Study On Possible Genetically Engineered Vaccine Against The Columnaris Disease Caused By Flavobacterium Columnare

Flavobacterium columnare, a Gram-negative bacterium, is the causative agent of columnaris disease in fish. It affects wild and cultured freshwater fish, causing often mass mortality and leading to severe economic losses in the fish farming industry. Two strategies involving chemicals and vaccinations are normally applied to control the infection caused by F. columnare. It is well known that the use of chemicals has some negative and side effects, such as the existence of drug residues in aquatic products, the negative impact on aquatic ecosystem, and the emergence of resistant strains of pathogens. Therefore, vaccine can be an alternative strategy to control the columnaris diseaseThe outer membrane proteins（OMPs） were regarded as good candidates for vaccine antigens because of their exposed epitopes on the cell surface of Gram-negative bacteria, which allows them to be easily recognized as foreign substances by host immune systems. Five proteins were identified from outer membrane proteins（OMPs） of F. columnare using a Western blotting approach in two-dimensional electrophoresis map gels with antibacterial sera from grass carp, Ctenopharyngodon idella（Valenciennes）, and then anti-grass carp-recombinant Ig（r Ig） monoclonal antibodies. These protein spots were identified conclusively by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry（MAL-DI-TOF/TOF MS）. The 5 proteins are considered as immunogenic molecules of F. columnare, including gliding motility lipoprotein（Gld J）,hypothetical protein FCOL₀9735（fcol）,F0F1 ATP synthase subunit beta（f0f1）,lipoprotein（lip） and outer membrane efflux protein precursor（omepp）.The genes encoding the 5 immunogenic proteins were amplified from the genomes of F. columnare G4 by PCR. Then, the gene fragments were linked to the prokaryotie expression vector p ET28 a, constructing recombinant plasmids p ET28a-gld J, p ET28a-fcol, p ET28a-lip, p ET28a-f0f1 and p ET28a-omepp. Recombinant proteins were induced with IPTG in Escherichia coli BL21 harbouring these recombinant plasmids and purified by affinity chromatograph respectively. Grass carp were then immunized with these purified recombinant proteins, including rgld J, rfcol, rf0f1, rlip and romepp respectively, and specific serum antibodies were detected and significantly enhanced expression of the immune-related genes were observed including Interleukin 1β（IL-1β）, IL-8 and IL-10, IFN-I and IFN-γ, Immunoglobulin M（Ig M）, Ig D and Ig Z, major histocompatibility complex I（MHC Iα） and MHC IIβ and C-reactive protein（CRP）. The relative protective rate（RPS） of grass carp immunized with rgld J, rfcol, rf0f1, rlip and romepp were 72%, 8%, 32%, 64% and 68% respectively. Furthermore, the serum of grass carp immunized with rgld J, rlip, and romepp significantly restrained the reproduction of F. columnare G4. It is shown that gld J, lip, and omepp were protective antigens and good candidate molecules for the development of genetically engineered vaccine against columnaris disease.On the other hand, virulence factors may serve as good candidate antigens for vaccine development. In order to increase immunogenicity and immune-protective ability, the antigen region of five possible virulence factor genes, encoding zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase were fused and linked to the prokaryotie expression vector p ET32 a, constructing the recombinant plasmid p ET32a-zptcc. Recombinant fusion protein was expressed and purified. Grass carp were immunized with the recombinant protein, producing 39% relative protection rate against F. columnare challenge infection. Specific antibodies and significantly enhanced expression of immune-related genes including IL-1β, IL-8, IL-10, Ig M, IFN-I, CRP and MHC Iα were detected in the blood of grass carp immunized with the recombinant fusion protein. It is indicated that the recombinant fusion protein provided a certain level of protection for grass carp against the challenge infection of F. columnare.