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齐若含, 陈燕, 郭志来, 张芬, 方郑, 黄凯, 于海宁, 王义鹏. 2019: 两种大绿臭蛙(Odorrana livida)来源新型cathelicidins分子的发现和表征. 动物学研究, 40(2): 94-101. DOI: 10.24272/j.issn.2095-8137.2018.062
引用本文: 齐若含, 陈燕, 郭志来, 张芬, 方郑, 黄凯, 于海宁, 王义鹏. 2019: 两种大绿臭蛙(Odorrana livida)来源新型cathelicidins分子的发现和表征. 动物学研究, 40(2): 94-101. DOI: 10.24272/j.issn.2095-8137.2018.062
Ruo-Han Qi, Yan Chen, Zhi-Lai Guo, Fen Zhang, Zheng Fang, Kai Huang, Hai-Ning Yu, Yi-Peng Wang. 2019: Identification and characterization of two novel cathelicidins from the frog Odorrana livida. Zoological Research, 40(2): 94-101. DOI: 10.24272/j.issn.2095-8137.2018.062
Citation: Ruo-Han Qi, Yan Chen, Zhi-Lai Guo, Fen Zhang, Zheng Fang, Kai Huang, Hai-Ning Yu, Yi-Peng Wang. 2019: Identification and characterization of two novel cathelicidins from the frog Odorrana livida. Zoological Research, 40(2): 94-101. DOI: 10.24272/j.issn.2095-8137.2018.062

两种大绿臭蛙(Odorrana livida)来源新型cathelicidins分子的发现和表征

Identification and characterization of two novel cathelicidins from the frog Odorrana livida

  • 摘要: 抗菌肽(antimicrobial peptides, AMPs)是一类基因编码的小分子多肽,在多细胞生物的宿主免疫系统中发挥关键作用。Cathelicidins是脊椎动物体内一个重要的抗菌肽家族,从哺乳类、鸟类、爬行类和鱼类中已经发现大量cathelicidins分子。但是到目前为止对两栖类动物来源的cathelicidins研究不多。在本研究中,我们从大绿臭蛙(Odorrana livida)体内发现两种新型cathelicidins分子(OL-CATH1和2)。首先,从构建的大绿臭蛙肺cDNA文库中成功克隆得到了编码OL-CATH1和2的cDNA序列(序列长度分别为780 bp和735 bp)。采用多序列比对分析了OL-CATHs前体和其他代表性cathelicidins的差异。预测了OL-CATH1和2的成熟肽序列(33个氨基酸残基),并测定了它们的二级结构(OL-CATH1为无规则结构,OL-CATH2为α-螺旋型结构)。进一步化学合成了OL-CATH1和2样品并检测了其体外活性。抗菌和杀菌动力学等研究结果表明OL-CATH2具有相对高效和快速的杀菌活性,同时具有较强的抗炎活性。在极低的浓度下(10 μg/mL)OL-CATH2就可以显著的抑制小鼠腹腔巨噬细胞中细菌脂多糖(lipopolysaccharide, LPS)诱导的促炎因子TNF-α、IL-1β和IL-6的转录和合成。与之相反,OL-CATH1并未表现出任何可以检测到的抗菌和抗炎活性。总之,大绿臭蛙来源OL-CATHs的发现能够丰富我们对cathelicidins在两栖类动物免疫系统中功能的认识。OL-CATH2所具有的较强的抗菌和抗炎活性表明其在抗感染药物研发中是一种具有潜力的新型候选分子。

     

    Abstract: Antimicrobial peptides (AMPs) are a group of gene-encoded small peptides that play pivotal roles in the host immune system of multicellular organisms. Cathelicidins are an important family of AMPs that exclusively exist in vertebrates. Many cathelicidins have been identified from mammals, birds, reptiles and fish. To date, however, cathelicidins from amphibians are poorly understood. In the present study, two novel cathelicidins (OL-CATH1 and 2) were identified and studied from the odorous frog Odorrana livida. Firstly, the cDNAs encoding the OL-CATHs (780 and 735 bp in length, respectively) were successfully cloned from a lung cDNA library constructed for the frog. Multi-sequence alignment was carried out to analyze differences between the precursors of the OL-CATHs and other representative cathelicidins. Mature peptide sequences of OL-CATH1 and 2 were predicted (33 amino acid residues) and their secondary structures were determined (OL-CATH1 showed a random-coil conformation and OL-CATH2 demonstrated a-helical conformation). Furthermore, OL-CATH1 and 2 were chemically synthesized and their in vitro functions were determined. Antimicrobial and bacterial killing kinetic analyses indicated that OL-CATH2 demonstrated relatively moderate and rapid antimicrobial potency and exhibited strong anti-inflammatory activity. At very low concentrations (10 μg/mL), OL-CATH2 significantly inhibited the lipopolysaccharide (LPS)-induced transcription and production of pro-inflammatory cytokines TNF-a, IL-1b and IL-6 in mouse peritoneal macrophages. In contrast, OL-CATH1 did not exhibit any detectable antimicrobial or anti-inflammatory activities. Overall, identification of these OL-CATHs from O. livida enriches our understanding of the functions of cathelicidins in the amphibian immune system. The potent antimicrobial and anti-inflammatory activities of OL-CATH2 highlight its potential as a novel candidate in anti-infective drug development.

     

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