湖北科技学院学报（医学版）2025，Vol.39Issue(1)：19-24,6.DOI:10.16751/j.cnki.2095-4646.2024062107
去唾液酸糖蛋白受体的原核表达、纯化及活性分析
Expression,Purification,and Functional Analysis of the Asialoglycoprotein Receptor in Prokaryotic Cells
摘要
Abstract
Objective To express and purify the asialoglycoprotein receptor(ASGPR)for subsequent use in investigating the role of glycosylation modifications.Methods Nde I and Xho I were chose as restriction enzyme cleavage sites.Primers were de-signed by SnapGene and cDNA were used as the template,the ASGPR gene was amplified via PCR.The PCR product and the en-zyme-digested plasmid pET-30a(+)were recombined by using a one-step cloning kit.The recombinant product was transformed into competent E.coli DH5α,then the transformed bacterial solution was plated onto LB agar plates containing kanamycin(LBK plates)to select for resistant colonies.Next,the recombinant plasmid was extracted and transformed into the expression strain BL21(DE3).The resistant colonies were selected and verified by sequencing.Subsequently,small-scale expression and large-scale pro-tein expression of ASGPR were induced,followed by the inclusion body refolding.Finally,different tumor cell lysates were collected as samples and the purified ASGPR protein was used as a carbohydrate recognition tool to verify the carbohydrate-binding activity of ASGPR through immunoblotting experiments.Results The recombinant plasmid pET-30a(+)-ASGPR was successfully con-structed,and immunoblotting experiments demonstrated that ASGPR had the binding activity to GalNAc-terminal glycosylation mod-ifications.Conclusion The variations based on O-GalNAc modification(Tn antigen)are closely associated with various diseases.Therefore,ASGPR with high purity and specific binding to Tn antigen will play a significant role in advancing the study of this modification's functions in the future.关键词
去唾液酸糖蛋白受体/糖基化修饰/糖识别工具/Tn抗原Key words
Asialoglycoprotein receptor/Glycosylation/Glycan recognition tool/Tn antigen分类
生物学引用本文复制引用
张峥嵘,陈邦航,乐壮,敖欣,张明珠,张振旺,苏延停..去唾液酸糖蛋白受体的原核表达、纯化及活性分析[J].湖北科技学院学报（医学版）,2025,39(1):19-24,6.基金项目
国家自然科学基金青年基金项目(32000906) （32000906）
湖北省自然科学基金面上项目(2024AFB1024) （2024AFB1024）
