人细胞角蛋白20的原核表达及其单克隆抗体的制备  

Prokaryotic expression of human cytokeratin 20 protein and preparation of monoclonal antibody

作　　者：宋军营 袁永 张钟允 闫敏 曾华辉 刘文弟 张振强 SONG Jun-ying;YUAN Yong;ZHANG Zhong-yun;YAN Min;ZENG Hua-hui;LIU Wen-di;ZHANG Zhen-qiang(Immunological Laboratory of Traditional Chinese Medicine, Henan University of Traditional Chinese Medicine, Zhengzhou 450046, Henan Province, Chin)

机构地区：[1]河南中医药大学中医药免疫学实验室,河南郑州450046

出　　处：《中国生物制品学杂志》2018年第4期383-387,共5页Chinese Journal of Biologicals

基　　金：河南省高校科技创新人才支持计划(13HASTIT027);河南省科技创新人才支持(科技创新杰出青年)(154100510019);2014年度河南中医学院创新团队(2014XCXTD01);河南中医学院省属科研业务费专项(2014KYY-WF-ZZCX3-06);河南中医药大学科研苗圃工程项目(MP2016-12)

摘　　要：目的原核表达人细胞角蛋白20(cytokeratin 20,CK20),并制备其单克隆抗体。方法利用CK20蛋白的cDNA扩增CK20的外显子基因,插入原核表达载体pET28a中,构建重组表达质粒pET28a-ck20,转化大肠埃希菌BL21中,IPTG诱导表达。表达的重组蛋白经亲和层析柱纯化后,免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体,通过间接ELISA法对杂交瘤细胞进行筛选,小鼠体内诱生腹水法批量制备单抗,并进行亚型、Western blot及免疫组化鉴定。结果重组表达质粒pET28a-ck20经双酶切及测序证明构建正确,纯化的重组CK20蛋白纯度在90%以上。制备的CK20单抗重链类型为IgG2b,轻链类型为Kappa链;能够很好地与CK20蛋白抗原产生结合反应,在1∶200稀释后能够较好地对结肠腺癌组织进行染色,且效果优于进口产品。结论利用重组表达的人CK20蛋白制备的单抗可用于检测相关组织中该蛋白的表达,为CK20的临床诊断及研究提供了有效的工具。Objective To express human cytokeratin 20（CK20）in prokaryotic cells and prepare its monoclonal antibody（McAb）.Methods The exon gene of CK20 was amplified by using the cDNA of CK20 and inserted into prokaryotic expression vector pET28 a.The constructed recombinant plasmid p ET28 a-ck20 was transformed to E.coli BL21 for expression under induction of IPTG.The expressed recombinant CK20 was purified by affinity chromatography,with which BALB/c mice were immunized to prepare McAb by hybridoma technique.Hybridoma cells were screened by indirect ELISA,and McAb was prepared,analyzed for subtype and identified by Western blot and immunohistochemical assay.Results Restriction analysis and sequencing proved that recombinant plasmid pET28 a-ck20 was constructed correctly.The purified CK20 reached a purity of more than 90%.The prepared McAb consisted of an IgG2b heavy chain and a Kappa light chain,which showed specific binding to CK20 antigen,even at a dilution of 1：200.In immunohistochemical assay,the color of colon cancer containing CK20 after reaction with the McAb was darker than that after reaction with the imported McAb.Conclusion The McAb prepared by the expressed human CK20 may be used for determination of expression of the protein in the relevant tissues,which provides an effective tool for clinical diagnosis and research of CK20.

关 键 词：细胞角蛋白20 基因表达 单克隆抗体 

分 类 号：R392.12[医药卫生—免疫学] R392-33[医药卫生—基础医学]