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目的探讨利用HIV反向转导结构域(TAT-PTD)系统表达的转导性Ag85B蛋白疫苗的抗结核分枝杆菌效应。方法构建p ET28a-Ag85B、p ET28a-TAT-Ag85B质粒,原核表达纯化和鉴定后获得Ag85B、TAT-Ag85B蛋白;将BALB/c小鼠随机分为3组:Ag85B组,TAT-Ag85B组,PBS组。重组蛋白经皮下免疫小鼠3次,末次免疫后1周处死部分小鼠,ELISA检测小鼠血清中Ag85B抗体滴度,以及脾细胞分泌的γ干扰素(IFN-γ)、白细胞介素2(IL-2)的水平;用流式细胞术检测巨噬细胞经Ag85B、TAT-Ag85B蛋白刺激后表面分子CD80、CD86变化情况;剩余小鼠经尾静脉感染H37Rv结核分枝杆菌,感染后第1、2、4、8周动态监测小鼠肺和脾内结核分枝杆菌载量,并在感染后8周取肺脏进行HE染色,观察肺组织病变情况。结果成功获得Ag85B、TAT-Ag85B蛋白;与Ag85B相比,TAT-Ag85B诱导小鼠表达高水平的Ig G和IFN-γ、IL-2,感染小鼠的脾、肺结核分枝杆菌载量明显减少,肺组织病变范围小、结核分枝杆菌数量少。另外,TAT-Ag85B增强巨噬细胞表面分子CD80/CD86的表达。结论 TAT-Ag85B蛋白疫苗增强巨噬细胞的抗原提呈能力,诱导小鼠产生强烈的Th1免疫应答,有一定的抗结核分枝杆菌保护作用。
Objective To investigate the anti-Mycobacterium tuberculosis effect of transduced Ag85B protein vaccine expressed by the HIV reverse translocation domain (TAT-PTD) system. Methods The plasmids p ET28a-Ag85B and p ET28a-TAT-Ag85B were constructed and purified by prokaryotic expression. Ag85B and TAT-Ag85B proteins were obtained. The BALB / c mice were randomly divided into three groups: Ag85B group, TAT- group. The mice were immunized subcutaneously three times with recombinant protein, and some mice were sacrificed one week after the last immunization. The titer of Ag85B antibody in sera of mice and the IFN-γ, IL- IL-2). The changes of CD80 and CD86 on macrophages stimulated by Ag85B and TAT-Ag85B protein were detected by flow cytometry. The remaining mice were infected with Mycobacterium tuberculosis H37Rv through tail veins. After infection, The 2,4-week, 8-week and 8-week dynamic monitoring of Mycobacterium tuberculosis in lung and spleen of mice was performed. HE staining was performed 8 weeks after infection to observe the lung tissue lesions. Results Ag85B and TAT-Ag85B proteins were successfully obtained. Compared with Ag85B, TAT-Ag85B induced high levels of Ig G, IFN-γ and IL-2 in mice, and markedly decreased the mycobacterium tuberculosis load in infected mice , A small range of lung lesions, a small number of Mycobacterium tuberculosis. In addition, TAT-Ag85B enhances the expression of macrophage surface molecules CD80 / CD86. Conclusion The TAT-Ag85B protein vaccine enhances the antigen-presenting ability of macrophages, induces a strong Th1 immune response in mice, and has certain protection against Mycobacterium tuberculosis.