人尿激酶原全长cDNA在中国仓鼠卵巢细胞中的稳定表达

Expression of Pro-urokinase cDNA in Chinese Hamster Ovary Cell Lines

通过磷酸钙共沉淀技术,将含有尿激酶原cDNA的表达载体(pSV_2-proUK)与含有二氢叶酸还原酶基因的表达质粒pSV_2-dhfr或MMTV-dhfr共转染中国仓鼠卵巢细胞二氢叶酸还原酶基因缺陷株(CHO-dhfr^-)。利用选择培养基选择出二氢叶酸还原酶基因表达阳性(dhfr^+)的转化细胞灶。用溶解圈法测定尿激酶型纤溶酶原激活剂(u-PA)的生物活性,其中两株显著高于其他株细胞,命名为CLF-14和CLF-8,表达水平依次为241U/10~6细胞/48h,16IU/10~6细胞/48h。用ELISA测定CLF-14和CLF-8细胞上清,表现为强阳性,阳性值比阴性对照值(P/N)大于10,CLF-14和CLF-8细胞表达量分别为0.14—0.22μg/10~6细胞/48h和0.08—0.14μg/10~6细胞/48h。分泌产物能被抗尿激酶(UK)血清抑制,而不被抗组织型纤溶酶原激活剂(t-PA)的抗血清和正常兔血清抑制。

Expression vectors containing the pro-UK cDNA (pSV_2-proUK) anda dihydrofolatc reductase cDNA (pSV_2-dhfr or MMTV-dhfr) were co-transfected into CHO-dhfr-cells by calcium phosphate precipitation tec- hnique. 1he dhfr^+ transformants were selected by fibrinolytic agaroseplate assay.Two colonies,named as CLF-14 and CLF-8, exhibited signi-ficantly higher expression level of u-PA activity. They reached morethan 24IU/10~6 cells/48h and 16IU/10~6 cells/48h,respectively.Examinationof the cell supernatants for u-PA using ELISA method also showedstrong positive results, the quantity of expression is about 0.14--0.22μg/10~6 cells/48h and 0.08--0.14μg/10~6 cells/48h, respectively. The u-PAsecreted by stable transformed cells can be completely inhibited by UKanti-serum,but not by t-PA anti-serum and normal rabbit serum.