[Preparation and Application of Recombinant Mycobacterium Tuberculosis CFP10-ESAT-6 Fusion Protein].

OBJECTIVE:To prepare the recombinant CFP10-ESAT-6 fusion protein, and to study its immunological characteristics, and its potential for serodiagnosis of tuberculosis.METHODS:The lhp-ESAT-6 fusion gene was amplified by Gene SOEing, and then cloned into pQE30 plasmid. The recombinant CFP10-ESAT-6 fusion protein was expressed and purified. Its antigenicity was confirmed by Western blot. Animal models infected with M. tuberculosis H(37)Rv strain and M. bovis BCG respectively were made to evaluate the potential value of the fusion protein in the serodiagnosis of tuberculosis.RESULTS:The sequence of recombinant plasmid pQE30-CFP10-ESAT-6 was identical to the predicted sequence. The recombinant protein (rCFP10-ESAT-6), about 26 000, existed in the cytoplasm of DH5alpha in soluble form and represented 40% of the total bacterial protein. The purity and concentration of the final product was 98% and 1.2 g/L, respectively. Western blot showed that the rCFP10-ESAT-6 had good immunoreactivity with sera from patients with active tuberculosis and rabbits immunized with CFP10 and ESAT-6 respectively. The positive cutoff value was A(490) plus 2 standard deviation from negative guinea pig sera detected by ELISA. Serological reactivity to rCFP10-ESAT-6 was observed in 11 of the serum samples from guinea pigs with tuberculosis and 1 of sera from guinea pigs infected with BCG, while the serological reactivity to PPD was observed in 11 of sera from guinea pigs with tuberculosis and in 11 of sera from guinea pigs infected with BCG.CONCLUSIONS:The rCFP10-ESAT-6 fusion protein was highly expressed in soluble form in E. coli. It had antigenicity of both CFP10 and ESAT-6, and could be used to differentiate infection with M. tuberculosis H(37)Rv strain from immunization with M. bovis BCG. The study provided experimental data for potential application of rCFP10-ESAT-6 in the diagnosis of tuberculosis.