| Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (M.tb). According to the World Health Organization (WHO) statistics, one-third of the world’s population was infected with mycobacterium tuberculosis, causing over3million deaths annually. Moreover, with the emergence of multidrug-resistant TB and the spread of HIV, tuberculosis became one of the main infectious diseases which destroyed people’s health. Understanding the drug resistant mechanism、 good vaccine development and early diagnosis followed by chemotherapy are the major control strategy. The only available vaccine BCG shows disputable protection efficacy against pulmonary tuberculosis, therefore it is urgently needed to develop more effective vaccines against TB. Furthermore, improved diagnostic reagents are needed for the detection of Mycobacterium tuberculosis infections, and the development of a serodiagnostic test would complement the diagnostic methods presently available.Quantitative proteomics is a strong tool to study M.tb drug-resistant mechanism, find new vaccine and diagnostic proteins. We use isobaric tag for relative and absolute quantitation (iTRAQ) to study the differently expressed protein between two extensively drug resistant tuberculosis (XDR-TB) with H37Rv. We found that Mtb9.9c up express1.7and21.3fold in two XDR-TBs, respectively. Mtb9.9c belongs to Mtb9.9protein family. Mtb9.9protein family consisting of five members Mtb9.9A (Rv1793), Mtb9.9B (Rv3619c), Mtb9.9C (Rv1198), Mtb9.9D (Rv1037c) and Mtb9.9E (Rv2346c), shows>90%aa sequence identity.Mtb9.9proteins were highly homologous and were predicted to encode94amino acids with molecular mass of9.9kD. Because of they are highly homologous and antigenic proteins, we study whether they have potential to be subunit vaccine and diagnostic antigen.First we cloned, expressed and purified Mtb9.9family proteins in E.coli. And then we immunize mouse with Mtb9.9family proteins and IFA, at the third week after three immunization, we gather mouse serum and spleen cells to do western blot, cellular immunity and humoral immunity tests. The result of western blot showed that Mtb9.9proteins have same and typical antigenic epitope. The Mtb9.9proteins induced an increased Th1type cellular and humoral immune response in mice, characterized by an elevated concentration of IFN-y and TNF-a in antigen stimulated splenocyte culture and a strong lgG2c antibody response.In order to evaluate whether Mtb9.9can be diagnostic antigen or not, we cloned, expressed and purified CFP-10protein as control. When tested in a conventional ELISA, the Mtb9.9a protein revealed statistically significant antigenic distinction between healthy BCG-vaccinated controls (n=61) and MDR tuberculosis patients. The panel of patient sera comprised sera from pulmonary tuberculosis patients (n=101) and extra-pulmonary tuberculosis patients (n=10) and MDR patients (n=60). It was shown that serum immunoglobulin G antibodies positive rate of recombinant protein antigen Mtb9.9a was75.0%which was higher than CFP-10in MDR pulmonary tuberculosis patients, the specificity of these proteins were83.6%and88.5%, respectively. This study demonstrates that Mtb9.9a protein can be a useful diagnostic marker in MDR pulmonary tuberculosis. In conclusion, Mtb9.9family proteins have the potential to be subunit vaccine because they have the Th1type cellular immunization. Besides Mtb9.9a can distinguish MDR patients and health control, so it also can be use as candidate antigen as serological test. |
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