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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 investigate the prokaryotic expression of anti-tumor related glycoprotein-72 (TAG-72) single-chain antibody and its specific affinity with four kinds of hepatoma cell lines and hepatocellular carcinoma. Methods The cDNA fragment of anti-TAG-72 single-chain antibody was inserted into the phagemid vector pCANTAB5E to obtain the phagemid vector TAG-72-scFv-pCANTAB5E. The latter was transformed into E. coli HB2151 and induced by β, D isopropylthiogalactoside (IPTG) to obtain anti-TAG-72 single-chain antibody protein. TAG-72 antigen expression in four hepatocellular carcinoma cell lines and paraffin-embedded hepatocellular carcinoma tissues was detected by cell culture and immunocytochemistry / histochemistry. Results SDS-PAGE and Western blot confirmed that anti-TAG-72 scFv protein was correctly expressed. Anti-TAG-72 scFv was able to bind to hepatocellular carcinoma cell lines SMMC7721, HepG2 and HHCC, indicating that these three kinds of cells expressed specific tumor antigens; anti-TAG-72 scFv was unable to bind to BEL7402 cells. The positive expression rates of TAG-72 in stage I, stage II-III were 23.08% (3/13) and 62.96% (17/27) respectively in 40 cases of HCC, without expression of TAG-72 antigen in normal liver tissues, The difference of TAG-72 antigen expression in normal liver tissue and liver cancer tissue was statistically significant (P <0.05). Conclusion The TAG-72 antigen is specifically expressed in hepatocellular carcinoma or hepatocellular carcinoma and may be a marker of liver cancer.