Construction Of Expression Systems Of Antimicrobial Peptide Cecropin A And Mutants Design And Function Study

With the abuse of traditional antibiotics in the process of production and life,a large number of super-resistant bacteria have emerged,which seriously threatens the health of human beings and the development of aquaculture industry.Therefore,the exploration and development of new antibacterial drugs has become a hot research area.Due to the broadspectrum antibacterial properties of antimicrobial peptides and the unique bactericidal mechanism,bacteria are difficult to produce drug resistance,and it is one of the most potential antibiotics to replace antibiotics.In recent years,more and more scientists have focused on the development of antimicrobial peptides.Cecropin A antibacterial peptide has strong antibacterial,fungal,viral and tumor activities,and has broad application prospects in the fields of medicine,health care,food and animal husbandry.At present,the preparation of antimicrobial peptides is mainly obtained by the following methods: separation and extraction from nature,artificial chemical synthesis and biorecombination expression.Due to the cumbersome operation steps,high cost of preparation and low yield,it is difficult to obtain a large number of high-purity antimicrobial peptides,which seriously hinders the further research of antimicrobial peptides.In recent years,due to the advantages of simple operation,low cost and high yield,the biorecombination method has gradually become a hot spot in the field of preparation of antimicrobial peptides.In this study,we explored and constructed several cecropin A expression systems based on E.coli.Moreover,the wild type cecropin A antibacterial peptide was molecularly modified to screen for the high antibacterial activity of cecropin A mutant peptide PEW300.And then its antibacterial property was measured and characterized.To reduce the toxic effect of cecropin A on the expression host,three E.coli expression systems were constructed: tag dependent expression system(AT-His system),non-tag dependent expression system(SA-ELK16 system)and cell free sytem(CF system).Then the three expression systems were systematically evaluated in terms of production cost,production cycle,and final yield of cecropin A antimicrobial peptide: AT-HIS expression system was found to be cumbersome and expensive,with long production cycles and lowyields(about 0.41 mg/g of wet cell pellet of cecropin A);In CF system,no cumbersome steps such as cell disruption are required,and there is no toxic effect of the cecropin A on the host cell and about 0.93 ?g/ml of the reaction mixture of cecropin A was successfully collected.However,the high cost,low yield,and the need of nickel ion affinity chromatography,CF expression system still cannot meet the requirements for large-scale preparation of cecropin A.In SA-ELK system,nearly 6.2 mg/g of wet cell pellet of cecropin A was successfully collected,and its purity was as high as 99.8 %,which greatly exceeds AT-HIS expression system and the CF expression system.More importantly,in the SA-ELK16 expression system,the cost is low,the operation is simple,and no complicated steps such as nickel ion affinity chromatography are required,and high-purity cecropin A can be obtained by simple centrifugation.However,cell disruption is still required,which seriously hinders the largescale preparation of cecropin A.In this study,we explored and constructed an extracellular secretion system based on E.coli.Through gene knockout and construction of secretory plasmid with cecropin A and the fibrin-like protein Sup35 NM to realize the cecropin A peptide displayed on the surface of E.coli.According to fluorescence microscopy and transmission electron microscopy,cecropin A could be successfully secreted to the surface of E.coli and aggregates in the form of "Curi" pili.The secretion system does not require complicated steps such as cell disruption and metal affinity chromatography steps.The cells can be collected by simple centrifugation,and highpurity cecropin A can be obtained by adding DTT to induce the cleavage of fusion protein.According to the MIC test,the antibacterial property of the cecropin A was consistent with that of the chemically synthesized antimicrobial peptide.To obtain cecropin A mutant with high antibacterial activity,we rationally designed several mutants by comparing and analyzing the primary sequence and secondary structure characteristics of the wild cecropin A.According to the results of the online simulation tool:the positive charge and hydrophobic properties of the design mutants increased to varying degrees.The mutants were initially screened by constructing mutant expression vectors in a manner that induces expression.The results showed that the PEW300 mutant exhibited strongantibacterial activity.Therefore,we successfully expressed and purified PEW300 through the pre-constructed SA-ELK16 expression system,and finally obtained high-purity PEW300 mutant peptide with a yield of 7.38 mg/g wet cell pellet.Subsequently,the purified PEW300 was tested for the antibacterial activity and results showed that PEW300 showed strong antibacterial activity,and the antibacterial activity was improved by 4-7 times compared with the wild type cecropin A.In addition,high concentration(224 ng/?l)PEW300 has no hemolysis characteristics;PEW300 exhibits good stability in neutral and alkaline environments;PEW300 has good thermal stability,and has good resistance against proteinase K,snail enzyme,stomach Protease and trypsin.These results show that PEW300 mutant peptide has good application prospects in the fields of medicine and pharmacy.