Study On High-level Expression And Antibacterial Activity Of Antimicrobial Peptides In Pichia Pastoris

Antimicrobial peptides have the characteristics of good thermal stability,broad antibacterial spectrum,fast sterilization,low drug resistance and low toxicity and side effects,and have gradually become a hot spot of people’s attention.In addition,some animal-derived antimicrobial peptides,as an important part of innate immunity,also have anti-inflammatory,multiple immunomodulatory activities and toxicity to cancer cells.Their natural and excellent biological activities have attracted widespread attention.Because the molecular weight of antimicrobial peptides is too small and has an inhibitory effect on most bacteria,the large-scale preparation of antimicrobial peptides has become a bottleneck in the development and application of antimicrobial peptides.In order to improve the expression level and antibacterial ability of antibacterial peptides,this study adopted the P.pastoris expression system.First,seven kinds of antibacterial peptides from different sources were expressed and screened,and then the antibacterial peptides were modified through fusion expression or artificial mutation to achieve high activity.The results of high-efficiency expression of antimicrobial peptides are as follows:(1)Heterologous recombinant expression and screening of antimicrobial peptides: the antimicrobial peptides bombesin(Adelin),hybrid cecropin(Cecropin AD),porcine marrow-derived antimicrobial peptide(PMAP-36)),bovine lactoferrin peptide(LFALFC),subtilisin(SUBA)gene sequence was inserted into p PIC9 K,constructed recombinant plasmid and introduced into P.pastoris X33,high antibacterial activity was screened by protein content determination and antibacterial plate experiment The expression strain of subtilisin SUBA which is resistant to Gram-positive bacteria,P.pastoris/p PIC9K-SUBA-2 was obtained;p PICZα was used as the expression vector,and avian defensins(AVBD)and human antibacterial were selected.Peptide(HAPD)and the above five antimicrobial peptide genes are connected with the modified yeast α signal peptide to form a gene fragment,inserted into p PICZα and transformed into P.pastoris X33,to construct a new round of antimicrobial peptide recombinant expression strains,and to screen high-activity antimicrobial peptides The antibacterial spectrum was determined,and the expression strains P.pastoris/p PICZα-opori SP(d4)-Adelin-4 and P.pastoris/p PICZα-opori SP(d4)-PMAP36-1.with broad-spectrum antibacterial activity of bombesin Adelin and porcine myeloid antimicrobial peptide PMAP-36 were obtained.(2)Cloning,expression and screening of antimicrobial peptide fusion proteins: human lysozyme,egg-derived lysozyme and phospholipase were selected as the partners for the fusion expression of porcine antimicrobial peptides,and the N-terminals of the three fusion partners were respectively combined with porcine antimicrobial peptides.The C-terminal was spliced with the modified yeast α signal peptide to form a complete gene sequence.The three fusion protein gene sequences were inserted into the p PICZα vector and transformed into P.pastoris to construct a pig-derived antimicrobial peptide fusion protein expression strain.Two strains with antibacterial activity were screened by the plate experiment,which are the human lysozyme-pig source antimicrobial peptide fusion protein expression strain P.pastoris/p PICZα-opori SP+HLMRK-PMPW-5 and the phospholipase-pig source antimicrobial peptide fusion protein expression Strain P.pastoris/p PICZα-opori SP(d4)-PLB-4DK-PMPW-3.(3)Cloning and expression of anti-degradation mutants of pig-derived antimicrobial peptides and optimization of expression strain induction conditions: site-directed mutagenesis was used to mutate the KR site in p PICZα-opori SP(d4)-PMAP36 to KK,and to construct recombinant P.pastoris X33 The strains were expressed and screened on plates and shake flasks,and an expression strain P.pastoris/p PICZα-opori SP(d4)-PMAP36(kr-kk)-10 that could produce mutants with high antibacterial activity was obtained.The antibacterial plates showed that the expression strain P.pastoris/p PICZα-opori SP(d4)-PMAP36(kr-kk)-10 The antibacterial activity of the fermentation broth of mutant PMAP-36(kr-kk)was higher than that of PMAP-36,which was 1.30 times that before the mutation;the induction conditions for the mutant PMAP-36(kr-kk)expression strain were further optimized,The optimal induction temperature is 28℃,the methanol induction concentration is 2% methanol added every 24 h,and the optimal induction time is 96 h.(4)PMAP-36(kr-kk)antibacterial stability: after using the ultra-low molecular weight characteristics of pig-derived antimicrobial peptides to crudely purify it,study its high temperature resistance and acid-base stability.The results show that the pig-derived antimicrobial peptide mutant has strong acid and alkali resistance stability,and has antibacterial activity in the p H range of 2-12,while the stability of high temperature and temperature is weak,and the activity is significantly reduced above 60°C.