Analysis Of Extracellular Product Characteristics Of Pseudoalteromonas And Cloning And Expression Of Its Antibacterial Protein

Part 1: Application of probiotics in aquacultureThe rapid development of aquaculture,frequent occurrence of aquatic diseases,the use of probiotics to improve the ecological environment,prevention and treatment of aquatic animal diseases have been increasingly recognized.This part provides a brief overview of the definition of aquatic probiotics,the probiotic species commonly use d in aquaculture and the mechanism of probiotics,and introduces Pseudoalteromonas sp.,Which secrete a variety of bioactive substances,including small molecules compounds,proteins,polysaccharides,etc.,in aquatic disease prevention and control,red tide control and so has a greater potential.Part 2: Comparison of antimicrobial activity of extracellular and intracellular products of Pseudoalteromonas m8 and a22In this part,the extracellular product and the intracellular product of the experimental strain were prepared by cover plate with cellophanes.The antibacterial activity of the extracellular product and the intracellular product was detected by the filter paper method.The results showed that the extracellular product and the intracellular product have inhibitory effect,and the extracellular product of the antibacterial effect is better than intracellular products.In order to optimize the culture time of the preparation of extracellular products,the extracellular products were prepared after incubation for 12 h,24h,36 h and 48 h respectively.The antibacterial activity of the extracellular product was determined by the filter paper method,and the total protein content of the extracellular product was quantified.The results showed that the antibacterial activity of the extracellular product was the best after 24 hours of culture and the total protein content reached the maximum at 24 h.Part 3: Study on the related prope rties of extracellular products of Pseudoalteromonas m8 and a22The effects of catalase on the antibacterial activity of extracellular products were tested by setting the concentration of catalase to three gradients at 100 ?g / mL,200 ?g / mL and 500 ?g / mL,respectively.The results showed that catalase could inhibit the antibacterial activity of extracellular products.The filter paper containing catalase could make the gap of the inhibition zone,but because of the concentration setting or the distance,there is no significant difference in the gap of the different concentrations of catalase.L-amino acid oxidase can catalyze the release of hydrogen peroxide from amino acids and produce antibacterial effects,but their substrates are diverse.The amount of hydrogen peroxide produced was measured by a modified spectrophotometer.20 kinds of L-amino acids were used as substrates,and the absorbance values were measured at 505 nm.The results showed that the amount of hydrogen peroxide produced by L-lysine as the substrate was higher than that of other L-amino acids.The addition of L-lysine to the extracellular protein of strain m8 produces a higher amount of hydrogen peroxide than a22.The extracellular product of m8 of the antibacterial activity is better than a22,suggesting that the antibacterial activity is mediated by hydrogen peroxide.By SDS-PAGE and in-gel antibacterial assay,it was found that the protein band with molecular weight of 70 KDa had antibacterial activity,formed a significant antibacterial band on the strip,and the antibacterial band was cut down for mass spectrometry.The results showed that the 70 KDa protein band of strain m8 was identified as TonB-dependent receptor [Pseudoalteromonas flavipulchra JG1] and 70 KDa protein band of strain a22 was identified as antibacterial protein [Pseudoalteromonas flavipulchra JG1].Part 4: Cloning and expression of antibacterial protein of a22According to the sequence of antibacterial protein published by Gene Bank,primers were designed by PrimerPremier5.0 software.The target gene was amplified by using genomic DNA as template.The purified target gene was ligated with expression vector pBAD / gIIIA and transferred into E.coli BL21.The recombinant plasmids BL21 / pBAD / gIII / 2-p-1 were expressed in the final concentration of 0.5% L-arabinose at 20 ?,and the expressed product was analyzed by SDS-PAGE.The results showed that the protein could be expressed in Escherichia coli,but the soluble part of the expressed product did not detect the antibacterial activity.It speculated that that the inclusion body may be formed,can not be modified after translation,resulting in the expression of no antibacterial activity.