High-level Expression In Pichia Pastoris SMD1168 Of Moronecidin,Epinecidin-1,Pardaxin-4,Misgurin And Smelly Frog Antibacterial Peptide

The advent of antimicrobial peptides has created a new avenues for resisting superbugs and bacteria resistance,bringing the gospel to human beings.Fish and amphibians are the most primitive vertebrate.Due to their extremely fragile acquired immune system,they have a powerful innate immune system to avoid the infection of the pathogen.Therefore,the antimicrobial peptides distributed in different parts have become the important components of first defense line of broad-spectrum in these lower organisms.At present,antimicrobial peptides with broad-spectrum antimicrobial activity and immunoregulatory activity found in low-level organisms,such as moronecidin,epinecidin-1,pardaxin,and misgurin distributed in Hybrid striped bass,Epinephelus coioides,Pardachirus marmoratus,Misgurnus anguillicaudatusis,as well as antimicrobial spectrum of smelly frog antimicrobial peptides.Studies have found that moronecidin can kill all gram positive bacteria and some gram negative bacteria;epinecidin-1 can effectively prevent grouper-infected Vibrio vulnificus and kill the nervous necrosis virus;pardaxin has the antitumor activities;misgurin has strong bactericidal ability;antimicrobial peptides from the smelly frog can kill Candida albicans.This paper reports the main results on the study of moronecidin,epinecidin-1,pardaxin-4,misgurin and smelly frog antimicrobial peptide which expressed in Pichia pastoris SMD1168.The aim of this paper was to provide a theoretical basis for green,safe and effective antimicrobial peptides drugs in Pichia pastoris expression and play a great role in various fields.1.Moronecidin gene was cloned into expression vector p PIC9 K in order to construct recombinant expression vector p PICMC and integrated into Pichia pastoris SMD1168.By comparing the diameter of bacteriostasis circle of Escherichia coli and Staphylococcus aureus,the strain p PICMC-4 with the better antimicrobial activity was screened out.After shaking flask fermentation,the antibacterial activity of the strain was highest at 72 h.The bioavailability of Escherichia coli was 1.5661×109 U/g,the bioavailability of Staphylococcus aureus was 1.957×108 U/g.2.Epinecidin-1 gene was cloned into expression vector p PIC9 K in order to construct recombinant expression plasmid p PICEC and integrated into Pichia pastoris SMD1168.By comparing the diameter of bacteriostasis circle of Escherichia coli and Staphylococcus aureus,the strain p PICEC-7 with the better antimicrobial activity was screened out.After shaking flask fermentation,the antibacterial activity of the strain was highest at 72 h.The bioavailability of Escherichia coli was 1.5661×109 U/g,the bioavailability of Staphylococcus aureus was 5.812×108 U/g.3.Pardaxin-4 gene was cloned into expression vector p PIC9 K in order to construct recombinant expression plasmid p PICPD and integrated into Pichia pastoris SMD1168.By comparing the diameter of bacteriostasis circle of Escherichia coli and Staphylococcus aureus,the strain p PICPD-17 with the better antimicrobial activity was screened out.After shaking flask fermentation,the antibacterial activity of the strain was highest at 96 h.The bioavailability of Escherichia coli was 1.4816×109 U/g,the bioavailability of Staphylococcus aureus was 3.9646×109 U/g.Induced expression was performed in a 100 L fermentor and antimicrobial activity assay was performed 72 h later.This active substance of p PICPD-17 strain was identified as pardaxin-4 by mass spectrometry analysis.In comparison with shaking flask fermentation 96 h,the bioavailability of Escherichia coli and Staphylococcus aureus in fermentor fermentation 72 h increased by 5.49 fold,2.42 fold,respectively;pardaxin-4 has weak antimicrobial activity to Acinetobacter baumannii and obvious hemolytic activity;when the temperature of water bath reaches 100 °C,1 h,the antimicrobial activity of fermentation broth is obviously weakened,the p H value of fermentation broth at 6,it has better antimicrobial activity;the antimicrobial activity was slightly weakened after adding trypsin,pepsin and protease K.4.Misgurin gene was cloned into expression vector p PIC9 K in order to construct recombinant expression plasmid p PICMG and integrated into Pichia pastoris SMD1168.By comparing the diameter of bacteriostasis circle of Escherichia coli and Staphylococcus aureus,the strain p PICMG-22 with the better antimicrobial activity was screened out.After shaking flask fermentation,the antibacterial activity of the strain was highest at 48 h.The bioavailability of Escherichia coli was 1.062×109 U/g,the bioavailability of Staphylococcus aureus was 5.804×108 U/g.Then the activity was detected 48 h after induction in a 100 L fermentor.Tricine-SDS-PAGE protein gel analysis and mass spectrometry analysis showed that the active substance expressed by p PICMG-22 was Misgurin.In comparison with shaking flask fermentation,the bioavailability of Escherichia coli and Staphylococcus aureus in fermentor fermentation increased by 1.47 fold,1.43 fold,respectively;misgurin has weak antimicrobial activity to Acinetobacter baumannii and Salmonella and weak hemolytic activity;when the water bath of temperature reaches 90 °C,the antimicrobial activity of fermentation broth is obviously weakened,the p H value of fermentation broth at 1-12,everyone has antimicrobial activity;the antimicrobial activity is weakened after adding trypsin,pepsin and protease K.5.Smelly frog antimicrobial peptide gene was cloned into expression vector p PIC9 K in order to construct recombinant expression plasmid p PICSFA and integrated into Pichia pastoris SMD1168.By comparing the diameter of bacteriostasis circle of Escherichia coli and Staphylococcus aureus,the strain p PICSFA-17 with the better antimicrobial activity was screened out.After shaking flask fermentation,the antibacterial activity of the strain was highest at 96 h.The bioavailability of Escherichia coli was 6.017×108 U/g,the bioavailability of Staphylococcus aureus was 2.1246×109 U/g.