Cloning,Prokaryotic Expression And Antimicrobial Activities Of Gene Multimers From Human Antimicrobial Peptide LL-37 And Its Variant Based On Sequence Reformation

Objective:Antimicrobial peptides(AMPs)show great potential application value in medicine and agriculture as it have low molecular weight,broad antimicrobial spectrum,good water-soluble,unique antibacterial mechanism molecular and as an important part of the body's innate immune defense system,AMPs is considered to be the best substitute for antibiotics and have become the focus in the field of biological pharmacy.LL-37 was the only human member of the cathelicidin family.In addition to antimicrobial and anti-septic effects,LL-37 also possesses other functions such as chemotaxis,wound healing,cell differentiation,apoptosis,and innate immune response.Therefore,LL-37 will be have a great application value,However,most AMPs encountered the difficulties of low antibacterial activity and high producing cost.So LL-37 and its variant gene multimers were clond and expressed in E.coli to promote the application of LL-37 in medicine and agriculture areas more early.Methods:In this study,The LL-37 variant gene was obtained by bioinformatics tools.Glutamic acid was substituted glutamine from 16th sequence,aspartic acid was substituted lysine from 26th sequence,part of the rare codons was replaced by the prokaryotic cell preferred codons.Then LL-37 and its variant gene were synthesized and amplified by overlap PCR.The dimmer was cloned into vector PET-28(a)+(PET-28(a)-dLL)and transformed to E.coliBL21(DE3)pLysS expression system.The expressed product was purified by nickel ion affinity chromatography.The antimicrobial effects was studied through liquid growth inhibition method.Results:The two-dimensional structure,three-dimensional structure and chemical characters of LL-37 and its variant were analyzed by ExPASy?SWISS-MODEL and softer ware(Anthepro5.0,Raswin).The results shown the theoretical isoelectric point was increased from 10.61 to 11.51,the stability and half-life in E,coli were significantly improved,the N-terminal hydrophilic and C-terminal hydrophobic,two-dimensional helix,antigenicity and solubility were basically unchanged.The three-dimensional structure had some diversification.The gene multimers PET-28(a)-dLL was proved by sequencing.The recombinant LL37 and its variant peptide were expressed as an inclusion body in E.coli BL21.The relative molecular mass of recombinant peptide was about 11000,the concentration reached 0.47mg/mL after purification.It was able to inhibit the growth of Staphylococcus aureus and Salmonella.Conclusion:The human antimicrobial peptide LL-37 and its variant were successful cloned and expressed in E.coli BL21(DE3)pLysS.And it was purified by affinity binding chromatography and protein dialysis.The recombinant peptide have proved that it could inhibit the growth of Staphylococcus aureus and Salmonella.It lay a foundation for further study on biological activity of LL-37 and its variant.