非糖基化尿激酶原突变体的研究.docVIP

非糖基化尿激酶原突变体的研究 作者:杨波,李天德,王广义,陈练 作者单位：1解放军总医院心内科 北京市 100853；2西藏军区总医院心内科，西藏 拉萨市 850007 【摘要】 　　目的：构建一种非糖基化、抗凝血酶激活的尿激酶原突变体，在CHO-dhfr-（二氢叶酸还原酶缺陷型-中国仓鼠卵巢）细胞中表达，收取无血清培养上清，并鉴定其活性。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CHO-dhfr-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量略大于1 200道尔顿(dalton)，与理论分子量1 296道尔顿符合。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58 IU/ml。结论：非糖基化、抗凝血酶的单链尿激酶型纤溶酶原激活剂突变体构建成功。转染后细胞生长良好，表达水平较高。 【关键词】 尿纤溶酶原激活物；凝血酶；糖基化 　　Study for an non|glycosylated pro|urokinase mutant construct/YANG Bo,LI Tian|de,WANG Guang|yi,CHEN Lian//Chinese Journal of Cardiovascular Rehabilitation Medicine,2007，16（2）: Abstract：Objective:To construct non|glycosylated,thrombin resistant pro|urokinase mutantTo express pro-UK gene in CHO-dhfr cellsSerum|free culture supernatant was harvestedTo identify the activity of pro-UK mutantMethods:With PCR amplification,glycosylation site was deleted by Asn302-Ala302 mutagenesisThrombin cleavage site was eliminated by Arg156-Lys 156 mutagenesisPro-UK gene was introduced into DHFR-deficient (dihydrofolate reductase,DHFR) CHO 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 1200 daltons as determined by agarose gel electrophoresis,which was consistent with theoretical molecular weight 1296 daltonsThe successfully transfected clone was selected for amplification productionThe fibrinolytic activity of supernatant was 29558IU/ml by FAPAConclusion:Non|glycosylated,thrombin resistant pro-UK mutant was constructedThe cells glowed well after transfectionThe expression is comparatively high. Author′s　address：Department of Cardiology,PLA General Hospital,Beijing,100853,China Key words：Urine plasminogen activator;Thrombin;Glycosylation 心肌梗塞、脑栓塞及其它血栓病是严重危害人类健康的常见病，我国每年至少有500万人需要溶栓治疗[1]，因此研制高效、特异、副作用小的新型溶栓制