Cloning, sequencing and expression of a cDNA encoding an antigen from the Myxosporean parasite causing the proliferative kidney disease of salmonid fish.
A pool of monoclonal antibodies (MAbs) raised against the unknown organism causing the proliferative kidney disease of salmonid fish (PKX) has been used to screen a cDNA expression library constructed from poly (A+) RNA extracted from PKX infected kidney. Four immunopositive lambda ZapII recombinant phages were selected. Sequencing of the cDNAs revealed identical 3' ends. The longest cDNA clone (2652 nucleotides) had an open reading frame (ORF) of 872 amino acids and encoded a protein with a predicted size of 101 kDa (PKX101). Sequence analysis of PKX101 revealed two leucine zipper motifs, a putative transmembrane region and a microbody targeting signal at its C-terminal end. Three cDNA fragments were subcloned in pET-14b expression vector and the ORF verified by an in vitro transcription/translation procedure. Recombinant clones were expressed in Escherichia coli and the antigenicity of fusion proteins was studied by Western blotting using monoclonal antibodies directed against PKX cells and a pool of serum from preimmune or PKX-infected rainbow trout. Western blotting of enriched PKX cell antigen probed with one MAb or with sera from infected trout revealed a single protein with relative mobility of 13 kDa (PKX13). This discrepancy between PKX101 and PKX13 observed in Western blot suggests post-translational modifications of the full-length PKX antigen.
Saulnier
D
D
Unité de Virologie et Immunologie Moléculaires, Pathologie Infectieuse et Immunité des Poissons, INRA, Jouy-en-Josas, France.
Brémont
M
M
de Kinkelin
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005393:Fish Diseases / Q000469:parasitology*; D015870:Gene Expression; D007674:Kidney Diseases / Q000469:parasitology / Q000662:veterinary*; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000469:parasitology*; D011528:Protozoan Infections / Q000469:parasitology; D011529:Protozoan Infections, Animal; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000953:Antigens, Protozoan; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins