柞蚕蛹表达人促红细胞生成素的糖基化修饰分析  

Analysis of Glycosylation Modification of Human Erythropoietin Expressed in Pernyi Pupae

作　　者：刘晓黎 刘宇博[1,2] 李学臣[3] 陈秋实 范琦 张嘉宁[1] 李文利[1] LIU Xiaoli;LIU Yubo;LI Xuechen;CHEN Qiushi;FAN Qi;ZHANG Jianing;LI Wenli(School of Life and Pharmaceutical Sciences,Dalian University of Technology,Panjin 124221,China;Key Laboratory of Bio-Intelligent Manufacturing,Ministry of Education,Dalian University of Technology,Dalian 116023,China;Department of Chemistry,State Key Laboratory of Synthetic Chemistry,the University of Hong Kong,Hong Kong SAR 999077,China;Laboratory for Synthetic Chemistry and Chemical Biology Limited,Hong Kong Science Park,Hong Kong SAR 999077,China;Liaoning Ocean and Fisheries Science Research Institute,Dalian 116023,China)

机构地区：[1]大连理工大学生命科学与药学系,盘锦124221 [2]大连理工大学生物智能制造教育部重点实验室,大连116023 [3]香港大学化学系,合成化学国家重点实验室,中国香港特别行政区999077 [4]合成化学暨分子生物学有限公司,香港科学园,中国香港特别行政区999077 [5]辽宁省海洋水产科学研究院,大连116023

出　　处：《高等学校化学学报》2025年第4期133-142,共10页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金(批准号:32171282);中央高校基本科研业务费(批准号:DUT23YG114)资助。

摘　　要：利用DNA重组技术在柞蚕蛹中表达重组人促红细胞生成素(Recombinant human erythropoietin,rhEPO),经过亲和层析纯化后,用SDS-PAGE分离纯化各组分,并通过电喷雾电离串联质谱技术(ESI-MS/MS)检测其糖基化修饰.结果显示,在柞蚕蛹中表达的rhEPO比活性约为1190 U/μg,其糖基化修饰位点与人体表达的EPO一致,有3个N-糖基化位点和1个O-糖基化位点.凝集素杂交实验结合质谱结果表明,柞蚕蛹表达的rhEPO的糖链中缺乏唾液酸修饰,而缺少唾液酸修饰的EPO通过鼻腔给药后在多种神经系统疾病的治疗中发挥着重要的作用.所得结果为进一步研究外源蛋白在柞蚕蛹-柞蚕核型多角体病毒(Anthraea pernyi nucleopolyhedrorirus,ApNPV)宿主载体表达系统表达后的糖基化与生物活性提供了依据.Recombinant human erythropoietin(rhEPO)was expressed in pernyi pupae using DNA recombination technology and gel purified.The purified components were separated by SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis),and the rhEPO gel strips were cut and its glycosylation modification was detected by electrospray ionization tandem mass spectrometry(ESI-MS/MS).The mass spectrometry results showed that the glycosylation modification sites of rhEPO expressed in pernyi silkworm pupae were consistent with EPO expressed in humans,with three N-glycosylation sites and one O-glycosy-lation site.Although the specific sugar chains at the glycosylation sites cannot be determined based on ESI-MS/MS,its results combined with lectin experiments can help determine the specific sugar chains at the glycosylation modification sites.According to the results,the overall sugar chain lacks sialic acid modification.The results of cell experiments showed that rhEPO expressed by pernyi pupae had certain biological activity,with a specific activity of 1190 U/μg.Therefore,pernyi pupae can express rhEPO without sialic acid modification with certain biological activity,and low sialylated EPO can play an important role in the treatment of central nervous system diseases after nasal administration.The results provide a basis for further studying the glycosylation and biological activity of exogenous proteins after expression in the pernyi pupae-Anthraea pernyi nucleopolyhedrorirus(ApNPV)host vector expression system.

关 键 词：电喷雾质谱 促红细胞生成素 柞蚕蛹 糖基化 

分 类 号：Q51[生物学—生物化学] O657.6[理学—分析化学]