非糖基化尿激酶原的研究

Study on Non-Glycosylated Pro-Urokinase

目的:构建一种非糖基化、抗凝血酶激活的尿激酶原突变体,并观察其表达及活性情况。方法:使用PCR扩增的方法,将302位天冬酰胺(Asn302)突变为丙氨酸(Ala302),去掉糖基化位点;将156位精氨酸(Arg156)突变为赖氨酸(Lys156),去掉凝血酶作用位点。将突变体基因转染到哺乳动物细胞中。收取无血清培养上清,并用纤维蛋白板溶解圈法鉴定活性。结果:PCR产物经琼脂糖电泳鉴定分子质量略大于1200bp,与理论分子质量1296bp基本符合。转染成功的细胞株用于扩大培养。纤维蛋白板溶解圈法测定上清活性为295.58IU/mL。结论:非糖基化、抗凝血酶的尿激酶原突变体构建成功。转染后细胞生长良好,表达水平较高。

Objective： To construct non-glycosylated, thrombin resistant pro-urokinase mutant and identify it＇s expression and activity. Methods： Glycosylation site was deleted by Asn302-Ala302 mutagenesis using PCR amplification. Thrombin cleavage site was eliminated by Arg156-Lys156 mutagenesis. Pro-urokinase gene was introduced into mammalian cells. Serum-free supernatant was collected. Fibrinolytic agarose plate assay （FAPA） was used to identify the activity. Results： DNA molecular weight of PCR product was a little above 1 200 bp as determined by agarose gel electrophoresis, which was consistent with theoretical molecular weight 1 296 bp. The successfully transfected clone was selected for amplification production. The fibrinolytic activity of supernatant was 295.58 IU/mL by FAPA. Conclusions： Non-glycosylated, thrombin resistant scu-PA mutant was constructed. The cells grew well after transfection. The expression was comparatively high level.