Expression Of Recombinant Chicken β-defensin-13 And Granulysin In Aspergillus Niger And Analysis Of Their Antibacterial Effects

Antibiotics have good antibacterial and bactericidal activities,and irregular use of antibiotics can induce bacterial mutation and resistance,threatening public health safety.Therefore,the research and development of antibiotic alternatives has become one of the most urgent problems to be solved.Antimicrobial peptides have a promising future because of their rich variety and efficient antibacterial activity.Chicken β-defensin-13(Gal-13)and chicken granulysin(GNLY)are natural antimicrobial peptides in chickens with broad-spectrum antimicrobial activity and thermal stability.At this stage,there is a challenge of extraction and purification for antimicrobial peptide synthesis,which makes industrial production difficult to achieve.The technology to promote the expression of recombinant antimicrobial peptides in heterologous hosts will solve the problem of its industrial production.Aspergillus niger can be used as a feed additive because of its high efficiency in exocytosis of heterologous proteins and high safety of its expression products.When heterologous proteins are expressed in Aspergillus niger,preferential codon optimization of heterologous protein amino acid sequences and fusion expression of endogenous proteins with heterologous proteins that promote efficient expression in Aspergillus niger are common solutions.In this study,Gal-13 and GNLY were codon optimized in accordance with the coding preference of Aspergillus niger,and subsequently fused with p SZHA6R-Xyn B to form two recombinant strains,and the recipient bacteria were Aspergillus niger(TH-2),and the effect of fusion protein codon optimization on the secretory expression of Gal-13 and GNLY in Aspergillus niger was investigated to achieve the efficient expression of chicken-derived antimicrobial peptides in Aspergillus niger.The main research results are as follows:(1)Construction and screening of recombinant strains of Aspergillus NigerAfter codon optimization of the sequence of Gal-13 and GNLY genes by Aspergillus niger encoding preference,the recombinant expression vectors p SZHA6R-Xyn B and p SZHA6R-Xyn BGal-13-M and p SZHA6R-Xyn B-GNLY-M were constructed by ligating and recombining them with p SZHA6R-Xyn B,and then the two recombinant expression vectors were transformed into TH-2 by Agrobacterium freeze-thaw method to obtain two recombinant strains GA and GN.(2)Detection of recombinant strains of Aspergillus niger expressing Gal-13 with GNLYGA and GN were inoculated into the fermentation medium for shake flask fermentation,and the gene expression of Gal-13 and GNLY was detected in both the fermentation supernatant and mycelium on days 4-11.The fermentation supernatants were detected by Tric ine-SDS-PAGE and SDS-PAGE,and the target bands could be observed at 4.8 and 14.5 KD.In addition,ELISA assay revealed that the protein concentration of Gal-13 and GNLY reached the highest level on day 11,54.20 and 73.65 μg/m L,respectively.similar results were detected by RT-q PCR on mycelium,and there was a significant elevation of transcript levels of Gal-13 and GNLY on days 4-11 of fermentation.The above results indicated that recombinant strains GA and GN of Aspergillus niger were able to express Gal-13 and GNLY efficiently,respectively.(3)Growth status of recombinant strains of Aspergillus niger and results of unfolded protein assayThe growth status of recombinant strains p SZHA6R-Xyn B-Gal-13-M and p SZHA6R-Xyn BGNLY-M of Aspergillus niger in fermentation solid medium was examined using TH-2 as a control,and it was found that there was no difference between the growth of the 2 recombinant strains and TH-2.The results of unfolded protein response(UPR)assay showed that the m RNA transcript levels of bip A,pdi A and hac A were significantly higher in GA and GN strains than in the control,but there was no significant difference between 4 and 11 days of fermentation.The above results indicated that the unfolded protein response was involved in the exp ression of target genes in the recombinant strains,but there was no growth inhibition in the 2 recombinant strains.(4)Identification of Gal-13 and GNLY inhibitory activity expressed by recombinant strains of Aspergillus nigerThe inhibition range assay and MIC assay were performed on the supernatant of the two expressed recombinant strains.The results of inhibition range assay showed that the inhibitory ability of GA was,from strong to weak,E.coli,Salmonella,S.aureus,and B.subtilis.the inhibitory ability of GN was,from strong to weak,Salmonella,S.aureus,E.coli,and B.subtilis.In the MIC assay,the MICs of GA against E.coli,Salmonella,Bacillus subtilis and Gluconococcus aureus were38.74,42.62,56.73 and 51.57 μg/m L,respectively.the MICs of GN against E.coli,Salmonella,Bacillus subtilis and Gluconococcus aureus were 46.48,38.41,51.13 and 42.25 μg/m L.This indicates that the secreted expression of Gal-13 and GNLY from Aspergillus niger showed antibacterial activity against E.coli,Salmonella,Bacillus subtilis and Staphylococcus aureus.