In order to identify
immunodominant antigens of Mycobacterium tuberculosis that may be used in the serodiagnosis of active
tuberculosis (TB), we designed an M.
tuberculosis fusion
protein consisting of CFP-10 (10-kDa culture filtrate
protein), ESAT-6 (6-kDa early secreted antigenic target), and the extracellular domain fragment of PPE68 (PPE68'). Then, the coding sequences of the three
proteins were inserted into a prokaryotic expression vector, pET-32a(+). To enhance the immunological response, the
proteins were linked together. The fusion
proteins with a 6 × His tag were successfully overexpressed in Escherichia coli BL21 and purified. The purified
proteins were applied for detection of the total
IgG titer by using an
enzyme-linked
immunosorbent assay (ELISA) with human sera from well-characterized TB cases and the control cases, and results were compared to those with purified
protein derivative
tuberculin (
PPD). The ELISA results showed that among 140 cases of confirmed active TB and 70 control cases, CFP-10-ESAT-6-PPE68' had a sensitivity of 73.3% and specificity of 94.3%, compared to a sensitivity of 66.7% and specificity of 74.3% for
PPD and a sensitivity of 65% and specificity of 91.4% for CFP-10-ESAT-6. In addition, the fusion
protein CFP-10-ESAT-6-PPE68' stimulated a higher level of
antigen-specific
gamma interferon (IFN-γ) release for active-TB patients than
PPD and CFP-10-ESAT-6. After immunization of C57BL/6 mice, the findings indicated that the total
IgG titers and the concentrations of IFN-γ in mice immunized by CFP-10-ESAT-6-PPE68' were high and induced strong, long-term humoral immunity compared to results with
PPD and CFP-10-ESAT-6. Thus, our study indicates that the fusion
protein CFP-10-ESAT-6-PPE68' may be useful as an immunodominant
antigen for the serodiagnosis of active TB.