LC3-LpqH自噬靶向性抗结核DNA疫苗的制备  

Preparation of an autophagy-targeted antituberculosis DNA vaccine encoding LpqH fused with LC3

作　　者：胡东[1] 杨小康[1] 吴静[1] 王干勋 王文洋[1] 祁真玉 陈功[1] 张荣波[1] 

机构地区：[1]安徽理工大学医学院免疫与检验教研室免疫与感染研究所 [2]淮南东方医院集团总院病理科,安徽淮南232001

出　　处：《细胞与分子免疫学杂志》2014年第6期581-584,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(61170172;81202294;81172778);安徽省自然科学基金(1208085QH162);国家级大学生创新创业训练计划项目(201210361121;201210361112)

摘　　要：目的制备结核杆菌脂蛋白抗原前体LpqH基因与微管相关蛋白轻链3(LC3)基因融合的抗结核DNA疫苗,探究其诱导与靶向自噬的效应。方法构建pCMV-LpqH与pCMV-LC3-LpqH重组质粒并转染RAW264.7细胞,免疫印迹法检测LC-3与LC3-LpqH的表达。转染pCMV-LpqH或pCMV-LC3-LpqH至GFP-LC3-RAW264.7细胞,免疫荧光法观察LC3-LpqH蛋白的定位。结果细胞转染pCMV-LpqH后LC-3表达水平增高。pCMV-LC3-LpqH在RAW264.7细胞的表达水平与质粒浓度呈剂量依赖性,并且LC3-LpqH与GFP-LC3体外表达后共定位于自噬体上。结论 pCMV-LC3-LpqH的表达能够增强自噬,并将LpqH抗原靶向定位于自噬体,为设计新型抗结核疫苗提供了新思路。Objective To construct an autophagy-targeted vaccine harboring the genes encoding lipoprotein antigen precursor LpqH from Mycobacterium tuberculosis and microtubule-associated protein light chain-3 （ LC3 ）, and to investigate its efficacy of inducing and targeting autophagy. Methods The expressions of LC-3 and LC3-LpqH in RAW264.7 cells were detected by Western blotting after transfected with pCMV-LpqH and pCMV-LC3-LpqH plasmids respectively. The pCMV-LC3-LpqH or pCMV-LpqH plasmids were transfected into GFP-LC3-RAW264.7 cells to analyze the localization of LC3-LpqH by immunofluorescence staining. Results After transfected with pCMV-LpqH DNA, RAVV264.7 cells showed a significant increase of LC-3 amount. The LC3-LpqH fusion protein was also detected in RAW264.7 cells after pCMV-LC3-LpqH transfection and in a dose-dependent manner. Interestingly, LpqH was found to be transported to autophagosomes through the fusion protein, which was demonstrated by the co-localization of GFP-LC3 and LC3-LpqH on autophagosomes. Conclusion The recombinant plasmid encoding pCMV-LC3-LpqH could enhance the autophagy in vitro, and facilitate the localization of LpqH on autophagosomes. Our study provides a new practical strategy for the development of improved vaccines against Mycobacterium tuberculosis.

关 键 词：自噬 结核分枝杆菌 LC3 LpqH 疫苗 

分 类 号：R392-33[医药卫生—免疫学] R392.11[医药卫生—基础医学]