Groppelli, ElisabettaAljanabi, Walla2025-07-092024https://hdl.handle.net/20.500.14154/75780Biomedical Science – Infection and Immunity Focus This research falls under the field of Biomedical Science, with a specific focus on Infection and Immunity. The study aimed to develop a novel and affordable vaccine against Hepatitis A virus using virus-like particles (VLPs) expressed in plant systems. It involved molecular cloning, protein expression in vitro and in mammalian cells, and highlighted the potential of VLP-based vaccines in global public health, especially in low-income regions.This project aimed to develop an inventive and affordable vaccine against hepatitis A virus (HAV) by producing HAV virus-like particles (VLPs) in plant systems. The main objective was to generate the expression plasmids for the structural protein P1 and the non-structural protein 3C, with the addition of an HA-tag, and to validate their expression through several laboratory experiments. The workflow begins with primer designing specific to the P1 and 3C regions of the HAV genome, followed by PCR amplification and the creation of the expression constructs pCMVTnT-P1 and pCDNA3.1-HA-3C. Verification of these plasmid constructs was confirmed via restriction enzyme digestion and ligation. Primary protein expression was carried out in vitro using Rabbit Reticulocyte Lysates (RRL). At around 27 kDa bands were observed in both the HA-3C and control lanes, referring to unexpected bands and non-specific rather than successful protein expression, as shown and confirmed by Western blotting. These findings suggest that while the TnT RRL system did not generate enough amounts of the desired proteins, in mammalian cells the constructs were successful and detectable. The observations of this study integrate with past research on applying heterologous systems for viral protein expression and support the possibility of converting to plant-based expression systems to produce VLPs. This technique has implications of significance for generating affordable and expandable HAV vaccines, specifically for low-income areas where hepatitis A has a high incidence rate. Further studies will focus on enhancing expression protocols and evaluating and analysing the immunogenicity of plant-produced VLPs to progress toward clinical practice.69enHepatitis A Virus Virus-Like Particles (VLPs) Vaccine Development Plant-Based Expression Biomedical Science Protein Expression Infectious Diseases Molecular CloningThesis