Krätzschmar J, Haendler B, Bringmann P, Dinter H, Hess H, Donner P, Schleuning W D
Research Laboratories of Schering AG Berlin, Germany.
Gene. 1992 Jul 15;116(2):281-4. doi: 10.1016/0378-1119(92)90526-u.
The cDNAs coding for the four Desmodus rotundus salivary plasminogen activators (DSPAs) were subcloned into the mammalian expression vector, pMPSV/CMV, which carries the myeloproliferative sarcoma virus promoter and the cytomegalovirus enhancer. These constructs were transfected, together with plasmids harbouring Geneticin (G418)-resistance and puromycin-resistance genes, into baby hamster kidney cells. Through the selective pressure of both antibiotics, cell clones constitutively overexpressing the DSPA alpha 1, DSPA alpha 2, DSPA beta or DSPA gamma cDNAs were obtained. Secretion of active DSPAs was confirmed by zymographic analysis and quantified using a fibrin plate assay and ELISA.
编码四种圆叶吸血蝠唾液纤溶酶原激活剂（DSPAs）的cDNA被亚克隆到哺乳动物表达载体pMPSV/CMV中，该载体携带骨髓增殖性肉瘤病毒启动子和巨细胞病毒增强子。将这些构建体与携带遗传霉素（G418）抗性和嘌呤霉素抗性基因的质粒一起转染到幼仓鼠肾细胞中。通过两种抗生素的选择压力，获得了组成型过表达DSPAα1、DSPAα2、DSPAβ或DSPAγ cDNA的细胞克隆。通过酶谱分析证实了活性DSPAs的分泌，并使用纤维蛋白平板试验和酶联免疫吸附测定（ELISA）进行定量。
