N-糖基化修饰对热休克蛋白gp96免疫学功能的影响  

N-glycosylation modification of heat shock protein gp96 affects its immunological function

作　　者：郭鹏 李长菲[1,2] 鞠莹 刘二龙[3] 张含 胡俊 孟颂东 Peng Guo;Changfei Li;Ying Ju;Erlong Liu;Han Zhang;Jun Hu;Songdong Meng(CAS Key Laboratory of Pathogenic Microbiology and Immunology,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;University of Chinese Academy of Sciences,Beijing 100049,China;Heat Shock Biotechnology Co.,Ltd.,Beijing 100123,China;Cominghealth Biotechnology Co.,Ltd.,Beijing 100101,China)

机构地区：[1]中国科学院微生物研究所中国科学院病原微生物与克疫学重点实验室,北京100101 [2]中国科学院大学,北京100049 [3]北京热休生物技术有限公司,北京100123 [4]北京康明海慧生物科技有限公司,北京100101

出　　处：《生物工程学报》2021年第11期4036-4046,共11页Chinese Journal of Biotechnology

基　　金：国家自然科学基釐(Nos.81761128002,81621091,81871297,81672815,31700803);中国科学院B类先导项目(No.XDB29040000);中国科学院“一带一路”科技合作专项(No.153211KYSB20170001)资助。

摘　　要：文中旨在以N-糖基化位点突变的重组热休克蛋白gp96为对象,研究N-糖基化修饰对其免疫功能的影响。首先利用昆虫表达系统表达野生型和突变型gp96蛋白,并检测其糖基化水平。进一步通过体外和体内实验,利用流式细胞术和酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)检测小鼠CD8^(+)IFN-γ^(+)T细胞亚群和IFN-γ的分泌,查明糖基化对gp96抗原呈递功能的影响,进一步用ATPase试剂盒检测gp96的ATPase活性。最后通过小鼠免疫实验探究糖基化对gp96疫苗佐剂功能和活化流感疫苗特异性T细胞的影响。结果显示,N-糖基化修饰位点突变后,重组gp96蛋白总含糖量下降了27.8%。与野生型重组蛋白相比,突变gp96的抗原呈递能力减弱,同时ATPase活性明显降低。同时与野生型重组gp96相比,突变gp96佐剂活化流感疫苗特异性T细胞水平也明显减少。这些结果表明,N-糖基化修饰参与调节gp96的ATPase活性和抗原呈递功能,进而影响其疫苗佐剂功能,为开发基于gp96的佐剂疫苗提供了依据。N-glycosylation modification,one of the most common protein post-translational modifications,occurs in heat shock protein gp96.The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites.Firstly,wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected.To determine the effect of N-glycosylation on gp96 antigen presentation function,the IFN-γ^(+)CD8^(+)T cells in gp96-immunized mice and secretion level of IFN-γwere examined by flow cytometry and ELISA.The ATPase activity of gp96 was further detected by the ATPase kit.Finally,the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice.It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%.Compared to the wild type recombinant gp96,mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96.Furthermore,influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96.These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96,thereby affecting its adjuvant function.The results provide the technical bases for development of gp96-adjuvanted vaccines.

关 键 词：热休克蛋白GP96 N-糖基化修饰 佐剂 ATPASE 抗原呈递 

分 类 号：R392[医药卫生—免疫学]