Effect Of Hepcidin On The Growth Of Aeromonas Veronii By Regulating Iron Metabolism In Yellow Catfish(Pelteobagrus Fulvidraco)

Hepcidin is an antibacterial peptide with iron regulation function.It binds to membrane iron transporter ferroportin(FPN)on the surface of hepatocytes,intestinal epithelial cells and other cells,resulting in the internalization and degradation of FPN,so as to regulate iron homeostasis.Iron is not only the basic element to maintain the function of organisms,but also an important nutrient for pathogens to compete after infecting the host.Human beings have developed a variety of calmodulin related drugs to treat various congenital immune diseases and other diseases.Pelteobagrus fulvidraco is one of the most common species in aquaculture in recent years and plays an important role in aquaculture in China.In its breeding process,P.fulvidraco is easy to be infected with ascites disease caused by Aeromonas veronii,resulting in large-scale death and huge economic losses to the breeding industry.At present,there are few studies on the function and application of fish ferritin.The previous research of our research group found that feeding grass carp ferritin protein can effectively inhibit the growth of Flavobacterium columnatus and improve the immune protection rate of grass carp.This study focuses on the function of iron calmodulin gene of P.fulvidraco,which can provide new ideas for the prevention and control of bacterial diseases of P.fulvidraco.The relevant experimental results are as follows:1.The recombinant P.fulvidraco Hepcidin protein rPf Hep(recombinant P.fulvidraco Hepcidin protein)was successfully expressed using the prokaryotic expression system,and it was verified in vitro that rPf Hep has iron-regulating activity,which can inhibit bacterial proliferation and improve cell viability.The p GEX-KG-Hepcidin recombinant plasmid was constructed and transferred into BL21(DE3)p Lys S competent cells to successfully express rPf Hep.The protein was purified by affinity chromatography.The size of rPf Hep was 35 k Da by SDS-PAGE,which was detected by Western-Blot.The target band,the higher purity P.fulvidraco Hepcidin recombinant protein was obtained.After incubating 100 μM rPf Hep with primary hepatocytes of yellow catfish for 6 hours,the m RNA level of Hepcidin gene in hepatocytes increased significantly,and the content of ferritin protein in hepatocytes increased,indicating that rPf Hep can promote Hepcidin downstream related molecules in hepatocytes express.Hepatocytes were pretreated with 100 μM rPf Hep for 6 hours and then infected with A.veronii for 3 hours.It was found that the number of bacteria in the rPf Hep group was less than that in the control group,and the number of bacteria survival was higher.It shows that the addition of rPf Hep limits the available iron for the proliferation of A.veronii and protects cells from bacteria.2.Screen out Hepcidin agonist-Berberine hydrochloride(BBR)and antagonist-Fursultime(FSL),BBR and FSL can regulate the expression of Hepcidin and affect the growth of bacteria;feeding rPf Hep and BBR can increase the immune resistance of P.fulvidraco to bacteria and reduce mortality.From the cell level,the Hepcidin m RNA level was significantly higher than that of the control group after 50 μM BBR treatment of hepatocytes for 6 hours,and the Hepcidin m RNA level of the 10 μM FSL treatment group was significantly lower than that of the control group.BBR and FSL pretreated A.veronii-infected hepatocytes.The proliferation level of A.veronii was consistent with the change trend of Hepcidin m RNA level.At the individual level,after feeding rPf Hep and BBR 24 h,the serum iron content in the treatment group was lower than that in the control group,and there was obvious iron accumulation in the liver of rPf Hep group.On the third day after challenge,rPf Hep and BBR treatment groups significantly reduced the bacterial load in gill,liver,intestine,kidney and spleen.It alleviated the pathological damage of gill and liver,reduced the serum iron content of P.fulvidraco and increased the iron storage concentration of liver.The mortality of P.fulvidraco seven days after infection was counted.It was found that rPf Hep and BBR reduced the mortality of P.fulvidraco;the relative percentage survival(RPS)of BBR group was 55.98%.The growth of bacteria needs the participation of iron.Reducing the available iron content of bacteria can inhibit the proliferation of bacteria.This study focused on the Hepcidin-related related pathway of P.fulvidraco,expressed the prokaryotic recombinant fusion protein rPf Hep,and verified its function in cells and individuals.The agonist BBR and antagonist FSL were screened on cells.The individual level proved the in vivo protective effect of rPf Hep and BBR,which provided a new idea for the control of ascites disease of P.fulvidraco caused by A.veronii in aquaculture production.