【作者】 李超；

【导师】 谷劲松；

【作者基本信息】 济南大学 ， 生物工程（专业学位）， 2016， 硕士

【摘要】 在感染病领域,耐药细菌的感染是目前全球关注的重要问题之一。现阶段采取的相应策略主要有:(1)通过合理使用抗生素以降低细菌耐药的变异速度;(2)探讨新的抑菌生化途径,研发新型的抗菌药物,使之用于对抗耐药菌。以甲基化修饰为途径,进而使抗菌药物靶点的结构发生改变,这是常见致病菌的一种重要耐药机制。S-腺苷高半胱氨酸核苷酶(SAHN)作为一种重要的酶参与甲基循环,对S-腺苷甲硫氨酸(SAM)依赖的甲基转移反应产生的通用产物S-腺苷高半胱氨酸(SAH)进行裂解,并促使甲硫氨酸(Met)和腺嘌呤(Adenine)进行再生,进而产生通用群感效应信号分子AI-2。群感效应所产生的生物膜作为细菌生长分裂的一种重要的保护模式,能保证细菌在恶劣环境下可以生存,也会使细菌对抗抗生素的能力提高,生物膜的形成成为细菌耐药的重要机制之一,这也是大多数慢性传染性疾病难以控制的主要原因。首先,SAHN酶活性下降会造成菌体内S-腺苷高半胱氨酸的累积,作为SAM所依赖的甲基转移酶的有效反馈抑制剂,菌体内SAH的积累就会使甲基转移反应受到全面抑制,进而以影响基因表达调控和蛋白修饰的方式来降低病原菌的适应性,使细菌的生长和分裂得到抑制。其次,抑制SAHN酶蛋白会使群感效应信号分子的前体缺失,使病原菌无法顺利合成信号分子,可使细菌群感效应,如:病原菌毒力因子的表达、菌膜的形成等得到有效控制。综上,SAHN酶蛋白活性变化会对大肠杆菌生长分裂以及群感效应产生影响。由于哺乳动物细胞中并不存在这种核苷酶,因此,SAHN酶在致病菌中的功能分析和研究成为发展新型广谱杀菌剂的一种新思路。本论文以大肠杆菌的全基因组DNA做为模板,利用聚合酶链式反应(Polymerase Chain Reaction,PCR)的方法扩增目sahn基因,构建重组表达载体。以IPTG为诱导剂进行诱导表达,并通过Ni-NTA亲和层析对SAHN酶蛋白进行纯化。将纯化后的蛋白经除盐浓缩后用考马斯亮蓝法测其蛋白含量约为348μg/m L。并进一步建立了黄嘌呤氧化酶的酶偶联分析检测技术对SAHN酶蛋白进行了酶活性测定。通过设计梯度下pH及温度反应体系对SAHN酶蛋白酶促反应最佳pH及温度进行分析,实验表明SAHN在广泛的pH条件下均具有酶活性,并发现该蛋白热稳定性较差。最后通过比较SAHN酶蛋白在不同表达水平下,对其群感效应的表征信号分子AI-2合成量也是不同。在大肠杆菌生物膜实验中,发现SAHN酶蛋白在过量表达水平下,使大肠杆菌生物膜的生成量及致密性上升。在实验结果中可以发现,SAHN酶蛋白在过量表达下,会加强菌体的群感效应,从而为发展以SAHN为药物靶点的新型抗感染药物提供理论和实验上的依据和参考。更多还原

【Abstract】 In the field of infectious diseases, drug-resistant bacteria infection is one of the important issues throughout the world. At present, in addition to the reasonable application of antibiotics to reduce the drug resistance mutation rate of pathogenic bacteria, corresponding strategies involve exploring new antibacterial biochemical pathways and developing novel antimicrobial agents for the resistance against pan-drug resistant bacteria.Through methylation modification, changing the structure of antibacterial drug targets is a significant drug-resistance mechanism for E.coli(as a representative of pathogenic bacteria). As a important enzyme, S-adenosylhomocysteine nucleosidase(hereafter referred to as SAHN) participates in activated methyl cycle. Furthermore, in the methylation reaction regulated by S-adenosine methionine(hereafter called SAM), methionine and adenine are regenerated and the signal molecule Autoinducer 2(AI-2) of Quorum sensing(QS) is produced.As an important protection mode of bacteria growth ad division, biofilm affected by quorum sensing can guarantee the survival of bacteria in the harsh environment and enhance the resistance of bacteria against antibiotics. Biofilm formation becomes one of the significant mechanisms of bacterial resistance, which is the main reason for repeated episodes and uncontrollability of numerous chronic infectious diseases. Besides, the declining activity of SAHN exerts serious influence on bacteria growth and division in the cells. Firstly, SAHN activity decrease will cause the accumulation of intracellular S-adenosylhomocysteine(hereafter called SAH). However, as an effective feedback inhibitor of SAM-dependent methyltransferase, intracellular SAH accumulation of low concentration will thoroughly suppress methyl transfer reaction. As a result, gene expression regulation and protein modification are affected. On the other hand, pathogenic bacteria adaptability is reduced to stop bacterial cells growth and division. Secondly, SAHN inhibition will lead to the fact that downstream AI-2 precursor is deleted and pathogens cannot synthesize AI-2 and therefore bacterial quorum sensing effects can be effectively controlled, such as pathogen virulence factors expression and bacterial biofilm formation. In addition, SAHN inhibition will cause methyl cycle interruption and important compounds(including methionine and SAM) are unable to regenerate in the methyl cycle. Provided that no methionine is supplied in the environment, SAM cannot penetrate bacterial cell membranes and pathogens simply cannot grow. Consequently, bacteria growth and division may be influenced by different mechanisms. As this type of nucleosidase does not exist in mammalian cells, the analysis and research concerning the function of SAHN has provided a number of ideas for broad spectrum bactericide development.In this paper, the gene sahn was amplified with the genome of E.coli as template by PCR then was linked to vector to pET-28a-SAHN and transformed into Escherichia coli BL21. SAHN was expressed as IPTG-inducible His-tagged recombinant enzymes purified using Ni-NTA chromatography. After purification, it was concentrated to 348 μg/m L, which was measured by coomassie brilliant blue method. The determination of SAHN enzyme activity was carried out based on coupling of xanthine oxidase enzyme. By inducing E.coli that has been built, the effect of SAHN activity at different expression levels on E.coli growth, division and quorum sensing is investigated, which provides theoretical and experimental basis and reference for the study of developing novel SAHN-targeted anti-infection drugs.更多还原