KDP胞外502～764位氨基酸基因合成、表达及功能鉴定

应用基因搭桥法及 Taq酶聚合反应合成了编码人血管内皮生长因子受体 - ( h VEGFR- ,KDR)第 50 2～ 764位 2 62个氨基酸的基因片段 .DNA序列分析表明 ,合成的 786bp的基因片段与文献报道的 KDR相应 c DNA序列完全一致 .将该基因与原核融合蛋白表达载体 p GEX- 3X重组 ,在大肠杆菌 JM1 0 9中表达了 GST- KDR2 62融合蛋白 ,表达量约占菌体总蛋白的 35% .表达产物依次经包涵体分离、变性、复性、亲合层析纯化和 Xa因子酶解 ,获得了 KDR2 62目的蛋白纯品 .GST-KDR2 62融合蛋白和纯化产物经 Western blot分析 ,两者均可被 VEGF1 65特异性识别 ,前者分子量约 56k D,后者分子量约 30 k D;这两种蛋白用 VEGF1 65及其抗体进行的 ELISA分析结果均显示阳性 ,并有剂量依赖关系 ,而用 Xa因子酶解 GST- KDR2 62融合蛋白获得的 GST和空载体诱导产物对照均为阴性 .以上结果表明表达的 KDR2 62蛋白可特异性地与 VEGF结合 .

Gene Synthesis, Protein Expression and Function Identification of 502nd-764th Amino Acids in Extracellular Domain of KDR Human Vascular Endothelial Growth Factor Receptor

Gene encoding 502nd 764th amino acids in the extracellular domain of KDR human vascular endothelial growth factor receptor is synthesized using recursive PCR. The sequence analysis indicates that the sequence of 786 bp gene fragment is consistent with that documented previously. After insertion of the gene fragment into the prokaryotic fusion protein expression vector pGEX 3X, the fusion protein GST KDR262 is expressed in E. coli JM109 and its amount is about 35% of total bacterial proteins. KDR262 is obtained after inclusion body separation, denaturation, renaturation, affinity chromatography purification and Factor Xa digestion. The molecular mass of GST KDR262 and KDR262 are about 56 kD and 30 kD respectively. Western blot analysis shows that both GST KDR262 and KDR262 can be detected by recombinant human vascular endothelial growth factor. ELISA analysis using VEGF165 and anti VEGF antibody shows the conclusion that GST KDR262 and KDR262 can specificly bind to VEGF in a concentration dependent manner.