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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。
Objective: To construct human anti-hepatoma phage scFv library and scFv against it. Methods: Phage display antibody library was constructed by in vitro sensitization and EBV transformation combined with phage display technology. The primary antibody pools were affinity-enriched and ELISA screened, and the positive clones obtained were identified by immunohistochemistry and sequenced. Results: After the scFv gene was ligated with the vector, a primary phage antibody library with a capacity of 1.0 × 108 was obtained. After 3 cycles of positive and negative panning and enrichment of the library, 2798 clones were randomly selected and subjected to ELISA. Three clones showed a strong positive response to HepG2 but a weak positive or no positive correlation with QSG-7701 and other normal cell lines reaction. The clone SA3 was identified by immunocytochemistry and the results were consistent with ELISA. Immunohistochemistry showed that the positive rate of SA3 and liver cancer tissue and liver tissue was statistically significant difference. Conclusion: A full-body anti-hepatoma phage antibody library with a capacity of 1 × 108 was constructed. By panning enrichment, ELISA and immunohistochemical identification of specific phage clones, for the clinical diagnosis and treatment of liver cancer laid the foundation.