Immunological characterization of clonorchis sinensis venom allergen-like protein 13

Abstract

The SCP/TAPS (sperm coating protein/Tpx-1/Ag5/PR-1/Sc7) proteins are multifunctional protein found in eukaryotes. Venom allergen-like (VAL) proteins, members of the SCP/TAPS protein superfamily, have been reported from several parasitic helminths. However, little is known about their biological and immunological function. In this study, a VAL protein of the Chinese liver fluke Clonorchis sinensis was cloned and characterized. A cDNA encoding 25 kDaprotein was identified from EST database of C. sinensis. A BLAST search revealed that the protein shares 46% sequence identity with Schistosoma mansoni VAL 13 protein, and thus, the protein was named CsVAL13. Multiple sequence alignment indicated that SCP/TAPS domain of CsVAL13 shared 39~46% sequence identity with VAL proteins from parasitic helminths. His and Tyr residues, which help to stabilize protein structure, were highly conserved across the VAL protein sequences. Phylogenetic analysis showed that SCP/TAPS domain of CsVAL13 sequence clusters together with other group 2 VAL protein sequences. In the homology-modeled structure of CsVAL13, an α-β-α sandwichstructureand residues for a putative active site were highly conserved. PCR-amplified cDNA sequence of CsVAL13 was subcloned into pET32a bacterial expression plasmid vector. Recombinant CsVAL13 protein was produced bacterially and purified by Ni-NTA affinity chromatography. Immune sera were obtained from BALB/c mice immunized with the recombinant CsVAL13 protein.ELISA value of IgG1 in the immune sera against the recombinant CsVAL13 protein was six-times higher than that of IgG2a suggesting that CsVAL13 can induce Th2-polarized immune response. Immunohistochemical localization using the immune sera revealed that CsVAL13 was distributed mainly in the tegument and intrauterine eggs of adult C. sinensis. These findings suggest that CsVAL13 may be involved in host-parasite interactions and immune stimulation on the surrounding host environments.