【作者】 王栋；

【导师】 杨桂文； 何成强；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2009， 硕士

【摘要】 人组织型纤溶酶原激活剂(tissue-type plasminogen activator,t-PA)是人体内存在的多区域丝氨酸蛋白酶,它可以特异性地激活纤溶酶原转变为纤溶酶,后者能在血栓局部有效地溶解血栓中的纤维蛋白,使血管再通,是治疗血栓性疾病的特效药。正常人体组织内,t-PA分布广,但含量非常低。随着科学技术的发展,t-PA的生产也经历了从原组织中提取、原核生物表达、真核细胞表达和动物乳腺生物反应器表达等几个发展阶段。目前动物乳腺生物反应器制备t-PA已实现产业化,但是仍存在整合率低,转基因动物遗传不稳定,转基因动物生长周期长等问题。病毒载体是目前转移基因的最优手段之一。慢病毒载体因为可以介导外源基因在非分裂细胞中稳定高效地表达而成为转基因动物和基因治疗研究的重要工具。在本课题中,我们拟克隆人t-PA酶活性区域,然后构建慢病毒载体,通过四质粒包装体系生产慢病毒颗粒,从而为下一步研制转基因高等模式动物及基因功能的研究提供条件。本研究通过高效Trizol试剂快速从黑色素瘤细胞中提取总RNA,自行设计引物采用RT- PCR获得t-PA基因酶活性区域(K2区和P区) cDNA,经测序鉴定后与GenBank报道序列一致。为获得CMV强启动子,将该基因片段用BamHⅠ、HindⅢ双酶切后,连入pEGFP-N3质粒中,采用脂质体2000转染NIH 3T3真核细胞,经荧光显微镜观察,在转染后24 h有t-PA-EGFP蛋白表达,产生较强的绿色荧光,证实载体构建成功,为下一步构建慢病毒载体奠定基础。采用已构建的pEGFP-N3-t-PA质粒,经PCR克隆获得CMV-t-PA片段。应用基因重组技术,双酶切pLenti质粒和目的CMV-t-PA片段后进行连接,构建pLenti-CMV-t-PA慢病毒质粒。同时提取pLenti-CMV-t-PA质粒和其他三个包装质粒,并测定其浓度,使用磷酸钙共沉淀法将其转染293T细胞,在细胞中生产慢病毒颗粒,取上清用聚乙二醇均相沉淀法浓缩病毒。通过观察病毒感染293T细胞后细胞表达EGFP阳性克隆数,计算得知病毒滴度均在1.5×107 TU/mL左右。将纯化后的病毒感染293T细胞并传代,经荧光显微镜观察有绿色荧光,同时用t-PA基因特异性抗体经Western blot检测细胞中有t-PA蛋白表达,且分子量为35.93kDa符合预测的蛋白大小,未感染病毒的细胞组无表达。综上所述,利用RT-PCR方法克隆得到t-PA酶活性区域(K2区和P区) cDNA,构建的重组真核表达质粒能在特定细胞中表达外源基因。随后进一步制备的慢病毒载体经过病毒包装系统,成功包装出病毒颗粒,经GFP荧光法检测其具有较高的病毒滴度。将纯化的病毒颗粒感染细胞后,通过RNA水平和特异性抗体的Western blot检测,证实目的基因插入到染色体上,并随细胞传代而转录表达,说明慢病毒载体及病毒颗粒制备成功。这为研制转基因高等模式动物的建立提供了物质基础。更多还原

【Abstract】 Tissue-type plasminogen activator(t-PA) is a key enzyme in fibrinolysis system, the physiological role of which is to convert zymogen plasminogen into an active form of serine proteinase plasmin,which then initiates or accelerates the process of fibrinolysis and degrades the fibrin network of thrombi and blood clots, is the medicine for the therapy of thrombotic diseases. t-PA is widely distributed in the normal tissue. With the development of science technology, the manufacture of t-PA goes through many stages of development which included the original tissue extracting, prokaryotic expression, eukaryotic expression and animal mammary gland bioreactor expression. At present, although the manufacture of t-PA by animal mammary gland bioreactor expression has achieved industrialization, there were certain defect, for example the problem of the low rate of integration, transgenic animal genetic instability, genetic instability of transgenic animal, growth cycle length of transgenic animal and so on.Now optimal means for transgenic are the usage of virus vectors. As one of them, lentivirus provides effective means for expression of exogenous genes in mammalian cells, which had proved efficient and achieced stable long-term expression of the transgenes, so it was developed as an important transgenic vector for transgenic animals and gene therapy. In this research, we cloned the activity region of human t-PA cDNA, constructed t-PA lentiviral vector and produced high-titer lentivirus by four-plasmid system in 293T cells. These works provide the basis for generating t-PA transgenic animal.The t-PA activity regional cDNA was obtained from melanoma cell through RT-PCR and confirmed by DNA sequence analysis. In order to get strong promoter,we firstly construced pEGFP-N3-t-PA vectors. And then the t-PA lentiviral vectors were generated by subcloning recombinant technology and confirmed by restricition endonuclease analysis.In order to package virus, we extracted and purified recombinant plasmid and the else three packaging plasmids and measured the density of plasmids. The four-plasmid system was co-transformed into 293T cell for lentivirus production. The lentivirus in cell culture medium were collected, concentrated by PEG and quantified. The generated recombinant virus was used to infect 293T cell, detecting the number of positive clone of EGFP to calculate the virulence,which grossly was 1.5×107 TU/mL. The expression of t-PA in 293T cells was detected by RT- RCR and Western blot, and the molecular weight of the recombinated protein was 35.93kDa as our forecast. As a conclusion, the t-PA cDNA was successfully cloned using RT-PCR method from melanoma cell.The recombinant t-PA lentiviral vectors were successfully constructed by the method of molecular cloning and recombinant technics.The GFP protein was dectected by fluorescent microscope and t-PA protein was dectected by RT-PCR and Western blotting analysis in 293T cell.High-titer lentivirus was successfully produced and identified to be able to generate the transgenic animal.更多还原