【作者】 丁一；

【导师】 张钦宪；

【作者基本信息】 郑州大学 ， 病理与病理生理学， 2003， 博士

【摘要】 血管栓塞性疾病是当前危害人类健康导致死亡的主要原因之一。目前，国产正式应用于临床溶栓治疗的纤溶酶原激活剂主要有尿激酶(Urokinase，UK)和链激酶(Streptokinase，SK)，而尿激酶和链激酶的溶栓效率有限且因无纤维蛋白结合特异性，易发生治疗性出血。组织型纤溶酶原激活剂(tissue-type plasminogen activator，tPA)是人体内生理性纤溶酶原激活剂，与尿激酶和链激酶相比，人tPA与血栓基质有很高的特异性亲和力。tPA能在血栓局部激活纤溶酶原，使其转化成具有生物活性的纤溶酶，后者可水解血栓的基质—纤维蛋白，使凝血块溶解，血管再通，并且不引起血纤溶酶原的系统性激活，因而大大降低了治疗过程中出血的危险，是一种用于治疗急性心肌梗死等血栓性疾病的特效药品。tPA除了参与体内溶血和凝血的平衡调控外，还与其它一些重要的生理与病理过程密切相关，如组织再造、细胞迁移、排卵、补体激活、激肽产生、肿瘤侵袭等。 正常人体组织内，tPA分布广，但含量非常低，如在血液中只有4-20 μg／L。在人体内半衰期短，仅4～5 min。因此从天然材料中很难大量制备tPA，用于研究和治疗。通过基因工程手段获取大量tPA是一条很有希望的途径，但各有其优缺点。大肠杆菌缺乏修饰酶类，真核生物蛋白翻译后无法进行磷酸化、酰胺化等加工修饰，不能正确折叠，缺乏分泌作用形成无活性包含体。借助逆转录病毒载体将目的基因导入细胞有潜在致癌性，并有引起宿主病毒感染的可能性。酵母提纯工艺复杂、成本高，产品纯度达不到要求。而且，由于tPA的临床应用剂量(每次80—100mg)的要求，从高效表达tPA的细胞培养液中生产tPA也远远不能满足市场需要。郑州人学2004届博}刃「究生论文t以乳腺特异性表达载体的构建及其瞬时表达 乳腺生物反应器(mamlnary gland bioreactor)技术是利用哺乳动物乳腺特异性表达的启动子元件构建转基因动物，指导外源基因在乳腺中表达，并从转基因动物的奶液中获取重组蛋白。利用乳腺生物反应器生产药用蛋白具有表达体系简单、可进行翻译后修饰及费用低廉等优点。乳腺生物反应器已成为生产重组蛋白的首选手段。 建立乳腺生物反应器的关键在于构建乳腺特异性表达载体。乳腺特异性表达载体由乳腺特异性启动子、目的基因和加PolyA信号三部分组成。目前己克隆并用作构建载体的乳蛋白基因主要包括日一乳球蛋白基因 (BLG)、酪蛋白基因和乳清酸蛋白基因(WAP)等。本研究中，首先将克隆的乳清酸蛋白基因(wAP)2.4kb启动子插入pBluescriPt质粒，构建质粒pw;然后将克隆的0.37kb加p01yA信号插入wAP启动子的下游，构建可指导外源基因在乳腺特异性表达的载体pwA;最后将克隆的目的基因2.IkbtPA CDNA连接于wAP启动子和加p。lyA信号之间，构建tpA乳腺特异性表达载体pWTA。构建成功的pWTA表达载体通过脂质转染胺试剂LipofeC七amine山川()()转染乳腺癌细胞系，通过体外注射转移至妊娠期小鼠乳腺，以原位杂交技术检测tPA在乳腺癌细胞系和泌乳期小鼠乳腺中的瞬时表达，验证PWTA表达载体的表达能力。材料和方法:1.载体pw的构建:以含WAP启动子的质粒WapZhGH为模板，设计引物(上、下游引物分别带有Nhel和Hindlll酶切位点)，PCR扩增WAP启动子DNA片段;以Nhel和Hindm对扩增产物进行双酶切，琼脂糖凝胶电泳回收2.4kb酶切片段。以Xbal和Hindlll双酶切pBlueseript质粒，琼脂糖凝胶电泳回收2.9了kb酶切片段。Nhel和Xbal粘性末端互补。以体外连接试剂盒连接2.4kb WAP启动子片段和2.97kb pBlueseript质粒DNA片段，16oC，连接过夜。重组体转化感受态JMIOg细菌。将转化菌涂于含Amp的培养皿，37℃，培养过夜。随机挑选10个克隆，接种于含AlnP的LB培养基，37℃，培养过夜。小提质粒，分别进行HindIJI单酶切和Hindlll/Sacll双酶切鉴定。构建成功的载体命名为pw。2.载体pwA的构建:以pcDNA3.O质粒为模板，设计引物(上、下游引物郑州人学2004届博日口}:究生论文t以乳腺特异性表达载体的构建及其瞬时表达分别带有Xhol和Kpnl酶切位点)，PCR扩增加polyA信号DNA片段。以xhol和Kpnl对扩增产物和PW质粒进行双酶切，琼脂糖凝胶电泳回收0.37kb和5.4kb酶切片段。体外连接0.37kb加polyA信号DNA片段和5.4kb pw质粒DNA片段。获得含重组体DNA阳性菌的方法同上。小提质粒，分别进行Xhol单酶切、Xhol/Kpnl双酶切及Hindlll/Sae 11双酶切鉴定。构建成功的载体命名为pwA。3.载体pWTA的构建:以含t PA cDNA的质粒pETPFR为模板，设计引物(上、下游引物分别带Hindlll和Sall酶切位点)，PCR扩增tPA eDNA片段。以Hindln和Sall对扩增产物及pWA质粒进行双酶切，琼脂糖凝胶电泳回收2.Ikb和5.skb酶切片段。体外连接2.Ikb tPA cDNA片段和5.skb pWA质粒DNA片段。获得含重组体DNA阳性菌的方法同上。小提质粒，分别以Hindlll单酶切、Hindlll/Sall双酶切、Hindlll/Sae 11双酶切及KPnl/SaCn双酶切进行鉴定。对插入片段进行序列测定。构建成功的载体命名为pwTA。4.载体pWTA的瞬时表达鉴定:以随机引物法地高辛标记tPA CDNA探针并进行灵敏度检测，一20℃保存。将表达?更多还原

【Abstract】 Thrombosis is one of diseases resulting in death in mankind; and urokinase and streptokinase are currently applied to clinical therapy. However, they have side reaction, such as hemorrhage due to the limited solving efficiency and the unspecific properties of solving thrombus. Comparing with UK and SK, tissue-type plasminogen activator (tPA) is one kind of natural fibrinolysis substances and possesses high specific affinity to the thrombus. The tPA can convert locally the inactive plasminogen into plasmin which is capable of dissolving the fibrin in blood clots and renewing the circulation. Since tPA is not correlated with systemic activation of plasminogen, it significantly decreases the risk of haemorrhage during treatment. Therefore, it is a drug with special effect used to the treatment of acute thrombotic disorders, such as myocardial infarction. In addition to revolving in regulating the balance of hemolysis and coagulation, it is closely concerned to some important physiological and pathological courses, such as tissue reforming, cell translocation, ovulation, activated peptide production, tumor invasion and so on.tPA is widely distributed in the normal tissue, yet the serum level is very low with a content of 4-20ug/L and the half life is very short for only 4-5 minutes. Therefore it is very difficult to obtain tPA from natural materials for research and therapy. It will be a promising way of gaining plenty of tPAthrough genetic engineering. Each method for producing recombinant proteins has its own virtues and defects. The proteins translated in the prokaryote, such as Escherichia coli, can not be modified by phosphorylation and acetaminolation because of lacking the modifying enzymes. Thus the proteins can not be secreted because they are folded incorrectly and formed into inactivated occlusion body. Purification of recombinant protein expressed in yeast is complicated and costly. The purity of produc can not meet the demands. The retroviral vector can transfer the exogenous gene into cell. Yet, it may activate oncogene and cause virus infection. According to the clinical dosage of tPA (80-1 OOmg/each t ime), p roduction t PA from m edium o f t he c ell 1 ine f or highly effective expression of tPA also can’t satisfy the clinical demand.Mammary gland bioreactor technique is to establish the transgenic animals by using the mammalian mammary-specific promoter, which can express the target gene in mammary gland and produce the recombinant protein in milk. Using mammary gland bioreactor to produce recombinant protein possesses virtues of simple expressional system, post-translation modification, low cost and so on. The mammary gland bioreactor has been the first choice for production of recombinant protein.The key point of establishment of mammary gland bioreactor is the construction of mammary-specific vector. The mammary-specific vector is composed of mammary-specific promoter, target gene and poly adenylation signal. The milk protein genes which have been cloned and used to construct vector m ainly ine lude b eta-lactoglobulin gene (BLG), c asein gene a nd whey acidic protein (WAP) gene and etc. In this paper, the cloned 2.4kb WAP promoter was inserted into pBluescript plasmid to construct pW plasmid firstly. Then, the cloned 0.37kb polyadenylation signal was inserted into the downstream of WAP promoter to construct vector pWA. Finally, the cloned target gene 2.1kb tPA cDNA was ligated between WAP promoter and polyadenylation signal to construct mammary-specific vector pWTA. The successfully constructed pWTA expression vector was transfected intomammary carcinoma cell line MCF-7 mediated by Lipofectamine?000 and into mouse mammary glands by direct injection. The expression level of pWTA was identified by transient expression of tPA in MCF-7 cell line and in lactating mice mammary glands demonstrated in situ hybridization.Materials and methods:1. Construction of pW vector: The WAP promoter DNA fragment was amplified by PCR using the Wap2hGH plasmid containing WAP promo更多还原