结核分枝杆菌重组Ag85A、Ag85B蛋白免疫原性的研究

The studies on immunogenicity of Mycobacterium tuberculosis recombinant Ag85A and Ag85B protein

目的研究结核分枝杆菌重组Ag85A(rAg85A)和/或Ag85B(rAg85B)蛋白与母牛分枝杆菌菌苗(Mycobacteri-um vaccae,MV)联合免疫的免疫原性。方法 60只雌性BALB/c小鼠随机分为下列6组:(1)阴性对照组:磷酸盐缓冲液(PBS)组,(2)阳性对照组:卡介苗(BCG)组,(3)阳性对照组:MV组,(4)实验组:rAg85A-MV组,(5)实验组:rAg85B-MV组及(6)实验组:rAg85A-rAg85B-MV组。每2周免疫1次,共免疫3次。末次免疫结束后第14天杀鼠,用ELISA法检测血清中IgGI、gG1和IgG2a水平;ELISPOT检测脾脏分泌γ干扰素(IFN-γ)的T淋巴细胞斑点数;流式细胞术检测全血单个核细胞内Th1和Th2百分比。结果三个实验组小鼠血清抗体水平均明显高于三个对照组(P<0.001);三个实验组和两个阳性对照组小鼠的T淋巴细胞斑点数均明显高于阴性对照组(P<0.05);rAg85A-rAg85B-MV组小鼠Th1百分比明显高于其余五组(P<0.001),三个实验组Th1/Th2比值均明显高于三个对照组(P<0.05)。结论结核分枝杆菌rAg85A和rAg85B蛋白均具有很强的免疫原性,与MV联合免疫可增强其Th1型细胞免疫应答。

Objective To study the immunogenicity of Mycobacterium tuberculosis rAg85A and/or rAg85B protein joint MV.Methods 60 female BALB/c mice were randomly divided into the following 6 groups：（1）Negative control group：Phosphat buffer solution（PBS）,（2） Positive control group：Bacille Calmette-Guerin（BCG）,（3） Positive control group：MV,（4） Experimental group：rAg85A-MV,（5） Experimental group：rAg85B-MV,and（6） Experimental group： rAg85A-rAg85B-MV.3 times at 6 weeks intervals.After 6 weeks,all the mice were killed.rAg85A/rAg85B-specific antibodies of IgG,IgG1,and IgG2a were determined by ELISA,the number of T-lymphocytes spots which secreted IFN-γwere detected by ELISPOT,and the percentage of Th1 and Th2 in whole blood mononuclear cells were detected by Flow cytometry.Results The level of three specific antibodies of the three experimental groups were significantly higher than those of the three control groups（P0.001）;the number of T-lymphocytes spots of the three experimental groups and two positive control groups were higher than the negative control group（P0.05）;the percentage of Th1 of rAg85A-rAg85B-MV group was significantly higher than that of the other 5 groups（P0.001）,and the ratio of Th1/Th2 of three experimental groups were higher than the other three control groups（P0.05）.Conclusion Mycobacterium tuberculosis rAg85A and rAg85B protein have strong immunogenicity,joint with MV,they can strengthen its Th1-type immune response.