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目的评价表达Mtb Ag85B-ESAT-6融合蛋白的重组耻垢分枝杆菌(Ag85B-ESAT-6-rM.s)在小鼠中的免疫原性。方法6周龄BALB/c小鼠经背部皮下注射1×106 CFU(colony-forming unit,菌落形成单位)的Ag85B-ESAT-6-rM.s,同时设M.s免疫组(10只)、BCG免疫组(10只)、Ag85B-ESAT-6融合蛋白免疫组(10只)和生理盐水组(10只)。接种免疫后1～6周,尾静脉采血,检测小鼠外周血CD4+和CD8+T细胞所占百分比;免疫后6周,MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,innersalt]法检测小鼠脾淋巴细胞增殖和酶联免疫斑点检测(enzyme-linked immunospot assay,ELISPOT)检测脾淋巴细胞分泌γ干扰素(interferonγ,IFN-γ)、白细胞介素-2(interleukin-2,IL-2)和白细胞介素-4(interleukin-4,IL-4)等细胞因子的水平。结果Ag85B-ESAT-6-rM.s免疫小鼠后能够明显刺激小鼠外周血CD4+和CD8+T细胞的产生,其所占百分比[(59.6±1.5)%]明显高于BCG免疫组[(48.8±2.3)%](t=12.252,P<0.05)和原始M.s免疫组[(45.7±1.6)%](t=20.166,P<0.05)。Ag85B-ESAT-6-rM.s免疫小鼠的脾淋巴细胞增殖指数(3.23±0.31)与BCG免疫刺激的增殖指数(2.95±0.36)相当(t=1.864,P>0.05),但其诱生IFN-γ的能力[斑点形成细胞(spotforming cells,SFC)][(167.5±36.6)SFC/106]明显高于BCG[(98.5±26.9)SFC/106](t=4.804,P<0.05)。结论Ag85B-ESAT-6融合蛋白在耻垢分枝杆菌中的表达能够明显提高M.s的免疫原性,并能够刺激小鼠机体产生有利于抗Mtb感染的免疫反应,作为TB新型候选疫苗具有一定的研究前景。
Objective To evaluate the immunogenicity of recombinant M. smegmatis (Ag85B-ESAT-6-rM.s) expressing Mtb Ag85B-ESAT-6 fusion protein in mice. Methods 6-week-old BALB / c mice were injected subcutaneously with Ag85B-ESAT-6-rM.s of colony-forming unit (1 × 106 CFU) Group (10 mice), Ag85B-ESAT-6 fusion protein immunization group (10 mice) and saline group (10 mice). Blood samples were taken from the tail vein 1 to 6 weeks after inoculation and the percentages of CD4 + and CD8 + T cells in the peripheral blood of mice were measured. MTS [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, innersalt] method was used to detect the spleen lymphocyte proliferation and ELISPOT detection of splenic lymphocytes secreting γ interference Interferon gamma (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4) Results The immunization of mice with Ag85B-ESAT-6-rM.s could obviously stimulate the production of CD4 + and CD8 + T cells in peripheral blood of mice, which was significantly higher than that of BCG group [(59.6 ± 1.5)%] [( 48.8 ± 2.3)%] (t = 12.252, P <0.05) and the original Ms immunized group [(45.7 ± 1.6)%] (t = 20.166, P <0.05). The spleen lymphocyte proliferation index (3.23 ± 0.31) of Ag85B-ESAT-6-rM.s immunized mice was comparable to that of BCG immunostimulatory proliferation (2.95 ± 0.36) (t = 1.864, P> 0.05) The ability of IFN-γ (spotforming cells, SFC) [(167.5 ± 36.6) SFC / 106] was significantly higher than that of BCG [(98.5 ± 26.9) SFC / 106] (t = 4.804, P <0.05). Conclusion The expression of Ag85B-ESAT-6 fusion protein in Mycobacterium smegmatis can significantly enhance the immunogenicity of Ms and stimulate the mouse body to produce an immune response that is favorable to anti-Mtb infection. As a novel TB vaccine candidate, Ag85B- Research prospects