【摘要】 从人的黑素瘤细胞提取总RNA,进行RT-PCR,克隆了组织纤维溶酶原激活剂(tPA)基因,对其缺失突变,进一步克隆了溶栓药物瑞替普酶-reteplase(rPA)基因;以羊的β-乳球蛋白基因(BLG)作为调控序列,构建了瑞替普酶的乳腺表达载体BrPA。注射BrPA到乳腺动脉,收集乳汁进行鉴定和检测,体外溶栓活性分析发现瑞替普酶在羊的乳腺中获得了表达,最高表达量为8.52 mg/L,为转基因动物乳腺表达载体构件的检测建立了合理的方法,也为下一步制备转基因奶山羊乳腺生物反应器奠定了基础;该研究提示,对于临床给药剂量小,而价格昂贵的药物,如细胞因子类,也许可以用这种乳腺瞬时表达系统进行生产。更多还原

【Abstract】 Reteplase(rPA) is a recombinant non-glycosylated form of human tissue plasminogen activator(tPA) in which the molecule has been genetically engineered to contain 357 of the 527 amino acids of the original protein.Reteplase converts endogenous plasminogen to active plasmin,which in turn(degrades) the fibrin matrix of the thrombus.In this paper,total RNA extracted from human melanoma cells(Bowes cell line) were reversely transcripted to cDNA with the tPA specific antisense primer,and deletion mutation was made to clone rPA cDNA.The rPA fragment was inserted into mammary gland expression vector with β-lactoglobulin gene as a regulation sequence.Transient expression was carried on 7 milk goats by injecting the vetor BrPA to the vein of goats’ mammry glands.At the same time,hormone was injected to stimulate the goats producing milk and the milk was collected to test fibrinolysis bioassay in vitro.Experiments showed that the expression vector is reasonable,and r-PA was expressed in the milk of the goats.The milk showed high clot lysis activity and the activity lasted about 10 days.更多还原