| Musca domestica is a worldwide distributed insect.It has good adaptability to environment and strong innate immune system.It can carry many pathogenic microorganisms without being infected.A variety of antimicrobial peptides(AMPs)with bactericidal and antiviral abilities can be produced in the body of Musca domestica.They are important immune effectors of Musca domestica.Antimicrobial peptide is a kind of small molecular polypeptide produced by the host to resist the invasion of pathogens,which is an important component of innate immunity.It has broad-spectrum antibacterial activity and is not easy to produce drug resistance.Studies on antimicrobial peptides of Diptera,such as Attacin,cecropin,diptericin and defensin,are more in-depth.The research and development of new antibiotics and their separation and purification are the hot spots at present,such as muscin and domesticin,AMP-17,MLH,MAF-1 and so on.As an immune effector,the expression of antimicrobial peptide gene is often up-regulated after the host is stimulated by the pathogen.By searching the transcriptome data of Musca domestica,it was found that many unknown gene sequences were significantly up-regulated after bacterial infection.Combined with bioinformatics analysis,it was initially suspected that it was a new antimicrobial peptide.In this study,two of them AMP-5 and AMP-8 encoded proteins were recombined and expressed,and the antibacterial activity of their products was analyzed.The results showed that they are two novel antimicrobial peptide genes.The results are as follows:The N-terminal of AMP-5 peptide has signal peptide,and the theoretical molecular weight of mature peptide is 2.92 kDa;the isoelectric point is 6.23;it is rich in hydrophobic amino acids;the secondary structure is mainly ?-helix.Similarly,AMP-8 peptide also has signal peptide,the theoretical molecular weight of mature peptide is 5.43 kDa;the isoelectricpoint is 4.79;it is rich in hydrophobic amino acids and negatively charged amino acids;the secondary structure is mainly ?-helix.No homologous sequences of AMP-5 and AMP-8 were found in the blast sequence alignment in NCBI.It is speculated that they may be new genes.They were submitted to GenBank to obtain the login numbers GBAB01013207.1 and GBAB01028625.1,respectively.In order to further determine the biological functions of AMP-5 and AMP-8.In this study,the recombinant expression plasmid pPIC9K-AMP-5 / AMP-8 was constructed and expressed in Pichia pastoris KM71.The expression products were purified by Ni column affinity chromatography,and the antibacterial activity of the purified products was tested.It was found that the two antimicrobial peptides had strong inhibitory effect on Gram-positive bacteria and Gram-negative bacteria,including some resistant bacteria and pathogenic bacteria,such as Staphylococcus aureus,Pseudomonas aeruginosa,Salmonella typhi,etc.Then,we observed the morphological changes of Escherichia coli and Bacillus subtilis after the action of AMP-5 and AMP-8 by cold field scanning electron microscopy to study the antibacterial mechanism of antimicrobial peptides.Its effect on the permeability and integrity of bacterial cell membrane.The results showed that the two antimicrobial peptides play an antibacterial role by destroying the integrity of cell membrane.The cytotoxicity of AMP-5 and AMP-8 was further studied.It was found that their hemolytic activity to rabbit erythrocytes was relatively low.To sum up,two antimicrobial peptide genes,AMP-5 and AMP-8,were identified from Musca domestica,and pPIC9K-AMP-5 and pPIC9K-AMP-8 plasmids were successfully constructed and recombinant proteins were obtained.These two antimicrobial peptides have broad-spectrum antibacterial activity and low hemolytic activity.They play an antibacterial role by acting on the cell membrane of bacteria.This study enriches the research of insect derived antimicrobial peptides and provides theoretical reference for the treatment of drug-resistant bacteria. |
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