组织型纤溶酶原激活剂突变体基因转染细胞株的建立

Construction and Identification of t-PA Variants Gene Transfected Cell Lines

以脂质体介导法将含有t PA突变基因的真核表达载体pCSRA及pCSRK分别转染CHO dhfr ,经G4 1 8及DMEM双重选择 ,获得两者的稳定表达细胞株。结果表明 ,表达产物具有良好溶纤活性 ;Western印迹实验证明 ,产物具有特异抗原性 ;经多次传代 ,细胞株具有良好的稳定性。比较了无血清培养基CHO S SFMⅡ与常规培养基DMEM培养条件下pCSRK细胞株产物的表达量 ,前者约为后者的 2倍 ,表明无血清培养基SFM有可能用于药用性蛋白的生产。实验为t

The eucaryotic expression vector pCSRA and pCSRK, each of which included different t PA mutant gene, were transfected into CHO dhfr - independently by liposome mediated method; then through diplex choice of G418 and DMEM, the authors had got the stable cell lines of the two. Monitoring result showed that the expression products had good cellulolytic activity; the result of Western blotting showed that they had the specific antigenicity; and passaged for many times, the cell lines had heredity stability to a certainty. For the expression quantity of the target product of pCSRK in CHO S SFM Ⅱ was as twice as in regular medium DMEM, it showed that SFM would be used to produce the medicinal protein. Thus it had done spadework under certain degree for the usages of new type mutants of t PA in clinic.