利用噬菌体展示技术筛选人源抗肝癌单链抗体

Screening of Human Single-Chain Fv Antibody Against Hepatocellular Carcinoma by Phage Display

目的:利用噬菌体展示技术,从Griffin1Library人源噬菌体单链抗体库中筛选出人源抗肝癌单链抗体。方法:扩增Griffin1Library人源噬菌体单链抗体库后,以甲胎蛋白(AFP)为包被抗原,用免疫试管法富集筛选人源抗肝癌单链抗体,用ELISA法鉴定获得的阳性克隆,对其基因进行测序。将淘选出的阳性单链抗体噬菌体载体用CaCl2法转化到E.coli HB2151菌株中用于表达可溶性单链抗体,然后采用镍亲和层析柱对表达的蛋白进行纯化。结果:经过3轮富集和筛选,共获得含有单链抗体片段的阳性克隆11个,其中3个克隆与AFP结合为阳性,DNA序列分析表明3株中2个克隆(A3、F10)含有完整的scFv基因片段,1个克隆(C11)含有部分scFv基因片段。SDS-PAGE检测单链抗体蛋白质的分子质量为28kU。结论:成功从Griffin1Library人源噬菌体单链抗体库中筛选出人源抗肝癌单链抗体。

Objective：To screening out the human single-chain Fv（scFv） antibody from the Griffin1 Library against hepatocellular carcinoma by phage display technique.Methods：The Griffin1 Library was amplified first and then the AFP was used as antigen to screen the human scFv.The positive clone was identified with ELISA,then the sequence of scFv gene was detected before it was transfected into E.coli HB2151 by CaCl2 method to express scFv induced by IPTG,and its expression level was detected by SDS-PAGE.The scFv was purified from the bacterial lysates by immobilized metal affinity chromatography（IMAC）.Results：After three rounds of panning,96 clones were randomly picked out and assessed for binding ability to fibrin by phage ELISA and three with AFP binding ability designated A3,F10 and C11,were chosen for further analysis.The complete nucleotide sequences of the heavy-and light-chain variable regions from the three positive clones were determined.The result indicated that the sequence of A3 and F10 are identical,whereas C11 is different from them in many sites.Molecular weight of the whole soluble scFv was about 28 kDa.Conclusion：An scFv antibody against AFP was screened out from Griffin1 Library successfully.