可内化的人源抗Met基因工程抗体Fab的亲和力成熟与特性分析  

Affinity Maturation and Characterization of Internalized Human Anti-Met Recombinant Antibody Fab

作　　者：朱进[1] 赵萍[1] 焦永军[1] 王昕[1] 曹伯良[1] 冯振卿[1] 管晓虹[1] 

机构地区：[1]南京医科大学卫生部抗体技术重点实验室

出　　处：《生物化学与生物物理进展》2007年第1期73-79,共7页Progress In Biochemistry and Biophysics

摘　　要：应用噬菌体展示技术制备抗Met(HGF受体,一个与肿瘤发生、侵袭和转移相关原癌基因产物)特异性、高亲和力的全人Fab片段.Fab基因分三步合成,以从错配PCR突变库中筛选出的Fab基因可变区为模板,扩增VH和VL基因,分别与CH1、CL基因融合,合成Fd和L基因,再拼接合成Fab基因,克隆于pComb3XSS中,构建Fab次级抗体库.经细胞筛选和固相筛选,获得高亲和力阳性克隆.工程菌经IPTG诱导表达,SDS-PAGE和蛋白质印迹分析,在25ku和27ku出现预期大小蛋白质条带.Fab分子经流式细胞术、免疫沉淀、细胞免疫荧光检测,结果表明,Fab能够与S114和MKN45细胞膜上的Met胞外区特异性结合,而与阴性细胞NIH3T3不结合.抗体内化分析显示,Fab能够与标记肥皂草毒素(ZAP)的抗人IgG结合,并进入细胞内,抑制Met阳性细胞的生长,揭示该抗体能够与Met特异性结合,并且被细胞内化.该抗体有望成为肿瘤临床诊断或治疗的候选分子.High affinity, fully human antibody fragment Fab that specifically bind to HGF receptor, Met （a proto-oncogene product and a key molecule on tumorigenesis, invasion and metastasis）, was selected from phage display antibody library. To construct antibody library, the Fab gene fragment was constructed in 3 steps. The variable region genes were amplified from VH and VL of anti-Met Fab selected from error-prone PCR mutation library. Fab gene was assembled by overlap PCR and purified and digested with Sfi I , and inserted into pComb3XSS. Positive phage-displayed antibody clones were selected on live cell lines and immobilized protein. The purified Fab was verified by SDS-PAGE and Western blot, which showed two bands at about 25 ku and 27 ku at the expected sizes. To analyze the immunological characters of Fab for Met binding, flow cytometry and immunoprecipitation assays were set up and carried out with S114, MKN45 and NIH3T3 cell lines. The results demonstrated Fab could bind native Met specifically on the S 114 and MKN45 cell surface. In vitro cytotoxic assay showed that only Hum-ZAP/anti-Met Fab complex could inhibit the Met positive cell growth, it illuminated that anti-Met Fab bound the Met on Met positive cell surface and were internalized. For Met negative cell line NIH3T3, no significant inhibition among the different dosages of Fab and Hum-ZAP. The results showed that anti-Met Fab antibody fragments could recognize Met extracellular domain in native conformation with relatively high affinity. Importantly, these antibody fragments were able to be internalized into Met over-expressed tumor cell lines through binding to the Met receptor, and could be applied as a potential powerful reagents for clinical therapy.

关 键 词：肝细胞生长因子受体 噬菌体抗体库 亲和力成熟 内化抗体 

分 类 号：Q78[生物学—分子生物学]