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用RT—PCR方法 ,从分泌抗人CD3抗体的杂交瘤细胞中扩增抗体的重链和轻链可变区基因 ,并运用重叠延伸拼接法 (SOE)将其连接成单链抗体 (ScFv)基因 ,并克隆于带有Tac启动子的原核表达载体中 ,用斑点杂交方法筛选出阳性克隆。重组子在E .coilB1 2 1 (DE3)中获得高效表达且具有活性的单链抗体。
Antibody heavy and light chain variable region genes were amplified by RT-PCR from hybridoma cells secreting anti-human CD3 antibody and ligated into single chain antibody (ScFv) using overlap extension splicing (SOE) Gene and cloned into prokaryotic expression vector with Tac promoter. The positive clones were screened by dot blot hybridization. The recombinant protein obtained highly expressed and active single chain antibody in E. coli B 1 2 1 (DE3).