锌离子依赖的金属蛋白酶-1体内抑制分枝杆菌的抗原处理

Zinc metalloprotease-1 from Bacillus Calmette-Guerin inhibits the processing of mycobacterial antigen in vivo

目的:在小鼠体内观察卡介苗的锌离子依赖的金属蛋白酶-1(Zinc metalloprotease-1,Zmp1)对结核分枝杆菌抗原PPE68处理的影响。方法:6~8周龄BALB/c小鼠随机分为5组,分别为p Bud CE4.1空质粒对照组(n=7)、表达PPE68抗原肽的p BudCE4.1-PPE68p质粒组(n=9)、表达PPE68抗原的p Bud CE4.1-PPE68质粒组(n=9)、PPE68抗原和Zmp1共表达的p Bud CE4.1-PPE68-Zmp1质粒组(n=10)及PPE68抗原肽和Zmp1共表达的p Bud CE4.1-PPE68p-Zmp1质粒组(n=8)。各组质粒电转化减毒沙门菌株SL7207构建相应的重组沙门菌。以灌胃方式将各组沙门菌免疫小鼠3周。收集小鼠血液和肺肠灌洗液,采用ELISA法测定血清中IFN-γ、IL-12含量及肺与肠道s Ig A的含量;分离培养脾细胞,用CCK-8法检测脾淋巴细胞增殖情况。结果:与PPE68抗原组相比,PPE68和Zmp1共表达组的IFN-γ(117.29±16.86,P=0.034)、IL-12(40.46±7.11,P=0.0037)、淋巴细胞刺激指数SI(3.30±0.49,P=0.004)均降低;与PPE68抗原肽组相比,PPE68抗原肽和Zmp1共表达组相应因子含量下降不明显,IFN-γ(132.75±28.40,P=0.070)、IL-12(55.43±11.78,P=0.198)、SI(4.70±1.57,P=0.124)。结论:卡介苗的Zmp1影响结核分枝杆菌抗原PPE68的处理过程,这可能是影响BCG免疫效果的重要原因。

Objective：To observe the effect of Zinc metalloprotease-1（Zmp1）from Bacillus Calmette-Guerin（BCG）on processing Mycobacterium tuberculosis antigen PPE68 in mouse in vivo. Methods：BALB/c mice of 6 to 8 weeks were randomly divided into five groups：plasmid p Bud CE4.1 control group（n=7）,plasmid p Bud CE4.1-PPE68 p group expressing PPE68 peptide（n =9）,plasmid p Bud CE4.1-PPE68 group expressing PPE68 antigen（n = 9）,plasmid p Bud CE4. 1-PPE68-Zmp1 group co-expressing PPE68 anti gen and Zmp1（n=10）,and plasmid p Bud CE4.1-PPE68 p-Zmp1 group co-expressing PPE68 antigen and Zmp1（n=8）. Recombinant Salmonella was constructed through the electrotransformation of these plasmids to attenuated Salmonella strain SL7207. All mice were immunized with corresponding recombinant Salmonella for three weeks via gavage. Mice blood and douche fluid of lung and small intestine were collected and ELISA was used to analyze the level of IL-12,IFN-γ and secretory Ig A（s Ig A）. Mice splenocytes were separately cultivated and CCK8 was used to analyze the proliferation of splenic lymphocytes. Results ：Compared with those of the PPE68 antigen group,levels of IFN-γ（117.29±16.86,P=0.034）,IL-12（40.46±7.11,P=0.004）,and lymphocyte stimulation index（SI）（3.30±0.49,P=0.004）were decreased in co-expressing group of PPE68 and Zmp1. Compared with those of the PPE68 peptide group,levels of IFN-γ（132.75 ±28.40,P =0.070）,IL-12（55.43 ±11.78,P =0.198）,and SI（4.70 ±1.57,P =0.124） were not significantly decreased in co-expressing group of PPE68 peptide and Zmp1. Conclusion：Zmp1 from BCG may have effects on the processing of Mycobacterium tuberculosis antigen PPE68 in mouse in vivo,which may be an important factor influencing BCG immune.