Molecular cloning, expression of orange-spotted grouper goose-type lysozyme cDNA, and lytic activity of its recombinant protein.
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.
Yin
Zhi-Xin
ZX
State Key Lab for Biocontrol, School of Life Sciences, Zhongshan University, 135 Xingang Road West, Guangzhou, 510275, PR China.
He
Jian-Guo
JG
Deng
Wei-Xin
WX
Chan
Siu-Ming
SM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000662:veterinary; D015153:Blotting, Western / Q000662:veterinary; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D007113:Immunity, Innate; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D016366:Open Reading Frames; D010473:Perciformes / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary
D018076:DNA, Complementary; D011994:Recombinant Proteins; D009113:Muramidase