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抗转铁蛋白受体单链抗体的表达、纯化及活性测定
引用本文:赵莉霞,颜冰 许龙 蒋世卫 张莹莹 杨志新 周晓巍 黄培堂.抗转铁蛋白受体单链抗体的表达、纯化及活性测定[J].生物工程学报,2006,22(3):488-491.
作者姓名:赵莉霞  颜冰 许龙 蒋世卫 张莹莹 杨志新 周晓巍 黄培堂
作者单位:军事医学科学院生物工程研究所,北京,100071
基金项目:国家高技术研究发展计划(863计划);北京市自然科学基金
摘    要:根据转铁蛋白受体和转铁蛋白特异性结合的性质,利用亲和纯化的方法从胎盘中提取转铁蛋白受体。以转铁蛋白受体为抗原包被免疫管,从全合成人源噬菌体单链抗体库中筛选其抗体。对筛选到的抗体进行特异性鉴定后,将抗体基因插入表达载体pET22b( ),转化大肠杆菌BL21(DE3),IPTG诱导后获得可溶性表达。HeLa细胞的免疫组化结果显示,表达的抗体可以与转铁蛋白受体结合。表达产物经Ni-NTA金属螯和层析柱纯化、脱咪唑后,从尾静脉注射小鼠。1h后,去除血液及毛细血管的干扰,在脑实质中检测到了抗体的存在,说明该抗体可以通过血脑屏障。

关 键 词:转铁蛋白受体  单链抗体  表达  纯化  血脑屏障
文章编号:1000-3061(2006)03-0488-04
收稿时间:01 18 2005 12:00AM
修稿时间:02 17 2006 12:00AM

Expression, Purification and Activity Analysis of Antihuman Transferrin Receptor ScFv
ZHAO Li-Xia,YAN Bing,XU Long,JIANG Shi-Wei,ZHANG Ying-Ying,YANG Zhi-Xin,ZHOU Xiao-Wei,HUANG Pei-Tang.Expression, Purification and Activity Analysis of Antihuman Transferrin Receptor ScFv[J].Chinese Journal of Biotechnology,2006,22(3):488-491.
Authors:ZHAO Li-Xia  YAN Bing  XU Long  JIANG Shi-Wei  ZHANG Ying-Ying  YANG Zhi-Xin  ZHOU Xiao-Wei  HUANG Pei-Tang
Institution:Beijing Institute of Biotechnology, Beijing 100071, China
Abstract:Human transferrin receptor(TfR) was isolated from homogenates of placental tissues by affinity chromatography on transferrin-Sepharose, and then used to screen human scFv against it from a fully-synthesized phage scFv library. After verifying the specificity, gene fragment of one of the selected scFv was inserted into the plasmid pET22b( ) and transformed into E.coli BL21(DE3). Expression of scFv in transformant was induced with 0.5mmol/L IPTG. ELISA assay on HeLa cells showed that scFv protein could recognize and bind to TfR on the surface of HeLa cells.The scFv was purified by one-step affinity chromatography with Ni~ -NTA agarose, and injected into Kunming mouse via tail veins. This scFv was detected in brain tissues 1h later by capillary depletion method, which indicates that scFv protein can permeate through the blood brain barrier by mediation of the TfR receptor. Our works lay the foundation for the treatment of tumors and central nervous system diseases.
Keywords:transferrin receptor  scFv  expression  purification  blood brain barrier
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