Cloning, Expression Of Virulence-associated Gene InvA Of The Strong Virulent L. Interrogans Serovar Lai And Its Effects On Pathogenicity And Immunogenicity

Leptospira is the pathogen of leptospirosis that is one of the most widespread zoonotic diseases. The currently knowledge about pathogenic molecular mechanism of leptospira is not enough. After sequencing the genomics of L. interrogans serovar Lai 56601 and Copenhageni strains, about 60% predicted genes will be studied their functions. What kinds of genes which are associated with pathogenicity and immunity are urgently needed?An invA gene is present in pathogenic leptospira strains, including L.interrogans serovar Lai strain 56601 and leptospira interrogans serovar Copenhageni strain FiocruaLl-130. The gene has 43% identity with an invasion associated IalA gene of Bartonella bacilliformis. Morever, specific amplification wasobtained from Lai 017, but negative results were observed with non-pathogenic L. bif lexa serovar Patoc strain Patoc I . So the gene may be a pathogenic-associated gene and immunity candidate gene.To express virulence-associated protein InvA from a strong virulent L. interrogans serovar Lai strain 017 in E Coli BL21 (DE3) and to investigate its effects. The invA was cloned into prokaryotic expression vector pET32a(+). Then the recombination expression plasmid pETINVA was transformed into E. Coli BL21 (DE3). The InvA fused protein which had a 6-His tag was expressed after induction by IPTG. Then the InvA fusion protein was purified by His-affinity column. SDS-PAGE showed that the molecular weight of the protein was about 35KDa and The fused protein InvA was soluble in E. Coli BL21(DE3) cytoplast mainly.The effects on ANA-1, SPC-A-1, A549 and ECV304 cells proliferation to different concentrations of InvA were observed using the XTT assay. The XTT assay indicated that InvA promoted a time and concentration-depended cell proliferation of ANA-1 when the concentration of InvA was among 0 to 8.0pg/ml. But InvA has no significant change to SPC-A-1, A549 and ECV304 cells.To further study effects of InvA on pathogenisis and immunity, we prepared anti-InvA antibody. 200μg/kg InvA was inoculated into new Zealand white rabbits three times at 4 weeks intervals. The antibody titer was as high as 1:32000 measured by ELISA 10 days after the last boosted inoculation. Antigen-specificity of anti-InvA antibody was evaluated by western-blotting. The resultsshowed high-effective and specified anti-InvA antibody was prepared.Subsequently, we carried out an assay to measure IFN-y level of ANA-1 cell affected by InvA. The ANA-1 cells were washed and resuspended in complete RPMI 1640 medium supplemented with 10% fetal calf serum, and 1% penicillin-streptomycin. Cells were added to 96-well plates in the triplicate for culture with InvA proteins. Supernatants were harvested from cultures after 72 hours of incubation for the investigation of IFN-y . The result showed that InvA could not produce significantly higher IFN- Y levels than control group.We also made invasion assay to have insight into the InvA function in vivo. The BL21(DE3) bored pETINVA invaded human erythocytes at a significantly higher level than that of BL21 (DE3) bored pET32a(+). The results indicated that InvA had the invasive ability. We also constructed recombinant L. biflexa—Escherichia coli shuttle vector which inserted invA. The recombinant vectors were transferred into L. biflexa serovar Patoc strain Patoc I . The transformants obtained were resistant to kanamycin and product a specificity DNA by PCR, indicating that they are pGKINVA transformed mutants. These mutants would provid an experimental mutant to investigate potential pathogenic functions of InvA in vivo.These results suggested that invA was potential pathogenic factor. In addition, sera from rabbits had a strong antibodyspecificity and sensitivity response indicated which may be useful develop a new serological diagnosis kit of leptospirosis.