摘要
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.
出处
《世界华人消化杂志》
CAS
北大核心
2006年第18期1780-1784,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金项目
No.39900068西安市科技攻关计划项目
No.GG05168~~
关键词
胃癌细胞
单链抗体
原核表达
癌胚抗原
Gastric carcinoma
Single-chain fragment variable antibody
Prokaryotic expression
Carcinoembryonic antigen