抗TAG-72抗原单链抗体的原核表达及对人肝癌的检测  

Prokaryotic Expression of Anti-TAG-72 Single-Chain Variable Fragment and Its Value in Detection of Hepatocellular Carcinoma

作　　者：徐宏勇[1] 徐立[2] 李开宗[3] 窦科峰[3] 

机构地区：[1]解放军第451医院肝胆普外科,西安710054 [2]第四军医大学西京医院全军消化病研究所,西安710032 [3]第四军医大学西京医院肝胆外科,西安710032

出　　处：《中国普外基础与临床杂志》2010年第7期699-702,共4页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金项目(项目编号:39900068);陕西省自然科学基金项目(项目编号:2007C259)

摘　　要：目的探讨抗肿瘤相关糖蛋白-72(TAG-72)单链抗体的原核表达及与4种肝癌细胞系和肝癌组织的特异性亲和力。方法将抗TAG-72单链抗体的cDNA片段插入噬菌粒载体pCANTAB5E,获得噬菌粒载体TAG-72-scFv-pCANTAB5E。将后者转化入E.coliHB2151,经β,D异丙基硫代半乳糖苷(IPTG)诱导表达获得抗TAG-72单链抗体蛋白。采用细胞培养及免疫细胞/组织化学方法 ,检测4种肝癌细胞系及石蜡包埋的肝癌组织中TAG-72抗原的表达。结果 SDS-PAGE及Western blot法证实,抗TAG-72单链抗体蛋白分子得以正确表达。抗TAG-72单链抗体可与肝癌细胞系SMMC7721、HepG2和HHCC结合,表明这3种细胞表达了特异性肿瘤抗原;抗TAG-72单链抗体不能结合BEL7402细胞。40例肝癌组织中TAG-72抗原表达阳性率Ⅰ期、Ⅱ～Ⅲ期分别为23.08%(3/13)和62.96%(17/27),在正常肝组织标本中无TAG-72抗原表达,TAG-72抗原在正常肝组织及肝癌组织中表达的差异有统计学意义(P<0.05)。结论 TAG-72抗原在肝癌细胞或肝癌组织中有特异性表达,有可能成为判断肝癌的标志物。Objective To study the prokaryotic expression of the anti-tumor associated glycoprotein-72 （TAG-72） single-chain fragment variable （scFv） antibody and its specific affinity to hepatocellular carcinoma cell lines and tissues.Methods The cDNA of anti-TAG-72 scFv antibody was inserted into pCANTAB5E to obtain phage vector anti-TAG-72-scFv-pCANTAB5E.Isopropyl-β-D-thiogalactoside （IPTG） was used to induce the expression of anti-TAG-72 scFv antibody.SDS-PAGE and Western blot were used to identify the anti-TAG-72 scFv antibody.Human hepatocellular carcinoma cells were cultured,and TAG-72 was determined with the obtained scFv by immunohistochemistry in the cells and paraffin-embedded hepatocellular carcinoma tissues.Results SDS-PAGE and Western blot showed that the anti-TAG-72 scFv antibody was successfully expressed.Anti-TAG-72 scFv could bind to hepatocellular carcinoma cell lines SMMC7721,HepG2,and HHCC,but not to BEL7402,suggesting that SMMC7721,HepG2,and HHCC cells expressed TAG-72.For the 40 cases of hepatocellular carcinoma tissues,the positive rate of TAG-72 in stage Ⅰ and Ⅱ-Ⅲ was 23.08% （3/13） and 62.96%（17/27）,respectively.While no TAG-72 expression was found in the 10 normal cases.TAG-72 expression was significantly different between hepatocellular carcinomas and normal tissues （P〈0.05）.Conclusions The prokaryotic expression of anti-TAG-72 scFv antibody is successfully achieved,and can be used to identify TAG-72 antigen.TAG-72 is highly expressed in hepatocellular carcinoma,but not in normal liver tissue,which may be suggested as cancer marker of hepatocellular carcinoma.

关 键 词：肝细胞癌 单链抗体 原核表达 肿瘤相关糖蛋白-72抗原 

分 类 号：R735.7[医药卫生—肿瘤]