Molecular Cloning, Expression, Purification and Immunological Characterization of Three Low-Molecular Weight Proteins Encoded by Genes in Genomic Regions of Difference of Mycobacterium Tuberculosis

The aim of this study was to clone, express and purify three major antigenic proteins, i.e. Rv3874, Rv3875 and Rv3619c, encoded by genes located in regions of difference of Mycobacterium tuberculosis and characterize them for immunogenicity in rabbits. The respective genes were amplified using gene-specific primers and genomic DNA of M. tuberculosis by polymerase chain reaction. The amplified DNA were cloned into pGEM-T Easy and subcloned into pGES-TH-1 vector for high-level expression in Escherichia coli and efficient purification. The results showed that the three fusion proteins, i.e. glutathione-S-transferase (GST)-Rv3874, GST-Rv3875 and GST-Rv3619c, were expressed at high levels and were purified (free of the GST fusion partner) to homogeneity using glutathione-Sepharose and Ni-NTA agarose affinity matrix after cleavage of the column-bound fusion proteins by thrombin protease. The purified recombinant Rv3874, Rv3875 and Rv3619c proteins were immunogenic and induced antigen-specific antibodies in rabbits. Testing of the rabbit sera with overlapping synthetic peptides showed that the antibodies were induced to several epitopes that were scattered throughout the sequence of each protein. These results show immunogenicity of all the proteins for inducing antigen-specific antibodies in rabbits and demonstrate the usefulness of pGES-TH-1 vector for obtaining purified recombinant proteins of M. tuberculosis for immunological characterization.