鼠抗人纤维蛋白单链抗体-尿激酶杂合基因的构建及其在中国仓鼠卵巢细胞中的表达

采用 DNA重组技术构建了表达鼠抗人纤维蛋白单链抗体与低分子量尿激酶融合基因的真核表达载体。通过磷酸钙共沉淀法 ,将该表达载体转染到中国仓鼠卵巢细胞二氢叶酸还原酶基因缺陷株 ( CHO- dhfr-)中 ,利用选择培养基筛选出稳定表达的细胞株 ,溶解圈法测定融合蛋白的表达水平为每 1 0 6细胞每天 5 8IU。该融合蛋白保留了与纤维蛋白的结合活性和溶解纤维蛋白的溶纤活性。SDS- PAGE,Western印迹法分析证明融合蛋白的相对分子质量约为 70× 1 0 3

The construction of mouse against human cross-linked fibrin ScFv-urokinase hybrid gene and its expression in Chinese hamster ovary cell

The eukaryotic expression vector containing mouse against human cross linked fibrin ScFv urokinase fusion gene had been constructed by a series of DNA manipulation and was transfected into CHO dhfr - cells by calcium phosphate precipitation technique. After selection and subcloning, a few new cell strains that can express the fusion protein were obtained. The expression level of the fusion protein reached 58 IU/10 6 cells/d by FAPA (fibrin agarose plate assay). The expressed fusion protein remained the capacity to activate plasminogen and bind fibrin. SDS PAGE and Western blot analysis showed that the molecular weight of the fusion protein was about 70 kD.