Baculovirus-Mediated Expression and Functional Characterization of a Glycyrrhizin-Specific Single-Chain Variable Fragment in Sf9 Cells

Abstract Background Glycyrrhizin (GC) is a major bioactive saponin in licorice plants ( Glycyrrhiza spp.). A GC-specific single-chain variable fragment (GC-scFv) was constructed from anti-GC monoclonal antibody 2H2. To obtain a soluble recombinant recognition reagent for GC immunoassays, GC-scFv was expressed using a baculovirus–insect cell platform. Methods The variable heavy- and light-chain genes were amplified from 2H2 hybridoma-derived complementary DNA (cDNA) and assembled using a (Gly 4 Ser) 3 linker. The construct was transferred through pET-28a(+) and pCold I into pFastBac1Mel, which contains the honeybee melittin signal sequence for secretion in Sf9 insect cells. GC-scFv was recovered by nickel-chelate affinity chromatography and characterized using indirect competitive enzyme-linked immunosorbent assay (icELISA). The calibration curve was analyzed by semi-logarithmic regression using triplicate wells at each concentration. Results An anti-T7-tag Western blot detected GC-scFv at approximately 29 kDa in the eluate recovered from culture medium. Free GC produced concentration-dependent inhibition, with a half-maximal inhibitory concentration (IC 50 ) of 0.46 μg/mL and a semi-logarithmic calibration interval of 0.0488–3.125 μg/mL. The apparent limit of detection and apparent limit of quantification, defined as 10% and 20% inhibition, were 54.2 and 92.8 ng/mL, respectively. Cross-reactivity was 24.47% toward glycyrrhetinic acid 3- O -glucuronide, 6.52% toward glycyrrhetinic acid, and below 0.92% toward the remaining tested compounds. Conclusions The baculovirus–Sf9 platform produced GC-scFv with competitive binding activity toward GC. The recovered reagent and its assay characteristics provide a basis for developing recombinant antibody-based immunoassays for licorice-containing products.