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Recombinant Fasciola gigantica tegumental calcium binding protein-4 modulates host peripheral blood mononuclear cell
Areeba Yousaf1, Mirza Imran Shahzad2, Muhammad Ehsan3
1Department of Parasitology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, 63100 Punjab, Pakistan; Department of Biochemistry and Molecular Biology, Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, 63100 Punjab, Pakistan.
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Fasciolosis caused by Fasciola gigantica is a parasitic disease affecting livestock and occasionally humans in tropical and subtropical regions of Africa and Asia. Increasing reports of triclabendazole resistance have emphasized the need for alternative control strategies and vaccine development. Calcium-binding proteins are promising antigenic targets involved in host-parasite interactions. This study aimed to clone, express, and characterize tegumental calcium-binding protein-4 (TCBP-4) of F. gigantica, and to evaluate its immunomodulatory effects on host peripheral blood mononuclear cells (PBMCs). The TCBP-4 gene was amplified and cloned into the pET-28a(+) expression vector in Escherichia coli BL21(DE3). The recombinant protein (rFg-TCBP-4) was purified, and verified using SDS-PAGE and Western blotting. Bioinformatic analyses predicted physicochemical and structural properties. Interaction of rFg-TCBP-4 with PBMCs was examined by immunofluorescence. The immunomodulatory effects of rFg-TCBP-4 were evaluated by measuring cytokine production using enzyme-linked immunosorbent assay (ELISA), along with nitric oxide production, and PBMC proliferation and migration assays. The TCBP-4 gene (∼576 bp) was expressed as a recombinant protein with a molecular weight of ∼26.2 kDa. Bioinformatic analyses revealed conserved EF-hand calcium-binding motifs and predicted cytoplasmic localization. rFg-TCBP-4 specifically interacted with PBMCs and modulated cytokine production in a dose-dependent manner, characterized by decreased levels of pro-inflammatory cytokines (IFN-γ, IL-17, and TNF-α) and increased levels of regulatory cytokines (IL-4, IL-10, and TGF-β). In addition, rFg-TCBP-4 enhanced nitric oxide production, PBMC proliferation, and migration. rFg-TCBP-4 possesses immunomodulatory properties and may contribute to host immune regulation during F. gigantica infection. This protein represents a promising antigen for further investigation in vaccine development against fasciolosis.
