本文選題：rMPN668 + 有機(jī)氫過氧化物抗性蛋白��； 參考：《南華大學(xué)》2011年碩士論文

【摘要】：目的:研究肺炎支原體(Mycoplasma pneumoniae,Mp)重組MPN668蛋白是否具有降解有機(jī)氫過氧化物(Organic Hydroperoxide,OHP)的能力,并通過分子模擬解析rMPN668的結(jié)構(gòu),初步分析其結(jié)構(gòu)與酶活性的關(guān)系,以進(jìn)一步了解Mp的致病機(jī)制。 方法:以Mp129株基因組DNA為模板,PCR法擴(kuò)增目的基因mpn668,將其亞克隆至pGEX-6P-1載體中。經(jīng)鑒定后將其轉(zhuǎn)化至表達(dá)菌E.coli BL21中進(jìn)行誘導(dǎo)表達(dá)。采用SDS-PAGE和Western blotting等方法對(duì)表達(dá)產(chǎn)物進(jìn)行分析鑒定并純化GST融合蛋白;切除重組蛋白GST標(biāo)簽后,采用氧化鐵二甲酚橙(Ferrous Oxidation-Xylenol Orange ,FOX)實(shí)驗(yàn)檢測其氫過氧化物酶活性。固體培養(yǎng)Mp,隨后將其置于氧化應(yīng)激條件下培養(yǎng)3~5d,RT-PCR檢測mpn668 mRNA的表達(dá)水平。利用同源建模和分子動(dòng)力學(xué)等方法,通過軟件Expasy與Visual Molecular Dynamics(VMD)模擬rMPN668的分子結(jié)構(gòu),并對(duì)其已預(yù)測出的酶活性位點(diǎn)的氨基酸進(jìn)行定點(diǎn)突變,通過誘導(dǎo)表達(dá)獲得△rMPN668突變蛋白, FOX實(shí)驗(yàn)測定其氫過氧化物酶活性。 結(jié)果:成功擴(kuò)增出總長度為423bp的mpn668基因,所構(gòu)建的原核重組質(zhì)粒經(jīng)PCR、雙酶切以及測序鑒定與預(yù)期目的基因相符。SDS-PAGE顯示,IPTG可誘導(dǎo)一分子量約為41KD的可溶性GST融合蛋白,經(jīng)GST?Bind? Purification Kit純化后,純度可達(dá)95%以上。FOX實(shí)驗(yàn)顯示,經(jīng)Prescission protease切除GST標(biāo)簽的rMPN668能降解有機(jī)氫過氧化物叔丁基過氧化氫( tert-butyl hydroperoxide,t-BHP)以及無機(jī)H2O2,其降解能力與rMPN668濃度呈正相關(guān);催化t-BHP時(shí),20ng/μL rMPN668在2min內(nèi)降解約500μM t-BHP,而催化H2O2反應(yīng)時(shí),催化效率遠(yuǎn)低于此水平,200ng/μL rMPN668降解500μM H2O2需要35min以上。RT-PCR分析表明,Mp在不同濃度的t-BHP(0.05M~1.00M)和氫過氧化枯烯(cumene hydroperoxide,CHP)(0.05M~1.00M)條件下,mpn668基因表達(dá)水平隨著t-BHP和CHP濃度增大而升高;模擬的分子模型顯示,rMPN668的二級(jí)結(jié)構(gòu)序列為β1-β2-β3-α1-β4-β5-α2-α3-β6,Cys55可能位于其活性中心內(nèi)。Cys55錯(cuò)義突變后,表達(dá)產(chǎn)物的酶活性幾乎喪失。 結(jié)論: (1) rMPN668具有氫過氧化物酶活性,能降解有機(jī)及無機(jī)氫過氧化物,但降解有機(jī)氫過氧化物的效率高于無機(jī)過氧化氫。 (2)氧化應(yīng)激條件下能上調(diào)Mp中mpn668基因的表達(dá)。 (3) Cys55可能位于rMPN668的活性中心,在酶促反應(yīng)中可能發(fā)揮關(guān)鍵作用。
[Abstract]:Objective: to study whether the recombinant MPN668 protein of Mycoplasma pneumoniae pneumoniae (MP) has the ability of degrading organic hydroperoxide (OHP), and to analyze the structure of rMPN668 by molecular simulation, and to analyze the relationship between its structure and enzyme activity.To further understand the pathogenesis of MP.Methods: the target gene mpn668 was amplified by Mp129 genomic DNA and subcloned into pGEX-6P-1 vector.After identification, it was transformed into E.coli BL21 for induction expression.SDS-PAGE and Western blotting were used to identify and purify the GST fusion protein, and after the recombinant protein GST label was removed, the hydroperoxidase activity of the recombinant protein was detected by Ferrous Oxidation-Xylenol Orange (FOX) assay.The expression of mpn668 mRNA was detected by RT-PCR.By means of homology modeling and molecular dynamics, the molecular structure of rMPN668 was simulated by software Expasy and Visual Molecular Dynamics, and the amino acids of its predicted enzyme activity sites were mutated.RMPN668 mutant protein was obtained by induced expression, and its hydroperoxidase activity was determined by FOX assay.Results: the mpn668 gene with total length of 423bp was successfully amplified. The recombinant plasmid was identified by PCR, double enzyme digestion and sequencing. SDS-PAGE showed that the recombinant plasmid could induce a soluble GST fusion protein with a molecular weight of about 41KD.After purification by Purification Kit, the purity of rMPN668 was over 95%. Fox experiment showed that rMPN668, which was removed from GST label by Prescission protease, could degrade tert-butyl hydroperoxide-t-BHP2 (tert-butyl hydroperoxide-t-BHPP2) and inorganic H2O2. The degradation ability of rMPN668 was positively correlated with rMPN668 concentration.When t-BHP was catalyzed, 20ng / 渭 L rMPN668 degraded about 500 渭 M t-BHP in 2min, while in the case of H2O2 reaction,The catalytic efficiency was much lower than that of 200ng / 渭 L rMPN668 in degradation of 500 渭 M H2O2. RT-PCR analysis showed that the expression level of MPN668 gene increased with the increase of t-BHP and CHP concentration at different concentrations of t-BHP0. 05 MN 1. 00 M) and Cumene hydroperoxide1. 05 M0. 00 M).The simulated molecular model showed that the secondary structure sequence of rMPN668 was 尾 1- 尾 2- 尾 3- 偽 1- 尾 4- 尾 4- 尾 5- 偽 2- 偽 3- 尾 6Cys55, and the enzyme activity of the expressed product was almost lost after the missense mutation of Cys55.Conclusion:1) rMPN668 has the activity of hydroperoxidase and can degrade organic and inorganic hydrogen peroxide, but the efficiency of degradation of organic hydrogen peroxide is higher than that of inorganic hydrogen peroxide.2) oxidative stress could up-regulate the expression of mpn668 gene in MP.Cys55 may be located in the active center of rMPN668 and may play a key role in the enzymatic reaction.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Interactions between mycoplasma lipid-associated membrane proteins and the host cells[J];Journal of Zhejiang University Science;2006年05期

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本文編號(hào)：1771966