Cloning expression of antigens Rv2654c, Rv1985c and Rv3868 of Mycobacterium tuberculosis and value in serological diagnosis of tuberculosis

Objective To evaluate the value of three antigens Rv2654c, Rv1985c and Rv3868 of Mycobacterium tuberculosis in the serological diagnosis of tuberculosis (TB). Methods The whole genes of Rv2654c, Rv1985c and Rv3868 were amplified from the genomic DNA of M. tuberculosis strain H37Rv by using PCR and the recombinant plasmid was constructed with expression vector pET-32a. The proteins were expressed by prokaryotic expression and purified by affinity chromatography. The best reactive conditions of enzyme linked immunosorbent assay (ELISA) of each antigen were determined by chessboard titration method. The antibodies IgG against M. tuberculosis were tested with 190 serum samples by using ELISA and the diagnostic efficiency of each antigen was analyzed by means of receiver operating characteristic curve (ROC). Results The results showed that the recombinant antigens were cloned and expressed stably and the DNA sequences of the protein antigens were completely consistent with the sequences in Genbank of NCBI respectively by BLAST alignment. The serological results of the three antigens which had been purified were analyzed with statistical software. The diagnostic efficiency of Rv2654c, Rv1985c and Rv3868 was 73.16%, 56.84% and 71.05% respectively. The best combination from the three antigens was Rv2654c+Rv3868, the sensitivity, specificity and efficiency were 78.95%, 72.63% and 75.79%. Conclusion The recombinant antigens Rv2654c, Rv1985c and Rv3868 have good diagnostic value and can be applied as candidate antigen for TB diagnosis. The combination of antigens Rv2654c and Rv3868 has good performance in TB diagnosis and possesses a certain application value in clinical practice.