EFFECT OF KOZAK AND SIGNAL PEPTIDE SEQUENCES ON RECOMBINANT ANTIBODY EXPRESSION IN CHO CELLS TARGETING OPISTHORCHIS VIVERRINI ANTIGEN

Abstract

Opisthorchis viverrini (OV) infection is a major public health concern in Southeast Asia and is strongly associated with cholangiocarcinoma. Reliable production of monoclonal antibodies is essential for antigen-based diagnostic assays; however, conventional hybridoma systems may have limitations in stability and scalability. This study aimed to develop and optimize a recombinant antibody expression system targeting OV antigen using mammalian cells. Variable heavy-chain (VH) and light-chain (VL) sequences derived from the OV-03 hybridoma clone were cloned into the pTRIOZ-hIgG1 vector to generate two constructs: (+Kozak, +SP) and (−Kozak, −SP). Following transfection into CHO-K1 cells, gene expression was confirmed by PCR, demonstrating successful transcription in both constructs. However, Western blot analysis revealed intracellular expression of IgG heavy chain (~50 kDa), light chain (~28 kDa), and full-length antibody (~150 kDa) only in cells transfected with the (+Kozak, +SP) construct, highlighting the essential roles of the Kozak sequence and signal peptide in efficient protein expression. Additionally, increasing fetal bovine serum (FBS) concentration (1.25-10%) enhanced intracellular antibody levels. These findings demonstrate that optimized translational elements and culture conditions significantly improve recombinant antibody production. This study provides a foundation for scalable antibody production and supports the development of recombinant antibodies for diagnostic applications in opisthorchiasis.

Keywords: Recombinant Antibody, Opisthorchis viverrini, CHO-K1 cells