Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein
Section snippets
Animals
The sea cucumber S. japonicus was provided by Dalian Zhangzidao Island Fishery Group Corporation, Dalian, P. R. China. The fresh sea cucumbers were washed with deionized water, and dissected immediately. Tissues such as body wall, intestine, and digestive tract were collected, quick frozen in liquid nitrogen and stored at − 80 °C until use.
Total RNA
The frozen samples from S. japonicus tissues were ground under liquid nitrogen with a mortar and pestle. Total RNA was extracted using TRIzol Reagent
Sequence analysis of S. japonicus lysozyme cDNA
To obtain cDNA encoding the lysozyme of the sea cucumber S. japonicus (SjLys), two degenerate primers were designed from the analysis and comparison of selected known lysozyme and lysozyme-like gene sequences of other echinoderms and invertebrates, such as starfish, sea urchin, nematode, etc. As a result, a 220 bp fragment was amplified, cloned, and sequenced. The BLAST search showed that this sequence was highly homologous to lysozyme sequences. The complete cDNA sequence was then determined
Discussion
In the present study, the first cDNA encoding an i-type lysozyme was identified from S. japonicus (SjLys) by RT-PCR and RACE techniques. Like i-type lysozymes found in bivalves, SjLys contained a hydrophobic signal peptide at the N-terminus and a hydrophilic tail at the C-terminus. A characteristic of the mature peptide of SjLys in the primary sequence is the high content (8%) of cysteine residues (10 out of 125 residues), and it predictedly forms four disulfide bonds. Disulfide bonds formed
Acknowledgements
We thank Professor José E. García-Ararrás, Department of Biology, University of Puerto Rico, USA, for revision and suggestion on the manuscript. We also thank Dr. Zongshen Zhang and other laboratory members for technical advice and helpful discussions. This work was financially supported by the National Natural Science Foundation of China (NSFC; No. 30571449) and National Basic Research Program of China (973 Program; No. 2006CB708210).
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