Antibacterial Mechanism,Recombinant Expression And Characterization Of The Antimicrobial And Antiviral Hybrid Peptide Cecropin A(1-8)-LL37(17-30)

To develop novel,safe and environmental antibiotic alternative,we did research on hybrid peptide cecropin A(1-8)-LL37(17-30)(C-L)with potent antimicrobial and antiviral activity obtained from our earlier work.The effect of C-L on the potential bacterial targets(Including membrane,DNA and protein)and was carried out to explore the potential antibacterial mechanism using scanning electron microscope,gel electrophoresis and Tricine-SDS-PAGE methods.Results showed that C-L killed E.coli CVCC245 and S.aureas ATCC25923 through the same way(membrane disruption via pore formation)as the parental peptides(C&L),but did not affect the DNA and protein synthesis.In addition,synergy studies between C-L and five antibiotics(chloramphenicol,thiamphenicol,neomycin sulfate,penicillin G and kanamycin)showed that the antibacterial activity was enhanced when C-L used with chloramphenicol(0.5<FIC<1)and neomycin sulfate(FIC<0.5)against E.coli CVCC245,and when C-L used with chloramphenicol(FIC<0.5),thiamphenicol(FIC<0.5),neomycin sulfate(FIC<0.5)and kanamycin(0.5<FIC<1)against S.aureas ATCC25923.Taken all these results,C-L was safe and less prone to drug resistance,and can be used together with antibiotic alternative to enhance antibacterial activity,reduce the additive dosage of traditional antibiotics and improve safety.To find a feasible approach for producing C-L,E.coli expression system was applied to produce recombinant C-L.The C-L gene with proper codon for E.coliwas synthesized and cloned into pETSUMO vector by "TA clon" to construct recombinant expression vector pETSUMO-C-L.After verified by PCR amplification and sequencing,the recombinant plasmid was transformed into competent E.coli BL21(DE3).After 1.5mM IPTG induction,SUMO-C-L was expressed successfully in E.coli,and the maximum fusion protein amounting to 89.14 mg/L was observed after 5h induction.And 17.54 mg/L recombinant hybrid peptide C-L was recovered after cleavage of SUMO protease and purification of Ni-NTA sepharose.Furthermore,R-C-L exhibited similar antibacterial activity to synthesized hybrid peptide C-L(S-C-L),wide pH tolerance ranged from 5 to 12 and excellent temperature stability.But in the experiment,E.coli expression system demonstrated several disadvantages including low production,complicated purification technology and high cost and did not fit to large-scale production.Thus,the excellent Pichia pastoris(P.pastoris)expression system was applied to exogenous secretory expression of C-L.Similarly,the plasmid pPICZaA-C-L was constructed by cloning target genes with proper codon for P.Pastoris into pPICZ?A vector.Then the recombinant plasmid was transformed into competent P.pastoris GS115 to construct recombinant engineering bacteria named GS115-pPICZ?A-C-L after verification by PCR amplification and sequencing.And the recombinant hybrid peptide C-L was secretory expressed successfully in the supernatant of fermentation after 0.5%methanol induction,the maximum recombinant hybrid peptide C-L,amounting to 190.65 mg/L,was observed after 96h induction.Furthermore,recombinant hybrid peptide C-L displayed similar antibacterial activity to S-C-L,wide pH tolerance ranged from 5 to 12 and excellent temperature stability.In conclusion,the research supplied an effective method to explore the antibacterial mechanism,recombinant expression and characterization of antibacterial and antiviral peptides,deepened the theory of hybrid peptides,and paved the way for its process of industrialization.