Liver-expressed Antimicrobial Peptide 2(LEAP-2) And ?-defensin Of Trachinotus Ovatus Might Be Involved In Anti-pathogeny Immune Response

Liver-expressed antimicrobial peptide-2(LEAP-2)and ?-defensin are the members of antimicrobial peptides family.Mammalian researches demonstrated that LEAP-2 and?-defensin contain numbers of cationic and play a key role in the innate immune system of organism.In this study,we cloned and identified TroLEAP-2 and TroBD of golden pompano(Trachinotus ovatus),and analyzed their functions in vivo and in vitro.The results are as follows:(1)TroLEAP-2 contains a 321 bp open reading frame(ORF)that encodes 106 putative amino acids with a molecular weight of 11.65 kDa.The mature peptide of TroLEAP-2 possesses four conserved cysteine residues.The amino acids sequence has high identities(40.57%-83.02%)with known liver-expressed antimicrobial peptide-2 of other teleosts.TroLEAP-2 was observed to have the highest similarity(83.02%)with Miichthy milluy LEAP-2.The phylogenetic analysis showed that TroLEAP-2 clustered with the LEAP-2 of M milluy and Larimichthys crocea that belong to Perciformes.TroLEAP-2 exhibited the highest expression in liver,spleen and kidney.Furthermore,the expression of TroLEAP-2 was significantly upregulated under Edwardsiella tarda,Streptococcus agalactiae and viral neuro necrosis virus(VNNV)infection.In vitro,purified recombinant TroLEAP-2(rTroLEAP)could significantly inhibit the growth of E.tarda and S.agalactiae.In vivo,the results demonstrated that overexpression of TroLEAP-2 significantly reduced bacterial(E.tarda and S.agalactiae)colonization or viral(VNNV)copies of tissues.In contrast,knockdown of TroLEAP-2 expression resulted in the quantity of bacteria or viruses increasing in fish tissues.Moreover,the results of an in vivo study showed that the overexpression of TroLEAP-2 significantly improved the levels of acid phosphatase,alkaline phosphatase,and superoxide dismutase.In addition,TroLEAP-2 could improve the activity of respiratory burst activity in head kidney(HK)macrophage.These results indicate that TroLEAP-2 plays a significant role in the innate immune system of T.ovatus.(2)The complete cDNA sequence of TroBD is 592 bp,which contains a 192 bp ORF that encodes a 7.217 kDa molecular weight of putative peptide comprising 63 amino acids.The mature peptide of TroBD contains six conserved cysteine residues.TroBD shares high sequence identities(27.27-98.41%)with known(3-defensins of other teleosts.TroBD had the highest similarity(98.41%)with Siniperca chuatsi ?-defensins.The phylogenetic analysis showed that TroBD has the closest phylogenetic relationship with the ?-defensins of Oplegnathus fasciatus,which also belongs to Perciformes.The expression of TroBD was most abundant in the head kidney and spleen,and was significantly upregulated following infection by Vibrio harveyi and VNNV.Purified recombinant protein(rTroBD)inhibited the growth of Vharveyi,and its antibacterial activity was affected by salt concentration.In vitro,the results showed that TroBD had a chemotactic effect on HK macrophages.In vivo,we found that overexpression/knockdown of TroBD significantly decreased/increased the bacterial(V harveyi)colonization or virus(VNNV)copies in tissues.In conclusion,these results indicated that TroLEAP-2 and TroBD play an important role in both antibacterial and antiviral immunity and provide a new avenues for protection against pathogens infection in the golden pompano.