Fusion Expression Of Hybrid Antimicrobial Peptide BM16-R And Study On Its Biological Activity

Antimicrobial peptides?AMPs?is a kind of small molecular peptides the originate from organism immune system resistant to pathogen infection.AMPs,distributing worldwide in the nature,has a very wide range of sources and generally can be divided into natural AMPs and artificial AMPs.Natural AMPs are extracted from a variety of animals and plants naturally.Those endogenous peptides with antibacterial activity are,namely the AMPs.There are two main sources of artificial antibacterial peptides extracted by genetically engineering method and chemically synthesized method respectively.Due to the limited resources in nature,the extraction of AMPs in traditional way is complicated,time-consuming and inefficient,which cannot achieve large-scale acquisition of AMPs to meet human needs.Although preparation of antibacterial peptides by chemical synthesis is convenient and quick,it is costly and likely to cause environmental pollution.These shortcomings have resulted in fewer products currently used for large-scale production.However,using genetic engineering technology to prepare AMPs has many advantages such as low cost and convenient for mass production etc.,and has broad application prospects.Therefore,this tech is the most effective method to produce AMPs.Some AMPs have the disadvantages of low antibacterial activity and haemolytic to eukaryotes.Consequently,how to improve activity and reduce toxicity has become the priority for the study of artificial AMPs.The molecular modification of AMPs has been focused.In this study,the Musca domestica antibacterial peptide in the laboratory and several AMPs with strong antibacterial activity were selected as mother peptides.The active centers were intercepted separately for heterozygosis,and AMPs with strong antibacterial activity were screened out.Then,a large number of hybrid peptides were prepared by genetic engineering methods.The existing Musca domestica antibacterial peptide MDAP-2 in our laboratory,Buforin II antibacterial peptide from the frog stomach mucosa,Buforin?derived peptide Buforin?c,cecropin A,and Apidaecin I A were used as the parent peptides.The bioinformatics analysis and design of amino acid sequences were performed for selection of active sites and hybridization among different peptide fragments.Heterozygous peptides were sent to a biotechnology company for chemical synthesis and then tested for in vitro antibacterial activity.A hybrid peptide BM16 with comparable antibacterial activity to the parent peptide and low hemolytic activity was screened out.The structure of hybrid peptide BM16 was modified to improve antibacterial activity and reduce hemolytic activity.On the basis of the hybrid peptide BM16,the full-length BM16-R gene was directly amplified by PCR and connected with pET28a-Ub expressionvectortoconstructarecombinantexpressionplasmid pET28a-Ub-BM16-R.The plasmid was transformed into E.coli BL21?DE3?and induced to expression by IPTG.The expression of target protein was detected by SDS-PAGE electrophoresis and Western blot.The fusion protein was purified by Ni²⁺affinity chromatography and digested with SUMO protease to obtain an antimicrobial protein of high purity.The antibacterial activity of the purified protein was tested using modified 2-fold dilution method.The results showed that the antibacterial activity of the hybrid antibacterial peptide BM16-R was stronger than that of parent peptide,and the hemolytic activity was low.This provided data for the development of novel antibacterial peptides.The main results are as follows:1?Using bioinformatics analysis software,5 hybrid antibacterial peptides were successfully designed and synthesized.A hybrid peptide,BM16,with better activity was screened by in vitro antibacterial activity.2?In further modification of BM16 amino acids,arginine?R?was used to replace no.11 phenylalanine?F?and no.14 leucine?L?in BM16,respectively.Determination of bacteriostasis and hemolysis activity in vitro indicated that the hybrid peptide BM16-R had strong antibacterial activity and low hemolysis activity.3?The BM16-R full-length gene was amplified by PCR,and the prokaryotic recombinant expression vector pET28a-Ub-BM16-R was successfully constructed and transformed into E.coli BL21?DE3?to induce fusion expression.SDS-PAGE electrophoresis showed that the13 kDa target protein was obtained.4?The fusion antibacterial protein Ub-BM16-R was purified by Ni²⁺affinity chromatography.The antibacterial protein BM16-R was obtained after SUMO enzyme digestion and molecular sieve filtration chromatography.In vitro antibacterial assay showed that BM16-R had stronger bacteriostatic effect on gram-negative bacteria than gram-positive bacteria.The MIC value for E.coli and Salmonella were 2?g·mL^-1 and 4?g·mL^-1,respectively,whereas the MIC value for Staphylococcus aureus was 16?g·mL^-1.