CLONING, SEQUENCING AND EXPRESSION OF A CDNA-ENCODING AN ANTIGEN FROMTHE MYXOSPOREAN PARASITE CAUSING THE PROLIFERATIVE KIDNEY-DISEASE OF SALMONID FISH

A pool of monoclonal antibodies (MAbs) raised against the unknown orga nism causing the proliferative kidney disease of salmonid fish (PKX) h as been used to screen a cDNA expression library constructed from poly (A(+)) RNA extracted from PKX infected kidney. Four immunopositive la mbda ZapII recombinant phages were selected. Sequencing of the cDNAs r evealed identical 3' ends. The longest cDNA clone (2652 nucleotides) h ad an open reading frame (ORF) of 872 amino acids and encoded a protei n with a predicted size of 101 kDa (PKX101). Sequence analysis of PKX1 01 revealed two leucine zipper motifs, a putative transmembrane region and a microbody targeting signal al its C-terminal end. Three cDNA fr agments were subcloned in pET-14b expression vector and the ORF verifi ed by an in vitro transcription/translation procedure. Recombinant clo nes were expressed in Escherichia coli and the antigenicity of fusion proteins was studied by Western blotting using monoclonal antibodies d irected against PKX cells and a pool of serum from preimmune or PKX-in fected rainbow trout. Western blotting of enriched PKX cell antigen pr obed with one MAb or with sera from infected trout revealed a single p rotein with relative mobility of 13 kDa (PKX13). This discrepancy betw een PKX101 and PKX13 observed in Western blot suggests post-translatio nal modifications of the full-length PKX antigen. Copyright (C) 1996 E lsevier Science B.V.