Preparation Of Recombinant CFP-10 Protein And CFP10-ESAT6 Fusion Protein And Evaluation Of Their Immunodiagnostic Value

Objective To obtain the recombinant CFP-10 protein and recombinant CFP10-ESAT6 fusion protein, to study their immunological characteristics, and to evaluate its potential value for immunodiagnosis of tuberculosis.Methods The gene CFP-10 and the fusion gene CFP10-ESAT6 were amplified by PCR, and then cloned into pET-24b or pET-28a plasmid. The recombinant protein CFP-l0(rCFP-l0) and the recombinant fusion protein CFP10-ESAT6(rCFP10-ESAT6) were expressed and purified. Their antigenicity were evaluated by western blot. Tuberculosis patients serum and health human serum were respectively detected the antibodies against the three recombinant proteins(rESAT-6 -. rCFP-10 and rCFP10-ESAT6) by ELISA. The three recombinant proteins and PPD antigen induced delayed-type-hypersensitivity (DTH) reaction in guinea pigs respectively infected with M. tuberculosis H37Rv strains and other twelve mycobacterium strains. Skin reactions were measured at 24h, 48h and 72h after antigen injection.Results The sequence of recombinant plasmids were identical to the predicted sequence. The rCFP-10 and rCFP10-ESAT6 proteins were highly expressed in soluble form in E.coli BL21(DE3),and amounted to 20% and 40% of total bacterial proteins. The purity of final products got to 90%. They produced good immunoreactivity with serum from tuberculosis patient by western blot. The positive cutoff values were OD492 mean puls 2 standard deviation of negative serum detected by ELISA. Of 33 serums from tuberculosis patients, the specificity and sensitivity of rCFP-10 as antigen of ELISA were 100% and 15.1%, that of rESAT-6 were 97% and 18.2%, and that of rCFP10-ESAT6 were 97% and 36.4%. The three recombinant proteins elicited DTH responses similar in strength to that of 5IU PPD in the group of guinea pigs sensitized by M.tuberculosis H37Rv, but in other groups sensitized by BCG or other mycobacteria, the strength was weaker than 5IU PPD.Conclusions The recombinant CFP-10 protein and CFP10-ESAT6 fusion protein could be highly expressed in soluble form in E.coli BL21(DE3). They had good antigenic specificity and immunoreactivity, and could be used to differentiate infection with M.tuberculosis H37Rv strains from immunization with M.bovis BCG and other enviromental mycobacteria.