Production and characterisation of an antiserum raised against recombinant rainbow trout (Oncorhynchus mykiss) MHC class II beta-chain (MhcOnmy-DAB)

A rainbow trout (Oncorhynchus mykiss) cDNA fragment encoding most of the extracellular part of the MHC class II β chain (MhcOnmy-DAB1) was cloned into a bacterial expression vector. After its purification, the recombinant protein was used for the immunisation of rabbits. The resulting antiserum, which demonstrated specific reactivity with the recombinant Onmy-DAB product, recognised a protein band of 34 kDa apparent molecular weight in western blots of SDS-PAGE-separated cell membrane preparations from spleen of pronephros, but not from muscle tissue. Proteins with similar characteristics in SDS-PAGE analysis under reducing conditions could be immunoprecipitated with the antiserum from trout peripheral blood leukocyte cell membranes. Under non-reducing conditions the precipitate was shown to run with an apparent molecular weight of 48 kDa, whereas under reducing conditions this immunoprecipitate consisted of two protein bands. Finally, deglycosylation resulted in a decrease of 3 kDa of both protein as observed under reducing gel electrophoresis conditions, thus confirming the glycoprotein character as suggested by the analysis of the trout MHC class II α and β nucleotide gene sequences.