Construction And Detection Of Pichia Pastoris Recombinant Expression Vector Of Modifying Antimicrobial Peptide Eumenitin Sequence And Evaluation Of Its Antimicrobial Activity

When the pathogen infects the host of various ways,it will grow and reproduce in the host,causing dysfunction of the body.Antibiotics are the most powerful weapon to kill pathogenic bacteria and play an important role in protecting animal health.Because of the extensive use of antibiotics,bacterial resistance is widespread,especially the emergence of highly resistant strains,resulting in the clinical efficacy of most drugs to drug-resistant bacteria reduced or ineffective.In addition,the toxic and harmful substances produced by antibiotics in the process of metabolism are not easy to be cleared by the body,and can cause damage to tissues and organs,forces researchers to seek safer and more effective alternatives to antibiotics.The discovery of antimicrobial peptide has brought the dawn for the research and development of new antibiotics.Antimicrobial peptides are easy to be metabolized by the body,have high safety,and have a variety of biological activities.They are excellent alternatives to antibiotics.However,the antimicrobial activity of natural antimicrobial peptides are weaker than that of traditional antibiotics.At present,the design strategy of natural antimicrobial peptides is based on changing their charge number,amphiphilicity,hydrophobicity and spatial conformation,so as to improve their antimicrobial activity and reduce hemolysis and cytotoxicity.In this study,Eumenitin,a bee-derived antimicrobial peptide with broad antimicrobial spectrum and low hemolysis,was screened for the database of antimicrobial peptides.Eumenitin(E1)was used as the parent peptide to predict and modify its charge number,hydrophobicity and amino acid composition.Four novel heterozygous peptides with antimicrobial potential were designed and named E2,E3,E4 and E5.The gene sequence was optimized by Pichia pastoris codon.The target gene was cloned into p MD19T-simple vector and recombined into Pichia pastoris eukaryotic expression vector p GAPZαA,transformed into Pichia pastoris GS115.The positive transformant was screened by Zeocin-containing resistant plate.The target protein was detected by Tricine-SDS-PAGE after the high-copy recombinant was expressed on a small scale.The hemolytic and bacteriostatic activities of five AMPswere tested.The results showed that the hemolysis of Eumenitin was low,and the hemolysis rate of the new peptide was lower than 5% when the concentration of Eumenitin was 512 μM,which indicated that the hemolysis ability of the five antimicrobial peptides to erythrocyte was weak.By calculating the bacteriostasis rate,the bacteriostasis effect of AMPs was evaluated.E1 has bacteriostatic effect on Proteus mirabilis,Streptococcus agalactiae and Streptococcus dysgalactiae in a specific concentration.E2 has bacteriostatic effect on Staphylococcus aureus.E3 has certain bacteriostatic effect on Streptococcus agalactiae.E4 has bacteriostatic effect on Streptococcus dysgalactiae and Staphylococcus aureus,and the bacteriostatic effect on Staphylococcus aureus is the highest.E5 has bacteriostatic effect on Escherichia coli,Proteus mirabilis,Streptococcus agalactiae,Streptococcus dysgalactiae and Pasteurella multocida all have bacteriostasis effect.Among them,the bacteriostasis rate of Streptococcus agalactiae and Streptococcus dysgalactiae is higher.