| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 102 === Human Interferon gamma (IFNγ) is an important therapeutic cytokine, and plays a significant role in anti-viral, anti-tumor growth, and immunoreactions. In order to produce IFNγ protein, we generated viral-based expression system to express candidate protein in Nicotiana benthamiana plants or suspension cell line. By replacing coat protein gene of Bamboo mosaic virus (BaMV), Foxtail mosaic virus (FoMV), Tobacco mosaic?virus (TMV), and Potato virus X (PVX) with IFNγ sequence, four independent viral expression cassettes namely pKBdCIFNγ, pKFdCIFNγ, pKTdCIFNγ and pKPdCIFNγ were generated. These expression cassettes were transformed into Agrobacterium and infiltrated into N. benthamina leaves. The transient expression results showed that BaMV based vector produce the most high-level protein accumulation in infiltrated leaves. The expression of IFNγ does not cause deleterious effect on replication ef?ciency of BaMV genome. To improve the IFNγ production, the N terminal secretory sequence of IFNγ was truncated, named as Nd20IFNγ. The Nd20IFNγ not only expressed efficiently in E. coli, but also significantly increased IFNγ accumulation in N. benthamiana. In order to produce IFNγ large-scale with low cost, we developed secretory expression vectors carry signal peptides fused at N terminal of Nd20IFNγ, named as pKBdCSSNd20IFNγ. Furthermore, to increase Nd20IFNγ stability, 10 repeats of the AGP glycomodule Ser-Pro (SP) were fused at C terminal and generated as pKBdCSSNd20IFNγ(SP)10. Western blot analysis revealed that IFNγ formed incremental shifts in molecular weight, suggesting the results of secretion and glycosylation. In the future, the pKBdCSSIFN γ (SP)10 transformed N. benthamiana plants and stable transgenic cell lines to produce recombinant IFNγ protein secreted into cell-suspension medium will be established. This method will be facilitate the high-level production of IFNγ.
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