去唾液酸糖蛋白受体H1亚单位的表达、纯化及其单链抗体的筛选与鉴定  

Expression and purification of the human asialoglycoprotein receptor H1 subunit and selection of anti-CRDH1 scFv

作　　者：曹利民[1] 司进[2] 朱荫昌[2] 王炜煜[3] 赵晓蓉[1] 叶庆[1] 李文涵[1] 朱慧芬[1] 沈关心[1] 

机构地区：[1]华中科技大学同济医学院免疫学系,分子与免疫药理研究室,武汉430030 [2]江苏省寄生虫病防治研究所 [3]华中科技大学同济医学院附属同济医院普外科

出　　处：《中华微生物学和免疫学杂志》2005年第9期756-761,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家重点基础研究发展规化基金(2002CB513109);卫生部科研基金(WKJ2004-2-013)

摘　　要：目的去唾液酸糖蛋白受体(ASGPR)H1亚单位的重组、表达、纯化及其单链抗体的筛选与鉴定。方法将ASGPRH1亚单位糖识别区基因(CRDH1)定向克隆至原核表达载体pET-32c经IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,免疫印迹技术(WB)检测;用纯化的CRDH1对人源噬菌体抗体库进行4轮固相筛选,阳性克隆用表达标记(Trx-His-stag)进行差异筛选。取阳性噬菌体C3感染宿主菌HB2151,37℃振荡培养过夜,终浓度为1mmol/L的IPTG诱导表达,12%SDS-PAGE与Westernblot鉴定以及免疫组化鉴定纯化的单链抗体C3与肝细胞结合的特性。结果CRDH1/pET-32c在原核系统经诱导表达出相对分子质量(Mr)约35×103大小的融合蛋白,以包涵体的形式存在;通过Ni2+亲和柱纯化获得CRDH1融合蛋白;经4轮筛选和差异筛选后,有2株可分泌性表达与CRDH1特异性结合的单链抗体,DNA序列分析表明单链抗体重链基因和轻链基因分别属于人免疫球蛋白VH3家族和VKI家族。噬菌体C3经诱导表达出Mr约28×103大小的融合蛋白,可溶性分泌表达于培养基中;通过Ni2+亲和柱纯化获得单链抗体,ELISA、Westernblot表明单链抗体C3能较好结合rCRDH1,免疫组化显示该单链抗体可与肝癌细胞、肝细胞特异结合。结论成功表达ASGPR并筛选出其人源单链抗体。该单链抗体可与表达的rCRDH1以及肝细胞和肝癌细胞特异性结合,提示对肝脏疾病的靶向治疗有潜在的应用价值。Objective To recombine, express and purify the human asialoglycoprotein receptor （ ASGPR） H1 subunit, select and identify the anti-CRDH1 scFv. Methods The amplified CRDH1 cDNA was subcloned into prokaryotic vector pET-32c, and the recombinant protein expression was induced by IPTG. The recombinant CRDH1 was purified with Ni^2＋ chelating HiTrap HP column, and its specificity was evaluated by Western blot. After four rounds of bioparming（ solid-phase screening） of the human phage antibody library with the purified recombinant CRDH1, the positive clones were selected with Trx-His-s tag. The specific anti-CRDH1 phage clones （C3） were transfected into E.ooli HB2151, and induced for the expression of scFv antibody with IFI＇G. The immunoresponse of the anti-rCRDH1 scFv C3 was evaluated by Western blot, and its binding capacity to human asialoglyprotein receptor was observed with immunohistochemistry. Results The recombinant CRDH1 about 35.0 kDa was expressed in E. coli as inclusion body, prepared with Ni^2＋ column purification. After selecting, two of the colonies could be induced to produce soluble anti-rCRDH1 scFv. By DNA sequencing, V, gene and VL gene of the scFv antibody belong to Ig-VH 1 and Ig-VKI families, respectively. The C3 phage can be induced to produce 28 kDa recombinant protein that is. The results of ELISA and Western blot showed that rCRDH1 could be recognized by C3 scFv antibody, which has a specific combination character with liver cells and hepatoma cells by immunohistochemistry. Conclusion High-level expression of rCRDH1 was achieved in E. coli, and anti-rCRDH1 specific scFv has successfully identified. The C3 scFv antibody has the binding capacity to rCRDH1, liver cells and hepatoma cells, indicating that it has a potential value as a targeting molecule for biological therapy of hepatocellular carcinoma.

关 键 词：去唾液酸糖蛋白受体 人源噬菌体抗体库 单链抗体 去唾液酸糖蛋白受体 人源单链抗体 亲和纯化 差异筛选 亚单位 鉴定 H1 Western 噬菌体抗体库 

分 类 号：R392[医药卫生—免疫学]