| Hepcidin is a cationic antimicrobial peptide mainly expressed in the liver and rich in cysteine residues,including signal peptide,leader peptide and maure peptide.It is an important innate immune factor,exhibiting potent antibacterial activity and immunomodulatory functions.Hepcidin has been found in humans and other mammals,avians,reptiles,and amphibians.In addition,it has been reported that Hepcidin is widely presented in a variety of fish species,which is expected to have a promising prospects in aquaculture.In our previous studies,two isoforms(Hepcidinl and Hepcidin2)of the Hepcidin gene from Lateolabrax japonicus were cloned and the expression pattern of Hepcidin2 were also illustrated in vivo.Other researchers expressed Hepcidin2(LJ-hep2)gene in a prokaryotic system and found that the recombinant product only showed activity against Vibrio harveyi.In order to better understanding the antimicrobial activity and application value of LJ-hep2,the chemically synthesized LJ-hep2 mature peptide was used to preliminarily explore its antimicrobial activity and mechanism,and to verify its immunoprotective effect on medaka Oryzias melastigma infected by Aeromonas hydrophila.Meanwhile,LJ-hep2 was expressed with a eukaryotic expression system,in which two expression vectors were constructed including the precursor peptide of LJ-hep2,and the fusion peptide combined the precursor peptide with the mature peptide of Scygonadin originated from Scylla paramamosain.The antimicrobial activity and function of the recombinant proteins were investigated,which laid the foundation for large-scale fermentation and applacation in the future.The results obtained are presented as follows:1.Chemical synthesis of LJ-hep2 mature peptide LJ-hep2(66-86)and analysis of its antimicrobial activity.The results showed that LJ-hep2(66-86)exhibited antibicterial activity against several Gram-positive and Gram-negative bacteria,such as Corynebacterium glutamicum,Staphylococcus epidermidis,Bacillus subtilis,Micrococcus lysodeikticus,Aeromonas sobria,Shigella flexneri,Pseudomonas stutzeri,Escherichia coli,Pseudomonas aeruginosa,Aeromonas hydrophila,Edwardsiella tarda,and Pseudomonas fluorescens,and their minimal inhibitory concentration(MIC)is 3-6 μM,6-12 μM,6-12 μM,3-6 μM,24-48 μM,3-6 μM,6-12 μ,1.5-3 μM,6-12μM,6-12 μM,24-48 μM and 24-48 μM,respectively.In addition,LJ-hep2(66-86)also showed bactericidal activity against a variety of clinically drug-resistant bacteria,such as Acinetobacter baumannii(QZ18050),A.baumannii(QZ18055),Klebsiella pneumoniae(QZ18106),K.pneumoniae(QZ18107),P.aeruginosa(QZ19124),P.aeruginosa(QZ19125),E.coli(QZ18109),E.coli(QZ18110),Enterococcus faecium(QZ18080),E.faecium(QZ18081).Their minimal inhibitory concentration(MIC)is 1.5-3 μM,1.5-3 μM,6-12 μM,12-24 μM,6-12 μM,6-12 μM,3-6 μM,3-6 μM,6-12μM and 6-12 μM,respectively.The results of bactericidal kinetics curves showed that LJ-hep2(66-86)could kill all the bacteria within 2 h after incubated with P.aeruginosa,A.hydrophila,E.coli,and S.flexneri;within 1 h with S.epidermidis,within 2 min with B.subtilis.LJ-hep2(66-86)has good thermal stability,but its antimicrobial activity is easily affected by Na+.As the concentration of Na+ increased,the antibacterial activity of LJ-hep2(66-86)against E.coli,S.flexneri,S.epidermidis,and P.aeruginosa gradually weakened.When the Na+concentration reached 160 mM,LJ-hep2(66-86)exhibited no antibacterial activity except for P.aeruginosa.which still has certain activity.In a solution of 10 mM Na+ concentration.LJ-hep2(66-86)has no antibacterial activity against B.subtilis and P.fluorescens.What’s more,the safety of synthetic peptides as exogenous proteins was clarified,and the results showed that it has no obvious toxic effect on mammalian cells(293T,NCI-H460),but has a slight toxic effect on insect cells(Sf9)at high concentration(96 μM).2.The antibacterial mechanism of synthetic peptides was preliminarily studied.Scanning electron microscopy(SEM)observation results showed that after treated with LJ-hep2(66-86),the morphological characteristics of E.coli,P.aeruginosa,and S.epidermidis changed significantly over time,and the bacteria wrinkled shrinking and breaking,the contents leaked.Bacterial genome binding experiments showed that the bacterial genome could be combined by LJ-hep2(66-86).By analyzing the results of the bacterial agglutination activity assay and the polysaccharide binding experiment,it is speculated that LJ-hep2(66-86)might be the first interaction with the bacterial cell membrane,affecting the stability of the bacterial cell membrane,causing membrane rupture,and finally the bacteria lysis and death.However,the specific antibacterial mechanism needs further study.3.Obtain the recombinant protein of LJ-hep2 precursor peptide and Scy/LJ-hep2 fusion peptide.Several eukaryotic expression vectors were constructed using pPIC9K plasmid,including a tandem expression vector consisting of 5 LJ-hep2 mature peptide;an LJ-hep2 precursor peptide expression vector with a 6×His tag at the N termial;LJhep2 precursor peptide expression vector with 8 X His and 6 X His tag at the N and C terminal respectively;combined peptide expression vector of LJ-hep2 precursor peptide and Scygonadin mature peptide,with a linker peptide targeted by Kex2;LJhep2 precursor peptide and Trx combined peptide expression vector,and the peptide sequence specifically recognized by P3C protease is used as linker;LJ-hep2 precursor peptide and Scygonadin mature peptide combined peptide expression vector,with GGPGSG as linker After expression and purification,the recombinant protein of LJhep2 precursor peptide and Scy/LJ-hep2 combined peptide was successfully obtained.4.Clarify the antibacterial function of the recombinantly expressed LJ-hep2 precursor peptide.The results showed that the recombinant LJ-hep2 precursor peptide exhibited antimicrobial activity against S.epidermidis,B.subtilis,C.glutamicum,S.flexneri,P.fluorescens,and Cryptococcus neoformans.Their MIC is 1.5-3 μM,24-48μM,1.5-3 μM,1.5-3 pM,12-24 μM and 12-24 μM,respectively.Furthermore,LJ-hep2 precursor peptide had binding activity with bacterial polysaccharide such as LPS,LTA and PGN,and also showed good binding affinity with E.coli,while not C neoformans and B.Subtilis.5.The study found that the recombinant protein Scy/LJ-hep2 has good bactericidal activity against bacteria and fungi including S.epidermidis,B.subtilis,C.glutamicum,M.lysodeik,S.flexneri,P.stutzeri,E.coli and C.neoformans.The MIC is 3-6 μM,1224 μM,24-48 μM,3-6 μM,1.5-3 μM,24-48 μM,1.5-3 μM and 12-24 μM,respectively.The bactericidal kinetics curves showed that Scy/LJ-hep2 could kill C.neoformans within 12 h.6.LJ-hep2(66-86)could improve the survival rate of O.melastigma after infected with A.hydrophila.By co-injecting LJ-hep2(66-86)with A.hydrophila,the result showed a higher survival rate compared with the control group(A.hydrophila injection only),increased by about 40%. |
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