利用Red系统构建人组织型纤溶酶原激活剂突变体BAC乳腺特异表达载体

发布时间：2018-03-18 23:34

本文选题：人组织型纤溶酶原激活剂突变体(ht-PA_m)　切入点：转基因动物乳腺生物反应器　出处：《中国人民解放军军事医学科学院》2005年硕士论文　论文类型：学位论文

【摘要】：组织型纤溶酶原激活剂(t-PA)是治疗血栓栓塞性疾病的重要药物。本项目的t-PA突变体,活性提高,半衰期延长,已成功获得表达。目前t-PA主要来自于哺乳动物细胞大规模培养,成本高昂,价格昂贵。利用动物乳腺生物反应器来生产不仅维持生产的成本低,而且产量高,能够进行翻译后修饰、正确折叠。但位置效应的影响是其主要技术瓶颈之一。细菌人工染色体容量大,可装载乳蛋白基因的所有调控序列,乳蛋白基因座中的一些边缘序列具有绝缘子的功效,能使乳蛋白基因表达调控不受周围序列的影响,作为转基因载体就有可能克服外源基因的位置效应。本研究主要构建了由β-酪蛋白基因调控序列指导的t-PA突变体BAC转基因载体。采用了λ噬菌体Red同源重组系统介导的同源重组方法对含有鼠β-酪蛋白基因的RPCI23-440C1 BAC进行了快速改构。这种方法要明显优于传统的——利用微生物本身的RecA重组系统进行重组的方法。首先通过PCR方法,获得两端带有鼠β-酪蛋白基因同源序列的tPAm-Zeo和tPAm-BGHpA-Zeo同源重组片段,将其电击转化至已含有编码Red重组酶质粒和BAC的宿主菌。在λRed重组系统的帮助下,通过同源重组片段两端与β-酪蛋白同源的序列在体内与β-酪蛋白基因发生同源重组,将其置换。经Southern blot和测序鉴定,得到了t-PAm基因定点敲入的tPAm-Zeo/tPAm-BGHpA-Zeo BAC转基因载体。最后通过FLP位点专一性重组,利用Zeocin抗性基因两侧的FRT位点,将抗性基因剔除。Southern blot和序列分析鉴定,获得了无抗性基因且编码两种重组酶质粒丢失的t-PAm BAC载体。最后利用上述两种转基因载体分别从细胞和动物水平初步验证了其有效性。由此建立了快速高效的BAC重组技术,获得了转基因所需的BAC载体,为转基因动物以及动物乳腺生物反应器的制备提供了新的方法,这一方法的建立也为利用BAC研究基因调控和功能提供了新途径。
[Abstract]:Tissue plasminogen activator (t-PA) is an important drug in the treatment of thromboembolic diseases. The activity of t-PA mutants in this project has been increased, the half-life of t-PA has been prolonged, and t-PA has been successfully expressed. At present, t-PA is mainly derived from the large-scale culture of mammalian cells. Using animal mammary gland bioreactor not only to maintain the cost of production, but also high production, can be modified after translation, Correct folding. But the influence of position effect is one of the main technical bottlenecks. Bacteria artificial chromosome capacity, can carry all the milk protein gene regulatory sequence, milk protein gene in some of the edge sequence has insulator effect, It can make the regulation of lactoprotein gene expression not affected by the peripheral sequence. As a transgenic vector, it is possible to overcome the position effect of exogenous gene. In this study, t-PA mutant BAC transgenic vector guided by 尾 -casein gene regulatory sequence was constructed. RPCI23-440C1 BAC containing mouse 尾 -casein gene was transfected by RPCI23-440C1 BAC mediated by 位 phage Red homologous recombination system. This method is obviously superior to the traditional method of recombination using the RecA recombination system of the microorganism itself. First of all, through the PCR method, TPAm-Zeo and tPAm-BGHpA-Zeo homologous recombination fragments with mouse 尾 -casein gene homologous sequences were obtained and transformed into host bacteria containing recombinant plasmid encoding Red and BAC. With the help of 位 Red recombination system, 尾 -casein gene was homologous recombined with 尾 -casein gene at both ends of homologous recombination fragment in vivo, and was replaced by Southern blot and sequencing. The tPAm-Zeo/tPAm-BGHpA-Zeo BAC transgenic vector with t-PAm gene knockout was obtained. Finally, by the specific recombination of FLP locus, the resistance gene was eliminated by FRT locus on both sides of Zeocin resistance gene and identified by sequence analysis. The t-PAm BAC vector with no resistance gene and encoding two recombinant enzyme plasmids was obtained. Finally, the effectiveness of t-PAm BAC vector was preliminarily verified by the above two transgenic vectors at the cell and animal levels, respectively. Therefore, a rapid and efficient BAC recombination technique was established, and the BAC vector for transgenic animals was obtained, which provided a new method for the preparation of transgenic animals and mammary gland bioreactor. The establishment of this method also provides a new way to study gene regulation and function by using BAC.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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