Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

摘要

Flavobacterium columnare causes columnaris disease in freshwater fish.In the present study,the antigenic regions of five outer membrane proteins (OMPs),including zinc metalloprotease,prolyl oligopeptidase,thermolysin,collagenase and chondroitin AC lyase,were bioinformatically analyzed,fused together,and then expressed as a recombinant fusion protein in Escherichia coli.The expressed protein of 95.6 kDa,as estimated by 10％ sodium dodecyl sulfate-polyacrylamide gel electrophoresis,was consistent with the molecular weight deduced from the amino acid sequence.The purified recombinant protein was used to vaccinate the grass carp,Ctenopharyngodon idella.Following vaccination of the fish their IgM antibody levels were examined,as was the expression ofIgM,IgD and IgZ immunoglobulin genes and other genes such as MHC Ia and MHC Ⅱβ,which are also involved in adaptive immunity.Intedeukin genes (IL),including IL-1β,IL-8 and IL-10,and type Ⅰ and type Ⅱ interferon (IFN) genes were also examined.At 3 and 4 weeks post-vaccination (wpv),significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein,and an increase in the expression levels ofIgM,IgD and IgZ genes was also detected following the vaccinations,thus indicating that an adaptive immune response was induced by the vaccinations.Early increases in the expression levels ofIL and IFN genes were also observed in the vaccinated fish.At four wpv,the fish were challenged with F.columnare,and the vaccinated fish showed a good level of protection against this pathogen,with 39％ relative percent survival (RPS) compared with the control group.It can be concluded,therefore,that the five OMPs,in the form of a recombinant fusion protein vaccine,induced an immune response in fish and protection against F.columnare.