抗CD3单链抗体基因克隆表达及生物活性鉴定.doc

抗CD3单链抗体基因克隆表达及生物活性鉴定
【关键词】单链抗体
【关键词】 CD3分子; 单链抗体
【Abstract】 The genes encoding antibody heavy and light chain variable regions (VH and VL) were cloned by RTPCR from OKT3 hybridoma cells, which produced antiCD3 moleclonal antibody. The VH and VL genes were fused and e a single chain Fv (scFv). The scFv gene was cloned into pCANTAB5E vector and expressed on bacterial phage surface. By three panning rounds, we have obtained two singlechain antibodys that specific for CD3. The antiCD3 scFv will be a reagent for diagnosis and therapy of immunodisorder
1 实验资料
.质粒 pCANTAB5E,大肠杆菌TG1和HB2151,内切酶SfiI和NotI, DNA聚合酶,,采用Promega公司试剂盒,从大约107细胞中分离和纯化mRNA,(VH)和轻链可变区(VL)基因. 为了加强抗体轻、重链可变区(Fv)的稳定性和更好地维持抗体分子内结合抗原区的空间构象,在VH和VL基因之间用一段编码(Gly4Ser)3的DNA序列把二者连接起来, 质粒,我们以单链抗体基因为模板,引入一对含SfiI和 NotI 酶切位点的引物, 再次 PCR ,基因大小与预测的单链抗体基因相符合(图1).把单链抗体基因插入到表达载体pCANTAB5E所含的噬菌体外壳蛋白g3p基因中. 转化大肠杆菌TG1感受态细胞后,在辅助噬菌体M13K07的作用下,单链抗体和外壳蛋白g3p以融合蛋白的形式表达于噬菌体的表面,,, h,用PBS淋洗3次后,加入HRP标记的抗M13噬菌体抗体温育1 h, 再用PBS淋洗3次,,感染大肠杆菌,获得培养上清,-感染-扩增的免疫筛选过程,使CD3重组噬菌体抗体得到富极. ,经I