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

作　　者：张峥嵘 陈邦航 乐壮 敖欣 张明珠 张振旺 苏延停 ZHANG Zheng-rong;ZHANG Zhen-wang;SU Yan-ting(School of Pharmacy,Xianning Medical College,Hubei University of Science and Technology,Xianning Hubei 437100,China)

机构地区：[1]湖北科技学院医学部药学院,湖北咸宁437100 [2]湖北科技学院医学部基础医学院 [3]湖北科技学院医学部医药研究院

出　　处：《湖北科技学院学报(医学版)》2025年第1期19-24,共6页Journal of Hubei University of Science and Technology(Medical Sciences)

基　　金：国家自然科学基金青年基金项目(32000906);湖北省自然科学基金面上项目(2024AFB1024)。

摘　　要：目的表达与纯化去唾液酸糖蛋白(ASGPR),为后续作为糖识别工具研究糖基化修饰的功能奠定基础。方法选择Nde I和Xho I作为限制性内切酶切割位点,使用SnapGene设计的上下游引物,以cDNA为模板,通过PCR扩增ASGPR基因。将扩增产物与经酶切的质粒pET-30a(+)使用一步克隆试剂盒进行重组。将重组产物转化到感受态大肠杆菌DH5α中,随后,将转化后的细菌溶液涂布在含有卡那霉素的LB平板(LBK板)上,以筛选出抗性菌落。之后,提取质粒并将重组质粒转化到表达菌BL21(DE3),筛选出抗性菌落,并进行测序验证。随后,进行ASGPR的小量诱导和蛋白的大量表达,接着进行蛋白包涵体复性过程。最后,收集不同的肿瘤细胞裂解液作为样品,利用纯化后的ASGPR蛋白作为糖识别工具,通过免疫印迹实验验证ASGPR的糖结合活性。结果重组质粒pET-30a(+)-ASGPR构建成功,通过免疫印迹实验证明ASGPR具有对GalNAc末端糖基化修饰的结合活性。结论基于O-GalNAc修饰(Tn抗原)的变化与多种疾病密切相关,所以高纯度且具有结合Tn抗原的ASGPR在未来对研究这种修饰功能具有巨大的推动作用。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 designed by SnapGene and cDNA were used as the template,the ASGPR gene was amplified via PCR.The PCR product and the enzyme-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 protein 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 constructed,and immunoblotting experiments demonstrated that ASGPR had the binding activity to GalNAc-terminal glycosylation modifications.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 

分 类 号：Q558.4[生物学—生物化学]