Expression And Antibacterial Activity Analysis Of Porcine β-defensin-1 And Lysozyme In Aspergillus Niger

Today,the abuse of antibiotics is becoming more and more serious.It is urgent to find safe,efficient,broad-spectrum antibiotics and large-scale antibiotic substitutes.defensin and lysozyme,as important components of animal immune system,are natural substitutes for antibiotics Porcineβ defensin-1(PBD-1)is a cationic antimicrobial peptide,which can resist bacteria,fungi and coated viruses Sus scrofa lysozyme(SSL)is a kind of enzyme that can hydrolyze mucopolysaccharides in pathogenic bacteria.It has unique biological functions and pharmacological effects.It has strong bactericidal ability and is not easy to produce drug-resistant strains.Due to the low content of natural β-defensin and lysozyme,complex extraction and high cost of chemical synthesis,it is undoubtedly the best choice to use genetic engineering to produceβ-defensin and lysozyme in bulk.Aspergillus niger is one of the Aspergillus genera of filamentous fungi,which not only has a strong ability to secrete proteins,but also can carry out a high degree of post-translational processing.Therefore,in this study,PBD-1 and SSL genes were designed and artificially synthesized according to the codon bias of Aspergillus niger.The PBD-1 and SSL genes were integrated into the Gla A site by Agrobacterium-mediated method,and α-factor precursor peptides containing fusion protein Xyn B and Pichia pastoris source were constructed.αF)and the recombinant expression vector of α-factor precursor peptide(α-factor-CO1,αf-Co1)optimized by Aspergillus niger.Codon optimization and different expression vectors were selected to enhance the expression of alloprotein in Aspergillus niger,so as to seek a better strategy to promote the secretion of PBD-1 and SSL.In this experiment,PBD-1 and SSL genes optimized by the preferred codon table of Aspergillus niger were connected with the previously constructed expression vectors p SZHG6R-Xyn B,p SZHG6R-αF and p SZHG6R-αF-CO1.Six expression vectors p SZHG6R-Xyn B-PBD-1,p SZHG6R-αF-PBD-1,p SZHG6R-αF-CO1-PBD-1 and p SZHG6R-Xyn B-SSL,p SZHG6R-αF-SSL,p SZHG6R-αF-CO1-SSL were constructed.They are named XP,αP,αCP,XL,αL,αCL.The constructed expression vector was transformed into Agrobacterium AGL1 by freeze-thaw method,and six recombinant strains were obtained by co-culture with Aspergillus niger TH-2.The six recombinant strains were fermented in shake flask,and the supernatant was collected at 4-11 d.The protein expression levels of the recombinant strains were detected by SDS-PAGE and Tricine-SDS-PAGE.The results showed that the recombinant strains successfully secreted and expressed XP,XL,αL and αCL,which were significantly higher than those of the original strains.The maximum protein expression levels were 45.837,107.38,71.04,84.82 μg/m L,while no obvious target bands were found in recombinant strains αP and αCP.The antibacterial activity and MIC of the supernatant of 4 recombinant strains were determined.The results showed that XP had antibacterial activity against E.coli,Salmonella and Staphylococcus aureus,and the antibacterial activity was gradually weakened.XL,αL and αCL also had antibacterial activity against Salmonella,E.coli and Staphylococcus aureus,and the antibacterial activity was gradually weakened.In the determination of MIC,for Escherichia coli,the MIC of XP,XL,αL,αCL is about 360,2.25,3.5,3.25 μg/m L;for salmonella,the MIC of XP,XL,αL,αCL is about 400,2.5,3,2.75 μg/m L;for Staphylococcus aureus,The MIC of XP,XL,αL,αCL is about 400,2.5,3.5,3.25 μg/m L.The heat stability,acid-base stability and hemolytic stability of the supernatant of the four recombinant strains were also determined.The experimental results show that XP has general thermal stability,XL,αL,αCL has better thermal stability.XP,XL,αL and αCL have no obvious hemolytic properties and are easily stored in acidic environment.In addition,the enzyme activity of the supernatant of recombinant strains XL,αL and αCL was determined.The results showed that the maximum activity of XL,αL and αCL was about 387.9392,305.6755 and 338.6091 U/m L,respectively.In summary,PBD-1 and SSL recombinant expression vectors were constructed by Aspergillus niger expression system,which provided a new expression pathway for the secretion of PBD-1 and SSL,and also laid a theoretical foundation for the engineering production of PBD-1 and SSL and its application in animal husbandry industry.