Predictive,Clonal Expression And Preliminary Immunogenicity Analysis Of Outer Membrane Protein From Pseudomonas Plecoglossicida,A Pathogen Of Larimichthys Crocea

Pseudomonas plecoglossicida is the main pathogen bacteria causing visceral granulomas syndrome in large yellow croaker(Larimichthys crocea),this disease has differently degrees occurred frequently in in China's coastal cage cultured large yellow croaker,and the high mortality rate of this disease lead to a serious threat to the culture industry,and up till now,no effective control measures has been found.In this study,based on the whole genome sequencing sequence(GenBank Accession:NZ_ASJX00000000)of the NCBI-registered Pseudomonas plecoglossicida strain NB2011,and development thought of reverse vaccinology,prediction and screening outer membrane proteins of this bacteria from the genome through bioinformatics databases as candidate immunogens,cloned and expressed the recombinant protein in E.coli and analysis for obtained recombinant protein immunogenicity,the research shows the results as follows:1?In this study 28 outer membrane protein genes predicted and screened out as candidate immunogen from NB2011 strain genome.10 of them are outer membrane structural proteins,10 are virulence-secreting related proteins,3 lipoproteins,2 porins,and 3 other outer membrane proteins.2 ? Cloned 28 outer membrane proteins successfully by designing gene amplification primers,The sequence of 27 genes is 100% consistent with the NB2011 strain by designing gene amplification primers,and 1 of them named P10(EPB95217)has a base difference from NB2011 strain,repeated eperiments confirmed the existence of this differences,analysis shows that this base difference does not terminate translation and should be a valid coding sequence.3?In this study,28 outer membrane protein prokaryotic expression vector were was constructed(pET-30(a)-n).18 recombinant proteins were successfully expressed in E.coli by optimizing the expression conditions,the results of inclusion body detection shows that,2 of the proteins are soluble and the remaining 16 are in inclusions body.4?New Zealand rabbit anti Pseudomonas plecoglossicida polyclonal antibody with a titer of 1:6400 has been prepared,Apply this polyclonal antibody to immunogenic analysis the 18 recombinant proteins by Western Blot,results shows that : 10 recombinant proteins are binding to the polyclonal antibody strongly,8recombinant proteins are binding to the polyclonal antibody weakly.5?4 outer membrane proteins of P15,P16,A6 and B6 were selected for the fish immune challenge test form strongly immune response of recombinant outer membrane protein immunoassay.The result shows that,large yellow croaker begins to die in 6th day after challenge test,obvious visceral granulomas are found by anatomy,Until the 14 th day,Statistical the relative protection ratios(RPS)in immunization group,the RPS of P15 and P16 are 15.79% and 5.26%,the RPS of A6 and B6 are68.42% and 52.63%.In summary,this study uses the principle of reverse vaccine,start the prediction,preparation and preliminary screening wroking of Pseudomonas plecoglossicida from large yellow croaker outer membrane protein subunit vaccine antigen.the results provide a useful reference for the vaccine control of visceral granulomas syndrome disease in large yellow croaker.