表达结核分枝杆菌融合蛋白的DNA疫苗构建及免疫保护  

Construction and immunoprotection of the TB DNA vaccine expressing Mycobacterium tuberculosis Mtb8.4-38 000-Ag85B fusion protein

作　　者：朱中元[1] 王海波[1] 谢勇[1] 陈英兰[1] 郑冰冰[1] 张春发[2] 张颖[2] 

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

出　　处：《免疫学杂志》2006年第1期47-50,共4页Immunological Journal

基　　金：海南省自然科学基金资助项目(30221)

摘　　要：目的评价构建的结核DNA疫苗pVS3in1免疫的小鼠体外产生细胞因子能力和抵抗结核分枝杆菌H37Rv攻击的效果。方法将结核菌mtb8.4、Mr38 000和ag85b插入pVAX1载体,构建表达结核菌Mtb8.4-Mr38 000 CTL表位-Ag85B融合蛋白的DNA疫苗pVS3in1。将雌性C57BL/6小鼠分成5组,每组20只,分别用pVS3in1、pVAX1、pIL2S和PBS免疫3次,每隔2周。另一组用BCG(105CFU)皮内免疫1次。每组10只鼠在最后1次免疫后,培养脾细胞,检测上清液细胞因子。另10只用结核菌H37Rv经静脉攻击,2周后取脾、肝和肺培养结核菌并计数。结果pVS3in1组鼠脾细胞培养上清液mIL-2和mIFN-γ含量分别为(379.6±58.2)pg/mL和(529.7±63.7)pg/mL,显著高于3个阴性对照组(P<0.001),与BCG组无显著性差异(P>0.05)。5组的mIL-6和mIL-10无显著性差异。pVS3in1组的脾、肝和肺结核菌载量分别为(17 443.6±3 202.5),(19 047.2±3 395.5)和(14 822.2±2 882.2)CFU/g,低于pVAX1、pIL2S和PBS等3组相应器官的载量(P<0.001),仅脾菌落数显著高于BCG组。结论pVS3in1能够刺激机体产生抗结核菌所需的Th1型免疫应答,诱导小鼠抵抗H37Rv攻击的能力与BCG相当。Objective To evaluate the in vitro cytokine-producing ability and the immunoprotection of the mice immunized with the TB DNA vaccine pVS3in1. Methods Plasmid pVS3in1 expressing fusion protein Mtb8.4-38 000-Ag85B of Mycobacterium tuberculosis was constructed by inserting mtb8.4, CTL epitope of 38 000, and ag85b into the vector pVAX1. One hundred female inbred C57BL/6 mice were divided into 5 groups, and then immunized respectively with pVS3in1, pIL2S, pVAX1, PBS, and BCG for 3 times with 2 weeks intervals except BCG group （one injection only）. Sera and supematants of 10 mice in each group were tested for determining hIL-2, mIL-2, mIFN-γ, mIL-6, and mIL-10 by sandwich ELISA. The other 10 mice in each group were challenged with 10^6 CFUs M. tuberculosis H37Rv and sacrificed for culturing H37Rv from the spleens, hmgs,and livers. Results The average concentrations of mIL-2 and mIFN-γ in the supernatants of the mic from pVS3in1 immunized mice were （379.6 ± 58.2）pg/mL and （529.7 ± 63.7） pg/mL, respectively, which were significantly higher than those from the pVAX1, pIL2S, and PBS injected controls （P〈0.001）. No significant difference found in the mIL-6 and mIL-10 concentrations of the mice from all 5 groups. The average loads of M. tuberculosis in the spleens, livers and lungs in the pVS3in1 group were （ 17 443.6 ± 3 202.5） CFU/g, （ 19 047.2 ± 3 395.5） CFU/g and （ 14 822.2 ± 2 882.2） CFU/g, respectively. The average bacterial loads of the 3 organs in the pVS3in1 immunized group were significantly lower than those of their counterparts in pVAX1, pIL2S and PBS groups, hut bacterial load in the spleens was significantly higher than that of their counterparts in the BCG immunized controls. Conclusions TB DNA vaccine pVS3in1 can induce Thl immune response, which is necessary for prevention against TB, and its ability to enhance immunoprotection against H37Rv challenge in the immunized mice is equivalent to BCG vaccinization.

关 键 词：结核 DNA疫苗 融合蛋白 细胞因子 免疫保护 

分 类 号：R392[医药卫生—免疫学]