全人源抗肝癌噬菌体单链抗体筛选及鉴定.docVIP

全人源抗肝癌噬菌体单链抗体筛选及鉴定 　作者：黄建, 肖艳, 童永清, 郭锋杰, 周国华, 李跃辉, 胡锦跃, 李官成 【摘要】 　　目的: 构建人源抗肝癌噬菌体单链抗体库, 从中筛选抗肝癌单链抗体（scFv）, 并对其特异性进行鉴定。方法: 采用体外致敏法和EBV转化技术联合噬菌体展示技术构建噬菌体抗体库。对初级抗体库进行亲和富集及ELISA筛选, 获得的阳性克隆进行免疫组化鉴定并测序。结果: scFv基因与载体连接后得到库容量为1.0×108的初级噬菌体抗体库。对抗体库进行3轮正负淘选和富集后, 随机挑取2798个克隆进行ELISA, 发现3个克隆对HepG2呈强阳性反应, 而与QSG7701等人正常细胞系呈弱阳性或不反应。对克隆SA3进行免疫细胞化学鉴定, 结果与ELISA一致。免疫组织化学鉴定表明SA3与肝癌组织和肝组织阳性率的差别有统计学意义。结论: 构建了库容量达1×108的全人源抗肝癌噬菌体抗体库。通过淘选富集、 ELISA和免疫组织化学鉴定获得特异性较强的噬菌体克隆, 为肝癌的临床诊断及导向治疗奠定了基础。 【关键词】 EB病毒转化 噬菌体抗体库 肝肿瘤 单链抗体 　　[Abstract] AIM: To construct a fully human antihepatoma singlechain phage antibody library, select the antihepatoma scFv from it and identify its characteristics. METHODS: A fully human scFvdisplaying phage library was constructed by using phage antibody library technique in combination with in vitro immunization and EBV transformation. The library was subjected to three rounds of positive and negative cell panning and enrichment and then it was selected by ELISA. The binding specificity of phage antibodies with hepatoma carcinoma cells was confirmed by immunohistochemistry. RESULTS: After scFv genes being cloned into vector Fuse5 and transformed into E.coli MC1061 via electroporation, a phage antibody library containing 1.0×108 TU (transduced unit) was obtained through tetracyclineresistant screening. After panning, enrichment and testing by ELISA, 3 phage antibody clones reacting more strongly to HepG2 than QSG7701 were picked out of 2798 clones. One clone, SA3, was further analysed after DNA sequencing. The results of immunohistochemistry with cultured cells were similar to those of ELISA. SA3 reacted specifically to hepatoma cells in most human hepatoma tissue sections but in few human normal liver tissue sections. The distinction of positive rates is of a great statistical significance. CONCLUSION: A fully human antihepatoma scFv fusion phage library has been constructed. ELISA and immunohistochemistry results conform SA3 specifically bind with hepato