去唾液酸糖蛋白受体H1亚基CRD的原核表达、纯化及特性分析  

Cloning,Expression,Purification and Immunoresponse Evaluation of the Carbohydrate Recognition Domain of H1 Subunit of the Asialoglycoprotein Receptor

作　　者：乔健[1] 黄建萍[1] 曹利民[2] 司进[3] 

机构地区：[1]无锡市第三人民医院,江苏无锡214041 [2]华中科技大学同济医学院免疫学系,湖北武汉430030 [3]江苏省寄生虫病防治研究所,江苏无锡214064

出　　处：《苏州大学学报（医学版）》2006年第5期717-719,724,共4页Suzhou University Journal of Medical Science

基　　金：卫生部科研基金资助项目(WKJ2004-2-013)

摘　　要：目的研究去唾液酸糖蛋白受体H1亚基糖识别区(CRD)在大肠杆菌中的诱导表达,为从噬菌体抗体库中筛选其特异性抗体提供靶分子。方法以pET3-CRDH1为模板设计引物,通过PCR扩增CRDH1基因,采用分子克隆技术将其定向克隆至原核表达载体pET-32c中。将含CRDH1/pET-32c的BL21单菌落接种至LB肉汤培养基中培养,并通过IPTG诱导表达。表达产物经Ni2+螯合柱亲和纯化,采用免疫印迹技术(WB)分析融合蛋白的免疫反应性。结果表达产物经SDS-PAGE在35 kd左右显示条带,符合CRDH1与表达标签融合蛋白的理论值,并证明以包涵体的形式表达;通过Ni2+亲和柱纯化获得的CRDH1重组融合蛋白经WB证实,重组CRDH1融合蛋白能被羊抗人ASGPR血清特异性地识别。结论rCRDH1在原核系统获得高效表达,亲和纯化的rCRDH1具有较强的免疫反应性,为进一步从噬菌体抗体库中筛选特异性单链抗体奠定了基础。Objective The Carbohydrate Recognition Domain of the H1（CRDH1） subunit of the asialoglycoprotein receptor was induced to be expressed in E. Coll., and to provide the target antigen for screening by a large phage antibody library and to select single-chain antibodies directed against the human asialoglycoprotein receptor. Methods According to the gene sequence of CRDH1, the primers were designed. The full length eDNA encoding H1 amplified from pET-3CRDH1 by PCR was subcloned into an preukaryotic expression vector （pET-32c） . A single colony of E. coli BL21 containing the plasmid CRDH1/pET-32c was inoculated LB broth, then diluted 1/100 into 1000ml LB broth and induced with lmmol/L IPTG. The recombinant CRDH1 was purified with Ni^2＋ chelating HiTrap HP column. Its immunoresponse was evaluated with Western-blot. Results The recombinant CRDH1 protein about 35.2kd was expressed in E. coli as inclusion body; rCRDHlwas prepared with Ni^2＋ column purification; The result of western-blot showed that CRDH1 could be recognized by rabbit antiASGPR serum. Conclusions High-level expression of rCRDH1 was achieved in E. coli, the purified rCRDH1 has strong immunoresponse. This makes it possible for screening this specific scFv targeting to asialoglycoprotein receptor on the surface of hepatocyte by surface display phage antibody library technique.

关 键 词：去唾液酸糖蛋白受体 原核表达 免疫反应性 

分 类 号：R392-33[医药卫生—免疫学]