Molecular characterization of a putative protein disulfide isomerase from Babesia caballi.
We produced a mAb against the Babesia caballi extracellular merozoite termed mAb 2H2 and used it to screen a cDNA expression library prepared from B. caballi merozoite mRNA for highly expressed proteins. The complete nucleotide sequence of the cloned gene had 1547 nucleotides and contained a 36-nucleotide intron. The 1398 nucleotide open reading frame predicts a 51 kDa protein showing similarity to protein disulfide isomerase (PDI) from other species. The PDI gene had a predicted N-terminal signal sequence of 19 amino acids and a C-terminal tetrapeptide sequence (His-Thr-Glu-Leu; HTEL) for retention in lumen of the endoplasmic reticulum (ER). The recombinant protein expressed in baculovirus showed an apparent mass of 51 kDa, identical to that the native B. caballi protein. Moreover, the ER retention signal site (HTEL) of the recombinant protein retained its function in ER of insect cells. This 51 kDa protein was strongly expressed by extracelluar B. caballi merozoites in indirect immunofluorescence antibody tests, and was not expressed in the early phase of trophozoite development. Interestingly, detailed observation showed that the reaction of anti-P51 antibody and mAb 2H2 against pear-shaped forms was very erratic, some displaying one or two brightly fluorescent patterns.
Ikadai
H
H
Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan.
Tsukada
R
R
Sasaki
M
M
Takashiro
R
R
Yokoyama
N
N
Kudo
N
N
Igarashi
I
I
Oyamada
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D001403:Babesia / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D015723:Gene Library; D023061:Gene Order / Q000235:genetics; D006736:Horses; D007438:Introns / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D020539:Sequence Analysis, Protein; D018411:Spodoptera / Q000166:cytology
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases