SOD1启动子上调多肽筛选体系的建立

发布时间：2018-03-11 20:31

  本文选题：药物筛选　切入点：细胞穿透肽　出处：《南方医科大学》2008年硕士论文　论文类型：学位论文

【摘要】：超氧化物岐化酶(super oxide dismutase,SOD)是一种广泛存在于生物体内的金属酶,根据分布的不同主要分为SOD1(Cu/Zn SOD,主要分布于真核细胞胞浆),SOD2(Mn/Fe SOD,主要分布于真核细胞线粒体和原核细胞)和SOD3(EC-SOD,主要分布于细胞外基质),不同的SOD均有催化超氧阴离子自由基O_2~-发生歧化反应的功能,是体内重要的氧自由基清除剂。其中,SOD1是发现最早,研究最广泛的一类。 氧自由基是直接或间接由分子氧转化而成,外层轨道具有未配对电子的一类化学分子,包括超氧离子自由基、羟自由基、过氧化氢等。它们具有活泼的化学活性,极易发生得到或失去电子的反应,从而引发链式自由基反应。体内的氧自由基包括由于高温、辐射等原因导致某些含氧分子共价键断裂而产生的外源性自由基和体内各种代谢过程中产生的内源性氧自由基。氧自由基是机体代谢正常的中间产物,低浓度的氧自由基是机体执行正常生理功能所必需,但在氧自由基异常增多或机体抗氧化能力下降的情况下,氧自由基将通过链式自由基反应损伤细胞膜、DNA、蛋白质等多种细胞组分。目前已知氧自由基与衰老、炎症、缺血再灌注损伤、神经退行性病变等多种疾病相关,清除多余的氧自由基对延长动物寿命,增强其对氧应激的承受能力和改善炎症状况等多个方面具有显著的作用。 机体主要通过两阶段的反应来清除体内的氧自由基:第一阶段是超氧阴离子自由基在SOD的作用下转变为氧气和过氧化氢;第二阶段是过氧化氢在过氧化氢酶(catalase,CAT)的作用下转变为水和氧气。SOD在氧自由基的清除过程中起着至关重要的作用。SOD缺陷的大肠杆菌将无法在有氧条件下生存,而适量增加的SOD则能起到减少细胞内氧自由基含量,减轻脂质体过氧化乃至延缓衰老、控制炎症等多种作用。可见,通过控制SOD在体内的含量,可以调节氧自由基的浓度并从而改变多种相关的病理、生理状态,具有极其重要的临床意义。 皮肤皱纹的产生机制包括外源性因素和内源性因素两个方面。外源性因素主要有日光照射所致的光老化和其他环境因素如外伤、感染、空气污染等对皮肤的损伤;内源性因素主要有年龄增长导致的基因调控变化、内源性自由基对细胞的损伤和机体代谢的紊乱。目前国际上主要针对光老化和内源性氧自由基对皮肤的损伤这两个方面进行抗皱研究。光老化指日光长期照射所致的皮肤老化,日光中的紫外线可通过产生高度反应的外源性氧自由基损伤皮肤。此外,紫外线最早损伤的部位为调节SOD和过氧化氢酶的基因,使二者合成减少,致使氧自由基聚集而损伤细胞。由上述可见,在皮肤皱纹产生的内源性和外源性途径中,过高浓度的氧自由基都扮演着重要角色。因此,清除体内过高的氧自由基对于皮肤抗皱具有重要的意义。如果能适度提高皮肤细胞内SOD含量,则可以通过减少氧自由基的含量来减少对皮肤的损伤,从而保护皮肤,减少皱纹。 虽然针对SOD的研究已有多年,但目前还没有能够实际进入临床应用的SOD类药物,这是因为SOD作为一种蛋白质药物,具有制备价格昂贵,不能直接进入细胞发挥作用、半衰期短、易诱发免疫反应等缺陷,且具体使用剂量也不易控制。目前针对SOD药用方面的研究主要包括研究药物载体、发现SOD的小分子替代物和寻找能上调内源性SOD表达药物等途径,据此,我们设计了一个利用报告基因技术筛选具有上调内源性SOD1表达的随机多肽筛选系统,试图同时克服上述多种问题。 报告基因是指其表达产物易被检测且易与内源性背景蛋白相区别的基因。通过将特定基因的转录控制元件克隆到报告基因载体,可以通过报告基因的表达来直观地检测目标基因的表达情况。红色荧光蛋白(red fluorenscent protein,RFP)是一种分子量为28 kD的荧光蛋白,可自动发射荧光、无需任何辅助因子和底物的参与,且无需裂解细胞即可检测活体组织和细胞的基因表达,作为报告基因具有其特有的优越性。 本研究利用基因重组技术构建了含人SOD1启动子区的红色荧光蛋白报告基因质粒,并通过转染和抗生素筛选技术得到了能稳定表达该质粒的细胞系。此细胞系在适宜浓度的SOD1诱导剂卟啉醇肉豆蔻酸乙酸酯(phorbol myristateacetate,PMA)刺激下,红色荧光蛋白表达明显增加,红色荧光强度在一定范围内呈线性增长。这为筛选具有内源性SOD1表达上调功能的药物提供了一个方便、灵敏的工具。 为了克服蛋白质大分子常具有的免疫原性,我们使用连续的十二个随机氨基酸组成的多肽作为筛选的对象。由于细胞膜具有选择性通透性,大部分蛋白质分子无法直接进入细胞内部发挥生理学效应。我们选用Ⅰ型人类免疫缺陷病毒(human immunodeficiency virusⅠ,HIV-Ⅰ)Tat蛋白的蛋白转导域(proteintransduction domain,PTD)作为携带多肽入胞的载体。HIV-Tat是一种以非传统内吞途径、非能量依赖,非受体介导方式直接穿透细胞膜进入细胞的多肽,它能携带多种不同大小和性质的生物活性物质如核苷酸、多肽、蛋白质等进入细胞,是一种理想的多肽转运载体。此外,融合蛋白中的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)可作为蛋白入胞的指示剂,提示随机多肽在细胞内的分布情况。针对肽库的容量性和多样性问题,我们利用点突变技术引入36个随机碱基,形成随机表达文库,其理论容量达到20~(12),从而保证了随机多肽库的容量性和多样性。 为了能够进行高通量的多肽筛选,我们总结出一套切实可行的蛋白纯化及药物筛选流程。利用融合蛋白上的His标签纯化融合蛋白,将纯化后的融合蛋白与稳定表达报告基因质粒的细胞孵育一定时间后,融合蛋白进入细胞,通过测量细胞荧光强度的变化来判断多肽对SOD1启动子的影响,从而为后续大规模的筛选奠定了基础。 综上所述,本研究利用报告基因技术和细胞系筛选技术得到了能作为人SOD1启动子表达情况检测标准的细胞系,并利用基因重组和点突变技术得到了包含随机多肽序列、跨膜转运载体和示踪用绿色荧光蛋白的随机多肽库,在两者的基础上开发出一套能够用以筛选上调内源性SOD1表达的随机多肽药物的筛选流程,从而为后续的药物筛选工作提供了有力的支持。
[Abstract]:Superoxide dismutase (super oxide, dismutase, SOD) is a kind of metal enzyme widely existed in organisms, according to the different distribution of mainly divided into SOD1 (Cu/Zn SOD, mainly distributed in the cytoplasm of eukaryotic cells (Mn/Fe), SOD2 SOD, is mainly distributed in eukaryotic and prokaryotic cells and mitochondria) SOD3 (EC-SOD, mainly distributed in the extracellular matrix, SOD) can catalyze the superoxide anion free radical O_2~- dismutation reaction function is an important oxygen free radical scavenger in vivo. Among them, SOD1 was the first discovered and studied widely.
Oxygen free radicals are directly or indirectly by molecular oxygen into, the outer track has a class of chemical molecules with unpaired electrons, including superoxide anion free radical, hydroxyl radical, hydrogen peroxide. They are chemically active and lively, prone to gain or lose electrons reaction, causing a chain reaction of free radical oxygen. Including free radicals because of high temperature, endogenous oxygen free radicals of exogenous free radicals and various metabolic processes inside radiation causes some oxygen molecules resulting in the covalent bond. Oxygen free radicals are often intermediate body on behalf of Xie Zheng, a low concentration of oxygen free radical is the body's normal physiological function of the executive necessary, but the decline in the number of abnormal oxygen free radicals and antioxidant ability under the condition of oxygen free radicals by reacting damage to the cell membrane, free radical chain DNA, proteins and other cellular components. Previously known oxygen free radicals are related to many diseases, such as aging, inflammation, ischemia reperfusion injury, neurodegenerative diseases, etc., clearing excess oxygen free radicals plays a significant role in prolonging the life span of animals, enhancing their tolerance to oxygen stress and improving the inflammatory status.
The main body of oxygen free radicals through the two stage of the reaction to remove the body: the first stage is the superoxide anion radical into oxygen and hydrogen peroxide in the presence of SOD; the second stage is the hydrogen peroxide in catalase (catalase, CAT) under the action of water and oxygen gas into.SOD in the removal of oxygen free radicals in the process the role of.SOD critical defects of Escherichia coli cannot survive under aerobic conditions, and increase the amount of SOD can be used to reduce the content of oxygen free radical in cells, reduce lipid peroxidation and aging, control of inflammation and other role. Therefore, by controlling the content of SOD in the body, can adjust the oxygen concentration free radical and pathological changes, several related physiological condition, it has important clinical significance.
Mechanism of skin wrinkles includes two aspects of exogenous and endogenous factors. Exogenous factors are mainly caused by sunlight light aging and other environmental factors such as trauma, infection, injury of air pollution on the skin; the main factors of endogenous gene regulation changes age caused by endogenous free radical damage to cells and the body's metabolic disorders. The mainly injury to skin photoaging and endogenous oxygen free radicals in the two aspects of Anti Wrinkle research. Light aging refers to sunlight long-term exposure caused by skin aging, on the ultraviolet rays can be produced by highly reactive exogenous oxygen free radical damage to the skin. In addition, the earliest ultraviolet the site of injury to the regulation of SOD and catalase genes, two reduced the synthesis of oxygen free radicals, resulting in accumulation of damaged cells. It can be seen from the above, the skin wrinkles The endogenous and exogenous pathway, oxygen free radical concentration plays an important role. Therefore, to clear the body of excessive oxygen free radical has an important significance for the skin wrinkle. If can appropriately increase the content of SOD in skin cells, can reduce skin damage by reducing the content of oxygen free radicals. In order to protect the skin, reduce wrinkles.
Although the SOD has been studied for many years, SOD class of drugs but not yet able to enter the actual clinical application, this is because the SOD as a protein drug, which is prepared by the price is expensive, can not directly enter the cells, short half-life, easy to induce immune response and other defects, and the specific dosage is not easy to control. The present research on SOD mainly includes the study of pharmaceutical drug carrier, the discovery of small molecules and looking for alternative SOD could up regulate the expression of endogenous SOD drugs and other ways, accordingly, we design a report by gene technology screening of random peptide screening system expression of endogenous SOD1, while trying to overcome the various problems.
The report refers to the gene and its expression product is easy to detect and easy to distinguish background and endogenous protein gene. The gene specific transcriptional control element was cloned into the reporter gene vector, can directly detect the expression of target genes through the expression of a reporter gene. The red fluorescent protein (red fluorenscent, protein, RFP) is a fluorescent protein with a molecular weight of 28 kD, which can automatically emit fluorescence, without any cofactor and substrate involved, and the expression can be detected without cell lysis in vivo tissue and cell gene as a reporter gene has its unique advantages.
The establishment of red fluorescent protein reporter gene plasmid containing human SOD1 promoter by gene recombination technology, and through the transfection and antibiotic screening technology has been to the plasmid stable expression cell lines. This cell line induced phorbol myristate acetate in the appropriate concentration of SOD1 (phorbol myristateacetate PMA) stimulation. The expression of red fluorescent protein increased significantly, red fluorescence intensity increases linearly in a certain range. It provides a convenient for the screening of drugs with endogenous SOD1 expression function, sensitive tool.
In order to overcome the immunogenicity of protein molecules is, we use twelve random amino acid polypeptide composed of sequential as screening objects. Because the cell membrane with selective permeability, most protein molecules can not directly enter the cell play a physiological effect. We use the human immunodeficiency virus type 1 (human immunodeficiency, virus I, HIV- I) the protein transduction domain of Tat protein (proteintransduction domain, PTD.HIV-Tat) as a carrier carrying polypeptide into cells is a kind of non conventional endocytic pathways, non energy dependent, non receptor mediated directly through cell membrane and enter the cell polypeptide, it can carry a variety of different sizes and properties of bioactive substances such as nucleotide. Peptides, proteins into cells, is a kind of ideal peptide transporter. In addition, the fusion of enhanced green fluorescent protein (enha protein in nced green fluorescent protein,EGFP)鍙�浣滀负铔嬬櫧鍏ヨ優鐨勬寚绀哄墸�

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