Construction of multivalent DNA vaccines for Mycobacte-rium tuberculosis and its immunogenicity  

作　　者：CAI Hong PAN Yi LI Guoli ZHUANG Yuhui ZHU Yuxian 

机构地区：[1]College of Life Sciences,Peking University,Beijing 100871,China [2]Tuberculosis Research Laboratory,309 Hospital of PLA,Beijing 100091,China

出　　处：《Chinese Science Bulletin》2002年第19期1589-1593,共5页

基　　金：This work was supported by the Chinese National High-Tech "863" Project (Grant No. 2001AA213141).

摘　　要：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 theThe coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eukaryotic expression vector pJW4303, then transformed toE coli Top 10 strain for plasmid DNA extraction and further analysis. Plasmids containing the right insertion were sequenced to confirm their identity. COS7 cells were transfected 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 highest antibody levels of single DNA vaccine reported in literature after third injections. Antibody titer of MPT-64 was 1: 50 after the first injection and it reached 1: 200 after the second injection. No antigen-specific antibody against ESAT-6 was detected in sera harvested from immunized mice 21 days after both injections. Antigen-specific IFN-γ level of Ag85B was 110 pg/mL while no antigen-specific IFN-γ level of ESAT-6 and MPT-64 was detected even after third injections. To our knowledge, it is the first time that studies of polyvalent recombinant DNA vaccines against TB were carried out in C57BL-6 mice. Our results indicated that multiple DNA vaccines could be used to enhance protective responses against MTB.

关 键 词：M. TUBERCULOSIS SECRETED PROTEINS DNA vaccines immu-nogenicity HUMORAL and cell-mediated responses. 

分 类 号：R521[医药卫生—内科学]