人血管内皮生长因子受体Flt-1胞外配体结合域的筛选及其结构分析

 血管内皮生长因子受体 Flt- 1胞外区具有 7个免疫球蛋白样的袢 (Ig- like loop) ,氨基端 3个loop负责与其配体 VEGF的结合 .为了寻求能与配体结合的更小的 Flt- 1片段 ,在对 Flt- 1胞外前3个 loop氨基酸组成和晶体结构分析的基础上 ,应用酵母双杂交系统对 Flt- 1的配体结合域进行筛选 .利用 PCR技术 ,从人胎盘 c DNA文库扩增出 4个截短的 Flt- 1 c DNA,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融合表达质粒 ,并将 p GBT9/h VEGF165与 p GAD42 4 /Flt- 1 s两两配对转化酵母菌 SFY52 6,采用滤纸法和液体培养定量检测法对阳性克隆进行β-半乳糖苷酶活性分析 .结果显示 ,Flt- 1胞外 loop 2 - 3与 loop 1 - 3的配体结合能力相差不大 ,loop 1 - 2的结合力较弱 ,单独第 2个 loop无配体结合能力 .

Study on Ligand Binding Domains of Human Vascular Endothelial Growth Factor Receptor Flt-1 within Extracellular Domain and Its Structure Analysis

The vascular endothelial growth factor (VEGF) receptor Flt\|1 was characterized by seven Ig\|like loops within the extracellular domain and the first three loops were involved in high affinity binding of VEGF.In order to detect its minimal extracellular Ig\|like domains which were sufficient to achieve VEGF binding,amino acid component and crystal structure of the first three loops were analyzed.On the basis of this analysis,four cDNA clones coding truncated Flt\|1 mutants consisting of loop2,1\|2,2\|3 and 1\|3 were amplified by PCR from human placenta cDNA library and the interaction between Flt\|1 mutants and VEGF was investigated with yeast two\|hybrid system.Fusion expressio plasmids pGAD424/Flt\|1(2),pGAD424/Flt\|1(1\|2),pGAD424/Flt\|1(2\|3),pGAD424/Flt\|1(1\|3) and pGBT9/hVEGF 165 were constructed.Two kinds of plasmids were cotransformed into the yeast reporter strain SFY526.Transformants′ β galactosidase activities were detected by both filter assay for qualitative analysis and liquid culture assay for quantitative data.The data presented here suggested that loop2\|3 had almost the same hVEGF 165 binding ability as loop1\|3,but loop1\|2 had only faint ligand binding ability and loop2 could not bind hVEGF 165 by itself.