靶向B细胞并特异结合其分泌IL-10的重组融合蛋白的构建、表达和初步鉴定  

Construction,Expression and Identification of Recombinant Fusion Protein Specifically Targeting B Cells and Binding to IL-10

作　　者：赵荣 陈含宇 黄春 章晓联[1] 潘勤[1] ZHAO Rong;CHEN Han-yu;HUANG Chun;ZHANG Xiao-lian;PAN Qin(School of Basic Medical Immunology, Hubei Province Key Laboratory of AUergy and Immunology, and Department of Immunology, State Key Laboratory of Virology, Medical Research Institute, Wuhan University, Wuhan 430071, China)

机构地区：[1]武汉大学基础医学院免疫学系病毒学国家重点实验室湖北省过敏及免疫相关疾病重点实验室

出　　处：《中国生物工程杂志》2018年第2期1-6,共6页China Biotechnology

基　　金：国家自然科学基金(81471910;31370197;21572173;31770145);湖北省医学领军人才计划(523-276003);湖北省技术创新专项(2016ACA150;2016CFA062;2015CFA009);武汉市科技晨光计划(2016070204010127)资助项目

摘　　要：目的：CD19单链抗体可变区（single-chain fragment variable）sc Fv能特异性识别结合B细胞表面的CD19分子,白细胞介素-10（interleukin-10,IL-10）是调节性B细胞（Breg）的主要特征之一。旨在构建CD19sc Fv和IL-10受体1（IL-10R1）胞外段的重组融合蛋白,拟用该融合蛋白捕捉和封闭Breg细胞分泌的IL-10。方法：CD19sc Fv和IL-10R1胞外段克隆到p ET-28a原核质粒上,E.coli BL21（DE3）工程菌表达蛋白通过镍柱富集和纯化蛋白,SDS-PAGE和Western blot对表达蛋白进行鉴定,Pull down实验验证蛋白与CD19分子和IL-10细胞因子的结合。结果：成功表达CD19sc Fv-IL-10R1融合蛋白,该融合蛋白能在体外同时结合CD19和IL-10分子。结论：CD19sc Fv-IL-10R1融合蛋白在体外可以同时结合CD19分子和IL-10分子,具有潜在应用前景可作为特异性抑制Breg的生物小分子。Objective： CD19 scFv （single-chain fragment variable） specifically recognizes CD19 molecule expressed on the B cells surface. Interleukin-10 （IL-10） -producing B cells are a major subset of regulatory B cells （Breg）. The aim is to construct a recombinant fusion protein of CD19 scFv and IL-10 receptorl （IL- 10R1） , which potentially captures and blocks IL-10 secreted by Breg cells. Methods： CD19scFv and IL-10R1 were cloned into pET-28a prokaryotic plasmid. Recombinant proteins were enriched and purified by Ni column, identified by SDS-PAGE and Western blot. Pull down analysis was performed to identify the proteins captured by CD19scFv-IL-10R1 recombinant protein. Results： The CD19 scFv-IL-10R1 fusion protein is prepared and the protein binds to both CD19 and IL-10 molecules in vitro. Conclusion： CD19scFv-IL-10R1 fusion protein binds to both CD19 molecule and IL-10 molecule in vitro, and has potential application as a specific inhibitor for Bregs.

关 键 词：IL-10 调节性B细胞 IL-10受体1 CD19单链抗体可变区 

分 类 号：Q819[生物学—生物工程]