›› 2015, Vol. 58 ›› Issue (6): 617-624.

• 研究论文 • 上一篇    下一篇

家蝇新型抗菌肽Muscin的基因克隆、表达模式及抑菌活性

杨雪, 唐婷, 王一丽, 刘欣, 曹新茹, 倪志华, 柳峰松*   

  1. (河北大学生命科学学院, 河北保定 071002)
  • 出版日期:2015-06-20 发布日期:2015-06-20
  • 作者简介:杨雪, 女, 1989年生, 河南安阳人, 硕士研究生, 研究方向为无脊椎动物免疫学, E-mail: yangxuepphappy@126.com

Gene cloning, expression profiling and anti-microbial assay of Muscin, a new antimicrobial peptide in the housefly (Musca domestica)

YANG Xue, TANG Ting, WANG Yi-Li, LIU Xin, CAO Xin-Ru, NI Zhi-Hua, LIU Feng-Song*   

  1. (College of Life Sciences, Hebei University, Baoding, Hebei 071002, China)
  • Online:2015-06-20 Published:2015-06-20

摘要: 【目的】鉴定家蝇 Musca domestica  (Linnaeus)中一种新型抗菌肽(Muscin)基因,并分析其功能。【方法】通过数字基因表达谱和生物信息学分析,在家蝇转录组中筛选得到一条抗菌肽基因,命名为 muscin。以实时荧光定量PCR技术研究该基因的组织分布以及用大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus混合细菌刺激后的表达量变化。并对合成肽Muscin进行抑菌活性检测及溶血率测定。【结果】muscin基因cDNA序列全长379 bp,包含完整的开放阅读框153 bp。推导Muscin多肽序列由50个氨基酸残基组成,N端含有由25个氨基酸残基组成的信号肽。成熟肽中富含疏水性氨基酸残基和带正电荷的氨基酸残基,理论等电点为9.39。基因定量结果显示 muscin 基因在血细胞和脂肪体中表达量最高。通过细菌刺激进行免疫诱导后,幼虫体内该基因的表达水平明显上调,并在6 h达到高峰。抑菌和溶血实验显示c-Muscin对革兰氏阳性菌和革兰氏阴性菌具有广谱抑菌活性,且溶血活性较低。【结论】Muscin是一种新型的广谱抗菌肽,可能参与家蝇抗菌免疫反应,且具有一定药物开发潜质。

关键词: 家蝇, 抗菌肽, 基因克隆, 生物信息学分析, 表达量, 抑菌活性, 溶血实验

Abstract: 【Aim】 The objective of this research is to identify and characterize a new antimicrobial peptide (Muscin) gene from the housefly, Musca domestica (Linnaeus). 【Methods】 Based on the digital gene expression profiling (DGE) and bioinformatics analysis, a potential antimicrobial peptide gene, muscin, was identified from the housefly transcriptome database. Quantitative real-time PCR (qRT-PCR) was employed to investigate the tissue distribution of muscin mRNA and its temporal expression in housefly larvae challenged with the mixture of bacteria Escherichia coli and Staphylococcus aureus. The anti-microbial and hemolytic activities of the synthetic peptide were also tested. 【Results】 The full-length cDNA of muscin is 379 bp, with an open reading frame of 153 bp encoding a peptide of 50 amino acid residues. The deduced peptide contains a putative signal peptide of 25 amino acids. The deduced mature peptide is rich in hydrophobic and positively charged amino acid residues with a theoretical pI of 9.39. The qRT-PCR results showed that muscin was mainly transcribed in the hemocytes and fat body. Post immunity induction with the mixture of bacteria, the expression of muscin was up-regulated and reached the maximal level at 6 h. Anti-microbial assays and hemolytic test displayed that c-Muscin had broad-spectrum antibacterial activity against Gram positive and Gram negative bacteria, and a low hemolytic activity. 【Conclusion】 Muscin is a novel broad-spectrum antimicrobial peptide with potential medicinal value and may take part in immune defense against bacterial pathogens in the housefly.

Key words: Musca domestica, antimicrobial peptide, gene cloning, bioinformatical analysis, expression level, antibacterial activity, hemolytic test