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文章摘要
钟亨任,谢志鹏,魏涵淇,张淑霞,何佩兰,宋彦廷,张英霞.抗菌肽Brevinin-GR23抗金黄色葡萄球菌作用机制的初步研究[J].海南大学学报编辑部:自然科学版,2018,36(4):.
抗菌肽Brevinin-GR23抗金黄色葡萄球菌作用机制的初步研究
Mechanism of Brevinin-GR23 against Staphylococcus aureus
投稿时间:2018-10-09  修订日期:2018-10-23
DOI:
中文关键词: 抗菌肽;核酸泄露;DNA结合活性;抗菌机制;圆二色谱。
英文关键词: antimicrobial peptide; nucleic acid leakage; DNA-binding activity; antimicrobial mechanism; Circular Dichroism spectroscopy.
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目)
作者单位E-mail
钟亨任 海南大学热带生物资源教育部重点实验室 海南大学海洋学院药学系 海南 海口 570228 1164284269@qq.com 
谢志鹏 海南大学热带生物资源教育部重点实验室 海南大学海洋学院药学系 海南 海口 570228  
魏涵淇 海南大学热带生物资源教育部重点实验室 海南大学海洋学院药学系 海南 海口 570228  
张淑霞 海南大学热带生物资源教育部重点实验室 海南大学海洋学院药学系 海南 海口 570228  
何佩兰 海南大学热带生物资源教育部重点实验室 海南大学海洋学院药学系 海南 海口 570228  
宋彦廷 海南大学热带生物资源教育部重点实验室 海南大学海洋学院药学系 海南 海口 570228  
张英霞 海南大学热带生物资源教育部重点实验室 海南大学海洋学院药学系 海南 海口 570228 yingxiazhang@hotmail.com 
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中文摘要:
      Brevinin-2GHe是从海南沼蛙(Hylarana guentheri)皮肤中获得的抗菌肽。为进一步优化设计该抗菌肽,分析其结构与生物学活性之间的关系,去除了brevinin-2GHe的7个C-末端氨基酸,通过固相合成获得了含有23个氨基酸的brevinin-GR23。采用琼脂平板打孔法和二倍稀释法,检测抗菌肽brevinin-GR23的抗菌活性和最低抑制浓度;平板涂布法检测brevinin-GR23的时间杀菌曲线;通过测定核酸泄露判断brevinin-GR23对细菌细胞膜完整性的影响;通过凝胶电泳分析brevinin-GR23对细菌体内DNA的作用;测定其对兔红细胞的毒性作用;通过圆二色谱检测brevinin-GR23在不同溶液环境中的二级结构,在线软件分析3D结构。结果表明,抗菌肽brevinin-GR23对金黄色葡萄球菌(Staphylococcus aureus)的抗菌活性最好,最低抑制浓度为16.6 μM。在120 min时,brevinin-GR23的抑制细菌的效率最高。Brevinin-GR23对兔红细胞的溶血活性较低。核酸泄露实验结果显示,brevinin-GR23增加了S. aureus的细胞膜渗透性,从而使内容物外泄,brevinin-GR23还能与细菌DNA结合,干扰金黄色葡萄球菌DNA的复制从而发挥抑菌作用。二级结构分析表明,brevinin-GR23在PBS溶液中以不规则结构为主,而在膜模拟溶液中转变成α螺旋结构,从而有利于结合到细菌的细胞膜上改变细胞膜的完整性。在线软件分析其3D结构为α螺旋。经改造后的brevinin-GR23分子量更小,活性强和毒性小,化学合成成本降低,有利于进一步开发与应用。
英文摘要:
      Brevinin-2GHe is an antimicrobial peptide obtained from Hylarana guentheri. To further optimize the antimicrobial peptide and study the relationship between its structure and biological activity, the seven C-terminal amino acids of brevinin-2GHe were removed and brevinin-GR23 containing 23 amino acids was obtained. Brevinin-GR23 was synthesized by solid phase. The antibacterial activity and the minimum inhibitory concentration (MIC) of brevinin-GR23 were detected by the method of punching on the ager plate and double dilution method. The time kill curve of brevinin-GR23 was detected by spread plate method, and the bacterial membrane integrity was determined by measuring nucleic acid leakage of Staphylococcus aureus. The effect of brevinin-GR23 on the DNA of S. aureus in vivo was analyzed by DNA gel retardation assay, and toxic effect on rabbit red blood cells was also determined. Finally, the secondary structure of brevinin-GR23 in different solution environments was detected by Circular Dichroism spectroscopy. The results showed that brevinin-GR23 has antibacterial activity against S. aureus with the MIC of 16.6 μM, and the highest inhibitory efficiency against S. aureus achieved after bacteria were treated by brevinin-GR23 for 120 min. Brevinin-GR23 showed very low hemolytic activity. Nucleic acid leak assays, secondary structures, and online software analysis of 3D structures indicated that brevinin-GR23 has different structural forms in different solutions, which facilitates the binding of bacterial cell membrane to change cell membrane integrity. The modified brevinin-GR23 has a smaller molecular weight, stronger activity and less toxicity, and lower chemical synthesis cost, which is beneficial to further development and application in antibacterial agents.
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