Wei Shina, Huang Youhua, Huang Xiaohong, Cai Jia, Wei Jingguang, Li Pengfei, Ouyang Zhengliang, Qin Qiwei
Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China; College of Fishery, Guangdong Ocean University, Zhanjiang 524088, China.
Dev Comp Immunol. 2014 Oct;46(2):401-12. doi: 10.1016/j.dci.2014.05.006. Epub 2014 May 27.
Lysozyme acts as an innate immunity molecule against pathogen infection. In this study, a new G-type lysozyme gene with a typical G-type lysozyme domain (designated as Ec-lysG) was cloned and characterized from the orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysG cDNA contains 1419 bp and encodes a 256-residue protein containing a 25-residue signal peptide at the N-terminus. BLAST analysis reveals Ec-lysG shares 64% identity with Siniperca chuatsi, but 63% to another reported G-type lysozyme from orange-spotted grouper (OSG-lysG). The genomic DNA of Ec-lysG contains four exons and three introns, with a total length of 2062 bp. An amino acid sequence alignment showed that Ec-lysG shares the fundamental structural features of G-type lysozyme, including the catalytic residues, substrate binding sites, and soluble lytic transglycosylase domain. Quantitative PCR showed that Ec-lysG transcript is most abundant in the head kidney, and less abundant in the heart. The expression of Ec-lysG was differentially upregulated in the head kidney after stimulation with lipopolysaccharide, Vibrio alginolyticus, and Singapore grouper iridovirus (SGIV). A subcellular localization analysis showed that Ec-lysG is distributed predominantly in the cytoplasm. Recombinant Ec-lysG (rEc-lysG) has optimal activity at pH 7.5 and 35°C. rEc-lysG showed lytic activities against Gram-positive bacterium Streptococcus iniae, Staphylococcus aureus, and Micrococcus lysodeikticus, and the Gram-negative bacterium V. alginolyticus. Scanning electron microscopy (SEM) showed that rEc-lysG acts on M. lysodeikticus cell walls. The overexpression of Ec-lysG in grouper cells did not significantly delay the occurrence of the cytopathic effect (CPE) induced by SGIV, and did not inhibit viral gene transcription. In conclusion, Ec-lysG might be a potent antibacterial protein, with a role in innate immunity.
溶菌酶作为一种针对病原体感染的固有免疫分子。在本研究中，从斜带石斑鱼（Epinephelus coioides）中克隆并鉴定了一个具有典型G型溶菌酶结构域的新G型溶菌酶基因（命名为Ec-lysG）。Ec-lysG cDNA全长1419 bp，编码一个256个氨基酸的蛋白质，其N端含有一个25个氨基酸的信号肽。BLAST分析显示，Ec-lysG与鳜鱼（Siniperca chuatsi）的同源性为64%，但与另一个已报道的斜带石斑鱼G型溶菌酶（OSG-lysG）的同源性为63%。Ec-lysG的基因组DNA包含四个外显子和三个内含子，全长2062 bp。氨基酸序列比对表明，Ec-lysG具有G型溶菌酶的基本结构特征，包括催化残基、底物结合位点和可溶性溶菌转糖基酶结构域。定量PCR显示，Ec-lysG转录本在头肾中最丰富，在心脏中较少。在用脂多糖、溶藻弧菌（Vibrio alginolyticus）和新加坡石斑鱼虹彩病毒（SGIV）刺激后，Ec-lysG在头肾中的表达差异上调。亚细胞定位分析表明，Ec-lysG主要分布在细胞质中。重组Ec-lysG（rEc-lysG）在pH 7.5和35°C时具有最佳活性。rEc-lysG对革兰氏阳性菌海豚链球菌（Streptococcus iniae）、金黄色葡萄球菌（Staphylococcus aureus）和溶壁微球菌（Micrococcus lysodeikticus）以及革兰氏阴性菌溶藻弧菌具有溶菌活性。扫描电子显微镜（SEM）显示，rEc-lysG作用于溶壁微球菌的细胞壁。Ec-lysG在石斑鱼细胞中的过表达并未显著延迟SGIV诱导的细胞病变效应（CPE）的发生，也未抑制病毒基因转录。总之，Ec-lysG可能是一种有效的抗菌蛋白，在固有免疫中发挥作用。
