Molecular Characterization of a Novel Big Defensin from Clam Venerupis philippinarum

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.

Cloning and characterization of a sialic acid binding lectins (SABL) from Manila clam Venerupis philippinarum

“…[16]. BLAST analysis of all the 3226 EST sequences revealed that one EST of 508 bp was highly similar to the previously identified SABL.…”

“…The salinity for the supplied seawater was kept at 30&. The performance of V. anguillarum challenge experiment was according to our previous work [16,17]. There were five replicates for each challenge point and control group.…”

A defensin from clam Venerupis philippinarum: Molecular characterization, localization, antibacterial activity, and mechanism of action

“…Allam et al (2006) reported a rapid increase (within 72 h) of circulating hemocytes in V. philippinarum after injection of Vibrio tapetis and V. anguillarum. In general, expression of marine mollusk defensin transcripts is known to be induced by bacterial challenges (Adhya et al, 2012;De Zoysa et al, 2010;Rosa et al, 2011;Zhao et al, 2007Zhao et al, , 2010. However, there are some reports of decreased transcriptional responses of mollusk defensins against bacterial infection.…”

“…A Manila clam EST homologous to C. gigas defensin2 was identified through large scale EST sequencing of the cDNA library constructed from clam hemocytes (Zhao et al, 2010). To generate the full-length cDNA of VpDef, two reverse primers P1 and P2, and two forward primers P3 and P4 (Table 1), were designed based on the EST sequence.…”

“…Recombinant VpDef peptide (rVpDef) was obtained according to the method described previously with minor modifications (Zhao et al, 2010). The mature peptide of VpDef was amplified with genespecific primers P11 and P12 with Nde I and Xho I sites at their 5′ end, respectively (Table 1).…”