Abstract
Antimicrobial peptides (AMPs) are important mediators of the primary defense mechanism against microbial invasion. In the present study, a big defensin was identified from Venerupis philippinarum haemocytes (denoted as VpBD) by RACE and EST approaches. The VpBD cDNA contained an open reading frame (ORF) of 285 bp encoding a polypeptide of 94 amino acids. The deduce amino acid sequence of VpBD shared the common features of big defensin including disulfide array organization and helix structure, indicating that VpBD should be a new member of the big defensin family. The mRNA transcript of VpBD was up-regulated significantly during the first 24 hr after Vibrio anguillarum challenge, which was 7.4-fold increase compared to that of the control group. Then the expression decreased gradually from 24 hr to 96 hr, and the lowest expression level was detected at 96 hr post-infection, which was still 3.9-fold higher than that of control. The mature peptide of VpBD was recombined in Escherichia coli and purified for minimum inhibitory concentration (MIC) determination. The rVpBD displayed broad-spectrum inhibitory activity towards all tested bacteria with the highest activity against Staphyloccocus aureus and Pseudomonas putida. These results indicated that VpBD was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.
Highlights
The Manila clam, Venerupis philippinarum, is an important marine bivalve for commercial fisheries, accounting for about 80% of mudflat fishery production in China (China Bureau of Fisheries, 2004)
The gene-encoded cationic antimicrobial peptides (AMPs) are major humoral components of the innate defense systems [5,6], which were considered as promising therapeutic candidates for their innate advantages of less bacterial resistance and very specific targets [3]
The complete sequence of VpBD cDNA contained a 59 untranslated region (UTR) of 126 bp, a 39 UTR of 229 bp with a canonical polyA tail, and an open reading frame (ORF) of 285 bp encoded a polypeptide of 94 amino acids with the predicted molecular weight of 11.35 kDa and the theoretical isoelectric point of 9.46
Summary
The Manila clam, Venerupis philippinarum, is an important marine bivalve for commercial fisheries, accounting for about 80% of mudflat fishery production in China (China Bureau of Fisheries, 2004). Clam culture in China is sustaining a severe mortality problem, and suffering great economic losses [1]. The gene-encoded cationic antimicrobial peptides (AMPs) are major humoral components of the innate defense systems [5,6], which were considered as promising therapeutic candidates for their innate advantages of less bacterial resistance and very specific targets [3]. The discovery of antimicrobial peptides provides new clues for a fundamental understanding of the species immunity response and for further establishment of disease control in the practice of aquaculture industry. The presence of lower molecular mass AMPs started to be investigated from last decade [13]. Approximately 20 AMPs have been isolated or cloned from mollusks, mainly from mussels (for review see [14])
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