Screening,Heterologous Expression And Activity Analysis Of Antimicrobial Peptides From Penaeus Monodon

In recent years,the production of chinese aquaculture has leapt to the forefront of the world,accounting for 70%of aquaculture production around the world.However,disease in aquaculture frequently occurs with the scale of the aquaculture industry continued increasing.The popular mean of prevention is the application of chemical drugs and antibiotics,which ultimately led to a series of problems such as increased number of drug-resistant pathogens,increased environmental pollution and ecological risks.Antimicrobial peptides(AMPs)synthesized by organisms is a class of small molecule defense peptides and active substances of defensive peptide that produced by immune defense systems under the induction conditions against exogenous pathogens.It has the characteristics of high activity,strong specificity and broad-spectrum bactericidal effect.Meanwhile,it can neutralize endotoxin and enhance immunity.It is most likely to replace antibiotics as a new type of antibacterial.In this paper,some novel antimicrobial peptides were obtained through RNA-seq of Penaeus monodon,and then analysed by bioinformatics method.Prokaryotic expression system was used for soluble expression of antimicrobial peptides,then they were separated and purified by affinity chromatography.Finally,the function and activity of antimicrobial peptides were studied.The main research contents and results are as follows:(1)The expression of antibacterial peptides in Penaeus monodon were improved after Vibrio parahaemolyticus challenge for 3h,which caused death after 6h.After Penaeus monodon was injected Vibrio parahaemolyticus at the concentration of 1×10~7 CFU/mL for 3 h,the total RNA was extraced and then proceeded transcriptome sequencing project;(2)27.91 Gb of data and 40,040 unigenes with an average length of 1104 bp were obtained by RNA-seq.By annotating and enriching unigenes,the GO enrichment result showed that there were 103 genes related to immunity and virus.The KOG enrichment result showed 255 genes associated with defense mechanisms,and the KEGG enrichment result showed 5399 disease-related genes.Nine new antimicrobial peptide genes were predicted after comparing with the antimicrobial peptide database.Four of them belonged to the ALFs family,three belonged to Crustin families and two were lysozyme families.These antimicrobial peptides were analysized by bioinformatics methods and named as ALFPm9、ALFPm10、ALFPm11、ALFPm12、CarcininPm1、CarcininPm3、CrsutinPm11、Pmlyzi3 and Pmlyzi4,respectively;(3)The results of the structure prediction of the above antimicrobial peptides showed that four ALFs contained an LBD functional region which was consisted of 22 amino acid residues,three crustins contained a cysteine-rich region and WAP binding domain,and both lysozymes contains a cysteine-rich LYZ1 domain.Results of homology analysis showed that four ALFs,two Crustins and two lysozymes possessed homology with their near-source species,while Carcinin Pm3 did not find.(4)Eexpression of these antibacterial peptides with prokaryotic expression system was carried out by fused with SUMO protein.Results showed that the antimicrobial peptides were successfully expressed as soluble proteins except for ALFPm9,and the optimal induction time and IPTG concentration of these antimicrobial peptides were obtained.The higher purity antimicrobial peptides which were separated and purified by affinity chromatography were obtained with the concentration of protein up to 560μg/mL.The SUMO fusion tag was excised by SUMO protease.The results showed that 100μg of the target protein could be cleaved by treating with one unit of SUMO protease for 16 h at 4℃.The antimicrobial peptides were identified as target protein by liquid chromatography-mass spectrometer(LC-MS)after the fusion tag were excised.(5)Finally,we conducted a preliminary study on the antibacterial activity and mechanism of the recombinant antimicrobial peptide of ALFPm10.The antibacterial activity was analyzed by Optical Density(OD)method.The results showed that Bacillus sp.T2,Aeromonas hydrophila,and Vibrio Parahaemolyticu were significantly inhibited by ALFPm10 at the concentration of 12μM,and the bacterial density decreased significantly.The minimum inhibitory concentration experiment showed that the minimum inhibitory concentration of recombinant antimicrobial peptide of ALFPm10 on Aeromonas hydrophila was 6μM,and the antibacterial effect was better with increasing concentration.The effect of recombinant antimicrobial peptide of ALFPm10 on Bacillus sp.T2 was observed by scanning electron microscopy.It caused the cells of Bacillus sp.T2 to rupture and the morphogenesis to change severely.In summary,this experiment screened antimicrobial peptides from Penaeus monodon,which can inhibit the pathogens of aquaculture such as Vibrio Parahaemolyticu at the lower concentration.It is expected to be used in aquaculture and food preservation industries to achieve green farming.