Screening Of Differential Proteins Between Biofilm And Planktonic Cells And Construction Of GlpD Gene Mutant Of Aeromonas Veronii

Aeromonas veronii(AV)is a man-animal-fish pathogen,which has been widely concerned by people because of its high separation rate in recent years.Many studies have reported that most microorganism exist by biofilm in the environment,which is a floating with the corresponding growth mode.Biofilm enhanced resistance to external environmental stress to a certain extent,and the bacterial biofilm state can release free bacteria causing infection persistent and recurrent.If we can effectively prevent and control the biofilm infection,it will reduce the risk of bacterial infection,which is of great significance in the treatment of clinical infection.Therefore,screening of differential proteins between biofilm and planktonic cells of Aeromonas veronii has a certain significance in exploring the mechanism of the formation of Aeromonas veronii biofilm and screening of drug targets for inhibiting the formation of biofilm.In this study,Aeromonas veronii TH0426 was taken as the research object.First,96-well polystyrene microtiter plate assay was used to estimate the ability of biofilm formation in vitro under different conditions,and the conditions were optimized.The results showed that the biofilm formation ability of Aeromonas veronii TH0426 was not significantly different cultured in LB,THB and TSB medium,respectively.When cultured in culture medium PH 6,Nacl concentration 6g/L placed in 20 incubator for 24 hours,the biofilm formation ability ℃is better.In addition,the biofilm formation was detected by scanning electron microscopy.On the basis of above-mentioned study,iTRAQ technology was used to analyze the difference expression protein between planktonic cells and biofilm of Aeromonas veronii TH0426 and was verified by MRM.The results showed that 277 differential proteins in the planktonic cells and biofilm,including 130 up-regulated proteins and 147 down regulated proteins.Above-mentioned study,biological information preliminary analysis of proteins is conducted.In this study,5 differentially expressed proteins were selected for MRM validation,and randomly selected 20 up and down regulated proteins for fluorescence quantitative PCR,and the results were consistant with those of iTRAQ.The up-regulated protein gene glpD mutant and complementary strain was constructed by homologous recombination method.By comparing the biofilm formation ability of wild plants and missing strains,it was found that the biofilm formation ability of the glpD gene deletion strain decreased compared with the wild strain.K-B paper method was used to detect the drug resistance of the mutant strain and wild strain,and there was no significant differences.The results of the study will contribute to investigate the immune mechanism and pathogenesis of Aeromonas veronii biofilm at the protein level,and provide scientific basis for its development in vaccine production and drug development.