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Zoological Research
Kunming Institute of Zoology, Chinese Academy of Sciences
ISSN: 2095-8137
Vol. 35, No. 4, 2014, pp. 294-299
Bioline Code: zr14034
Full paper language: English
Document type: Research Article
Document available free of charge

Zoological Research, Vol. 35, No. 4, 2014, pp. 294-299

 en Characterization and expression of sweetfish ( Plecoglossus altivelis check for this species in other resources ) cathepsin D
JIAO, Yu; LI, Chang-Hong; LU, Xin-Jiang & CHEN, Jiong

Abstract

Cathepsin D (CTSD) is a lysosomal acidic endoproteinase that plays an important role in immune response. In this study, we obtained sweetfish ( Plecoglossus altivelis check for this species in other resources ) CTSD (PaCTSD) via de-novo transcriptome sequencing of sweetfish macrophages. The full length cDNA sequence of PaCTSD was 1 955 bp encoding a propeptide of 397 amino acids. The deduced protein had a calculated molecular weight of 43.17×103. Multiple alignment with other known CTSD amino acid sequences revealed amino acid conservation through the teleosts. Phylogenetic tree analysis showed that PaCTSD grouped tightly with other fish CTSD, and was close to that of Atlantic salmon and rainbow trout. Subsequently, PaCTSD was prokaryotically expressed and refolded by the urea gradient method on a nickel-nitrilotriacetic acid column. Enzyme activity analysis showed that PaCTSD exhibited pH-dependent proteolytic activity. Quantitative real-time PCR showed that PaCTSD mRNA was expressed in all detected tissues in healthy sweetfish. The highest expression was observed in the spleen and white blood cells, followed by liver, head-kidney, kidney, intestine, gill, and muscle. After Listonella anguillarum check for this species in other resources infection, PaCTSD transcripts were up-regulated significantly in liver, spleen, white blood cells, and head-kidney of sweetfish. In summary, PaCTSD has proteolytic activity and is closely involved in the immune response of sweetfish.

Keywords
Cathepsin D; Plecolossus altivelis; Bacterial infection; Prokaryotic expression; qRT-PCR

 
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