Molecular cloning, expression, and serological evaluation of an 8-kilodalton subunit of antigen B from Echinococcus multilocularis

Abstract

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.

FIG. 1.

Complete nucleotide and deduced amino acid sequences of EmAgB8/1. An asterisk indicates a stop codon. A polyadenylation addition signal is boxed. P₁, gene-specific primer used for RT-PCR; P₂ and P₃, primers were used for probe synthesis.

FIG. 2.

Multiple alignment of amino acid sequences deduced from four different cDNAs of EmAgB8/1 with EgAgB8/1 and Ag1V1 of T. solium. Amino acid residues identical to those of EmAgB8/1 are indicated by colons. Dashes are introduced to maintain alignment. The signal sequence is shown in boldface, and an arrow indicates the cleavage site of the signal peptide. The amino acids found in the NH₂-terminal amino acids of native AgB are underlined. The boxed sequence highlights the immunodominant region of EgAgB8/1. The cDNA and amino acid sequences are deposited in the DDBJ/EMBL/GenBank database under the accession numbers provided in Materials and Methods. The sequences of EgAgB8/1 and Ag1V1 of T. solium were obtained from the EMBL data bank (accession numbers Z26336 and AB044081, respectively).

FIG. 3.

SDS-PAGE analysis of purified rEmAgB8/1 and rEgAgB8/1. Approximately 1 μg of each sample were separated on a 4 to 20% polyacrylamide gradient gel and stained with Coomassie brilliant blue. Lane 1, prestained protein markers (Bio-Rad, Richmond, Calif.); lane 2, purified rEmAgB8/1; lane 3, purified rEgAgB8/1.

FIG. 4.

Comparison of antigenicities of rEmAgB8/1 and rEgAgB8/1 by WB. Representative data are shown. (A) rEmAgB8/1; (B) rEgAgB8/1. The membranes were probed with serum samples from patients with CE (lanes 1 to 18) or AE (lanes 19 to 35). The arrows show the locations of the recombinant antigens that reacted with serum samples.

FIG. 5.

Comparison of antigenicities of rEmAgB8/1 and rEgAgB8/1 by ELISA. The reactivity of rEmAgB8/1 or rEgAgB8/1 was assessed by using serum samples from 50 and 45 patients with CE (•) and AE (○), respectively. The broken lines indicate the cutoff values for rEmAgB8/1 (A₄₀₅ = 0.120) and rEgAgB8/1 (A₄₀₅ = 0.170), respectively. The cutoff values were set at four times the optical density for the negative pooled serum sample.