Molecular characterization and diagnostic value of Taenia solium low-molecular-weight antigen genes

Abstract

Neurocysticercosis (NCC) caused by infection with the larvae of Taenia solium is an important cause of neurological disease worldwide. In order to establish an enzyme-linked immunosorbent assay (ELISA) for this infection using recombinant proteins, we carried out molecular cloning and identified four candidates as diagnostic antigens (designated Ag1, Ag1V1, Ag2, and Ag2V1). Except for Ag2V1, these clones could encode a 7-kDa polypeptide, and Ag2V1 could encode a 10-kDa polypeptide. All of the clones were very similar. Except for Ag2V1, recombinant proteins were successfully expressed using an Escherichia coli expression system. Immunoblot analysis of NCC patient sera detected recombinant proteins, but because reactivity to recombinant Ag1 was too weak, Ag1 was not suitable as an immunodiagnostic antigen. So, Ag1V1 and Ag2 were chosen as ELISA antigens, and the Ag1V1/Ag2 chimeric protein was expressed. Of 49 serum samples from NCC patients confirmed to be seropositive by immunoblot analysis, 44 (89.7%) were positive by ELISA. No assays of serum samples from patients with other parasitic infections recognized the Ag1V1/Ag2 chimeric protein. The Ag1V1/Ag2 chimeric protein obtained in this study had a high value for differential immunodiagnosis.

FIG. 1

Alignment of amino acid sequences of four diagnostic-antigen candidate clones. The amino acid sequences predicted from cDNA clones were aligned by using the CLUSTAL V program (7). Asterisks indicate residues which are identical to the Ag1 sequence, while gaps introduced by the CLUSTAL V program are symbolized by dashes. Features within the sequences are denoted as follows: underlined letters at the N terminal, the putative signal sequences; boxed letters, N-linked glycosylation sites; circled letters, cysteine residues; the amino acid sequence conserved among all four clones is italicized.

FIG. 2

(A) Analysis by SDS-PAGE of purified recombinant proteins stained with Coomassie blue. (B) Immunoblot analysis using a pooled serum from NCC patients (left panel) or AE patients (right panel). Molecular size markers are indicated on the left. Lane 1, TRX; lane 2, rAg1; lane 3, rAg1V1; lane 4, rAg2.

FIG. 3

(A) The scheme of PCR mutagenesis for production of an Ag1V1/Ag2 chimeric gene. Details of the PCR mutagenesis to obtain Ag1V1/Ag2 chimeric gene are described in Materials and Methods. (B) Analysis of the resultant PCR products in a 2.0% agarose gel. Molecular size markers are indicated on the left. Lane 1, first PCR product of the Ag1V1 gene; lane 2, first PCR product of the Ag2 gene; lane 3, second PCR product of the Ag1V1/Ag2 chimeric gene. (C) Amino acid sequence of Ag1V1/Ag2 chimeric protein. The underlined amino acids were derived from an Ag1V1 (²⁰E to ⁷⁸C) clone, and the remains were derived from an Ag2 (²⁰K to ⁸⁵A) clone.

FIG. 4

(A) Analysis by SDS-PAGE of purified Ag1V1/Ag2 chimeric protein stained with Coomassie blue. (B) Immunoblot analysis using a pooled serum from NCC patients. Molecular size markers are indicated on the left. Lane 1, TRX; lane 2, Ag1V1/Ag2 chimeric protein.

FIG. 5

Results of ELISA using Ag1V1/Ag2 chimeric protein with sera from 50 patients with neurocysticercosis (NCC), 35 with alveolar echinococcosis (AE), 10 with cystic echinococcosis (CE), and 70 with other parasitic diseases (OP; 10 clonochiasis, 10 sparganosis, 8 fascioliasis, 32 paragonimiasis, 10 schistosomiasis) and from 20 healthy humans (NH). The dotted line shows the cutoff value (0.17).