摘要
可溶性血管内皮细胞生长因子受体 ( Flt- 1 )具有受体拮抗剂作用 ,可竞争结合血管内皮细胞生长因子 ,( VEGF)并与其膜表面受体 Flt- 1及 KDR形成异源二聚体 ,最终阻断 VEGF的生物学活性 .利用 RT- PCR技术从人脐静脉内皮细胞扩增出 Flt- 1胞外 ～ 区 c DNA片段 ,通过基因重组将该片段克隆于谷胱甘肽转移酶 ( GST)融合蛋白表达载体 PGEX2 -T中 ,连接产物转化大肠杆菌XL1 - blue,经 IPTG诱导可获大量稳定表达的 Flt- 1 - GST融合蛋白 .该表达产物经变性复性处理后 ,可特异性结合 12 5 - VEGF.大量具有活性的可溶性 Flt- 1的获得有助于新的抗肿瘤血管形成方法的探索 .
Due to its high affinity to VEGF,Flt 1 is expected to be a prospective candidate for soluble receptor used in dominant negative way.The increasing data show that soluble Flt 1 expressed by eucaryotic system has the ability to block the biological activity of VEGF.From cultured human umbilical vein endothelial cells,cDNA fragments of Flt 1Ⅰ Ⅳ extracellular domains were amplified through RT PCR with a pair of specific primers and cloned to P GEX2 T and the fusion protein was expressed stably in E.coli XL1 blue.SDS PAGE showed that the fusion protein was remarkably expressed after induced by IPTG,and its molecular weight was identical to expectation.After denaturation and refolding of recombinant Flt 1Ⅰ Ⅳ GST protein,soluble Flt 1 was obtained.Its direct binding activity with 125 Ⅰ VEGF was identified by solid phase binding assay and competitive solid phase binding assay.Getting a large quantity of soluble Flt 1 expressed by procaryotic system makes it possible for researchers to screen the VEGF inhibitors on a large scale.
出处
《中国生物化学与分子生物学报》
CSCD
2000年第1期62-66,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家杰出青年科学基金!资助 ( 3 95 2 5 0 2 1)
关键词
FLT-1
原核表达
结合活性
VEGF受体阻断剂
Vascular endothelial cell growth factor
Soluble receptor
Flt 1
Procaryotic expression
Binding activity
