Plant-Based Expression and Structural Characterization of M2e-HBc Virus-Like Particle in Nicotiana benthamiana

Department of Pharmocognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Science, Chulalongkorn University, Bangkok 10330 Thailand

^* Corresponding author: maykantkawbo@gmail.com

Abstract

Virus-like particles (VLPs) derived from hepatitis B core antigen (HBcAg) are high immunogenicity and self-assembling capabilities, making them ideal platforms for vaccine development. This study focuses on the plant-based production and purification of M2e-HBc VLPs in Nicotiana benthamiana. The M2e-HBc construct was engineered with a tandem M2e ectodomain fused to HBcAg, incorporating an N-terminal signal peptide and C-terminal His-tag SEKDEL. The gene was cloned into the pBY2ek vector, transformed into Escherichia coli and Agrobacterium tumefaciens, and agroinfiltrated into N. benthamiana leaves at OD₆₀₀ = 0.2. Leaves harvested at 3 days post-infiltration (DPI) were homogenized in IMAC10 buffer at a 1:2 (w/v) ratio for protein extraction. The crude was purified by nickel affinity chromatography and protein expression was evaluated by SDS-PAGE and Western blot under both reducing and non-reducing conditions. Protein concentration was determined via Bradford assay and further concentrated using Amicon® Ultra centrifugal filter. Sucrose gradient ultracentrifugation (10%, 30%, 70%) at 150,000 g for 4 hours was used to isolate VLP-containing fractions. The presence of assembled VLPs was confirmed by SDS-PAGE, Western blot, and transmission electron microscopy (TEM), which revealed spherical particles. This study demonstrates the feasibility of producing M2e-HBc VLPs in plants, providing a scalable platform for influenza vaccine development.