Stable expression of bioactive recombinant pleurocidin in a fish cell line

Abstract

Pleurocidin (Ple), a linear cationic peptide of 25 amino acids, is a member of a larger family of antimicrobial peptides present in flatfish. Previous studies have shown that Ple displays a strong antimicrobial activity against a broad spectrum of bacteria and appears to play a role in innate host defence. In this work, the genomic sequence encoding the Ple prepropeptide has been isolated from Limanda limanda and cloned in a vector under the control of a non-viral promoter (the carp beta-actin promoter). By using this construction, expression of bioactive Ple was demonstrated in transformed fish cell lines continuously growing for more than 2 years. Furthermore, the study of Ple processing, maturation and secretion (by using fusion with green fluorescence protein) and the high bactericidal activity of the secreted recombinant Ple (detectable in cell supernatants without any concentration) are all reported here, as no other recombinant Ple or fish antimicrobial peptide have been expressed before to that extent. Such an overexpression of recombinant Ple or any other related antimicrobial peptide might improve the chances to develop new antibiotic agents, as well as to provide essential information about the mechanism of action, range of activity and the role in the innate immune response of antibiotic peptides.

Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus)

“…Cell transfection assays were performed as previously described [27,28]. Briefly, EPC cell monolayers grown in culture flasks of 75 cm 2 were detached using trypsine (Sigma), washed, resuspended in culture medium supplemented with 10% of FCS and dispensed into 24-well plates at a concentration of 2.5 Â 10 5 cells per well in a final volume of 250 ml.…”

“…Two hour post-infection at 14 C, infected cells were washed, 100 ml/well of fresh cell culture medium (RPMI Dutch modified) supplemented with 2% FCS added and plates further incubated for 24 h at 14 C. To evaluate VHSV 860 infectivity, the EPC cell monolayers were fixed for 10 min in cold methanol and airdried and the immunostaining focus assay was performed as previously described byMas et al (2006). Brown foci of DAB stained cells (VHSV-infected cell foci) were counted with an inverted microscope with a 10 Â ocular eye grid.…”

In vitro and in vivo differential expression of rainbow trout (Oncorhynchus mykiss) Mx isoforms in response to viral haemorrhagic septicaemia virus (VHSV) G gene, poly I:C and VHSV

“…For cell transfection, RTG-2 cells, grown in culture flasks of 75 cm 2 , were detached using trypsin (Sigma), washed, resuspended in MEM supplemented with 10% FCS and dispensed into 24-well plates at a concentration of 6 Â 10 4 cells per well in a final volume of 300 ml. Then 0.5 mg of pMCV 1.4-G complexed with 1 ml of FuGene 6 (Roche, Barcelona, Spain) were added to each well and the plates were further incubated at 20 C for 24, 48, 72 and 96 h as previously described [47].…”

In vivo modulation of the rainbow trout (Oncorhynchus mykiss) immune response by the human alpha defensin 1, HNP1

“…The purity of the peptide was >98%. The mature sequence of Limanda (Limanda limanda) pleurocidin (LmPle) (GWKKWFKKATHVGKHVGKAALDAYL) [17] was used as a control to verify that the effects of HNP1 on the trout immune response were specific of HNP1 and not only due to the injection of a foreign peptide. Synthetic LmPle was obtained from Diverdrugs (Diverdrugs S.A., Barcelona, Spain).…”