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The coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eu-karyotic expression vector pJW4303, then transformed to E. coli Top 10 strain for plasmid DNA extraction and further analysis. Plasmids containing the right insertion were se-quenced to confirm their identity. COS7 cells were trans-fected with a mixture containing serially diluted plasmid DNA encoding three secreted proteins and Lipofectin (Gibco). The supernatants and pellets prepared from various cell lines were run on SDS-PAGE gel and the expression of these proteins in COS7 cells were demonstrated by immunoblot using polyclonal or monoclonal antiserum of M.TBH37Rv. 21 days after first vaccination of C57BL-6 mice by all three recombinant eukaryotic expressing vectors, antibody titer for Ag85B reached 1:3200. 21 days after second vaccination, the antibody titer reached 1:102400. The highest antibody levels induced by multivalent vaccines after the second injection were equal to or even greater than the hi
The coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eu-karyotic expression vector pJW4303, then transformed into E. coli Top 10 strain for plasmid DNA extraction and further analysis. Plasmids containing the right insertion were se-quenced to confirm their identity. COS7 cells were trans-fected with a mixture containing serially diluted plasmid DNA encoding three secreted proteins and Lipofectin (Gibco). The supernatants and pellets prepared from various cell lines were run on SDS-PAGE gel and the expression of these proteins in COS7 cells were demonstrated by immunoblot using polyclonal or monoclonal antiserum of M.TBH37Rv. 21 days after first vaccination of C57BL-6 mice by all three recombinant eukaryotic expressing vectors, antibody titer for Ag85B reached 1: 3200. after second vaccination, the antibody titer reached 1: 102400. The highest antibody levels induced by multivalent vaccines after the second injection were equal to even greater than the hi