Identification Of RD5-encoded Mycobacterium Tuberculosis Proteins As B-Cell Antigens Used For Serodiagnosis Of Tuberculosis

[Background] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis) and is still a serious global health care problem. In2010, there were8.8million incident cases of TB with1.45million deaths. Until now, there is no simple, rapid, sensitive, and specific test that can be used for diagnosis of tuberculosis.[Objective] In this study, we cloned, expressed, and purified the RD5-encoded M. tuberculosis recombinant proteins and aimed at revealing other potential candidate antigens to improve diagnostic sensitivity and specificity for TB.[Methods] The ORFs corresponding to RD5region were amplified by PCR from the genomic DNA of H37Rv. The PCR products were cloned into N-terminal or Cterminal His-tagged expression vector pET-21a, pET-32a, or pET-28b at the restriction sites indicated, and the generated recombinant plasmids were transformed into E. coli BL21(DE3) pLysS for expression. After DNA sequencing, individual transformant was cultured overnight in LB medium and then the target proteins were induced with IPTG. The recombinant proteins were purified by IMAC. ELISAs were performed to check the B-cell immune response in humans to RD5-encoded M. tuberculosis recombinant proteins and to the well-known antigen ESAT-6. For statistical analysis, the differences between different TB patients and healthy controls were calculated by the one-way ANOVA and independent-sample t-test using the Statistics Package for Social Science17.0.[Results] In this study, we successfully prepared five RD5-encoded recombinant proteins and evaluated by ELISA for their diagnostic potential in detecting serum antibodies comparison with ESAT-6antigen which have been used for the diagnosis of TB in a population of active TB patients and healthy controls. the sensitivity for detecting antibody responses to ESAT-6, Rv3117, Rv3118, Rv3119, Rv3120, and Rv3121was21.7%,25.0%, 11.7%,11.7%,31.7%and5.0%; respectively, and the specificity was90.6%,96.9%,90.6%,96.9%,96.9%, and87.5%, respectively. Most importantly, the sensitivity and specificity of antibody detection by Rv3117and Rv3120were higher than those of antibody detection by ESAT-6antigen. Furthermore, all the OD450values of antibodies against RD5-encoded recombinant proteins and ESAT-6antigen were used for the generation of ROC curves. The AUC of ESAT-6antigen is0.680and those of Rv3117-Rv3121recombinant proteins were0.757,0.656,0.632,0.735, and0.609, respectively (figure not shown), suggesting Rv3117and Rv3120have a better overall diagnostic performance than any other individual antigen.[Conclusion] In summary, the results identified two immunodominant antigens, that is, Rv3117and Rv3120, which show promise for use in the serodiagnosis of M. tuberculosis. They both revealed a statistically significant antigenic distinction between healthy controls and TB patients. Further studies are in progress to investigate the CMI response to recombinant protein Rv3117and Rv3120, with the aim of evaluating its use in the cellular immunity-based immunodiagnosis of TB.