Cloning, Expression And Purification Of Fusion Protein 19KD-ESAT6 Of Mycobacterium Tuberculosis And Its Value In Serodiagnosis

Objective: To construct the recombinant plasmid of fused protein 19KD-ESAT6 of Mycobacterium tuberculosis,express and purify 19KD-ESAT6 in E.coli. The antigenicity of the fusion protein was analyzed and its potential value for TB serodiagnosis was evaluated.Methods: The gene coding 19KD and ESAT6 were amplified by polymerase chain reaction from genome of the standard Mycobacterium tuberculosis H37Rv, and then cloned into the vector pMD18-T followed by the subclone into the expression vector pET-21a, respectively. Recombinant 19KD-ESAT6 was expressed in E.coli and then purified by Ni-NTA. The antigenicity of the fusion protein was analyzed by Western blot. Sera from 161 cases clinical sera were tested by ELISA.Results: Recombinant plasmid of 19KD-ESAT6 was constructed, and could be expressed efficiently in E.coli BL21(DE3). The relative molecular mass of the fusion protein was about 29KD by SDS-PAGE. The fusion protein was confirmed by Western blot with specific immunogenicity. The ELISA assay was established. Altogether, 40% of TB case patients had detectable antibodies while 2 of the healthy controls were positive, 1 of the other lung disease controls were positive, its total specificity was 96.8%.Conclusion: The fusion 19KD-ESAT6 protein showed the specific immunogenicity, and will provide evidences for the development of serodiagnostic reagents. The fusion protein presented more favorable sensitivity than that of each dominant antigen .