Prokaryotic Expression, Purification, Antigenicity Analysis Of Mycobacterium Tuberculosis Chimeric Protein Ag856A2 And The Preparation Of Its Monoclonal Antibodies

Objective To construct a recombinant prokaryotic expression vector harboring the Mycobacterium Tuberculosis chimeric gene ag856a2, purify the protein of Ag856A2 expressed in E. Coli BL21 (DE3) and analyze its antigenicity; To prepare and charaeterize the monoclonal antibodies(McAbs) against chimeric protein Ag856A2. Methods The chimeric gene ag856a2 was amplified by PCR, and cloned into pET28a vector, the recombinant plasmid was transformed into E. coli BL21 (DE3). After induced with IPTG, the expressing form was analyzed by SDS-PAGE. The target protein was purified by Ni2+ affinity chromatographyand, then its antigenicity was analyzed by Western-blotting with murine serum of anti-Ag85A or anti-ESAT-6. Using DNA vaccine HG856A and purified protein Ag856A2 immuned BALB/C mice, SP2/0 Myeloma cells were fused with mice spleen B lymphoeytes. The hybridoma cell was cultured in the HAT selective medium. The positive clone was screened by indirected ELISA (IELISA). After subcloning, gathered supernatant of hybridoma strain and identified with IELISA. Finally hybridoma cell strains that secreted monoclonal antibody were obtained. Using IELISA identify the subclasses, the potency and the noetic antigen of monoclonal antibodies, then, analyze the specificity of anti-ESAT-6 monoclonal antibody by Western Blotting. Results The chimeric protein was highly expressed in an insoluble form, accounted for about 35% in the total proteins; The target protein was purified successfully by the Ni2+ affinity chromatography; Western-blotting result showed that the chimeric protein Ag856A2 had good immuno-reactivity with murine serum of anti-Ag85A or anti-ESAT-6. After fusion, screening and subcloning, 6 hybridoma cell strains which secreted monoclonal antibody were obtained. Five monoclonal antibodies which showed positive reaction with protein Ag856A2,Ag85A belong to IgGl. One monoclonal antibodies which showed positive reaction with protein Ag856A2,ESAT-6 belongs to IgG2b, and its potency that react with ESAT-6 was 6400 and react with Ag856A2 was 12800. Conclusions The chimeric protein Ag856A2 was highly expressed in an insoluble form, and it has both antigenicity of protein Ag85A and ESAT-6. One hybridoma cell strain which secreted anti-ESAT-6 monoclonal antibody was obtained. All of this lay a foundation for further study of the subunit vaccine against tuberculosis and early diagnosis of TB.