摘要
分段合成大鼠抗转铁蛋白受体单链抗体基因 (ox2 6_scFv) ,经三轮PCR拼接成 794bp的完整片段。测序后构建pTIG_Trx ox2 6_scFv表达载体 ,在大肠杆菌BL2 1(DE3)中获得了可溶性表达 ,经Ni_NTA金属鏊合层析柱纯化 ,在2 9kD处可见单一条带。大鼠GH3细胞免疫组化显示 ,该单链抗体能识别并与转铁蛋白受体结合。将单链抗体尾静脉注射大鼠 ,于鼠脑组织切片上可见阳性染色 ,尤其是脑血管处着色明显 ,说明该单链抗体对脑血管具有较好的靶向作用 ,并在受体的介导下通过了血脑屏障。
The single-chain antibody gene(ox26-scFv) to transferrin receptor(TfR) was synthesized and amplified by three-step PCR. After sequencing, the gene was cloned into prokaryotic expression vector pTIG-Trx which carried thioredoxin (Trx) gene and a C-terminal His.tag. The Ox26-scFv proteins achieved 31% yields of total bacteria proteins at 20℃, after 0.02mM IPTG induction using the strain E.coli BL21(DE3). The soluble scFv proteins in cytoplasm suspension were about 35% and the inclusion bodies were about 65%. The soluble products were purified by immobilized metal chelation affinity chromatography(Ni-NTA), a single band with molecular weight 29 kD appeared on SDS-PAGE gel. Rat GH3 cell immunocytochemistry staining showed that Ox26-scFv protein could recognize and bind to transferrin receptor. Injected SD rats with Ox26-scFv proteins by tail veins, the antibodies were detected from brain tissues specially on the brain capillaries 4 h later which indicate that Ox26-scFv proteins have a good target function to brain capillaries and can permeate the blood-brain barrier mediated by the transferrin receptors.
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第3期342-347,共6页
Chinese Journal of Biotechnology
基金
国家自然科学基金项目资助 (No .3 0 3 0 0 10 2
北京市自然科学基金资助 (No .5 0 42 0 2 2 )~~
关键词
转铁蛋白受体
单链抗体
血脑屏障
可溶性表达
药物转运
transferrin receptor(TfR), single-chain Fv(scFv), blood-brain barrier(BBB), soluble expression, drug delivery
