Harnessing the manufacturing power of the plant system: Production of recombinant human interleukin-2 in Nicotiana benthamiana.

摘要

Various expression systems are employed for the mass production of recombinant proteins to be used for pharmaceutical, diagnostic and industrial purposes. In this thesis, I have explored the feasibility of producing recombinant human interleukin-2 (IL-2) in the green tissues of transgenic Nicotiana benthamiana (tobacco). IL-2 is a biopharmaceutical of great importance as it is the standard immunotherapeutic treatment for late-stage metastatic melanoma and renal cell cancers. Using the whole-plant expression system, rather than relying on the current production platform of microbes would not only be cheaper, but the IL-2 might show greater biological activity due to processing in a eukaryotic host. The human IL-2 gene was codon optimized to maximize expression in this host system. Five DNA constructs were developed using this optimized gene and used to create stable transgenic plants. Each of the five constructs targeted IL-2 accumulation to different sub-cellular compartments. An additional construct fusing red fluorescent protein to the IL-2 protein was developed and used for a transient expression assay. The correct folding of this IL-2 fusion protein was confirmed via confocal microscopy. Western blotting of the stably transformed lines demonstrated maximum accumulation of the appropriately sized protein in the endoplasmic reticulum and chloroplasts. Protein extract samples were tested against murine splenic CD4+ T-cells from C57BL/6 mice and were shown to have specific biological activity. This research has demonstrated the efficacy of using tobacco as an expression system for the production of human IL-2.