卵巢癌抗独特型单链抗体3D_5ScFv的高效表达

对 3D5卵巢癌抗独特型单链抗体进行原核系统的高效表达 .采用DNA重组技术 ,将 3D5ScFv克隆到原核表达载体pTMF中 ,重组质粒转化大肠杆菌BL2 1(DE3) ;IPTG诱导表达 ,SDS PAGE鉴定蛋白质的分子量约为 2 8kD ;通过直接ELISA、竞争抑制实验和Western印迹分别检测其活性 .3D5ScFv以包涵体的形式获得高效表达 ,占菌体总蛋白 36 % ,变性复性后蛋白的纯度大于 90 % ,表达蛋白能与卵巢癌抗体OC12 5特异性结合 ,并能有效抑制卵巢癌抗原CA 12 5与卵巢癌抗体OC 12 5的特异性的结合 .3D5ScFv在原核系统中获得了高效表达 ,并具有较好的活性 ,为抗独特型抗体的人源化改造提供必要的元件

High Level Expression of 3D_5 Ovarian Carcinoma Anti-idiotypic Single-Chain Antibody (3D 5ScFv) in E.coli

Using DNA recombinant technique, 3D 5ScFv gene was cloned into the prokaryotic expression plasmid pTMF. E.coli BL21(DE3) was transformed by the recombinant plasmid. Expression was induced by isopropylthio-beta-D-galactoside (IPTG). The protein identified by SDS-PAGE has a molecular weight about 28 kD.The activity of protein was determined by direct ELISA, competition inhibiting test and Western blotting. 3D 5ScFv was highly expressed in the form of an inclusion body, which was 36 percent of the total bacterial proteins. The purity of protein was more than 90% after denaturation and renaturation. It was demonstrated that expressed 3D 5ScFv protein could specially bind OC125 and could effectively inhibit the special binding of CA-125 (an antigen of ovarian carcinoma) to OC-125 ( an antibody of ovarian carcinoma).