抗胃癌单抗3H11的单链抗体表达载体的构建和表达  

Construction and expression of ScFv from anti human gastric cancer McAb 3H11

作　　者：李竞[1,2,3,4] 王琰[1,2,3,4] 李全喜[1,2,3,4] 王雅明[1,2,3,4] 徐建军[1,2,3,4] 王力民[1,2,3,4] 董志伟[1,2,3,4] 

机构地区：[1]北京市肿瘤防治研究所 [2]北京医科大学临床肿瘤学院 [3]解放军海军总医院 [4]中国医学科学院中国协和医科大学肿瘤研究所肿瘤医院

出　　处：《中华肿瘤杂志》1998年第2期85-87,共3页Chinese Journal of Oncology

基　　金：国家863项目

摘　　要：目的构建抗胃癌鼠单抗３Ｈ１１的单链抗体（ＳｃＦｖ），以利于临床应用。方法采用噬菌体抗体库技术和ＰＣＲ介导的定位点突变技术，构建和改造抗胃癌ＳｃＦｖ基因。结果从３Ｈ１１杂交瘤制备抗体库，以胃癌细胞系（ＭＧＣ８０３）进行筛选，未获阳性克隆。再对３Ｈ１１可变区基因以ＰＣＲ错配技术进行随机突变，构建次级突变抗体库，仍未筛出阳性克隆。最后用ＰＣＲ定点突变技术，将ＳｃＦｖ可变区基因氨基端序列恢复成３Ｈ１１的原始序列，终于得到可与胃癌细胞结合的ＳｃＦｖ克隆。Objective To construct single chain antibody (ScFv) from anti gastric cancer McAb 3H11. Methods Phage display technology was used to construct ScFv library from 3H11 hybridoma cells. Site directed mutagenesis was used to resume the N terminal sequences of 3H11 ScFv. Results The library was screened against human gastric cancer cells MGC803 but no positive clone was obtained. Then, a random mutated ScFv library was constructed by error prone PCR method, and panning selection was performed. Again the identification of positive clone was failed. Finally the N terminal sequences of ScFv variable region was resumed to 3H11 original sequences by site directed mutagenesis via PCR, and the expressed ScFv in bacterial culture supernatant showed binding activity to gastric cancer cells. Conclusion The N terminal of the variable region introduced by PCR primers may seriously affect binding activity of the antibody.

关 键 词：胃肿瘤 单克隆抗体 单链抗体 载体 噬菌体抗体库 

分 类 号：R735.203[医药卫生—肿瘤] R392－33[医药卫生—临床医学]