华西口腔医学杂志 ›› 2016, Vol. 34 ›› Issue (3): 295-301.doi: 10.7518/hxkq.2016.03.016

• 口腔微生物学专栏 • 上一篇    下一篇

针对变异链球菌的人源特异性靶向抗菌肽C16LL-37的生物学特性

车春晓1,姜科宇1,马媛媛1,曾飒1,周建业2,李志强2,何祥一1   

  1. 1.兰州大学口腔医学研究所,兰州730000; 2.甘肃省口腔疾病研究重点实验室(西北民族大学),兰州730030
  • 收稿日期:2015-12-05 修回日期:2016-03-02 出版日期:2016-06-01 发布日期:2016-06-01
  • 通讯作者: 何祥一,教授,博士,E-mail:hexy@lzu.edu.cn
  • 作者简介:车春晓,硕士,E-mail:chechx13@lzu.edu.cn
  • 基金资助:
    国家自然科学基金地区科学基金(31160124/C0309);甘肃省自然科学基金(1204FKCA16,1308RJZA248)

Biological characteristics of a human specifically targeted antimicrobial peptide C16LL-37 against Streptococcus mutans

Che Chunxiao1, Jiang Keyu1, Ma Yuanyuan1, Zeng Sa1, Zhou Jianye2, Li Zhiqiang2, He Xiangyi1   

  1. 1. School of Stomatology,Lanzhou University, Lanzhou 730000, China; 2. Key Laboratory of Oral Diseases of Gansu Province, Northwest Universityfor Nationalities, Lanzhou 730030, China)Supported by: Fund for Less Developed Regions of Natural Science Foundation of China (31160124/C0309); Natural ScienceFoundation of Gansu Province (1204FKCA16, 1308RJZA248). Correspondence: He Xiangyi, E-mail: hexy@lzu.edu.cn.
  • Received:2015-12-05 Revised:2016-03-02 Online:2016-06-01 Published:2016-06-01

摘要: 目的 研究针对变异链球菌(S. mutans)的人源特异性靶向抗菌肽C16LL-37的生物学特性。方法 通过标准固相合成技术(Fmoc保护法)合成人源抗菌肽LL-37、S. mutans感受态刺激肽(CSP)C端16个氨基酸组成的多肽CSPC16及二者的重组多肽C16LL-37;以LL-37、CSPC16为对照组,采用平板菌落计数法检测C16LL-37对S. mutans、黏性放线菌、大肠埃希菌、嗜酸乳杆菌、金黄色葡萄球菌的抗菌活性及其对S. mutans的靶向性。通过扫描电子显微镜(SEM)观察,浓度为32 μmol·L-1的C16LL-37作用后S. mutans的细胞形态学变化;利用酶联免疫吸附法测定C16LL-37的红细胞溶血率以及不同条件对C16LL-37抗菌活性的影响。结果 1)C16LL-37对S. mutans的最小抗菌浓度为16 μmol·L-1,最小杀菌浓度为64 μmol·L-1。2)C16LL-37浓度为64 μmol·L-1时,作用30 min后S. mutans存活率为3.46%,作用60 min后细菌存活率降至0%,其他4种细菌在各时间的存活率均大于60%(P<0.05)。3)SEM观察:经C16LL-37(32 μmol·L-1)作用后,部分S. mutans细胞形态出现不规则改变,部分细胞的细胞膜粗糙、细胞质外溢或出现细胞裂解。4)C16LL-37浓度不超过64 μmol·L-1时,红细胞溶血率低于0.33%,与阴性对照组无明显差异(P>0.05)。5)不同温度、pH值、盐浓度及低浓度胰蛋白酶处理后,C16LL-37的抗菌活性无明显变化(P>0.05)。结论 C16LL-37对变异链球菌具有靶向特异性,并且具有较强的抗菌活性、较高的生物安全性及良好的稳定性。

关键词: 变异链球菌, 感受态刺激肽, 抗菌肽, 特异性靶向抗菌肽

Abstract: Objective This study aimed to evaluate the biological characteristics of a human specifically targeted antimicrobialpeptide C16LL-37 against Streptococcus mutans (S. mutans). Methods In this study, an antimicrobial peptide LL-37, apeptide derived from CSPC16 (S. mutans competence stimulating peptide), and recombinant peptide C16LL-37 were synthesized byFmoc-chemistry-based strategy. The selectivity and antibacterial activity of C16LL-37 were identified by the colony countingmethod on microbial culture plates. After treatment of C16LL-37 at 32 μmol·L-1, the morphological changes in S. mutanswere observed by using scanning electron microscopy (SEM). In addition, enzyme-linked immunosorbent assay was used toevaluate the hemolytic activity and antibacterial activity of C16LL-37 under different conditions. Results 1) The minimuminhibitory concentration of C16LL-37 was 16 μmol·L-1, and the minimum bactericidal concentration was 64 μmol·L-1. 2) The survival rate of S. mutans was 3.46% after C16LL-37treatment at 64 μmol·L-1 for 30 min, whereas it was 0% at64 μmol·L-1 for 60 min. The survival rates of four otherkinds of bacteria were more than 60% at any time (P<0.05). 3) The morphological change in S. mutans was observedafter C16LL-37 treatment at 32 μmol·L-1 by using SEM. S. mutans presented an irregular shape, rough surface, and evidentsplitting. 4) The hemolysis rate of C16LL-37 (≤64 μmol·L-1) was less than 0.33%. 5) This study showed no significant influenceon the antibacterial activity of C16LL-37 under different conditions, such as temperature, pH, salinity, and trypsin atlow concentration (P>0.05). Conclusion C16LL-37 exhibited obvious specificity for S. mutans, strong antibacterial activity,low toxicity, and high stability. Thus, C16LL-37 has good potential in caries research and clinical application.

Key words: Streptococcus mutans, competence stimulating peptide, antimicrobial peptide, specifically targeted antimicrobial peptide

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