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Diseases of Aquatic Organisms

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DAO 55:117-123 (2003)  -  doi:10.3354/dao055117

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

Zhi-Xin Yin1, Jian-Guo He1,*, Wei-Xin Deng1, Siu-Ming Chan2

1State Key Lab for Biocontrol, School of Life Sciences, Zhongshan University, 135 Xingang Road West, Guangzhou, 510275, PR China
2Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR
*Corresponding author. Email:

ABSTRACT: 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.

KEY WORDS: Goose-type lysozyme · Orange-spotted grouper · Lytic activity · cDNA library

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