfected by recombinant plasmid pcDNA3.1-mip with Lipofection strategy. Transient and stable products of mip gene were detected by immunofluorescence and Western-blot. RESULTS: It was found that there was high green fluorescence on the cell membrane and inside the cell. It showed that NIH3T3 cell was transfected by pcDNA3.1-mip successfully. Rabbit serum antibody of Legionella pneumophila detected the NIH3T3 cell transfected with pcDNA3.1-mip. There was the protein in relative molecular weight 24 X 10(3), whereas no evidence for the protein in NIH3T3 cell transfected with pcDNA3.1(+) was seen. The protein expression of mip gene was shown. CONCLUSION: We have successfully constructed the recombinant plasmid of Legionella pneumophila mip gene and detected the relative molecular weight 24 X 10(3) Mip protein in NIH3T3 cells.