Study On Endogenous Antimicrobial Peptides Of Apostichopus Japonicus Based On Peptiomics

In recent years,the abuse of antibiotics has led to the emergence of drug-resistant strains,which makes the prevention and treatment of bacterial diseases once again a difficult problem.Antimicrobial peptides are a kind of small defensive peptides produced by organisms when they resist the invasion of external microorganisms.They are not easy to produce drug resistance and cross resistance of pathogenic bacteria.They have the advantages of small molecular weight,high water solubility,good thermal stability,wide antibacterial spectrum and so on.In recent years,the antimicrobial peptides from Marine sources have received extensive attention,but there are few researches on the antimicrobial peptides from sea cucumber.In this thesis,the immune system of Apostichopus japonicus was stimulated to produce antimicrobial peptides by scratch,and the endogenous peptides were identified and analyzed by polypeptomics and bioinformatics tools.Then the endogenous antimicrobial peptides were predicted and screened by the antimicrobial peptide database,and the antibacterial activity,stability,safety and antibacterial mechanism of the antimicrobial peptides were analyzed and studied.The aim of this study was to screen out the antibacterial peptides of Apostichopus japonicus with strong antibacterial activity,good stability and high safety.The main conclusions are as follows:The endogenous peptides of healthy Apostichopus japonicus were identified by the polypeptomics technique and the endogenous peptides of Apostichopus Japonicus after cutting for 12 and 24 h.Bioinformatics analysis showed that cell proliferation and nerve cell differentiation were underway in Apostichopus japonicus after cutting for 24h.791 endogenous antimicrobial peptides were predicted using CAMP_R3 antimicrobial peptide database.The predicted 28 suspected antimicrobial peptides were synthesized.Seven endogenous antimicrobial peptides were obtained using Escherichia coli,Pseudomonas aeruginosa,Bacillus subtilis,Staphylococcus aureus and Candida albicans as test strains.SC-1,SC-6,SC-7,SC-8,SC-10,SC-13 and SC-20 showed antibacterial activity against Escherichia coli,but not against Pseudomonas aeruginosa and Candida albicans.SC-8 and SC-13 showed antibacterial activity against Bacillus subtilis and Staphylococcus aureus.By NCBI comparison,SC-1,SC-10 and SC-13were derived from hypothetical protein,while SC-6,SC-7,SC-8 and SC-20 were derived from major yolk protein 1.The antimicrobial peptides of Apostichopus japonicus were basically produced by cutting induction.The results showed that the minimum inhibitory concentrations of acanthopepin against Escherichia coli were SC-8（64μg/mL）,SC-6/SC-13/SC-20（128μg/mL）and SC-1/SC-7/SC-10（256μg/mL）,respectively.The minimum inhibitory concentration of SC-8 and SC-13against Bacillus subtilis was 512μg/mL.ExPASy Protparam tool,HeliQuest,HNN,TpxinPred and HemoPI in NP@were used to predict the antiseptic peptides of Apostichopus japonicus.The results showed that 7 antiseptic peptides of Apostichopus japonicus had positive charge,and SC-6,SC-7 and SC-8 had certain amphiphilicity.SC-1 had a high proportion ofα-helix,and7 antimicrobial peptides were non-toxic,but had certain hemolytic activity.The results of bactericidal kinetics showed that SC-1,SC-6,SC-7,SC-8 and SC-13 could kill all Escherichia coli within 30 min and SC-20 could kill all Escherichia coli within 240min when the final concentration of antiseptic peptides was 4×MIC.It takes 300 min for SC-10 to completely kill Escherichia coli.The results of stability test showed that SC-8 and SC-20 had good thermal stability,SC-6,SC-7,SC-10 and SC-13 were very sensitive to heat,and the antibacterial rate decreased to 28.85%,55.49%,63.87%and67.97%,respectively.SC-10 is stable within 10 times of freezing and thawing.SC-6,SC-7,SC-8,SC-10,SC-13 and SC-20 have good stability under UV irradiation for 120min.The stability of SC-1,SC-7,SC-8 and SC-13 is better when stored at room temperature for 10 days.The hemolytic activity results showed that no hemolytic activity was observed at the maximum concentration of 1 024μg/mL.The cytotoxicity of Apostichopus japonicus antimicrobial peptides to BJ cell line and Hela cell line was determined by CCK-8 method.SC-8 and SC-10 showed no cytotoxicity to BJ cell line,while SC-1,SC-6,SC-7,SC-13 and SC-20 showed certain toxicity to BJ cell line at the test concentrations of 32～1 024μg/mL.SC-6,SC-7 and SC-13 showed no cytotoxicity to Hela cell line,while SC-1,SC-8,SC-10 and SC-20 showed some cytotoxicity to Hela cell line.The survival rate of Hela cell line was 47.60±12.04%when SC-1 was treated at 1 024μg/mL.The results showed that SC-8 had good antibacterial activity,high stability,no hemolytic activity and no cytotoxicity.SC-8 was found to have membrane damage by electron microscopy.SC-8 and ethidium bromide competitive binding DNA tests showed that SC-8 can bind to bacterial DNA in vitro,possibly with non-membrane damage.