抗periostin人源化单克隆抗体的制备及鉴定  

Preparation and identification of humanized monoclonal antibody against periostin

作　　者：李雪娇 赵航 张朔 康慧颖 周艳楠[1] 贾爽[1] 路旭 赵洪礼 海洋 LI Xuejiao;ZHAO Hang;ZHANG Shuo;KANG Huiying;ZHOU Yannan;JIA Shuang;LU Xu;ZHAO Hongli;HAI Yang(School of Basic Medicine,Liaoning HE University,Shenyang 110163,China;Shenyang Baifa Technology Co.,Ltd,Shenyang 110163,China)

机构地区：[1]辽宁何氏医学院基础医学院,沈阳110163 [2]沈阳百发科技有限公司,沈阳110163

出　　处：《中国免疫学杂志》2024年第12期2628-2633,共6页Chinese Journal of Immunology

基　　金：辽宁省民生科技计划项目(2021JH2/10300028)。

摘　　要：目的:制备抗periostin人源化单克隆抗体,建立稳定的细胞株。方法:以本实验室自主研发的抗periostin鼠单抗为基础,提取细胞总RNA,经RT-PCR扩增VH和VL基因测序获得可变区序列,将小鼠抗体CDR区域移植到人源抗体框架受体区域,基因合成后亚克隆到表达载体PATX-GS2,稳定转染CHO细胞,经MSX加压筛选、有限稀释获得单克隆细胞株。结果:经RT-PCR扩增V_(H)和V_(L)基因,确定轻重链可变区序列,通过CDR移植方法,成功对鼠源单抗进行人源化改造,并构建了真核质粒载体,经转染至CHO细胞表达,成功获得人源化抗periostin抗体,ELISA和Western blot结果证明,制备的人源化抗体具有良好的活性和亲和力。结论:本研究成功制备了抗periostin人源化单克隆抗体,且该人源化抗体可以与体内periostin蛋白特异性结合,具有生物学活性,为精准治疗视网膜纤维化病变、组织器官纤维化病变及恶性肿瘤提供了科学数据。Objective:To prepare a humanized monoclonal antibody against periostin and establish a stable cell line.Meth⁃ods:Based on anti-periostin mouse monoclonal antibody developed by our laboratory,total RNA was extracted,and variable region sequences were obtained by RT-PCR amplification of VH and VL genes.The mouse antibody CDR region was transplanted into the human antibody framework receptor region,and the gene was subcloned into the expression vector PATX-GS2,and stably transfected into CHO cells.Monoclonal cell lines were obtained by MSX pressure screening and limited dilution.Results:V_(H)and V_(L)genes were amplified by RT-PCR,and the sequence of the light and heavy chain variable region were determined.Antibody humanization were successfully stablished by CDR transplantation method a murine antibody to a human framework,and a eukaryotic expression plasmid was constructed,which was transfected into CHO cells for expression,and human anti-periostin antibody was successfully obtained.ELISA and Western blot results showed that the humanized antibody had good anti-periostin activities and binding affinity.Conclu⁃sion:In this study,anti-periostin humanized monoclonal antibody has been successfully prepared,which can specifically bind to periostin proteins in vivo and have biological activity,providing scientific data for the precise treatment of retinal fibrosis,tissue and organ fibrosis,and malignant tumors.

关 键 词：PERIOSTIN 人源化单克隆抗体 视网膜纤维化 

分 类 号：R392.33[医药卫生—免疫学]