结核分枝杆菌抗原MPT83和MPT64二价基因疫苗的研究

Immunogenicity and protection of divalent DNA vaccine encoding antigens MPT83 and MPT64 of Mycobacterium tuberculosis

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 imunogenicity and protective efficacy of the divalent DNA vaccine encoding the antigens MPT83 and MPT64 of Mycobacterium tuberculosis . Methods Two genes of the M. tuberculosis , MPT83 and MPT64, were amplified and cloned into the vector pJW4303. The vector containing the fusion gene DNA-MM and pJW4303 blank vector were transfected into CO57 cells. The expression of DNA-MM in the supernatant was detected by Western blotting. Thirty-six C57BL/6 mice were divided into 3 equal groups to be injected subcutaneously with the vector containing the fusion gene DNA-MM, pJW4303 blank vector, or bacillus of Calmette-Guerin vaccine (BCG) once a week for 3 weeks. Twelve mice were used as non-immunized controls. Blood samples were collected from the mice before and after every immunization to detect the titer of antibody by ELISA. Three weeks after the 3rd immunization 3 mice from each were killed and their spleens were taken out. The splenocytes were cultured and stimulated by the recombinant antigen to detect the concentrations of interferon (IFN)-γ and interleukin (IL)-4. Six weeks after the 3rd immunization M. tuberculosis of the line H37Rv was injected intravenously into the mice. Eight weeks later, the mice were killed and their lungs and spleens were taken out. The tissues were cultured to calculate the numbers of M. tuberculosis . Results Western blotting showed that the CO57 cells transfected with the vector containing the fusion gene expressed DNA-MM. ELISA showed that no antibody titer was detected before immunization in every group. The titer of MPT83 and MPT64 were increased to 1∶200, 1∶800, and 1∶6400 3 weeks after the 1st, 2nd, and 3rd immunization respectively in the divalent group, increased to 1∶800 3 weeks after the 3rd immunization in the BCG group, and remained 0 at any time points in the blank vector group. The IFN-γ titer of the divalent group was 7520 pg/ml, significantly higher than that of the BCG group (6675.6 pg/ml, P <0.05). The IL-4 concentration was basically the same and at the ng level in all groups. After challenge of H37Rv the bacterial loading in the lung was (2.21±0.032) ×10 5 in the divalent group, one thousandth of that in the blank vector group, and one tenth of that in the BCG group. Conclusion The divalent vaccine containing the antigens MPT83 and MPT64 effectively heightens the protective efficiency of vaccine against tuberculosis.