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目的探讨结核杆菌两种抗原MPT83和MPT64基因疫苗的免疫原性和免疫保护性。方法用成对引物扩增MPT83和MPT64两种抗原基因,构建到同一载体pJW4303中,测序正确和体外表达验证后免疫小鼠,间接酶联免疫吸附试验(ELISA)方法测定3次免疫后小鼠的抗体滴度。对3次免疫后的小鼠脾细胞进行培养,抗原刺激,用夹心ELISA方法测定细胞因子IFN-γ和IL-4的含量。免疫小鼠攻毒,进行细菌培养,检测肺脏和脾脏的载菌量。结果每次免疫后抗原的滴度分别为1∶200、1∶800、1∶6400,而卡介苗(BCG)组的抗体滴度为1∶800,空载体组未检测到抗体滴度。融合的二价基因疫苗免疫小鼠,主要诱发Th1型细胞因子,IFN-γ占优势,二价组中的含量为7520pg/ml,IL-4的含量仅为ng级。H37Rv攻毒后,二价组小鼠的肺脏载菌量为(2.21±0.03)×105,比空载体免疫小鼠低103倍,比BCG免疫小鼠低10倍。结论抗原MPT83和MPT64二价基因疫苗有效提高了机体保护效力,对于结核病的防治具有一定的借鉴价值。
Objective To investigate the immunogenicity and immunoprotection of Mycobacterium tuberculosis two antigen MPT83 and MPT64 gene vaccines. Methods Two pairs of primers were used to amplify the two antigens of MPT83 and MPT64, and then constructed into the same vector pJW4303. The recombinant plasmid pJW4303 was verified by sequencing and the expression of MPT64 protein was verified. The indirect immunosorbent assay (ELISA) Antibody titer. The spleen cells of three immunized mice were cultured and antigen stimulated. The contents of cytokines IFN-γ and IL-4 were measured by sandwich ELISA. Immunized mice were challenged with bacteria, cultured in bacteria, and the lung and spleen inoculated were tested. Results The titer of antigens after each immunization was 1: 200, 1: 800, 1: 6400, respectively. The antibody titers in BCG group were 1: 800. No antibody titers were detected in empty vector group. The mice immunized with the bivalent gene vaccine mainly induced Th1-type cytokines. IFN-γ was predominant. The content of bivalent group was 7520 pg / ml and the level of IL-4 was only ng-level. After challenge with H37Rv, the lungs of bivalent mice were (2.21 ± 0.03) × 105, 103-fold lower than that of empty vector mice and 10-fold lower than that of BCG-immunized mice. Conclusions The antigens MPT83 and MPT64 bivalent gene vaccines can effectively improve the protective efficacy of the organism and have some reference value for the prevention and treatment of tuberculosis.