Cloning, Expression And Serodiagnostic Evaluation Of Four Specific Antigens From Mycobacterium Tuberculosis

Tuberculosis (TB) has been being diagnosed by clinical symptoms together with bacteriology methods (including sputum smear microscopy, bacteria culture), tuberculin skin test (TST, PPD skin test), and X-ray examination results in China. But the positive rate of the sputum smear is very low, the bacteriology test needs very long time to get the results, PPD test exists serious false positive, and X-ray examination is nonspecific, so these methods greatly reduce the efficiency of tuberculosis diagnosis. In recent years the use of some new laboratory testing technology for TB diagnosis makes a TB test more quickly, for example, serological detection methods have the advantages of simple operation, rapid, good stability, no need of special precision instrument and easy to promote. So the serological detection has become a good auxiliary method for TB diagnosis. While the antigens of M. tuberculosis have a common characteristic of low sensitivity, and a single antigen cannot fulfill the requirement of serodiagnosis for TB. In this research, four kind specific antigenic proteins of M. tuberculosis were cloning, expression, purification, of which the detection performance for TB serodiagnosis were evaluated by the evaluation method early established in our laboratory.The evaluation system has been established by the enzyme-linked immunosorbnent assay (ELISA) and the receiver operating characteristic curve (ROC) to analyze the diagnostic effects of the four antigens. The sensitivity and specificity of every possible antigen combination were calculated, by which the best combination of the antigens has been achieved.By means of literature retrieval and antigen epitope analysis target antigenic proteins, CFP10-ESAT6,38kDa, Ag85B, and HspX, were selected in this research. With molecular clone and protein purification technologies the four recombinant proteins were successfully cloned, expressed and purified. Then their diagnosis value was evaluated with ELISA. The optimal concentration of the recombinant proteins CFP10-ESAT6,38kDa, Ag85B and HspX used in the ELISA was5.58μg/ml,5.45μg/ml,9.00μg/ml and7.60μg/ml respectively and the best serum dilution was1:200except the Ag85B (1:400). The detective effects of every antigen and any possible combination of CFP10-ESAT6,38kDa, Ag85B and HspX were evaluated respectively. The results showed that the diagnostic effect of38kDa was the best among the four antigens, which sensitivity was55.9%, specificity was87.8%, Youden index was0.437; and the effect of any antigenic combination was much better than any singal antigen, the combination (38kDa+CFP10-ESAT6+HspX) was the best of all, of which the sensitivity and specificity were75.3%and68.9%respectively.This study has conducted successfully on cloning, expression, purification and detection performance evaluation of the four antigenic proteins of M. tuberculosis. The results further supplemented the data of previous research and some rational suggestions for more adaptive combination of antigens in serological diagnosis method need to be research deeper in the future.