Prokaryotic Expression Of Hybrid Antimicrobial Peptide MLH And Preliminary Study Of Its Activity

Antibacterial peptides(AMP),which is widely found in organisms in nature,is an important part of the organisms immune system.AMP has stable physical and chemical properties,broad-spectrum antibacterial activity and has resistance to target strains.AMP has different targets and mechanisms of action from antibiotics,and it also has a good antibacterial effect on antibiotic-resistant bacteria.AMP is expected to replace the use of antibiotics and has good application prospects of the fields of medicine,foodstuff and other fields.In recent years,it has been found that the novel hybrid antimicrobial peptides formed by more than two kinds of antimicrobial peptides can improve the antibacterial effect of AMP.In this study,a new hybrid antimicrobial peptide MLH with antibacterial potential was screened by using musca domestica antimicrobial peptide(Cec-Med),human antimicrobial peptide LL-37 and helicobacter pylori antimicrobial peptide(Hp)as parent peptides.We had synthesized three primers with complementary ends.The MLH gene was amplified by overlap extension amplification technology(SOE-PCR),and then ligated with the expression vector p ET-32a(+),successfully constructed the recombinant expression vector p ET-32a-MLH.The recombinant vector were transformed into E.coli BL21(DE3)expression strains to construct the genetically engineered bacteria p ET-32a-MLH / E.coli BL21(DE3),and the best conditions of IPTG concentration,induction time and temperature were determined.The fusion protein was purified and refolded to obtain the hybrid antimicrobial peptide MLH powder.Finally,the antibacterial activity,hemolytic activity and stability of the hybrid antimicrobial peptide MLH were studied.The research and conclusion are as follows:(1)Design of the heterozygous peptide gene sequence.Bioinformatics methods were used to predict the primary,secondary structure,helicity,hydrophobicity,and antibacterial potential of the hybrid peptide MLH.The results are: The molecular mass is 5216.39,the number of amino acid residues are 43,the theoretical isoelectric point of PI is 11.88,and the total level of hydrophobicity is-0.914,the coefficient of instability of 15.90.The half-life in E.coli and yeast are greater than10 and 20 h,respectively,which indicated the stability of hybrid peptides;The hybrid peptide has ?-helical and amphipathic structure.It has the potential to be an antimicrobial peptide.(2)Synthesis of the hybrid peptide genes.The gene sequence of MLH encoded by E.coli codon preference.Through the synthesis of the three primers that overlap each other,The MLH gene was successfully synthesized after PCR based on overlap extension amplification(SOE-PCR)technology.(3)Construction of the expression vector.The gene MLH and the expression plasmid p ET-32 a were digested with Eco R I and Xho I.And after overnight connection,the recombinant vector of p ET-32a-MLH were successfully formed.(4)Prokaryotic expression of the hybrid peptides.The recombinant plasmid were transfered to the competent E.coli BL21(DE3).The positive recombinant strains were screened with ampicillin-resistant plates.The target protein was expressed by adding IPTG,collected and sonicated the cells.The results of SDS-PAGE and Western-blot proved the correctness of the expressed fusion protein,and the protein mainly existed in the sediment;(5)Fermentation culture of the engineering bacteria.The optimal fermentation conditions are as follows: the initial induction time was 3.0h,the concentration of IPTG is 1.0 m M,the induction time is 4.0 h,the induction temperature is 37 ?.Purification and renaturation of the fusion protein were carried out by Ni2+ affinity chromatography and dialysis renaturation.And the purified protein accounted for 95.2 % of the total bacterial cells.After digestion with enterokinase,the yield of the hybrid peptide MLH reached 60.1 mg / L.(6)Antibacterial activity of hybrid peptides.The antibacterialactivity,hemolytic activity and stability of the hybrid antimicrobial peptide MLH were studied.The results of antibacterial test showed that the hybrid peptide MLH had a good antibacterial activity against Gram-positive bacteria,Gram-negative bacteria and fungi.The minimal inhibitory concentrations of the hybrid peptides against Staphylococcus aureus,Escherichia coli,Bacillus subtilis,and Saccharomyces cerevisiae were 5,12,3,and 17 ?M,respectively.The hybrid antimicrobial peptide MLH had lower hemolytic activity than the parent peptide Md-Cec,and the hybrid antimicrobial peptide MLH had better thermo stability and p H stability.In this study,the hybrid peptide MLH was fusion expressed in E.coli.The antibacterial peptide MLH powder with high purity,strong antibacterial activity and low hemolytic activity was obtained,which have certain applications in the fields of food,medicine and animal husbandry value.