Cloning And Expression Of Mip Gene Of Legionella Pneumophila

Objective:To construct the expression recombinant by cloning the mip gene of Legionella phiIadephia 1(macrophage infectivity potentiator ,mip) into cytoplasmic vector pET30a(＋).The recombinant was transformed into E.coli BL21(DE3) by CaCl₂ and the expressed fusion Mip was studied primarily.This research will play a basis for Rapid Sreening Technology and Products for Respiratory Tract Diseases.Methods:Primers were designed and PCR amplifying system was optimized.The target mip gene was ligated with pET30a(＋) to construct the recombinant pET-mip.Then the recombinant plasmid pET-mip was transformed into E.coli DH5αand the positive clones were screened by kanamycin resistance selection.The recombinant was confirmed by double restrictive endonuclease digestion and sequence analysis.And then the recombinant pET-mip was transformed into E.coli BL21(DE3).The expression of fusion Mip in E.coli BL21(DE3) was observed by SDS-PAGE,Western-blotting,purification and quantitation.Results:The optimimal conditions for the amplification of target gene with PCR and the expression and purification of target protein Mip. The expression recombinant of the target gene mip was constructed successfully.The confirmed recombinant pET-mip was transformed into the E. coli DH5αand E.coli BL21(DE3) competent cells with high efficiency.And the recombinant pET-mip was expressed in the cytoplasm of the E.coli BL21(DE3) with the concentration of 1mg/ml.Conclusions:In this research,the target mip gene of Legionella philadephia 1 was cloned successfully into the expression vector pET30a(＋).The expression of the target gene in E.coli BL21(DE3) were studied primarily.The result demonstrated that the fusion Mip protein was expressed and the express level was high.