| Larimichthys crocea,an important economic species in China offshore,also a distinct cultured fish species in southeast coast of China,composes the largest yield for a single species in marine net-cage farming.The industry of crocea is a integrated and wide geographical reach range in the fish fisheries of China.However,disordered fish farming which seeks maximum benefits had led to various diseases outbreak,especially the marine white spot disease caused by ectoparasitic protozoa Cryptocaryon irritans.Its high incidence and mortality have resulted in one serious threat to many marine fish,could cause 75%mortality of L.crocea.Chemical drugs treatment,such as antibiotics,was difficult to prevent or cure seasonal epidemics.Above all,they could bring about more serious drug tolerance,drugs residue,and destruction of microecosystem.Recent studies have focused on natural active components from organism itself.Antimicrobial peptides(AMPs)are one of the key components in the innate immune system.The diverse biological properties of teleost AMPs have been the focus of researches,and a large number of novel AMPs have been isolated from fish.The piscidins constitute a kind of AMP family,studies with in vitro assays have confirmed the piscidin’s potency against infectious agents through multiple mechanisms,including bactericidal activity,inactivation of viruses,damage to fungal membranes,and parasiticidal activity.The differential expression AMPs were screened from hepatic transcriptome of C.irritans-immunized L.crocea in this study.Both upregulation expression and fold change of piscidin 5 like were the greatest among the candiated AMPs,it was selected to characterize its functional features.Both induction expression and tissue and cell localization changes in the healthy and C irritans-immunized L.crocea were analyzed by quantitative real-time PCR(qRT-PCR)and in situ hybridization(ISH)on the level of mRNA,respectively.The prokaryotic expression system was constructed successfully,the induction expression and purification conditions were optimized,and its resistance charaterisitics were studied on the level of protein.The main results were as follows:1.Excavation of differential expression AMPs from hepatic transcriptome of C.irritans-immunized L.croceaTaking advantage of the hepatic transcriptome of C.irritans-immunized L.crocea at 3d post infection,seven kinds of differential expression AMPs were screened,including liver expressed antibacterial peptide 2 like(LEAP 2 like),LEAP-2A,hepcidin,hepcidin-like,piscidin 5 like,piscidin 5 like type 4 and bactericidal permeability increasing protein(BPI).Hepcidin,hepcidin-like,piscidin 5 like,piscidin 5 like type 4 and BPI were up-regulated to protect L.crocea from being damaged by C.irritans infection;while LEAP 2 like and LEAP-2A were down-regulated,they might be as a negative-feedback regulator,or involved in some other regulatory mechanisms to adjust the excessive immune response during the defense.The differential expression changes were verified by qRT-PCR to verify the reliability of the RNA-seq data.Interestingly,the alternative splice of LEAP-2A mRNA existed,and whether the phenomenon was caused by C.irritans infection still needed to be conducted.Therefore,the data showed that these AMPs were involved in the immune response to the C.irritans infection.2.The tissue and cell localization of Lc-P5L mRNA before and after C.irritans infectionISH was used to detect tissue and cell localization of Lc-P5L mRNA in healthy and C.irritans-immunized L.crocea.Positive signals were distributed mainly in the head kidney,followed in gill,intestine,spleen and liver,while there were no any signals in the muscle.In addition,the transcription of Lc-P5L mRNA was in the mast cells(MCs).Positive signals in the head kidney were radially distributed around melano-macrophage center,they were only distributed in submucosa of intestine,and sparsely distributed throughout the liver of healthy individuals;after infected by C.irritans,only signals strength were increased in these three tissues.In the normal gill,positive signals mainly distributed on both sides of blood vessels in gill filaments,it reached maximum in the top of the filaments,and there were random distributions in the secondary filaments.In the C.irritans-immunized L.crocea,the number and strength of positive signals increased,a lot of positive signals clustered in the gill arch,while the aggregation degree in the top filaments became unapparent.Positive signals mainly distributed in the margin of spleen in healthy individuals,while increased positive signals number and strength distributed throughout the spleen.The results were in accordance with the previous qRT-PCR result and other positioning reports.The infection of C.irritans coild result in the upregulation of Lc-P5L mRNA either by enhancing transcription capacity,or increasing number of MCs.3.The induction expression of Lc-P5L mRNA by C.irritans infectionAfter infected by C.irritans,Lc-P5L mRNA was significantly upregulated in all detected tissues.The upregulated time points were highly consistent with the critical developmental stages of C.irritans,which illustrated infective stage,parasitic and secondary bacterial infection stages of C.irritans could cause strong immune response of hosts.Therefore,Lc-P5L was involved in the immune response against C.irritans infection.4.The optimization of induction expression and purification conditionsThrough constructing prokaryotic expression systen with pET-28a vector and E.coli BL21,the induction expression conditions were explored,OD600 reached 0.5 in modified LB medium with 0.5%glucose to add 0.3mM IPTG for 4h at 28℃,or 37℃for 6h in simplified autoinduction medium could obtain maximum induction expression level.The induced rLc-P5L existed in the inclusion bodies,purification conditions were also optimized through graded imidazole.The purified rLc-P5L was confirmed by western blot and mass spectrum identification.Secondly,heat treatment results indicated rLc-P5L owned high thermal stability.The recombinant system provided technology support for future potential to be a novel drug,or an addition agent.5.The resistance characteristics of rLc-P5LIn vitro assay showed rLc-P5L was of widely spectrum antibacterial activity in concentration-and time-dependent manners,it could cause gathering of plenty of bacteria,generated filament or floccule twining or coating bacteria.rLc-P5L was able to bind to bacteria through targeting to some pathogen-associated molecular patterns(PAMPs),such as LPS,LTA.Moreover,rLc-P5L could also bind to bacterial genomic DNA.In addition,Lc-P5L mRNA was involved in the immune response against infection of C.irritans.rLc-P5L exhibited strong parasiticidal activity to C.irritans theronts and trophonts,it could cause cilia inactivation or falling off,macronucleus swell and depolymerized,membranes rupture and contents leakage at last.Moreover,rLc-P5L could not cause eukaryotic cells hemolysis under effective concentrations of antibacterial and antiparasitic activity.In summary,Lc-P5L was of potential to be listed in the novel candidates for drug or addition agent development. |
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