结核分枝杆菌Ag85B-ESAT-6融合蛋白重组耻垢分枝杆菌对小鼠的免疫原性研究

Immunogenicity of the recombinant M.smegmatis expressing Ag85B-ESAT-6 fusion protein of M.tuberculosis in mice

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 the recombinant M.smegmatis expressing Ag85B-ESAT-6 fusion protein of M.tuberculosis（Ag85B-ESAT-6-rM.s） in mice.Methods Six-week-old BALB/c mice were immunized by subcutaneous injection on the back with 1×10^6 colony-forming unit（CFU） of Ag85B-ESAT-6-rM.s per mouse.The mice immunized by M.smegmatis（M.s）,BCG,Ag85B-ESAT-6 fusion protein and saline used as control.The percentages of CD4＋ and CD8^＋ T cells in the peripheral blood mononuclear cells（PBMC） of mice were detected once a week for six weeks after immunization.The proliferation of splenic lymphocytes of mice was assayed by MTS[3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium,inner salt],and the numbers of splenic lymphocytes secreting interferon γ（IFN-γ）,interleukin-2（IL-2） and interleukin-4（IL-4） were detected by enzyme-linked immunospot assay（ELISPOT） at 6 weeks after immunization.Results The Ag85B-ESAT-6-rM.s could significantly stimulate the generation of CD4＋ and CD8^＋ T cells in the PBMC from immunized mice,and its percentages of CD4＋ and CD8^＋ T cells [（59.6±1.5）%] were significantly higher than that in BCG group [（48.8±2.3）%]（t=12.252,P〈0.05） and M.s group [（45.7±1.6）%]（t=20.166,P〈0.05）.Ag85B-ESAT-6-rM.s could stimulate the proliferation of splenic lymphocytes in immunized mice,and its stimulation index（3.23±0.31） was equivalent to that in BCG group（2.95±0.36）.However,the level of IFN-γ in mice immunized by Ag85B-ESAT-6-rM.s [（167.5±36.6） spot forming cells（SFC）/10^6] was significantly higher than that in BCG group [（98.5±26.9）SFC/10^6 ]（t=4.804,P〈0.05）,and the level of IFN-γ was also higher than levels of IL-2 and IL-4（t=6.174 and 6.449,P〈0.05） in mice immunized by Ag85B-ESAT-6-rM.s.Conclusion The expression of Ag85B-ESAT-6 fusion protein in M.smegmatis could improve the immunogenicity,and Ag85B-ESAT-6-rM.s could induce immune response preventing M.tuberculosis infection in mice.Ag85B-ESAT-6-rM.s as a TB candidate vaccine is worthy of further study.