Isolation,Identification And Immune Regulation Of Piscidin-1 Natural Peptide From Grouper Antimicrobial Peptide

Piscidins,the teleost antimicrobial peptides play an important role in fish non-specific immune system.Piscidin is a type of small molecule amphipathicand cationicpoly peptide,containing 20-50 amino acid residues,in alpha helix structure,with a broad spectrum of bactericidal,anti-tumor,anti-viral and anti-protozoa activity.Studies have shown that piscidin has an immunoenhancing effect,but the mechanism of the immunomodulation is far from elucidation.The present research team has previously carried out transcriptome study using RNA-Seq in splenic lymphocytes of orange-spotted grouper(E.coioides)stimulated by piscidins.Differentially expressed genes were analyzed on KEGG-based signalling pathway enrichment,showing that piscidin regulated the immune signallin pathway.However,the results have not yet been verified.In this study,the mature peptide of piscidins-1 in orange-spotted grouper(E.coioides),ecPis-1S,was identified from the skin mucus of orange-spotted grouper(E.coioides)for the first time with gel filtration chromatography followed by high performance liquid chromatography and nano-LC-ESI-MS/MS.It was revealed that orange-spotted grouper piscidins-1 will undergo at least two steps of cleavage to remove the signal peptide and prodomain at COOH terminal of the peptide respectively.In this study,lymphocytes were isolated and purified from the spleen tissue of orange-spotted grouper(E.coioides).The mature peptide ecPis-1S and the full length peptide ecPis-1L were chemically synthetized and stimulated the splenic lymphocytes of the grouper in vitro to study the immunomodulatory activity.As results,both ecPis-1S and ecPis-1L enhanced significantly splenic lymphocyte proliferation of grouper at the concentration of 0.25 ?mol/L,0.5 ?mol/L,2.0 ?mol/L,and 4.0 ?mol/L(p<0.05).However,no dose-dependent effect was observed for both of the peptides.In addition,the effects of ecPis-1S and ecPis-1L on immune gene expression in splenic lymphocytes of thegrouper were studied at transcriptional level in this study by realtime fluorescence quantitative reverse transcripase polymerase chain reaction(RT-qPCR).The results showed expression of various genes,including IL-1,TNF?,was up regulatedin splenic lymphocytes stimulated by at the concentration of 4.0 ?mol/L for 24 h.Compared with the control group,the expression of interleukin(IL-1),tumor necrosis factor(TNF?),transforming growth factor-?(TGF?),Drosophila similar to mothers against decapentaplegic(Smad),cyclin-dependent kinases(cdk),cell division cycle protein(cyc),major histocompatibility complex(MHCI,MHCII)and other related immune genes were significantly increased(P<0.05).The study demonstrated that grouper antimicrobial peptide ecPis-1S and ecPis-1L possessed immunomodulatory activities to regulate innate signaling pathway and stimulate the proliferation of splenic lymphocytes.