结核分枝杆菌Mtb8.4DNA疫苗构建及免疫保护研究  

Construction and protective efficacy of the TB DNA vaccine expressing Mycobacterium tuberculosis secretory Mtb8.4 protein

作　　者：朱中元[1] 刘建兵[1] 王海波[1] 谢勇[1] 张春发[2] 张颖[2] 

机构地区：[1]海南医学院附属新华医院,海南海口570311 [2]南热带农业大学国家生物技术重点实验室,海南海口571101

出　　处：《中国热带医学》2005年第8期1595-1599,1624,共6页China Tropical Medicine

基　　金：海南省自然科学基金资助项目(编号:30221)

摘　　要：目的评价构建结核菌DNA疫苗pVS84免疫小鼠体外产生细胞因子的能力和抵抗结核分枝杆菌H37Rv攻击的效果。方法利用基因技术将结核菌Mtb8.4插入pVAXl载体,构建表达结核菌Mtb8.4蛋白(分泌型)的DNA疫苗pVS84。将雌性C57BL/6小鼠分成5组,每组20只,分别用pVS84、pVAXl、plL2S和PBS分别各免疫3次,每次均间隔2周,以等剂量加强免疫2次。另一组用BCG(105CFU)皮内注射免疫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只鼠则用结核菌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pVS84免疫组鼠脾淋巴细胞培养上清的mIL-2和mlFN-Y平均含量分别为(290.0±112.9)pg/ml和(501.7±79.4)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。pVS84免疫组小鼠的脾、肝和肺的平均结核菌载量分别为(39176.9±3702.6)、(18780.1±2535.8)和(24410.3±1846.8)CFU/g,分别低于pVAXl、plL2S和PBS三个阴性对照组的相应器官的结核菌载量(P<0.001),仅脾结核菌载量显著高于BCG免疫对照组。结论构建的DNA疫苗pVS84能够刺激机体产生抗结核菌所需的Thl型免疫应答,免疫鼠抵抗H37Rv攻击的能力与BCG接近。Objective To evaluate the capacity in production of cytokines in vitro and the protective efficacy of the mice immunized with the TB DNA vaccine pVS84 we constructed. Methods Plasmid pVS84 expressing secretory protein of Mycobacterium tuberculosis Mtb8,4 was constructed by inserting mtb8.4 into the vector pVAX1. 20 female C57BL/6 inbred mice in 5 groups immunized with the 100ug pVS84, pIL2S, pVAX1, PBS and BCG（105CFU） for 3 times with 2 weeks intervals except BCG（only one vaccnization without boost）. Sera and supematants of 10 mice in each group were determined for hIL- 2, mIL- 2, mIFN - γ, raiL - 6 and raiL - 10 by sandwich ELISA. The other 10 mice in each group were challenged with 10^6 CFUs Mtuberculosis H37Rv and killed for culturing H37Rv from the spleens, lungs and livers. Results The average concentrations of raiL - 2 and mIFN - γ in the supematants from pVS84 immunized mice were 290. 0 ＋ ll2.9pg/mL and 501.7 ＋ 79.4pg/mL respectively, significantly higher than those from the pVAX1, pIL2S, and PBS injected controls （P 〈 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 Mtuberculosis loads in the spleens, livers and lungs in the pVS84 immunized group were 39176.9 ＋ 3702.6 CFU/g, 18780. 1 ＋ 2535.8 CFU/g and 24410. 3 ＋ 1846.8 CFU/g respectively. The average bacterial loads of the 3 organs in the pVS84 immunized group were significantly lower than those of their counterparts in pVAX1, pIL2S and PBS injected groups, but bacterial load in the spleens was significantly higher than that of their counterparts in the BCG immunized controls. Conclusion TB DNA vaccine pVS84 we constructed can induce Thl immune response which is necessary for prevention against TB, and it can evoke protective immunity to H37Rv challenge in the immunized mice equal to BCG immunization.

关 键 词：结核分枝杆菌 DNA疫苗 分泌蛋白 细胞免疫 免疫保护 

分 类 号：R378.91[医药卫生—病原生物学]