Study On The Expression Of Rv0341 In Rifampicin-dependent Mycobacterium Tuberculosis And The Clinical Application Evaluation Of Rv0341

BackgroundRifampin-dependent Mycobacterium tuberculosis (RFP-dependent M.TB )is Mycobacterium tuberculosis that grow depending on rifampicin(RFP), this biological phenomenon is more serious than resistance variation. Patients infected with RFP-dependent M.TB have severe state of an illness , multi-drug resistance, poor effect of chemotherapy and poor prognosis of clinical features. If continuing to use RFP , the patient's condition may be more serious .It is one importance reason that occurred the refractory tuberculosis and the failure of chemotherapy. It is very important to treat of tuberculosis caused by RFP-dependent M.TB.ObjectiveWe studied on the high expressed protein Rv0341 of RFP-dependent M.TB.The polymorphism of encoding gene iniB was analyzed by restriction enzyme fragment polymorphism (RFLP). Then we cloned, expressed and purified Rv0341 and established the Dot immunogold filtration assay (DIGFA) to test anti-Rv0341 serumbodies of patients. To study the application value of diagnosis in tuberculosis caued by RFP-dependent M.TB, this study will provide theoretical and experimental basis for the further research.Methods1. Through SD-PAGE and western-blot analyzed the protein Rv0341of RFP-dependent M.TB strains, RFP-resistant M.TB strains and RFP-sensitive M.TB strains. Using RFLP analyzed iniB gene.2. The iniB gene were amplified from Mycobacterium tuberculosis H37Rv strains complete genome by PCR, and the PCR product was cloned into prokaryotic expression vector to generate recombinant plasmid pET22b(+)-iniB. The recombinant vectors were correspondly transformed and expreesed in E.coli BL21(DE3). The recombinant protein were purified with AKTA-explore 100 system. Purified recombinant protein Rv0341 had good immunogenicity and antigenicity.3. Establish the Dot immunogold filtration assay (DIGFA) that testing anti-Rv0341 serumbodies of patients.4. Evaluation of the clinical quality of DIGFA method.Results1. RFLP results show that iniB gene is conservative in M.TB strains.2. Constructed the high performanceing expression vector pET22b(+)-iniB.The recombinant plasmid were transformed in E.coli BL21(DE3) and induced with IPTG at 37℃. SDS-PAGE analysis showed that the expressing ratio was indicated as 25% of total bacterial protein by UVP scan. The expression form of Rv0341 was proved to be inclusion body. After purification by AKTA-explore , we got the target protein Rv0341 with purity of 95%. Western blot proved that the recombinant protein Rv0341 can specifically react with positive serum, but not with negatine serum. Rv0341 protein has the immunocompetence and immunogenicity .3. Establish the Dot immunogold filtration assay (DIGFA) that test anti-Rv0341 serumbodies of patients.4. Use DIGFA to detece the anti-Rv0341 serumbodies of 95 tuberculosis patients , the test results showed 95.8% of sensitivity; 81.7% of specificity, 85.3% of fidelity.Conclusion1. RFLP results show that iniB gene is conservative in M.TB strains. Expression of Rv0341 protein are not caused by gene mutation, may be caused by differences in transcription levels.2. Constructed the high expression of recombinant protein Rv0341, found out the best purification conditions to obtain the purified target protein.The recombinant protein Rv0341 have the immunocompetence and immunogenicity.3. Establish the Dot immunogold filtration assay (DIGFA) to test anti-Rv0341 serumbodies of patients.The methods showed 95.8% of sensitivity; 81.7% of specificity, 85.3% of fidelity. Laid the theoretical and experimental basis for the research and development of diagnose the tuberculosis caued by RFP-dependent M.TB...