Cloning And Expression Of CFP10-ESAT-6-CFP21 Fusion Protein Of Mycobacterium Tuberculosis And Whole Blood IFN-γ Assay Based On The Fusion Protein

Objective: To construct a recombinant plasmid of CFP10-ESAT-6-CFP21 (CE21) fusion protein of Mycobacterium tuberculosis and evaluate the diagnosis value of CE21 fusion protein to tuberculosis based on IFN-γdetecting.Methods: The genes encoding CFP21 and CFP10-ESAT-6(CE) protein were amplified by PCR, cloned into vector pMD18-T respectively and then cloned into expression vector pET21a(+) twice. Recombinant pET21a(+)-CFP10-ESAT-6-CFP21 vector was obtained and transformed into E.coli BL21(DE3).After expression, purification, refolding and removing endotoxin, CE21 fusion protein was used as a stimulating antigen to detect the concentration of IFN-γin whole blood samples of TB patients (n=48), other pulmonary disease patients (n=30) and healthy subjects (n=32) by ELISA.Results: The relative molecular weight of the CE21 fusion protein was approximately 41kDa analyzed by SDS-PAGE, consistent with anticipation. After purification, concertration assay and refolding, the concertration of the recombinant protein is 2.42mg/mL. According to the results of IFN-γdetection in whole blood samples, the detecting positive rate of CE21 fusion protein in TB patients was 72.92%, and that in disease and healthy controls were 3.33% and 3.13%.Conclusions: Recombinant CE21 fusion protein was obtained successfully in E.coli BL21 (DE3). It can be concluded that the fusion protein had a promising diagnosis value of tuberculosis based on the results of IFN-γdetection.