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

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, inner salt］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（interferon γ，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(spot forming cells，SFC) ］［(167.5±36.6)SFC/10.6］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。

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

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 1×106 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 MTS3-(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)/106] was significantly higher than that in BCG group (98.5±26.9)SFC/106 ](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.