|Wiens, Gregory - Greg|
Submitted to: American Fisheries Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/14/2005
Publication Date: 7/29/2005
Citation: Cain, K., Sudheesh, P.S., Lapatra, S.E., Wiens, G.D., Lafrentz, B.R., Call, D.R. 2005. Identification and expression of an immuno-reactive heat shock protein from flavobacterium psychrophilum. Annual Meeting of the Fish Health Section/American Fisheries Society. (abstract).
Technical Abstract: Immunogenic antigens associated with cellular and extracellular products (ECPs) of Flavobacterium psychrophilum were compared between a virulent and non-virulent strain. Western blotting with trout immune sera identified several immuno-reactive antigens following single or 2-dimensional PAGE. A number of cellular antigens were shared between the two strains, while 11 antigens appeared to be unique to the virulent strain and 3 were present only in the non-virulent strain. Separation of ECPs also showed numerous shared and differentially expressed antigens. Mass spectrometry (MALDI-TOF-MS and LC/MS-MS) of two highly immuno-reactive cell-associated antigens identified two heat shock proteins, HSP 60 and HSP 70. Following sequence comparison to a draft F. psychrophilum genome, the genes encoding HSP 60 and Hsp 70 were identified as GroEL and DnaK, respectively. Specific primers were designed and genes were amplified from genomic DNA of F. psychrophilum to obtain PCR products of approximately 1.5 kb in size. Genes have been cloned into the pET TOPO expression vector (Invitrogen) and high-level protein expression has been achieved for HSP 60. Immunization trials are currently underway to determine the efficacy of this recombinant product as potential subunit vaccine for coldwater disease.