title: Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides.
abstract: Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and "GSTDYGIFQINS" motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.
authors.affiliation: Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, PR China.
authors.forename: Shina
authors.initials: S
authors.lastname: Wei
authors.forename: Youhua
authors.initials: Y
authors.lastname: Huang
authors.forename: Jia
authors.initials: J
authors.lastname: Cai
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Huang
authors.forename: Jing
authors.initials: J
authors.lastname: Fu
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Qin
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D001492:Bass / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D060226:Head Kidney / Q000276:immunology; D017994:Iridovirus / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment; D044149:Vibrio alginolyticus / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D000900:Anti-Bacterial Agents; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D009113:Muramidase