人源抗CEA噬菌体抗体库的构建及筛选  

CONSTRUCTION AND SCREENING OF HUMAN CEA SPECIFIC PHAGE ANTIBODY COMBINATORIAL LIBRARY

作　　者：范健[1] 赵刚[2] 俞扬[3] 沈宇清[3] 

机构地区：[1]扬州大学医学院,江苏扬州225009 [2]东南大学医学院普外科,江苏南京210009 [3]东南大学遗传学研究中心,江苏南京210009

出　　处：《实用临床医药杂志》2006年第3期16-21,共6页Journal of Clinical Medicine in Practice

基　　金：江苏省应用基础基金资助项目(BΚ2001008)

摘　　要：目的构建人癌胚抗原(CEA)特异性噬菌体抗体库,制备人源抗CEA单克隆抗体(mAb)。方法用常规RT-PCR法,直接从10例结肠癌患者肠系膜淋巴结中的淋巴细胞克隆出免疫球蛋白Fd段和κ链基因,建成噬菌体抗体库。对抗体库进行5轮吸附-洗脱-扩增的亲和选择后,以ELISA法鉴定抗CEA噬菌体。结果RT-PCR可有效地扩增出Fd和κ基因并以此构建成容量为5.2×106的噬菌体抗体库。经5轮亲和选择可使特异性噬菌体抗体得到高度富集,并成功获得抗CEA噬菌体抗体阳性克隆。结论抗CEA噬菌体抗体库的构建和人源抗CEA mAb的制备,为CEA阳性表达肿瘤的靶向治疗奠定基础。Objective To construct human phage antibody library and produce human monoclonal antibodies to CEA. Methods Example of human phage antibody library obtained from lymphonode of ten patients with colon carcinoma. The concentration of plasma CEA of every patient was more than 10ng/mL. Wuth routine RT-PCR with sets of oligonucleotide primers designed by Kang, the genes of human immunoglobulin Fd and κlight chains we：re amplified and cloned into phagemids （p3MH） to generate human phage antibody library. The affinity selection and ELISA were adopted for identification of specific phase antibodies to CEA. Results A phage antibody library of gchain containing 1.4×10^7members was constructed and a plhage antibody library of Fab had 5.2×10^6 members. The recombinant frequency of the Fah was 30% determined by digesting DNA extracted from 10 clones. Five rounds of panning produced 30-fold amplification in eluted phage, indicating enrichment for specific antigen-blnding clones and four positive clones showed clear reactivity to CEA but not with BSA or HBsAg by ELISA, Conclusion The success of isolating human anti-CEA Fab proves the usefulness of phage display system in human antibody preparation. Human monoclonal antibodies to CEA may be useful as molecular tools to study the pathogenesis of tumor expressing CEA, as well as for diagnostic and therapeutic purposes.

关 键 词：噬菌体抗体库 癌胚抗原 人源性单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]