脑药物转运载体——抗转铁蛋白受体单链抗体的克隆表达及鉴定

为构建特异性的脑药物转运载体 ,分段合成了抗大鼠转铁蛋白受体的单链抗体基因 (Ox2 6 scfv) .经重叠PCR拼接成完整片段 ,克隆入pUC19载体中 ,测序正确后克隆到大肠杆菌表达载体pET 15b E .tag上 .IPTG诱导 ,表达产物分子量为 2 9kD ,约占菌体总蛋白量的 4 0 % .包涵体经 6mol L盐酸胍变性后 ,过SephacrylS 30 0HR分子筛柱复性蛋白 .免疫酶染色实验表明 ,该单链抗体能与转铁蛋白受体特异性结合 ,为建立以转铁蛋白受体为介导的血脑屏障转运载体打下了基础

Brain Drug Delivery Vector-Cloning, Expression and Identification of the Single-chain Antibody to Transferrin Receptor

In order to construct a special brain drug delivery vector, the gene of single chain antibody (Ox26 ScFv) to rat transferrin receptor was synthesized by 18 fragments. Full gene was obtained by overlap PCR, sequenced and inserted into the prokaryotic expression vector pET 15b/E.tag. After IPTG induction, the products (molecular weight 29 kD) were obtained with the 40% yield of total bacterial proteins. Inclusion bodies were dissolved into 6 mol/L guanidine HCl, refolded and purified by Sephacryl S 300 HR gel filtration chromatography. Immunohistochemical staining demonstrated that Ox26 ScFv protein could combine with transferrin receptor properly. It suggests that the genetically engineered single chain antibody may be used for a non invasive neurotherapeutic delivery across the blood brain barrier in vivo .