人血管内皮生长因子受体Flt-1和KDR胞外区的表达及活性测定

Expression and Activity Determination of Human VEGF Receptor Flt-1 and KDR Extracellular Domains

血管新生在肿瘤发生过程中起着重要作用,VEGF通过与其特异性受体Flt-1、KDR结合参与了这一过程。该研究在大肠杆菌中分别表达了Flt-1 D2、KDR D3以及Flt-1 D2与KDR D3的融合蛋白FK,表达率依次占菌体中蛋白的24%,39%,35%,随后使用SPFF阳离子层析柱对溶解后的包涵体蛋白进行了纯化,并通过透析的方法进行复性获得活性蛋白。Flt-1 D2、KDR D3、FK与VEGF的结合都具有剂量依赖性,解离常数Kd值分别为13.8,38.8,2.7 pmol/L,表明FK比Flt-1及KDR更容易结合VEGF。使用HMEC和HUVEC两种内皮细胞测量了重组蛋白的生物活性,Flt-1 D2的IC50分别为12.51和15.77 nmol/L,KDR D3的IC50分别为24.37和28.83 nmol/L,而FK的IC50分别为1.46和2.63 nmol/L,表明FK能明显抑制VEGF165刺激的HMEC和HUVEC细胞增殖。该研究为肿瘤治疗提供了一个新的候选药物。

Angiogenesis plays an important role in solid tumor growth and tumor metastasis. Vascular endothelial growth factor （VEGF） acts as a highly specific mitogen directly involving in the process by binding to special receptors. Flt-1 and KDR are the major VEGF receptors. Both VEGFRs contain seven Ig-like domains in their extracellular NH2-terminal regions. In this paper, Flt-1 domain 2, KDR domain 3 and recombinant FK comprising Flt-1 domain 2 and KDR domain 3 were constructed for the feature comparison. The recombinant proteins were expressed as inclusion body in E. coli after induction with IPTG. The dissovled inclusion body proteins were purified with SPFF gel at pH value 7.2, and the target proteins were completely eluted with buffer 2. Then, the eluents were refolded by dialysis to get biological activity. The binding affinitiy of VEGF receptor functional domains was measured by an enzyme-linked immunosorbent assay （ELISA） procedure. HMEC and HUVEC cells were used to measure the activity of Flt-1 D2, KDR D3 and FK. The results showed that the expression levels of Flt-1 D2, KDR D3 and FK were estimated to be 24% ,39%, 35% by SDS-PAGE, respectively. Flt-1 D2, KDR D3 and FK were able to bind to VEGF165 in a dose-dependent manner with a disocciation constant （ Kd ） of 13.8,38.8,2.7 pmol/L respectively,which revealed FK with higher affinity to YEGF than VEGFR1 （ Kd = 16 pmol/L） and VEGFR2 （Kd = 75 pmol/L）. In addition,FK specifically inhibited the proliferation of human microvascular endothelial cell （HMEC） and human umbilical vein endothelial CeU （HUVEC） stimulated by VEGF165. The IC50 of FK protein was 1.46 nmol/L for HMEC and 2.63 nmol/L for HUVEC. FK is a potent inhibitor of angiogenesis, which could make FK a potential drug candidate in anti-tumor therapy.