A Study On Cloning And Expression Of A Virulence Gene MviN Of L. Interrogans Serovar Lai And Its Effects

Leptospira is a kind of special microbe in organic structure and genetic character which forms during the period of development and evolution. Leptospirosis is a widespread zoonosis caused by leptospira. The whole genomic sequencing of Leptospira Interrogans Serovar Lai strain 56601 has being finished by the sciences of China in 2003, changing its research orientation from genomics to proteomics. The fuctional studies on Virulence related Genes of Leptospira Interrogans plays a very important role on clarifying the pathopoiesis mechanism and immunological principle of leptospirosis because the interaction between leptospiro and host is the critical step in the mechanism of Leptospirosis.The bioinformatical analysis on the whole genome has found that the mviN gene, which exists in variety of disease-causing microorganisms, is a virulent gene related to adhering and invasion. In comparison with other virulent gene , mviN, with 1596 nucleotides in its expression frame in Leptospira Interrogans has its own unique characteristics, expressing a trans-membrence protein of 531 amino acids.With the experimental material of genomic DNA of Leptospera interrogans serovar Lai strain 017 , the mviN gene was amplified through the use of PCR method and then was inserted into prokaryotic expression sector pET32a(+) to construct recombinant plasmid pET-mviN. Transformed into E. Coli. BL21(DE3), the recombinant plasmid pET-mviN could, after induction by IPTG, solubly express about 80kD fusion protein whose validity was checked by SDS-PAGE and further by western-blot. Then the fusion protein was purified by His-affinity column.The recombinant eukaryotic expression plasmid pcDNA3. 1-mviN has also been constructed. The mviN gene was successfully cloned into vector pcDNA3. 1(+). After confirming the correctness of the recombinant eukaryotic plasmid pcDNA3. 1-mviN by restrication enzyme analysis , PCR and sequencing it was transfected into COS7 cell by liposome,then its expression was analyzed by RT-PCR.The effects of the MviN fusion protein on cells have been studied deeply. The XTT method was applied to assay the inhibitory effects of MviN fusion protein on cell proliferation of ECV304 and A549 and flow cytometry (FCM) was applied to measure the apoptotic rate of ECV304 and A549 after incubation with the fusion protein. These experimental results demonstrate that , compared with the control group, the MviN fusion protein can significantly inhibit cell proliferation and promote apoptosis of ECV304 and A549.This study, in a certain extent, elucidates the role of mviN in the pathopoiesis mechanism and immunological principle of leptospirosis, which builds the foundation in the further researches.