转基因小鼠乳腺表达长效组织纤溶酶原激活剂

Production of longer acting human tissue plasminogen activator in transgenic mouse milk

组织纤溶酶原激活剂(tPA)是一种较理想的溶血栓药物,本研究采用其突变体长效组织纤溶酶原激活剂(LAtPA)的cDNA作为目的基因,将其插入羊β-乳球蛋白(BLG)基因起始密码之前,使LAt-PA的转录、翻译受控于BLG基因的5′、3′序列,将此构建BLG-LAtPA用显微注射方法建立转基因鼠,经点杂交筛选和DNA印迹鉴定,获得2只LAtPA基因整合阳性鼠,并在阳性母鼠乳汁检测到有溶纤活性的LAtPA表达,表达水平1.5μg/ml。在这两只转基因鼠的F1代子鼠中,9只中有5只是阳性的,tPA表达水平维持在1-2μg/ml,说明BLG-tPA融合基因整合到小鼠基因组,能稳定地遗传给子代。为今后利用牛、羊作为生物反应器表达LAtPA奠定了基础。

An exogenous gene was expressed in the mammary epithelium of transgenic mice in the hope that the encoded protein be able to secrete into milk. The transgenic mice carrying the promoter and upstream regulatory sequences from the sheep BLG gene which were fused to cDNA encoding human tissue plasmmogen activator (tPA) with its endogenous secretion signal sequence were generated. The hybrid genes were microinjected into mouse embryos. Two mouse were identified as being transgene by Southern blot. Milk obtained from lactating females contains biologically active tPA. and concentration was calculated to be about 1. 5 μ/ml. This result establishes the feasibility of secretion into the milk of transgenic animals for production of biologically active tPA proteins, and may provide a powerful method to produce such proteins on a large scale.