Bacteriostatic Mechanism Of Heterozygous Peptide MB7R And Tandem Expression In Sf9 Cells

Antimicrobial peptides（AMPs）are short peptides with antimicrobial activity produced by organisms.It usually consists of 6-60 amino acids,exhibits acid-base stability and thermal stability,and has a wide antimicrobial spectrum.Heterozygosity was an emerging strategy for the modification of antimicrobial peptides in recent years.Fusion of two or more peptides with different properties to produce new antimicrobial peptides,it can not only lead into the characteristics of new antimicrobial peptides,but also increase the antimicrobial activity and reduce the toxicity to host cells.Therefore,the modification of the amino acid sequence of antimicrobial peptides by heterozygous method to improve the antimicrobial activity and reduce cytotoxicity has become a hot and difficult point in the research of antimicrobial peptides.In this study,theα-helix region of the antimicrobial peptide MDAP-2 from Musca domestica（KFFTLLA）was used as the parent peptide,heterozygous with theα-helix region（3×RVVR）of the Buforin II-derived peptide.The heterozygous results were analyzed by bioinformatics tools and the dominant peptide MB19（KFFTLLARVVRRVVRRVVR）was screened out.Design of heterozygous peptides by amino acid substitution MB5R[Leucine（L）at position 5 was replaced by arginine（R）in the amino acid sequence of MB19]、MB7R[Alanine at position 7（A）and leucine at position 5（L）in the amino acid sequence of MB19 were replaced by arginine（R）]、MB19n（Amidation of C-terminal of MB19）.By increasing positive charge content,reducing hydrophobicity and hydrophobic moment,the bacteriostatic activity and cytotoxicity were improved.Four dominant peptides were synthesized by chemical method,the heterozygous peptide MB7R with strong bacteriostatic activity,lower cytotoxicity and better stability was screened through in vitro bacteriostatic activity,cytotoxicity,hemolytic activity and stability experiments;MB7R can kill bacteria by destroying bacterial cell membranes and binding bacterial DNA by means of outer membrane permeability,inner membrane permeability,scanning electron microscopy,transmission electron microscopy and DNA blocking experiments.On the basis of obtaining a heterozygous peptide MB7R with strong antimicrobial activity and low cytotoxicity,MB7R gene was linked by isocaudarner method to increase the length of target gene.Using Bac-to-Bac system transposition principle,the target gene 12MB7R was inserted into the expression box of pFast-Bac,the recombinant pFastBac-12MB7R was inserted into Bac mind’s Tn7 expression box site by transposon Tn7,the shuttle vector Bacmid-12MB7R was obtained by blue and white spot screening.The recombinant plasmid was transfected into Sf9 insect cells by liposome transfection.The recombinant plasmid was successfully expressed with a molecular weight of about 28.38 kDa tandem protein 12MB7R.High titer virus was obtained by repeated infection.Western Blot was used to optimize the infection time and expand the sample culture,which provided ideas for the development of new antimicrobial peptides.The main experimental results are as follows:1.Heterozygous antimicrobial peptides were designed by heterozygous method.A dominant peptide MB19 was screened by bioinformatics analysis.Three derivative peptides（MB5R,MB7R,MB19n）were optimized by amino acid substitution method.Four heterozygous peptides were synthesized by chemical synthesis.A heterozygous peptide MB7R with good activity was screened by in vitro bacteriostasis test,cytotoxicity test,hemolytic activity test and stability test.2.It was confirmed by NPN and ONPG that heterozygous peptides MB19,MB5R and MB7R have destructive effects on bacterial cell membrane;Further observation of cell membrane and its contents by scanning electron microscopy and transmission electron microscopy showed that the target of heterozygous peptide MB7R was bacterial cell membrane;DNA blockade experiments showed that heterozygous peptides destroyed cell membranes and entered cells,which could interact with intracellular DNA and kill bacteria.3.The pFast-12MB7R donor plasmid was successfully constructed and Bacmid-12MB7R recombinant baculovirus was produced.The Sf9 insect cells were successfully infected by liposome transfection,and the molecular weight of 12MB7R was successfully expressed.Western Blot was used to optimize the infection time and expand the sample culture,the highest protein expression was observed in Sf9 cells infected by virus for 72 hours,purification of tandem protein 12MB7R by Ni²⁺affinity chromatography.Lay a foundation for the preparation of heterozygous peptide MB7R and its clinical application in veterinary medicine.