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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 fragments of 622 amino acids encoding human VEGF receptor - (h VEGFR-, KDR) at position 50 2 to 764 were synthesized by gene-intron method and Taq polymerase polymerization.The DNA sequence analysis showed that the synthesized 786 bp The gene fragment was identical with the corresponding cDNA sequence of KDR reported in the literature.It was recombined with the prokaryotic fusion protein expression vector pGEX-3X and expressed GST-KDR262 fusion protein in E. coli JM109, 35% of total protein.The expression product was purified by inclusion body, denatured, renatured, purified by affinity chromatography and digested with factor Xa to obtain pure product of KDR2 62.GST-KDR2 62 fusion protein and purified product Western blot analysis showed that both of them could be specifically recognized by VEGF1 65, the former had a molecular weight of about 56kD and the latter had a molecular weight of about 30 kD. The results of ELISA analysis of both proteins with VEGF165 and their antibodies showed positive results and Dose-dependent manner, whereas both GST and empty vector-induced product controls obtained by digesting GST-KDR262 fusion protein with factor Xa were negative.The above results indicate that the expressed KDR262 protein specifically binds to the VEGF Together