Molecular cloning, expression, and serological evaluation of an 8-kilodalton subunit of antigen B from Echinococcus multilocularis.
Full-length cDNA and genomic DNA encoding an 8-kDa subunit of antigen B from Echinococcus multilocularis (designated EmAgB8/1) were isolated from an E. multilocularis metacestode cDNA library and a protoscolex genomic DNA library, respectively. The open reading frame of the cDNA clone encodes a polypeptide comprising 85 amino acids with a 20-amino-acid NH(2)-terminal signal sequence, which was confirmed following N-terminal sequencing of the native antigen. Reverse transcription-PCR analysis revealed that the clone encoding EmAgB8/1 is predominantly transcribed in larval E. multilocularis. The gene consists of two exons (encoding the signal sequence and mature protein) separated by a 91-bp intron. The mature form was expressed in Escherichia coli, and its antigenic reactivity was compared with that of a counterpart, an 8-kDa subunit of antigen B from Echinococcus granulosus (EgAgB8/1) by Western blotting and enzyme-linked immunosorbent assay (ELISA) with serum samples from patients confirmed to have cystic echinococcosis (CE) and alveolar echinococcosis (AE). Recombinant EmAgB8/1 showed positive reactions in Western blots with 81.3% (65 of 80) of serum samples from CE patients and 40.6% (26 of 64) of serum samples from AE patients, while recombinant EgAgB8/1 showed positive reactions with 86% (43 of 50) and 42% (19 of 45) of the serum samples from these CE and AE patients, respectively. By the ELISA, both EmAgB8/1 and EgAgB8/1 exhibited similar positive reactions with 88% (44 of 50) of serum samples from CE patients and 37.8% (17 of 45) serum samples from AE patients. Statistical analysis revealed that the sensitivity of EmAgB8/1 was comparable to that of EgAgB8/1 for the serodiagnosis of echinococcal diseases. There was no cross-reaction with sera from patients with cysticercosis, which often cross-react when native antigens are used for serodiagnosis.
Department of Parasitology, Asahikawa Medical College, Asahikawa, Japan.
Wulamu
W
Mamuti
Hiroshi
H
Yamasaki
Yasuhito
Y
Sako
Minoru
M
Nakao
Ning
N
Xiao
Kazuhiro
K
Nakaya
Naoki
N
Sato
Dominique A
DA
Vuitton
Renaud
R
Piarroux
Marshall W
MW
Lightowlers
Philip S
PS
Craig
Akira
A
Ito
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000032:analysis*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D004443:Echinococcosis / Q000175:diagnosis*; D004446:Echinococcus / Q000254:growth & development / Q000276:immunology*; D015723:Gene Library; D005849:Gerbillinae; D006801:Humans; D007814:Larva / Q000276:immunology; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D000947:Antigens, Helminth; D017931:DNA Primers; D018076:DNA, Complementary; D018509:DNA, Helminth; D021122:Protein Subunits