Molecular cloning and characterization of an antigenic protein with a repeating region from Clonorchis sinensis

Abstract

In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography. Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one year. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.

Fig. 1

Partial cDNA and its deduced amino acid sequences of a clone, CsRP12, from Clonorchis sinensis. A stretch of bold characters represents a repetitive unit. An asterisk (*) indicates a translational termination codon. Oligonucleotide primers used for DNA sequencing are underlined.

Fig. 2

multiple alignment of Clonorchis sinensis repetitive peptide, CsRP12-1, with surface proteins of animals. Residues identical to CsRP12-1 were shaded. A gap (-) was introduced for maximal alignment. Cs44, C. sinensis antigen 44 (Yong et al., 1998); XP2pre, Xenopus laevis skin secretory protein XP2 precursor (Hauser et al., 1992); CePro, Caenorhabditis elegans proline and glycine rich protein (Wilson et al., 1994); TcSA, Trypanosoma cruzi surface antigen (Buschiazzo et al., 1992); LdA2pre, Leishmania donovani amastigote-specific protein A2 precursor (Charest and Matlashewski, 1994); PfSAg, Plasmodium falciparum surface antigen protein precursor (Brown et al., 1987).

Fig. 3

Purification of recombinant CsRP12-1 and -2 proteins. Recombinant proteins bacterially produced were charged in a denaturation condition onto Ni-NTA column and eluted with a buffer containing 250 mM imidazole. The recombinant proteins purified and deployed in SDS-polyacrylamide gel were detected by Coomassie blue staining (A) and immunoblotting with anti-Xpress antibody (B). Lane 1, induced bacterial lysate; lane 2, flow-through; lane 3, eluate.

Fig. 4

Immunoblots of purified recombinant CsRP12-1 to helminth-infected patients' sera. A. Immunoblot against clonorchiasis patients' sera. B. Other helminth-infected patients' sera. Pw, paragonimiasis; Sp, sparganosis; Cy, cysticercosis; N, normal human sera; C, control by anti-Xpress antibody.

Fig. 5

An immunoblot of recombinant CsRP12-1 protein to Clonorchis sinensis-infected rabbit sera.

Fig. 6

Purification and antigenicity of bacterial recombinant C-terminus peptides of CsRP12. A. Purification of the recombinant proteins by Ni-NTA affinity chromatography. Lane 1, induced lysate; lane 2, pass-through fraction; lanes 3 and 4, buffers B and C, respectively; lanes 5, 6, 7 and 8, eluates with buffers C, D, E and F containing 250 mM imidazole, respectively. B. Immunolots against C. sinensis-infected patient sera. Lanes 1 to 7, clonorchiasis patients sera; lane 8, anti-RGS His antibody.