抗癌胚抗原单链抗体的原核表达及对人胃癌的检测

Prokaryotic expression of anti-carcinoembryonic singlechain variable fragment and its value in detection of gastric carcinoma cells

目的:探讨T84．66单链抗体的原核表达及对6 种胃癌细胞系及胃癌组织的特异性亲和力．方法:将抗CEA单链抗体T84．66的cDNA插入噬菌粒载体pcANTAB5E,获得噬菌粒载体T84．66-scFv-pCANTAB5E．将后者转化入 E．coli HB2151,经β,D异丙基硫代半乳糖苷 (IPTG)诱导表达．采用细胞培养及免疫细胞／组织化学方法,检测胃癌细胞中及石蜡包埋的胃癌组织中的癌胚抗原表达．结果:SDS-PAGE及Western blot证实,T84．66 单链抗体蛋白分子正确表达．T84．66单链抗体可结合KATOIII,HGC-27和MKN45,表明这3种细胞表达了特异性肿瘤抗原;单链抗体不能结合SGC7901,GC803,BGC823．42例胃癌组织癌胚抗原阳性率早期和进展期分别为55％(6／11)和61％(19／31),在正常胃黏膜组织标本中无表达,二者之间存在显著性差异 (P<0．05)．结论:KATOIII等胃癌细胞系可表达癌胚抗原．后者在胃癌组织表达水平较高,而正常胃组织不表达．

AIM： To study the prokaryotic expression of the anti-carcinoembryonic antigen （CEA） singlechain fragment variable （scFv） antibody T84.66 and its specific affinity to gastric cancer cell lines and tissues. METHODS： The cDNA of anti-CEA scFv antibody was inserted into pCANTAB5E to obtain phage vector T84.66-scFv-pCANTAB5E,and then the vector was transferred into E. coli HB2151. Isopropyl-β-D-thiogalactoside （IPTG） was used to induce the expression of anti-CEA scFv antibody. SDS-PAGE and Western blot were used identify the anti-CEA scFv antibody. Human gastric cancer cells were cultured, and CEA was determined with the obtained scFv by immunohistochemistry in the cells and paraffinembedded gastric carcinoma tissues. RESULTS： SDS-PAGE and Western blot showed that the anti-CEA scFv antibody T84.66 was successfully expressed. T84.66 could bind to gastric carcinoma cell lines KATOⅢ, MKN45 and HGC-27, but not to SGC7901, GC803 and BGC823, suggesting that KATOⅢ, MKN45 and HGC-27 cells expressed CEA. For the 42 cases gastric carcinoma tissues, the positive rate of CEA in the early and progressive stage was 55% （6/11） and 61% （19/31）, respectively, but no CEA expression was found in the 10 normal cases. CEA expression was significantly different between gastric cancer and normal tissues （P 〈 0.05）. CONCLUSION： The prokaryotic expression of anti-CEA scFv antibody T84.66 is successfully achieved, and can be used to identify CEA. CEA is highly expressed in gastric cancer, but not in normal mucosa.