转铁蛋白受体单链抗体与BDNF融合蛋白的表达及活性鉴定

Studies of the Expression and Biologic Activity of an Anti- transferrin Receptor ScFv-BDNF Fusion Protein

脑源性神经营养因子(BDNF)对中枢神经系统的多种神经元具有营养、修复和保护功能,但因无法通过血脑屏障限制了其应用。利用抗转铁蛋白受体(TfR)的单链抗体(ox26scFv)作为脑转运载体,分别扩增单链抗体和BDNF基因,插入pTIGTrx载体,构建融合基因表达载体pTIGTrx/scFvBDNF,在大肠杆菌BL21(DE3)中实现了高效表达。经NiNTA金属螯合层析柱纯化后,在41kDa处可见目的纯化条带。大鼠GH3细胞免疫酶染色显示,ScFvBDNF融合蛋白能与转铁蛋白受体特异性结合,同时能够促进鸡胚背根节神经突起的生长,具备了BDNF的生物学活性。为使BDNF能够跨越血脑屏障成为中枢神经系统的治疗药物打下了实验基础。

The brain-derived neurotrophic factor had nutrition, repair and protection functions to neurons of central nervous system, but its utilization was restricted by the blood-brain barrier. The single-chain antibody （ox26-scFv） to transferrin receptor（TfR） was used as a vector to transport BDNF. The ox26-scFv gene and BDNF gene were amplified by PCR from plasmid PUC19/ ox26-scFv and plasmid PUC19/BDNF, inserted into the prokaryotic expression vector pTIG-Trx which carting a thioredoxin （ Trx ） gene and a C-terminal His-tag separately, constructed a pTIG-Trx/scFv-BDNF recombinant plasmid was constructed. The ScFv-BDNF fusion proteins were expressed at 20℃, after 0.02mmol/L IPTG induction in the strain E. coli BI21 （ DE3 ）. The soluble ScFv proteins in cytoplasm suspension were purified by immobilized metal chelation affinity chromatography （Ni-NTA）, a single band with molecular weight 41 kDa appeared on SDS-PAGE gel. Immunoeytochemistry staining showed that ScFv-BDNF fusion proteins could recognize and bind to transferrin receptors on the surfaces of rat GH3 cells, and the fusion proteins could promote growth of the chick embryo dorsal root ganglion （ DRG）, which indicated that the fusion proteins Not only could combine with transferrin receptors specially but also had biological activity of BDNF. These results would lay groundwork to transport BDNF across BBB as a CNS therapeutics.