PRODUCTION AND CHARACTERIZATION 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 t he extracellular part of the MHC class II beta chain (MhcOnmy-DAB1) wa s cloned into a bacterial expression vector. After its purification, t he recombinant protein was used for the immunisation of rabbits. The r esulting antiserum, which demonstrated specific reactivity with the re combinant Onmy-DAB product, recognised a protein band of 34 kDa appare nt molecular weight in western blots of SDS-PAGE-separated cell membra ne preparations from spleen of pronephros, but not from muscle tissue. Proteins with similar characteristics in SDS-PAGE analysis under redu cing conditions could be immunoprecipitated with the antiserum from tr out peripheral blood leukocyte cell membranes. Under non-reducing cond itions the precipitate was shown to run with an apparent molecular wei ght of 48 kDa, whereas under reducing conditions this immunoprecipitat e consisted of two protein bands. Finally, deglycosylation resulted in a decrease of 3 kDa of both protein as observed under reducing gel el ectrophoresis conditions, thus confirming the glycoprotein character a s suggested by the analysis of the trout MHC class II alpha and beta n ucleotide gene sequences. (C) 1998 Academic Press Limited.