Prokaryotic Expression And Functional Analysis Of The Mb1514Gene In Mycobacterium Bovis

Bovine tuberculosis (BTB) is a chronic zoonosis caused by Mycobacterium bovis (Mb), posing a great risk to public health besides a major economic problem. The ability of M. bovis to invade and survive in the macrophages is the key component in the pathogenesis of BTB. Therefore, analysis of the proteins responsible for cell invasion will help us to combat BTB. The Mb1514gene in Mb encodes a hypothetical invasion protein (designated here as MbINV protein), whose function has not yet been directly identified. In this study, the Mb1514gene from Mb was cloned, expressed in E. coli and purified. The prokaryotic recombinant MbINV protein was successfully obtained for the first time and its function was initially analized using a murine macrophage cell line, RAW264.7. For further study of the Mb1514gene, a Mb1514-EGFP fusion protein expressing shuttle vector was constructed and successfully transformed into Mycobacterium smegmatis by electroporation. The detailed research contents were described as follows:1. The Mb1514gene was amplified from the DNA template of M. bovis Beijing strain by PCR and cloned into pEASY-T1simple vector. Sequencing result showed that the gene cloned in this study is100%identical with that in M. bovis AF2122/97published in GenBank. The Mb1514gene was then sub-cloned into the prokaryotic expression vector pET-28a(+).The recombinant plasmids were transformed into E. coli BL21(DE3) to express the recombinant MbINV protein by IPTG induction. SDS-PAGE analysis and Western blotting assay showed that the protein was successfully expressed as an inclusion body in the cell lysis pellets, with a molecular weight of approximately28kDa. Recombinant proteins were purified by Ni Sepharose column and renatured for function analysis.2. The specific recognition of the recombinant MbINV proteins by the rabbit-anti-Mb polyclonal antibody demonstrated that the recombinant protein is immunogenic, which sμggests that MbINV is a potential candidate for a diagnostic biomarker or a subunit vaccine.3. To investigate the virulence of Mb1514, the RAW264.7cells were stimulated with the recombinant MbINV protein at various concentrations and the cell viabilities were detected at different time points with CCK-8Kit. The results demonstrated that recombinant MbINV protein significantly inhibited the viability of RAW264.7macrophages in a dose-dependent manner (P<0.05).4. Cell death type assay was carried out using Apoptosis and Necrosis Assay Kit at24h post stimulation of the RAW264.7cells with the recombinant MbINV protein at the final concentration of lOμg/mL. The result showed that MbINV caused cell necrosis, compared with the PBS control, indicating that this protein is toxic.5.To investigate the effect of MbINV protein on TNF-a, NOS2, and IL-1β mRNA expression, the RAW264.7cells were stimulated with recombinant MbINV (10μg/mL) or MbPPD (10μg/mL). As a control, cells were incubated with an equal volume of PBS. Total RNA was extracted at each time point after stimulation and the inflammatory cytokine mRNA expression was detected by quantitative RT-PCR. The results showed that the mRNA levels of the three factors were all up-regulated significantly (P<0.01) in a time-dependent manner, with the most pronounced effect observed at24h post-stimulation, compared with the PBS control (P<0.01). MbPPD stimulation also resulted in up-regulation of the mRNA levels of the three factors to various degrees, but is significantly lower than that induced by MbINV after24h post stimulation(P<0.05).6. A cell invasion inhibition assay was performed to investigate the invasion capability of MbINV. The result demonstrated that the cell invasion rates by Mb is markedly inhibited by MbINV in a concentration-dependant manner at24-h post infection, indicating that MbINV is cell invasive.7. For further study of the Mb1514gene, Mb1514-EGFP-pLAM12shuttle expression vectors were successfully constructed and transformed into the competent cells of Mycobacterium smegmatis Mc2155by electroporation. This provides a basis tool for further characterization of this gene.