表达结核分枝杆菌Ag85B及GST融合蛋白DNA疫苗构建及免疫保护研.doc

表达结核分枝杆菌Ag85B及GST融合蛋白DNA疫苗构建及免疫保护研 　 作者：朱中元1，刘建兵１，王海波１，谢 勇１，张春发２，张 颖 【摘要】 目的 评价构建的结核DNA疫苗pVS85BG免疫的小鼠体外产生细胞因子和抵抗结核分枝杆菌H37Rv攻击的能力。方法 利用基因操作技术将结核菌Ag85B基因和谷胱甘肽S转移酶基因插入pVAX1载体构建表达结核菌Ag85B和GST融合蛋白的DNA疫苗pVS85BG。将雌性C57BL/6小鼠分成5组，每组20只，分别用pVS85BG、pVAX1、pIL2S和PBS分别免疫，以相同的剂量加强免疫2次，均间隔2周。另一组用BCG(105CFU)皮内注射免疫1次。每组的10只鼠在最后一次加强免疫后，无菌取脾培养，上清供检测细胞因子用。另外10只小鼠则用结核菌H37Rv经静脉攻击，于2周后取脾、肝和肺进行培养结核菌并进行菌落计数。结果 pVS85BG免疫组的鼠脾淋巴细胞的培养上清中的mIL-2和mIFN-γ的平均含量分别为(339.9±59.4)pg/ml和(432.1±74.4)pg/ml，显著高于3个阴性对照组（Plt;0.001），与BCG免疫组差异无显著性(P＞0.05)。5个组的平均mIL-6和mIL-10差异无显著性。pVS85BG免疫组小鼠的脾、肝和肺的平均结核菌菌落数分别为(32954.2±4457.5)CFU/g，(32748.0±3634.4)CFU/g和(31741.7±7293.7)CFU/g，分别低于pVAX1、pIL2S和PBS等3个阴性对照组的相应器官的结核菌载量（Plt;0.001），同时也显著高于BCG免疫对照组。结论 构建的DNA疫苗pVS85BG能够刺激机体产生抗结核菌所需的Th1型免疫应答，免疫鼠抵抗H37Rv攻击的能力显著增强。 【关键词】 结核； DNA疫苗； 细胞因子； 免疫保护 【Abstract】 Objective To evaluate the ability to produce cytokines in vitro and the protective efficacy of the mice immunized by the TB DNA vaccine pVS85BG we constructed.Methods Plasmid pVS85BG expressing fusion protein of Mycobacterium tuberculosis Ag85B and glutathione S-transferase were constructed by inserting ag85b and gst into the vector pVAX1. 20 female C57BL/6 in bred mice in 5 groups immunized with the 100μg pVS85BG， pIL2S， pVAX1， PBS and BCG(105 CFU) for 3 times with 2 weeks interval except BCG(only one injection without boost). Sera and supernatants of 10 mice in each group were determined for hIL-2， mIL-2， mIFN-γ， mIL-6 and mIL-10 by sandwich ELISA. The other 10 mice in each group were challenged by 106 CFUs M tuberculosis H37Rv and killed for culturing H37Rv from the spleens， lungs and livers.Results The average concentrations of mIL-2 and mIFN-γ in the supernatants from pVS85BG immunized mice were (339.9±59.4)pg/ml and (432.1±74.4)pg/ml， significantly higher than those from the pVAX1， pIL2S， and PBS injected controls (Plt;0.001). No significantly difference has been observed for the mIL-6 and mIL-10 concentrations from the mice in all 5 groups. The average M tuberculosis