抗人釉原蛋白单克隆抗体的制备及鉴定  

Preparation and characterization of monoclonal antibodies against human amelogenin

作　　者：程岚[1] 束蓉[1] 夏一如 宁航 CHENG Lan;SHU Rong;XIA Yiru;NING Hang(Department of Periodontology,Shanghai Ninth People's Hospital,College of Stomatology,Shanghai Jiao Tong University School of Medicine,National Clinical Research Center for Oral Diseases,Shanghai Key Laboratory of Stomatology&Shanghai Research Institute of Stomatology,Shanghai 200011,China)

机构地区：[1]上海交通大学医学院附属第九人民医院.口腔医学院牙周病科国家口腔疾病临床研究中心上海市口腔医学重点实验室上海市口腔医学研究所,上海200011

出　　处：《口腔生物医学》2019年第1期17-21,共5页Oral Biomedicine

基　　金：国家自然科学基金青年基金(81600863);上海市科学技术委员会青年基金(16ZR1418900)

摘　　要：目的:制备针对人釉原蛋白的单克隆抗体,并对其免疫学特性进行鉴定。方法:用原核表达系统表达纯化的重组人全长釉原蛋白免疫BALB/c小鼠,利用杂交瘤技术融合免疫后小鼠脾细胞和小鼠骨髓瘤SP2/0细胞,采用有限稀释法和间接酶联免疫吸附测定法筛选出能够稳定分泌单克隆抗体的杂交瘤细胞株。小鼠体内诱导制备腹水,通过饱和硫酸铵沉淀法和蛋白G亲和层析柱纯化抗体。结果:筛选到3株能稳定分泌抗人釉原蛋白单克隆抗体的杂交瘤细胞株,分别命名为H10G1、H3G1、G5H3。腹水单克隆抗体效价分别为1∶243 000、1∶729 000、1∶729 000,纯化后单克隆抗体效价为1∶81 000、1∶243 000、1∶729 000。3株单克隆抗体抗原表位分析显示均识别人釉原蛋白45～149位的氨基酸序列。结论:成功制备出针对人釉原蛋白的特异性单克隆抗体,为进一步研究探讨釉原蛋白在牙根发育及牙周再生中的功能奠定了基础。Objective:This study aimed to prepare monoclonal antibodies against human amelogenin and characterize their immunological properties.Methods:BALB/c mice were immunized with the recombinant full-length human amelogenin expressed in prokaryotic expression system.The spleen cells of immunized mice were fused with mouse myeloma SP2/0 cells,and hybridoma cell lines capable of stably secreting monoclonal antibodies were screened out by the limited dilution method and indirect enzyme-linked immunosorbent assay(ELISA)method.Ascites was produced in mice,and then purified by saturated ammonium sulfate precipitation and protein G affinity chromatography.Results:Three hybridoma cell lines stably secreting monoclonal antibodies against human amelogenin were screened and named as H10G1,H3G1 and G5H3.The titers of antibodies in ascites were 1∶243000,1∶729000,1∶729000,and the titers of monoclonal antibody after purification were 1∶81000,1:243000,1∶729000 respectively.The results of Western blotting showed that all of 3 monoclonal antibodies recognize the 45-149 amino acid of human amelogenin.Conclusions:The specific monoclonal antibody against human amelogenin was successfully prepared for further study on the amelogenin in root development and periodontal regeneration.

关 键 词：釉原蛋白 融合蛋白 单克隆抗体 细胞融合 

分 类 号：R780.2[医药卫生—口腔医学]