Heterologous Expression And Safety Evaluation Of Cecropin AD In Aspergillus Niger

Antimicrobial peptides（AMPs）are small peptides with antimicrobial activity that are induced by organisms.They play an important role in the immune system of most organisms,effectively killing pathogens and warding off invasion by foreign bacteria and viruses.Cecropin,the first antimicrobial peptide discovered,is also one of the most potent antimicrobial peptides to date.Cecropin AD（CAD）is a heterogeneous peptide composed of the N-terminal peptide 1～11 of cecropin A and the C-terminal peptide 12～37 of cecropin D,which has potent antibacterial and antiviral activities.The synthetic heterogeneous peptide CAD has significantly better performance than the natural antimicrobial peptide,so it is widely used in feed additives and food preservatives.At present,most of the synthetic peptides are obtained by chemical synthesis,but the synthesis process is too complicated and costly,so it is important to use genetic engineering technology to realise the efficient expression and industrial production of CAD.The recombinant pure strains were obtained and the fermentation conditions of CAD in the Aspergillus niger expression system were first optimised,and finally the antibacterial effect and biological properties were investigated.The main results are presented below:1.The single-copy CAD gene and the double-copy CAD gene with restriction endonucleases NheⅠand HindⅢenzymatic sites were amplified by overlap extension PCR technique and ligated with vector p SGH6R containing the same enzymatic sites to construct Aspergillus niger expression vectors p SGH6R-CAD and p SGH6R-（CAD）₂.2.The Aspergillus niger expression vectors p SGH6R-CAD and p SGH6R-（CAD）₂ were transformed into Agrobacterium tumefaciens AGL1 by the freeze-thaw method and Aspergillus niger TH-2 by the Agrobacterium mediated method,and the mycelial genome was extracted for PCR identification to obtain a pure recombinant strain of Aspergillus niger.3.The obtained pure recombinant strains were shaken and fermented,and the supernatants were collected from 4 to 11 d.The fermentation products were identified by Tricine SDS-PAGE small molecule protein gel,and a band with a molecular weight of about 3.9 k D was detected,and the protein content of the double-copy recombinant strain was significantly higher than that of the single-copy recombinant strain.The actual theoretical molecular weight of CAD was 3950.73 Da,and the molecular weight size obtained from the assay was basically in agreement with the theory.4.The carbon source and initial p H of the fermentation medium were optimised and maltose,soluble starch and glucose were used as the only carbon sources for the fermentation medium,and the test results proved that the best carbon source for the fermentation medium was glucose,and the initial p H was 6.0 with an antibacterial potency of 8.94×10⁸ U/L and the best inhibition.5.CAD has antibacterial activity against Staphylococcus aureus,Salmonella,Escherichia coli and Bacillus subtilis,and its inhibitory effect on Gram-negative bacteria is better than that on Gram-positive bacteria.6.CAD is highly resistant to high temperatures and remains active after a 3 hour water bath at100°C.CAD was also very resistant to protease and its antibacterial activity was only slightly inhibited after treatment with protease.In the haemolysis test,CAD showed non-haemolytic properties.In summary,this experiment has completed the construction of Aspergillus niger expression vector,transformed it into Aspergillus niger by Agrobacterium mediated method and screened out the pure strain,tested the fermentation products of the obtained pure strain and optimised the fermentation conditions,and obtained the pure Aspergillus niger which can be genetically stable.In this study,CAD was successfully expressed in Aspergillus niger using genetic engineering techniques,with potential applications in animal breeding,food and pharmaceutical industries..The results of this study will provide a new method for the production of artificial antibacterial peptides and a new expression system for the safe and efficient production of recombinant proteins.