pCMV-LC3-Ag85B重组质粒DNA疫苗对结核的免疫保护作用  

Immunoprotective effect of a plasmid DNA vaccine pCMV-LC3-Ag85B against Mycobaterium tuberculosis

作　　者：胡东[1,2] 杨小康[1] 王婉[1] 邢应如[3] 王文洋[1] 杜昭弘 尤其[1] 章力[1] 陈丽萍[1] 吴静[1,2] 张荣波[1,2] 

机构地区：[1]安徽理工大学医学院免疫与检验教研室,安徽淮南232001 [2]安徽理工大学免疫与感染研究所,安徽淮南232001 [3]安徽理工大学附属肿瘤医院检验科,安徽淮南232001

出　　处：《细胞与分子免疫学杂志》2014年第11期1133-1136,1141,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(61170172;81202294;81172778;81302524);安徽省自然科学基金(1208085QH162;KJ2013A105);大学生创新创业训练计划项目(AH201310361188;AH201310361189;AH201310361190;201310361096)

摘　　要：目的构建结核分枝杆菌Ag85B抗原基因与微管相关蛋白轻链3(LC3)基因融合的DNA疫苗,探究其抗结核分枝杆菌保护效应。方法构建pCMV-LC3-Ag85B重组质粒并转染RAW264.7细胞,Western blot法检测目的蛋白的表达水平。分别以pCMV、pCMV-Ag85B、pCMV-LC3-Ag85B质粒免疫BALB/c小鼠。末次免疫2周后,Ag85B刺激下培养各组小鼠脾脏淋巴细胞,ELISA检测IL-2、IFN-γ、IL-4、IL-10分泌水平。末次免疫3个月后,用H37Rv标准株尾静脉注射感染小鼠,计数肺和脾脏结核分枝杆菌载量。结果 pCMV-LC3-Ag85B在RAW264.7细胞的表达水平与质粒浓度呈剂量依赖性。与pCMV-Ag85B免疫组相比,pCMV-LC3-Ag85B免疫组的IL-2、IFN-γ分泌显著增加,而肺和脾脏内结核分枝杆菌载量降低。结论 pCMV-LC3-Ag85B可显著增强Th1型免疫应答,并显示较好的抗结核分枝杆菌免疫保护效应。Objective To construct an autophagy-targeted vaccine harboring the genes encoding Ag85 B and microtubule-associated protein light chain-3（ LC3） and to explore its immunoprotection against Mycobacterium tuberculosis（ MTB）. Methods The pCMV-LC3-Ag85 B plasmid was constructed and used to transfect RAW264. 7 cells. The level of LC3-Ag85 B was detected using Western blotting. Then,BALB /c mice were immunized with pCMV,pCMV-Ag85 B and pCMV-LC3-Ag85 B plasmid,respectively. In vitro,two weeks after the last immunization,the secretion of IL-2,IFN-γ,IL-4and IL-10 from Ag85B-stimulated lymphocytes was measured by ELISA. Three months after the last immunization,all mice were challenged with MTB H37 Rv via the tail vein and the bacterial loads in their spleens and lungs were determined by colony formation assay. Results The LC3-Ag85 B fusion protein was expressed in RAW264. 7 cells that had been transfected with pCMV-LC3-Ag85 B and the expression level was in a dose-dependent manner. Compared with the pCMV-Ag85 B treatment group,pCMV-LC3-Ag85B-immunized mice showed a significant increase of IL-2 and IFN-γ levels and the lower loads of MTB in the spleens and lungs. Conclusion pCMV-LC3-Ag85 B can enhance a specific Th1-predominant immunity and a superior immunoprotection against MTB,which may provide a new practical strategy for the development of improved vaccines against MTB.

关 键 词：自噬 结核分枝杆菌 LC3 AG85B 免疫保护 

分 类 号：R392.33[医药卫生—免疫学] R378.911[医药卫生—基础医学]