Liao Tian-Jiang, Gao Jie, Wang Jin-Xing, Wang Xian-Wei
Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Sciences, Shandong University, Jinan, 250100, China; School of Chemistry Engineering, Gansu Industry Polytechnic College, Tianshui, Gansu, 741025, China.
Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Sciences, Shandong University, Jinan, 250100, China.
Dev Comp Immunol. 2018 Aug;85:134-141. doi: 10.1016/j.dci.2018.04.010. Epub 2018 Apr 11.
Lysozymes possess antibacterial activities, making them crucial defense proteins in innate immunity. In this study, a chicken-type (c-type) lysozyme (designated PcLyzc) was cloned and characterized from red swamp crayfish Procambarus clarkii. The full-length cDNA had an open reading frame of 435 base pairs encoding a polypeptide of 144 amino acid residues. Multiple alignments and phylogenetic analysis revealed that PcLyzc shared high similarity to the other known invertebrate c-type lysozymes. PcLyzc transcripts were steadily expressed in a wide range of tissues in healthy crayfish, and were prominently up-regulated in the hepatopancreas and gills after Vibrio anguillarum or Aeromonas hydrophila challenge. Recombinant PcLyzc showed inhibitory activity in vitro against both Gram-positive bacteria, including Staphylococcus aureus, Micrococcus luteus and Bacillus thuringiensis, and Gram-negative bacteria, including A. hydrophila, V. anguillarum and Escherichia coli. By overexpressing PcLyzc through introducing exogenous recombinant protein, or silencing PcLyzc expression through injecting double strand RNA, it was found that PcLyzc could help eliminate the invading bacteria in crayfish hemolymph and could protect crayfish from death, possibly by promoting the hemocytic phagocytosis. These results indicated that PcLyzc played a role in the antibacterial immunity of crustaceans, and laid a foundation of developing new therapeutic agents in aquaculture.
溶菌酶具有抗菌活性，使其成为先天免疫中至关重要的防御蛋白。在本研究中，从克氏原螯虾中克隆并鉴定了一种鸡型（c型）溶菌酶（命名为PcLyzc）。全长cDNA具有435个碱基对的开放阅读框，编码一个由144个氨基酸残基组成的多肽。多重序列比对和系统发育分析表明，PcLyzc与其他已知的无脊椎动物c型溶菌酶具有高度相似性。PcLyzc转录本在健康小龙虾的多种组织中稳定表达，在鳗弧菌或嗜水气单胞菌攻击后，肝胰腺和鳃中的表达显著上调。重组PcLyzc在体外对革兰氏阳性菌，包括金黄色葡萄球菌、藤黄微球菌和苏云金芽孢杆菌，以及革兰氏阴性菌，包括嗜水气单胞菌、鳗弧菌和大肠杆菌，均表现出抑制活性。通过引入外源重组蛋白过表达PcLyzc，或通过注射双链RNA沉默PcLyzc表达，发现PcLyzc可能通过促进血细胞吞噬作用帮助清除小龙虾血淋巴中入侵的细菌，并能保护小龙虾免于死亡。这些结果表明PcLyzc在甲壳类动物的抗菌免疫中发挥作用，为水产养殖中开发新的治疗剂奠定了基础。
