Construction, expression, and characterization of scFv fragment against Fasciola gigantica cathepsin L1H.

Abstract

Fasciola spp. infection is a significant zoonotic disease. Fasciola gigantica cathepsin L1H (FgCathL1H) is expressed across the life stages of Fasciola gigantica: newly excysted juvenile (NEJ), juvenile, and adult. An emerging tool for diagnosing fasciolosis in humans and cattle involves single-chain variable fragments (scFv) antibodies. These antibodies, consisting of linked variable regions of heavy chains (VHs) and light chains (VLs), retain binding specificity and affinity. This study aims to construct, express, and characterize an scFv antibody for use in a diagnostic kit for fasciolosis. The study successfully constructed and expressed recombinant scFv antibody genes derived from mouse spleen cells in Escherichia coli HB2151. Specific VH and VL fragments targeting recombinant FgCathL1H were amplified, inserted into a phagemid vector (pCANTAB5E), and transformed into E. coli TG1. Infection with the M13KO7 helper phage produced recombinant phages, and scFv clones with a high binding capacity were selected through three rounds of bio-panning. The expression of scFv proteins was induced with 1 mM IPTG, yielding antibodies detectable in the culture supernatant and periplasmic space. The indirect ELISA revealed strong binding in 10 scFv phage clones, which were sequenced and analyzed via computer-guided homology modeling and showed a similar classification to CDR1-3, consisting of VHs and VLs. The scFv DNA construct was approximately 747 bp in length. The SDS-PAGE, ELISA, and western blot confirmed the specificity of the scFv clone 1B, particularly at ~ 29 kDa. Docking studies showed epitopes on the scFv interacting with FgCathL1H. This scFv reacted specifically with F. gigantica antigens at 36 kDa (whole body (WB) of metacercaria and NEJ) and ~ 28 kDa (WB of 4-week-old juveniles and adults, and adult excretory-secretory protein (ES)). Immunolocalization showed positive staining in the cecal epithelium. Thus, scFv anti-rFgCathL1H shows promise for diagnosing fasciolosis.