Abstract:
Modified Viral Protein 7 (VP7) of African horsesickness virus (AHSV) is being investigated as a peptide display protein. The protein represents a good candidate for recombinant peptide display due to its tertiary structure, which contains flexible hydrophilic loops on the top domain of the protein where small peptides can potentially be inserted. In addition, wild type (WT) AHSV VP7 tends to form hexagonal crystals of predictable shape and size when expressed in a recombinant expression system. Previous research has resulted in a number of AHSV VP7 genes containing modified cloning sites where DNA representing immunologically relevant peptides can be inserted. When these chimeric proteins are expressed the peptides should be displayed on the surface of the VP7 platform. Several studies have tested the ability to insert peptides of varying lengths into these sites and successfully express the chimeric protein. In these past cases, successful expression of a recombinant chimeric protein was gauged by the observation of particles formed by multimers of VP7 proteins that resemble the one formed by WT-VP7. However, little is known about the ability of these chimeric proteins to act as successful peptide presentations vectors. Specifically, it is not known whether the fusion peptides would retain their correct tertiary structure, or indeed be displayed to the surrounding environment in order to generate a specific immune response. Furthermore, there has been no investigation to track these chimeric proteins’ expression in a heterologous expression system. This dissertation attempts to answer several of these questions through the use of a fluorescent protein, enhanced green fluorescent protein (eGFP), as a model peptide. The use of eGFP as a model peptide can prove correct tertiary structure of the fusion peptide via function of the protein (fluorescence), as well as act as a means of monitoring expression of chimeric VP7-eGFP proteins. Chapter 1 of this dissertation reviews literature that is relevant to AHSV VP7 and the use of fluorescent proteins as fluorescent markers. In addition, the recombinant expression of proteins is discussed, with a focus on solubility and expression levels of expressed proteins. Next, a brief overview is given with regards to vaccination strategies that can be undertaken, with a focus on subunit vaccines and their viability as successful alternatives to live-attenuated vaccines. Finally, the progress with regards to using modified AHSV VP7 as a peptide presentation vector is discussed. Chapter 2 investigates the chimeric protein VP7-177-eGFP, including its construction via a recombinant DNA cloning strategy, its expression in Insect cells using a recombinant Baculovirus expression system, and the ability of eGFP to act as a model fusion peptide on the top domain of a modified VP7 protein. Several experiments investigate whether the chimeric protein maintains its tertiary structure under a series of purification steps, and investigate the solubility of the chimeric protein throughout the expression cycle. Finally, purified forms of the chimeric protein are examined for their ability to generate an immune response specific to the fusion protein, eGFP.<p
