本文關(guān)鍵詞：兼具GPx2和ApSOD活性抗氧化酶的真核表達(dá)及活性研究　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 谷胱甘肽過氧化物酶 超氧化物歧化酶 雙功能酶 真核表達(dá)

【摘要】：活性氧(ROS)是機(jī)體生理代謝的產(chǎn)物,不同濃度的ROS可對機(jī)體產(chǎn)生有益或有害的影響。正常生理狀態(tài)下,ROS的產(chǎn)生和消除處于動態(tài)平衡狀態(tài),從而維持機(jī)體氧化和抗氧化的相對平衡。然而,隨著生態(tài)惡化、環(huán)境污染及現(xiàn)代化生活不規(guī)律的程度日趨加深,多種內(nèi)在及外在因素導(dǎo)致ROS的過量產(chǎn)生,當(dāng)過多的ROS不能及時清除,與之相伴的疾病也愈加嚴(yán)重和高發(fā),對人類的健康造成嚴(yán)重威脅。機(jī)體內(nèi)的抗氧化進(jìn)程是復(fù)雜而多變的,諸多抗氧化酶間的協(xié)同作用以維持該系統(tǒng)的平衡。其中超氧化物歧化酶(SOD)、過氧化氫酶(CAT)和谷胱甘肽過氧化物酶(GPx)這三種酶類抗氧化劑組成的酶學(xué)防御系統(tǒng),相互協(xié)同作用,通過不同的反應(yīng)有效地將機(jī)體內(nèi)多余超氧陰離子(O2·-)、過氧化氫(H2O2)和多種氫過氧化物清除。SOD可將O2·-轉(zhuǎn)化為H2O2,繼而被CAT、GPx等將其分解為水(H2O),從而使有害的O2·-和H2O2轉(zhuǎn)化為無害的水分子。如果兩種抗氧化酶的協(xié)同作用被破壞,可將ROS的損傷效應(yīng)放大,從而引發(fā)多種疾病。根據(jù)三者在清除ROS時的協(xié)同作用,我們認(rèn)識到獲得兼具兩種或多種抗氧化功能于一體的抗氧化酶,相比單一抗氧化酶,更有利于了解抗氧化酶協(xié)同體系的機(jī)制,且它也具有一定潛在的應(yīng)用價值。GPx對過氧化物類底物的廣泛識別和高效的催化使其在整個抗氧化系統(tǒng)中發(fā)揮著極其重要的作用,其活性中心是由終止密碼子UGA編碼的硒代半胱氨酸(Sec),Sec需要經(jīng)過特殊而復(fù)雜的翻譯機(jī)制才能插入到蛋白質(zhì)中。SOD作為機(jī)體重要的抗氧化酶,是機(jī)體對抗自由基的第一道防線,它可以專一性地催化O2·-發(fā)生歧化反應(yīng)生成O2和H2O2。因此,制備出兼具GPx2和ApSOD活性的抗氧化酶具有更好的科研價值和廣泛的應(yīng)用前景。本研究利用真核表達(dá)系統(tǒng)表達(dá)人胃腸道來源GPx(GPx2)和龐貝蠕蟲來源SOD(ApSOD)的融合蛋白hGPx2-linker-ApSOD,通過體外催化活性測定,確定所獲得的融合蛋白具有明顯的GPx活力和SOD活力。(1)融合蛋白編碼基因的設(shè)計。為了使hGPx2和ApSOD融合表達(dá)時,互相不干擾蛋白折疊從而避免高級結(jié)構(gòu)變化引起的活力喪失,我們通過一段柔性肽linker(GGGGSGGGGSGGGGS)的編碼基因?qū)GPx2和ApSOD的編碼基因連接,獲得融合蛋白hGPx2-linker-ApSOD的編碼基因,另外,在基因3’端設(shè)計含有His6標(biāo)簽編碼序列以方便蛋白表達(dá)后檢測及純化。(2)pSel Express1-hGPx2-linker-ApSOD表達(dá)質(zhì)粒的構(gòu)建分別以從人肝癌細(xì)胞株Hep G2的m RNA反轉(zhuǎn)錄獲取的c DNA和表達(dá)質(zhì)粒p Cold I-ApSOD作為模板,通過PCR分別獲得hGPx2-linker和linker-ApSOD的基因片段,再利用重疊PCR獲得hGPx2-linker-ApSOD的完整片段,使用限制性內(nèi)切酶Sal I/Xbal I將質(zhì)粒pSel Express1和hGPx2-linker-ApSOD基因片段雙酶切,再用T4 DNA Liagse將二者連接到一起構(gòu)建出pSel Express1-hGPx2-linker-ApSOD表達(dá)質(zhì)粒。測序鑒定所構(gòu)建質(zhì)粒序列與理論相符。(3)hGPx2-linker-ApSOD的表達(dá)與純化pSel Express1的多克隆位點下游包含一段硒代半胱氨酸插入序列(SECIS),目的基因轉(zhuǎn)錄后SECIS位于m RNA的3’非編碼區(qū),能夠有效的將hGPx2編碼區(qū)UGA指導(dǎo)翻譯為Sec。將重組質(zhì)粒轉(zhuǎn)染至HEK 293T細(xì)胞中進(jìn)行表達(dá)。利用金屬螯合親和層析(IMAC)法純化表達(dá)的可溶性重組蛋白hGPx2-linker-ApSOD,通過Western blot鑒定重組蛋白。(4)hGPx2-linker-ApSOD的GPx和SOD活力測定活力測定結(jié)果顯示,hGPx2-linker-ApSOD的GPx活力為14.9 U/μmol,為小分子模擬物Ebselen(0.99 U/μmol)GPx活力的15倍;該雙功能酶SOD的活力為149.3 U/mg,與本課題組在大腸桿菌中表達(dá)的ApSOD單功能酶的活力在同一數(shù)量級,但比SOD3的活力低一個數(shù)量級。綜上所述,本研究首次利用真核表達(dá)系統(tǒng)成功制備了兼具GPx和SOD活性的hGPx2-linker-ApSOD雙功能抗氧化酶,這為闡明雙功能酶的協(xié)同作用提供了分子基礎(chǔ),同時具有一定潛在的藥用價值。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R915

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