Molecular cloning, expression of orange-spotted grouper goose-type lysozyme cDNA, and lytic activity of its recombinant protein

Dis Aquat Organ. 2003 Jul 8;55(2):117-23. doi: 10.3354/dao055117.


Lysozyme acts as a non-specific innate-immunity molecule against the invasion of bacteria pathogens. A leukocyte cDNA library of orange-spotted grouper Epinephelus coioides was constructed and the goose-type (g-type) lysozyme cDNA was isolated. The complete cDNA consists of an open reading frame of 585 bp encoding a protein of 194 amino acids. This protein shows a 72.2% amino acid sequence identity with the flounder g-type lysozyme. Similar to most other species, the glu catalytic residue in g-type lysozymes of the grouper is conserved. Furthermore, like the flounder and carp, the 4 conserved cysteine residues identified in avian and mammalian g-type lysozymes were also absent from the grouper. Northern blot analysis indicated that the g-type lysozyme was expressed in intestine, liver, spleen, anterior kidney, posterior kidney, heart, gill, muscle and leukocytes. In addition, RT-PCR analysis detected the g-type lysozyme transcripts in the stomach, brain and ovary. When an orange-spotted grouper was injected with Vibrio alginolyticus, the number of lysozyme mRNA transcripts detected in the stomach, spleen, anterior kidney, posterior kidney, heart, brain and leucocytes increased 72 h after injection. Recombinant grouper g-type lysozyme produced in the Escherichia coli expression system showed lytic activity against Micrococcus lysodeikticus, V. alginolyticus from Epinephelus fario, V. vulnificus from culture water, Aeromonas hydrophila from soft-shell turtle, A. hydrophila from goldfish and V. parahaemolyticus, Pseudomonas fluorescens and V. fluvialis from culture water.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Northern / veterinary
  • Blotting, Western / veterinary
  • Cloning, Molecular
  • DNA, Complementary / chemistry*
  • Electrophoresis, Polyacrylamide Gel / veterinary
  • Gene Expression Regulation, Enzymologic
  • Gene Library
  • Immunity, Innate
  • Molecular Sequence Data
  • Molecular Weight
  • Muramidase / biosynthesis
  • Muramidase / chemistry
  • Muramidase / genetics*
  • Open Reading Frames
  • Perciformes / genetics*
  • Perciformes / metabolism
  • Recombinant Proteins / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction / veterinary


  • DNA, Complementary
  • Recombinant Proteins
  • Muramidase