Design Of The Hybrid Antibacterial Peptide Me-HNP1 Gene And Its Expression In Pichia Pastoris

Antimicrobial peptides are a small molecular weight peptides that are resistant to high temperatures and have a wide range of antimicrobial properties.Most of these natural peptides are produced by the immune system when the organism defends against external pathogens.Compared with traditional antibiotics,antimicrobial peptides show more stable and wider antibacterial activity.In addition to the inhibition of bacteria and fungi,it can also inhibit some viruses and protozoa.Because of its remarkable antibacterial effects and the advantage of causing less drug resistance,it attracts more and more attention of scholars and experts.Hybrid antimicrobial peptides are new antimicrobial peptides that are made by splicing two or more different types of natural antimicrobial peptides using certain design principles via gene synthesis technology.The spatial structure and physical and chemical properties can be adjusted through artificial modification,so as to obtain new antimicrobial peptides more appropriate for application.Melittin is an alkaline cationic polypeptide with strong antimicrobial activity,but also has hemolytic side effects.Amino acids in the positively charged region of the C-terminal of bee venom peptides are an important part of the interaction with the cell membrane.The?-helical structure is a necessary condition for maintaining hemolytic activity,but it has little effect on antimicrobial activity,and a greater impact on its hemolytic activity.Human defensins are antimicrobial and viral antimicrobial peptides,As the first line of defense against microorganisms in the innate immune system,they have good resistance to foreign invaders including common gram-positive bacteria,Gram-negative bacteria,fungi and viruses,but do not have hemolytic activity.In this study,15 aa of C-terminus of melittin and human defensin?HNP-1?were used as maternal peptides.bioinformatics software was used to to predict their physical and chemical properties and analyzing structural functions,design and combine a hybrid peptide Me-HNP1.The aim is to obtain a novel hybrid antimicrobial peptide with antimicrobial activity similar to that of melittin,and its hemolytic toxicity is much lower than that of melittin.The results are as follows:Pichia pastoris favor codons were chosen to translated hybrid antimicrobial peptide Me-HNP1 gene sequence,Primer5.0 software was used to design its upstream and downstream primers.The polymerase chain reaction?PCR?amplified the hybrid antimicrobial peptide Me-HNP1 gene.The obtained hybrid antibacterial peptide Me-HNP1 gene was connected to the vector p PICZ?A to obtain a recombinant expression vector p PICZ?A-Me-HNP1.After Eco R I/Not I double digestion and PCR amplification and DNA sequencing identification,the eukaryotic expression vector p PICZ?A-Me-HNP1 has been successfully constructed.The single-digested linearized plasmid p PICZ?A-Me-HNP1 and transferred into GS115 Pichia pastoris cells by electric shock transformation,and then the recombinant expression vector p PICZ?A-Me-HNP1 was selected in medium containing Zeocin of yeast strains in BMMY medium,using methanol as an inducer to induce expression.The expression product was purified by nickel column affinity chromatography and detected by Tricine-SDS-PAGE protein electrophoresis.The detection result showed that there was a clear protein band at about 5.0KD,indicating that the Pichia pastoris successfully expressed the Me-HNP1 gene.The expression conditions of recombinant Pichia pastoris were optimized:A₃B₂C₁.When the induction time is 96 h,the induction temperature is 28?,and the methanol concentration is 0.5%,the expression of recombinant yeast can reach the optimal expression amount of 13.6 mg/L.The antimicrobial effect of hybrid antimicrobial peptide Me-HNP1 on Gram-positive bacteria?Staphylococcus aureus?and Gram-negative bacteria?Escherichia coli,Salmonella enteritidis?was determined by the tube-disc method.The results showed that the hybrid antimicrobial peptide Me-HNP1 had bacteriostatic effects on Staphylococcus aureus,E.coli and Salmonella enteritidis.The MIC values were:3?g/m L,4?g/m L,2?g/m L,compared with melittin,the antimicrobial ability of E.coli is increased by about 2 times,and the antimicrobial activity of Staphylococcus aureus is increased by more than 3 times,which is basically the same as HNP1activity.The animal cell hemolytic activity test showed that the red blood cell hemolysis rate of the hybrid antimicrobial peptide Me-HNP1 was reduced by about 400 times compared with 1?g/m L of melittin that occurred 100%hemolysis,and the content of the hybrid antimicrobial peptide was 100?g/m L,the hemolysis rate of red blood cells is about 47%,and when the content is 400?g/m L,the hemolysis rate of red blood cells reaches 100%.The research results of the stability of the hybrid antimicrobial peptide Me-HNP1?thermal stability,acid-base stability and genetic stability?show that the hybrid antibacterial peptide Me-HNP1 still has biological activity,when it be in the range of p H 2-10,after the p H exceeds 7,the antimicrobial activity shows a downward trend;after boiling for 40 minutes,the antimicrobial activity of the hybrid antimicrobial peptide disappears;after 10 subcultures,the expressed protein still has an inhibitory effect on the bacteria,PCR amplification a clear band was obtained at 149bp,indicating that the target gene was not lost.In summary,this study has constructed an eukaryotic expression vector p PICZ?A-Me-HNP1,and induced expression in the Pichia pastoris expression system.Multiple tests have shown that the hybrid antimicrobial peptide Me-HNP1 has good antibacterial activity and lower hemolytic toxicity.