抗胃癌单抗3H11的单链抗体表达载体的构建和表达

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。结论说明ＰＣＲ引物引入的可变区氨基端序列的改变有时可对ＳｃＦｖ的活性有很大影响

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

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.