重组新型冠状病毒受体结合区候选疫苗N-糖基化修饰对免疫原性的影响  

Effect of N-glycosylation Modification of Recombinant SARS-CoV-2 Receptor-binding Domain Vaccine Candidate on Immunogenicity

作　　者：李艾 孙鹏 王甜甜 张斌 侯旭宸 徐惠芳 任红英 巩新 刘波[2] 吴军 LI Ai;SUN Peng;WANG Tiantian;ZHANG Bin;HOU Xuchen;XU Huifang;REN Hongying;GONG Xin;LIU Bo;WU Jun(Institute of Physical Science and Information Technology,Anhui University,Hefei 230601,China;Institute of Bioengineering,Institute of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)

机构地区：[1]安徽大学物质科学与信息技术研究院,合肥230601 [2]军事科学院军事医学研究院生物工程研究所,北京100071

出　　处：《中国生物工程杂志》2024年第11期1-10,共10页China Biotechnology

基　　金：国家重点研发计划(2022YFC2104801)资助项目。

摘　　要：目的:蛋白质糖基化在调节疫苗免疫原性中发挥着重要作用。新型冠状病毒(SARS-CoV-2)的刺突蛋白(S蛋白)受体结合域(RBD)含两个N-糖基化位点(N331、N343),探讨RBD糖基化修饰对蛋白质表达水平及免疫原性的影响。方法:利用糖基工程酵母及哺乳动物细胞表达系统构建并表达了野生型RBD抗原和3种N-糖基化位点突变的RBD抗原,即野生型RBD-WT、去糖基化抗原(RBD-N1、RBD-N2、RBD-2N),经纯化得到目的抗原,免疫小鼠后比较重组RBD疫苗诱导产生的抗体水平。结果:RBD糖基化位点突变显著影响蛋白质表达量,在酵母细胞和哺乳动物细胞表达系统中具有相似特点,突变任一位点均导致表达量降低,尤其是突变N343糖基化位点,或两个位点同时突变时表达量降低更显著。RBD抗原免疫小鼠结果表明,用突变N343糖基化位点的抗原免疫小鼠,发现该糖基化位点的突变致使特异性抗体滴度和假病毒中和抗体滴度显著降低。结论:糖基化修饰对RBD蛋白表达水平及免疫原性至关重要,其中N343糖基化位点在调节蛋白质表达水平及免疫原性中发挥关键作用。Objective:Protein glycosylation plays an important role in regulating the immunogenicity of vaccines.The receptor-binding domain(RBD)of the spike protein of the novel coronavirus(SARS-CoV-2)contains two N-glycosylation sites(N331 and N343).This study aims to investigate the effect of RBD glycosylation on protein expression levels and immunogenicity.Methods:Wild-type RBD antigen and three N-glycosylation site mutations of RBD antigens,including wild-type RBD-WT and deglycosylated antigens(RBD-N1,RBD-N2,and RBD-2N),were constructed and expressed using glycosylation engineering yeast and mammalian cell expression systems.The purified antigens were obtained and the antibody levels induced by recombinant RBD vaccines were compared in immunized mice.Results:RBD glycosylation site mutations significantly affected protein expression levels,with similar characteristics in yeast and mammalian cell expression systems.Mutations at any N-glycosylation site resulted in decreased expression levels,especially when the N343 glycosylation site was mutated or both sites were mutated simultaneously.The results of RBD antigen immunization in mice showed that the mutation of the N343 glycosylation site resulted in a significant decrease in the titers of specific antibodies and neutralizing antibodies.Conclusions:Glycosylation modification is critical for the expression level and immunogenicity of the RBD protein,with the N343 glycosylation site playing a critical role in regulating protein expression level and immunogenicity.

关 键 词：SARS-CoV-2 受体结合域 糖基化 免疫原性 

分 类 号：Q939.91[生物学—微生物学]