Study On Heterologous Expression,Purification And Antimicrobial Activity Of Three Antimicrobial Peptides

Antimicrobial peptides(AMPS)are small molecular peptides which inhibit various of microorganism even cancer cells,produced by organism.Antimicrobial peptides display strong antimicrobial activity against G+ and G-,which result in food contamination.Antimicrobial peptides have great potential to be a novel food preservative because of solubility,stability and broad antibacterial spectrum.The purpose of this thesis is to express three antimicrobial peptides by genetic engineering technique.CFBD-1,UyCT5 and Hispidalin are isolated from Cynops fudingensis,Urodacus yaschenkoi scorpion and Benincasa hispida seeds,respectively.Molecular mass of three antimicrobial peptides are 4.5 KDa?.4 KDa and 5.7 KDa,respectively?The genes of CFBD-1 and UyCT5 are synthesized manually according to the animo acids sequence.CFBD-1 and UyCT5 fragments are cloned into pMAL-c2X,pGEX-2T vector.Recombinant plasmids are transformed to Esherichia coli BL21 competent cell and induced by IPTG The results indicate that GST-UyCT5 is expressed completely,while MBP-CFBD-1 is expressed incompletely with condition of 0.2 mmol/L IPTG,28? and 6 h inducing time.However,GST-UyCT5 plays no antimicrobial activity against Bacillus subtilis 151-1,Staphylococcus aureus,Listeria monocytogenes and Salmonella after purification.Then Hispidalin and CFBD-1 fragments are cloned into pPICZaA vector.Recombinant plasmids are transformed to GS115 competent cell after Sac I linearization and induced by methanol.The results show that His-Hispidalin is expressed successfully,while His-CFBD-1 is not expressed with condition of 1%methanol,25 mL liquid volume,28? and 144 h inducing time.The concentration of His-Hispidalin is 120.21 ?g/mL.His-Hispidalin is active to Bacillus subtilis 151-1,L300-1,LZZ-133 and Listeria monocytogenes.Minimum inhibitory concentration is 75,67,83 and 75 ?g/mL,respectively.The maximum concentration of His-Hispidalin is 133.87 ?g/mL after optimization of 0.5%methanol,50 mL liquid volume and 120 h inducing time.Comparing with previous productivity,the yield of His-Hispidalin is increased by 11.4%.