人血管内皮生长因子受体Flt—l胞外配体结合域的筛选及其结构分析

目的 应用酵母双杂交系统筛选人血管内皮生长因子受体Flt－1胞外最小配合结合域。方法 采用PCR技术，从人胎盘cDNA文库扩增出4个截短的Flt-1cDNA，分别含胞外第2、1、－2、2－3和1－3个loop，构建酵母双杂交系统配合表达质粒，并将pGB59／hVEGF165与pGAD424／Flt-ls两两配对转化酵母菌SFY526。采用滤纸法和液体培养法对阳性克隆进行β－半乳糖苷酶活性分析。结果 Flt－1胞外loop2－3与loop1-3的配体结合能力相关不大，loop1-2的结合力较弱，单独第2个loop无配体结合域－2－3loop，这对研制分子量更小、安全性高的可溶性Flt-1片段，开发其在肿瘤、糖尿病性网膜病变等血管生成相关疾病基因治疗中的应用具有重要的指导意义。

Screening and Constructional analysis of extracellular at the ligand binding domain of the vascular endothelial growth factor receptor Flt-1

Aim To screen the minimal extracellular ligand- binding domain of vascular endothelial growth factor (VEGF) receptor Flt - 1using yeast two - hybrid system. Methods Four turncated Flt - 1 cDNA fragments containing extracellular domain loops 2, 1 - 2, 2 - 3 and 1 - 3respectively were amplified from human placental cDNA library by PCR and used for constructing fusion expression plasmids in yeast two - hybrid system. Each of the four pGAD424/Flt - 1 plasmids was paired up with pGBT9/hVEGF165 and transformed into yeast SFY526, The 3- galac tosidase activities of positive colonies were analyzed by filter assay and liquid culture assay. Result The binding capacity of loop2 - 3 was close to that of loop1 -3. However, the binding capacity of loop1 -2 was weaker. Loop 2 alone showed no binding capacity. Conclusion The minimal extracellular ligand binding domains of Flt - 1 that were sufficient to achieve VEGF binding to loop2 - 3 were screened out using yeast two -hybrid system, which was of an important guiding significance in developing the more safe sFlt - 1 fragments with smaller molecular weight and studing the their application in the treatment of angiogenesis related diseases, such as tumor and diabetic retinopathy.