肺炎支原体Ohr样蛋白的结构及功能的初步研究

发布时间：2018-04-19 06:30

本文选题：rMPN668 + 有机氢过氧化物抗性蛋白　；参考：《南华大学》2011年硕士论文

【摘要】：目的:研究肺炎支原体(Mycoplasma pneumoniae,Mp)重组MPN668蛋白是否具有降解有机氢过氧化物(Organic Hydroperoxide,OHP)的能力,并通过分子模拟解析rMPN668的结构,初步分析其结构与酶活性的关系,以进一步了解Mp的致病机制。方法:以Mp129株基因组DNA为模板,PCR法扩增目的基因mpn668,将其亚克隆至pGEX-6P-1载体中。经鉴定后将其转化至表达菌E.coli BL21中进行诱导表达。采用SDS-PAGE和Western blotting等方法对表达产物进行分析鉴定并纯化GST融合蛋白;切除重组蛋白GST标签后,采用氧化铁二甲酚橙(Ferrous Oxidation-Xylenol Orange ,FOX)实验检测其氢过氧化物酶活性。固体培养Mp,随后将其置于氧化应激条件下培养3~5d,RT-PCR检测mpn668 mRNA的表达水平。利用同源建模和分子动力学等方法,通过软件Expasy与Visual Molecular Dynamics(VMD)模拟rMPN668的分子结构,并对其已预测出的酶活性位点的氨基酸进行定点突变,通过诱导表达获得△rMPN668突变蛋白, FOX实验测定其氢过氧化物酶活性。结果:成功扩增出总长度为423bp的mpn668基因,所构建的原核重组质粒经PCR、双酶切以及测序鉴定与预期目的基因相符。SDS-PAGE显示,IPTG可诱导一分子量约为41KD的可溶性GST融合蛋白,经GST?Bind? Purification Kit纯化后,纯度可达95%以上。FOX实验显示,经Prescission protease切除GST标签的rMPN668能降解有机氢过氧化物叔丁基过氧化氢( tert-butyl hydroperoxide,t-BHP)以及无机H2O2,其降解能力与rMPN668浓度呈正相关;催化t-BHP时,20ng/μL rMPN668在2min内降解约500μM t-BHP,而催化H2O2反应时,催化效率远低于此水平,200ng/μL rMPN668降解500μM H2O2需要35min以上。RT-PCR分析表明,Mp在不同浓度的t-BHP(0.05M~1.00M)和氢过氧化枯烯(cumene hydroperoxide,CHP)(0.05M~1.00M)条件下,mpn668基因表达水平随着t-BHP和CHP浓度增大而升高;模拟的分子模型显示,rMPN668的二级结构序列为β1-β2-β3-α1-β4-β5-α2-α3-β6,Cys55可能位于其活性中心内。Cys55错义突变后,表达产物的酶活性几乎丧失。结论: (1) rMPN668具有氢过氧化物酶活性,能降解有机及无机氢过氧化物,但降解有机氢过氧化物的效率高于无机过氧化氢。 (2)氧化应激条件下能上调Mp中mpn668基因的表达。 (3) Cys55可能位于rMPN668的活性中心,在酶促反应中可能发挥关键作用。
[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- 伪 1- 尾 4- 尾 4- 尾 5- 伪 2- 伪 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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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