KDR酪氨酸激酶的克隆表达及纯化

Cloning, Expression and Purification of KDR Tyrosine Kinase

摘要:

VEGF作为一种血管内皮细胞有丝分裂原, 通过与内皮细胞表面特定受体结合, 从而导致新生血管的形成, 其中VEGFR-2(KDR/Flk-1)在肿瘤血管形成中起重要作用, 因此其抑制剂有可能发展成为治疗肿瘤的新药。采用大肠杆菌成功表达了具有酪氨酸激酶活性的KDR酪氨酸激酶催化域(KDR-CD)。采用RT-PCR从人脐静脉内皮细胞中提取总RNA, 获得KDR-CD的编码基因, 将其克隆至pET-30a载体, 在E. coli BL21(DE3)中进行了成功表达, 采用Ni-NTA亲和层析对其进行了纯化, Western blotting结果显示表达的KDR-CD蛋白自身磷酸化, 重组KDR-CD蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性。

关键词:血管生成, KDR酪氨酸激酶, 大肠杆菌, 酪氨酸激酶活性

Abstract:

The catalytic domain of KDR kinase (KDR-CD) was amplified from RNA of HUVCEs cells with RT-PCR and expressed in E. coli BL21(DE3) by plasmid pET30a as vector. The recombinant protein was purified with affinity chromatography (Ni-NTA). Western blotting showed that the recombinant KDR-CD was phosphorylated in E. coli BL21(DE3). The recombinant KDR-CD was identified to have kinase activity catalyzing the substrate phosphorylated with ATP in the enzymatic reaction.

Key words:angiogenesis, KDR tyrosine kinase, E. coli, kinase activity.