结核分枝杆菌DNA疫苗pVS85B的构建及免疫保护实验研究

Construction of the eukaryotic expression plasmid pVS85B expressing the protective antigen Ag85B of Mycobacterium tuberculosis and its immunoprotective studies

评价构建的结核DNA疫苗pVS85B免疫小鼠后脾细胞体外产生细胞因子和抵抗结核分枝杆菌H37Rv攻击的能力。利用基因操作技术将结核菌ag85b插入pVAX1载体,构建表达结核菌Ag85B分泌型蛋白的DNA疫苗pVS85B。将雌性C57BL/6小鼠分成5组,每组20只,分别用pVS85B、pVAX1、pIL2S和PBS分别免疫3次,均间隔2周,以相同剂量加强免疫2次。另一组用BCG(105CFU)免疫1次。每组10只鼠在最后一次加强免疫后,无菌取脾培养,检测上清细胞因子。另10只鼠用结核菌H37Rv经静脉攻击,2周后取脾、肝和肺培养结核菌并进行菌落计数。结果是pVS85B免疫组鼠脾淋巴细胞培养上清的mIL-2和mIFN-γ平均含量分别为(311.3±46.2)和(273.6±55.4)pg/ml,显著高于3个阴性对照组(P<0.001),与BCG免疫组无显著性差异(P>0.05)。5个组的平均mIL-6和mIL-10无显著性差异。pVS85B免疫组小鼠的脾、肝和肺的平均结核菌载量分别为(47 716.6±5 689.0)、(50 113.4±6 532.2)和(51 095.3±2 788.9)CFU/g,分别低于pVAX1、pIL2S和PBS三个阴性对照组的相应器官的结核菌载量(P<0.001)。pVS85B免疫组脾、肝和肺的结核菌载量显著高于BCG免疫对照组。提示,表达结核菌分泌型Ag85B的DNA疫苗pVS85B能够刺激机体产生抗结核菌所需的Th1型免疫应答,免疫鼠获得抵抗H37Rv攻击的能力。

The eukarotic expression plasmid pVS85B expressing the protective antigen of Mycobacterium tuberculosis was constructed by inserting ag85b gene of M. tuberculosis into vector pVAX1. Twenty female C57BL/6 inbred mice in each of 5 groups were immunized with 100 μg of pVS85B, pLI2S, pVAX1 and PBS for 3 times with 2 weeks intravals as well as vaccinated once with BCG（10^5CFU） respectively. The levels of cytokines in the supernatants of spleen cells in 10 mice of each group and after two weeks intravenous challenge with 10^6CFU of M. tuberculosis H37Rv, the colony counts of bacteria in cultures of spleen, liver and lungs were estimated in 10 mice of each groups. It was found that the average concentrations of mUL-2 and mIFN-γ in the supernatants of spleen cells from pVS85B-immunized mice were （311.3±46.2） pg/ml and （273.6±55.4） pg/ml respectively, that was significantly higher than those from the pVAX1, pIL2S and PBS injected controls. However, there was no significant difference in the levels of mIL-6 and mIL-10 from mice in all the 5 groups. In addition, the average bacterial loads in spleen, liver and lungs of the pVSSSB-immunized mice were （47716.6±5 689.0）, （50013.4±6532.2） and （51 095.3±2 788.9） CFU/g respectively. Meanwhile, the average bacterial loads in these 3 organs in the pVS85B-immunized group were significantly lower than those of their counterparts in pVAX1, pIL2S and PBS-injected groups, but those in the spleen, liver and lungs were significantly higher than that of their counterparts in the BCG-immunized control group. It is concluded from the above observations that the DNA vaccine by using the pVS85B plasmid can induce the Thl immune response which is necessary for the prevention of/tuberculosis and it is able to evoke protective immunity against the challenge with M. tuberculosis H37Rv strain.