Study On The Construction Of Engineering Bacillus Subtilis Strains Of Insect Antimicrobial Peptides

The abuse of antibiotics has caused a series of environmental and health risks,especially the emergence of drug-resistant bacteria,which threatens human health.Finding more efficient and new types of antibiotics has been urgent.Antimicrobial peptide(AMP)has attracted wide attention due to its high safety,good thermal stability,wide antimicrobial spectrum,no drug resistance and no toxicity to higher animal cells.AMPs are a class of short peptides,which contain 30-60 amino acids.Currently,there are 2600 kinds of natural antimicrobial peptides which have been found in plants,bacteria,insects and animals.Insect is an important source of antimicrobial peptides in the database of AMPs.The similarity of amino acid sequences of Defensin-like-peptide 4(DLP4)isolated from black soldier fly and Sand moth fly peptide(SMFP)isolated from Sand moth fly reached 75%,which both has a strong inhibitory effect on Gram positive bacteria.Cecropin is the first antimicrobial peptide isolated from Silkworm pupa,which has a strong inhibitory effect on both gram negative and gram positive bacteria.In this experiment,Tris-HCl(pH6.5),Tris-HCl(pH8.0),physiological saline and 0.1% trifluoroacetic acid(TFA),4 solutions were used to extract AMPS from the larvae of black soldier fly.The crude extracts showed inhibitory effect on Escherichia coli and Staphylococcus aureus,though the inhibitory effect on Escherichia coli was more obvious.Although the antimicrobial peptides are wide existence,it's low content,easily deactivated by digestive system and high cost of chemical synthesis,make it difficult to form a large-scale application.The construction of genetic engineering strains to get AMPs by microbial fermentation is an important and selective way.Cecropin AD gene,DLP4 gene and SMF gene were used in this experiment to construct 2 groups,total 6 strains of engineering Bacillus subtilis,GF101,(1)using the plasmid PMA5 to ligate with the 3 sections of AMPs gene respectively,constructing PMA5-AMP free expression plasmid and transforming into GF101,(2)using plasmid PAX01 and PMA5 to construct a PAX01-KANA plasmid,which is a integrated expression plasmid with kanamycin resistant activity,next connecting with the 3 sections of AMPs gene respectively,forming PAX01-KANA-AMP palsmid,and transformed into GF101.In this experiment,6 engineering strains were constructed successfully,and the inhibition zone test results showed that all of fermentation liquids of 6 strains had obvious antibacterial effect on Escherichia coli,whereas there was no inhibition effect on Staphylococcus aureus observed.The experiment sets an example for the construction of engineering Bacillus subtilis strain of antimicrobial peptide,and provides a feasibility for a large-scale application in animal husbandry of antimicrobial peptides.