Hashi H, Hatai M, Kimizuka F, Kato I, Yaoi Y
Biology Division, National Cancer Center Research Institute, Tokyo, Japan.
Cell Struct Funct. 1994 Feb;19(1):37-47. doi: 10.1247/csf.19.37.
We constructed a fusion protein of the cell-binding domain of human fibronectin and human basic fibroblast growth factor, and prepared a polypeptide with both cell-adhesive activity and growth factor activity. A human gene fragment coding for basic fibroblast growth factor was amplified by the polymerase chain reaction, and introduced into the expression vector pTF7520, which encodes the cell-binding domain of human fibronectin. The resulting plasmid encoded a fusion protein in which basic fibroblast growth factor was added covalently to the C-terminal end of the fibronectin fragment. The fusion protein was expressed in Escherichia coli JM109 cells and purified from the extract by heparin affinity chromatography. The purified fusion protein had cell-adhesive activity toward BALB/c 3T3 cells, and stimulated their DNA synthesis in serum-depleted cultures. The fusion protein gave maximum mitogenic activity at the concentration of 10 nM. The fusion protein adsorbed to culture dishes, or added to collagen gels, stimulated the growth of human umbilical-vein endothelial cells. The fusion protein stimulated the angiogenesis in chorioallantoic membranes of developing chick embryos.
我们构建了人纤连蛋白细胞结合结构域与人碱性成纤维细胞生长因子的融合蛋白，并制备了一种兼具细胞黏附活性和生长因子活性的多肽。通过聚合酶链反应扩增编码碱性成纤维细胞生长因子的人基因片段，并将其导入编码人纤连蛋白细胞结合结构域的表达载体pTF7520。所得质粒编码一种融合蛋白，其中碱性成纤维细胞生长因子共价连接到纤连蛋白片段的C末端。该融合蛋白在大肠杆菌JM109细胞中表达，并通过肝素亲和层析从提取物中纯化。纯化的融合蛋白对BALB/c 3T3细胞具有细胞黏附活性，并在无血清培养中刺激其DNA合成。融合蛋白在10 nM浓度时具有最大促有丝分裂活性。吸附到培养皿上或添加到胶原凝胶中的融合蛋白可刺激人脐静脉内皮细胞的生长。融合蛋白可刺激发育中鸡胚绒毛尿囊膜的血管生成。
