兼具GPx2和ApSOD活性抗氧化酶的真核表达及活性研究

发布时间：2018-01-18 02:10

本文关键词：兼具GPx2和ApSOD活性抗氧化酶的真核表达及活性研究　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：活性氧(ROS)是机体生理代谢的产物,不同浓度的ROS可对机体产生有益或有害的影响。正常生理状态下,ROS的产生和消除处于动态平衡状态,从而维持机体氧化和抗氧化的相对平衡。然而,随着生态恶化、环境污染及现代化生活不规律的程度日趋加深,多种内在及外在因素导致ROS的过量产生,当过多的ROS不能及时清除,与之相伴的疾病也愈加严重和高发,对人类的健康造成严重威胁。机体内的抗氧化进程是复杂而多变的,诸多抗氧化酶间的协同作用以维持该系统的平衡。其中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPx)这三种酶类抗氧化剂组成的酶学防御系统,相互协同作用,通过不同的反应有效地将机体内多余超氧阴离子(O2·-)、过氧化氢(H2O2)和多种氢过氧化物清除。SOD可将O2·-转化为H2O2,继而被CAT、GPx等将其分解为水(H2O),从而使有害的O2·-和H2O2转化为无害的水分子。如果两种抗氧化酶的协同作用被破坏,可将ROS的损伤效应放大,从而引发多种疾病。根据三者在清除ROS时的协同作用,我们认识到获得兼具两种或多种抗氧化功能于一体的抗氧化酶,相比单一抗氧化酶,更有利于了解抗氧化酶协同体系的机制,且它也具有一定潜在的应用价值。GPx对过氧化物类底物的广泛识别和高效的催化使其在整个抗氧化系统中发挥着极其重要的作用,其活性中心是由终止密码子UGA编码的硒代半胱氨酸(Sec),Sec需要经过特殊而复杂的翻译机制才能插入到蛋白质中。SOD作为机体重要的抗氧化酶,是机体对抗自由基的第一道防线,它可以专一性地催化O2·-发生歧化反应生成O2和H2O2。因此,制备出兼具GPx2和ApSOD活性的抗氧化酶具有更好的科研价值和广泛的应用前景。本研究利用真核表达系统表达人胃肠道来源GPx(GPx2)和庞贝蠕虫来源SOD(ApSOD)的融合蛋白hGPx2-linker-ApSOD,通过体外催化活性测定,确定所获得的融合蛋白具有明显的GPx活力和SOD活力。(1)融合蛋白编码基因的设计。为了使hGPx2和ApSOD融合表达时,互相不干扰蛋白折叠从而避免高级结构变化引起的活力丧失,我们通过一段柔性肽linker(GGGGSGGGGSGGGGS)的编码基因将hGPx2和ApSOD的编码基因连接,获得融合蛋白hGPx2-linker-ApSOD的编码基因,另外,在基因3’端设计含有His6标签编码序列以方便蛋白表达后检测及纯化。(2)pSel Express1-hGPx2-linker-ApSOD表达质粒的构建分别以从人肝癌细胞株Hep G2的m RNA反转录获取的c DNA和表达质粒p Cold I-ApSOD作为模板,通过PCR分别获得hGPx2-linker和linker-ApSOD的基因片段,再利用重叠PCR获得hGPx2-linker-ApSOD的完整片段,使用限制性内切酶Sal I/Xbal I将质粒pSel Express1和hGPx2-linker-ApSOD基因片段双酶切,再用T4 DNA Liagse将二者连接到一起构建出pSel Express1-hGPx2-linker-ApSOD表达质粒。测序鉴定所构建质粒序列与理论相符。(3)hGPx2-linker-ApSOD的表达与纯化pSel Express1的多克隆位点下游包含一段硒代半胱氨酸插入序列(SECIS),目的基因转录后SECIS位于m RNA的3’非编码区,能够有效的将hGPx2编码区UGA指导翻译为Sec。将重组质粒转染至HEK 293T细胞中进行表达。利用金属螯合亲和层析(IMAC)法纯化表达的可溶性重组蛋白hGPx2-linker-ApSOD,通过Western blot鉴定重组蛋白。(4)hGPx2-linker-ApSOD的GPx和SOD活力测定活力测定结果显示,hGPx2-linker-ApSOD的GPx活力为14.9 U/μmol,为小分子模拟物Ebselen(0.99 U/μmol)GPx活力的15倍;该双功能酶SOD的活力为149.3 U/mg,与本课题组在大肠杆菌中表达的ApSOD单功能酶的活力在同一数量级,但比SOD3的活力低一个数量级。综上所述,本研究首次利用真核表达系统成功制备了兼具GPx和SOD活性的hGPx2-linker-ApSOD双功能抗氧化酶,这为阐明双功能酶的协同作用提供了分子基础,同时具有一定潜在的药用价值。
[Abstract]:Reactive oxygen species (ROS) is a product of the body's physiological metabolism, different concentrations of ROS can produce beneficial or harmful effect on the body. Under normal physiological conditions, the generation and elimination of ROS in a state of dynamic balance, so as to maintain the relative balance between oxidation and anti-oxidation. However, with the ecological deterioration, environmental pollution and modern life the law degree deepening, excessive production of various internal and external factors leading to ROS, when too much ROS can not be cleared, associated with the disease has become more serious and frequent, poses a serious threat to human health. In vivo antioxidant process is complex and changeable, the synergistic effect between many antioxidant enzymes in to maintain the balance of the system. The superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) of the three enzymes antioxidant enzyme composed of defense system, coordinate The same effect by different reaction effectively in the body of excess superoxide anion (O2 -), hydrogen peroxide (H2O2) and hydrogen peroxide scavenging.SOD - O2 can be transformed into H2O2, then was CAT, GPx was decomposed into water (H2O), so that the harmful O2 - and H2O2 into harmless water molecules. If two antioxidant enzymes synergy is damaged, can be amplified ROS damage, which causes a variety of diseases. According to the three ROS in the removal of synergy, we recognize that both antioxidant obtained two or more antioxidant functions, compared to a single antioxidant more conducive to understanding the mechanism of anti oxidase system, and it also has a certain potential application value of.GPx wide recognition of peroxides and efficient catalytic substrate makes it play a very important role in the antioxidant system, its activity center Is a stop codon UGA encoding selenocysteine (Sec), Sec requires special and complex mechanisms can be translated into Anti oxidase to the protein.SOD as an important body, the body is the first line of defense against free radicals, which can selectively catalyze O2 - generating disproportionation reaction of O2 and H2O2. therefore, prepared with GPx2 and ApSOD activity of antioxidant enzyme has better research value and wide application prospect. This study used eukaryotic expression system for expression of human gastrointestinal GPx (GPx2) and Pompeii worm source SOD (ApSOD) fusion protein hGPx2-linker-ApSOD by catalytic activity in vitro was determined by the the fusion protein has obvious GPx activity and SOD activity. (1) the design of fusion protein encoding gene. In order to make the hGPx2 and expression of ApSOD fusion protein folding, do not interfere with each other so as to avoid advanced structural changes The loss of vitality, we through a flexible peptide linker (GGGGSGGGGSGGGGS) encoding gene encoding genes of the hGPx2 and ApSOD connection, obtained gene encoding fusion protein of hGPx2-linker-ApSOD gene 3 'end also in design with the His6 tag encoding sequence to facilitate protein expression after the detection and purification of pSel Express1-hGPx2-linker-ApSOD expression (2). To construct m obtained from human hepatocellular carcinoma cell line RNA G2 Hep C of reverse transcription and expression of DNA plasmid P Cold I-ApSOD plasmid as template respectively, were obtained through PCR hGPx2-linker and linker-ApSOD gene fragments, complete fragment by overlapping PCR hGPx2-linker-ApSOD, using restriction endonuclease Sal plasmid pSel Express1 and I/Xbal I the hGPx2-linker-ApSOD gene fragment was digested with T4 DNA Liagse two will be connected to construct the pSel expression of Express1-hGPx2-linker-ApSOD Plasmid sequencing. The constructed plasmid sequence consistent with the theory. (3) hGPx2-linker-ApSOD expression and purification of pSel Express1 multiple cloning sites downstream contains a selenocysteine insertion sequence (SECIS), gene transcription after SECIS in M RNA 3 'non encoding region, it can be effective to hGPx2 encoding region of UGA guidance to Sec. the recombinant plasmids were transfected into HEK 293T cells. Using metal chelate affinity chromatography (IMAC) method for expression and purification of soluble recombinant protein hGPx2-linker-ApSOD by Western blot identification of recombinant protein. (4) hGPx2-linker-ApSOD GPx and SOD activity assay and activity determination showed that the hGPx2-linker-ApSOD activity of GPx was 14.9 U/ mol. For the small molecule analogue Ebselen (U/ 0.99 mol) 15 times GPx activity; the bifunctional enzyme activity of SOD was 149.3 U/mg, and the expression of the Escherichia coli ApSOD in a single work The enzyme activity in the same order of magnitude, but the activity of SOD3 than an order of magnitude lower. In summary, this study is the first to use the eukaryotic expression system was successfully prepared with both GPx and SOD activity of hGPx2-linker-ApSOD Bi functional antioxidant enzymes, which provide a molecular basis for the synergistic effect of clarifying bifunctional enzyme, also has medicinal the value of a certain potential.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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