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Molecular cloning and characterization of a new G-type lysozyme gene (Ec-lysG) in orange-spotted grouper, Epinephelus coioides.
- Source :
-
Developmental and comparative immunology [Dev Comp Immunol] 2014 Oct; Vol. 46 (2), pp. 401-12. Date of Electronic Publication: 2014 May 27. - Publication Year :
- 2014
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Abstract
- 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.<br /> (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Subjects :
- Amino Acid Sequence
Animals
Anti-Bacterial Agents pharmacology
Cells, Cultured
Cloning, Molecular
Conserved Sequence
Fish Proteins biosynthesis
Fish Proteins chemistry
Fish Proteins pharmacology
Gene Expression Regulation, Enzymologic
Head Kidney enzymology
Head Kidney immunology
Iridovirus immunology
Lipopolysaccharides pharmacology
Molecular Sequence Data
Muramidase biosynthesis
Muramidase chemistry
Muramidase pharmacology
Organ Specificity
Phylogeny
Protein Transport
Vibrio alginolyticus drug effects
Virus Replication
Fish Proteins genetics
Muramidase genetics
Perciformes genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1879-0089
- Volume :
- 46
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Developmental and comparative immunology
- Publication Type :
- Academic Journal
- Accession number :
- 24877656
- Full Text :
- https://doi.org/10.1016/j.dci.2014.05.006