Cloning, Expression, Purification and Antiviral Activity Assesment of Recombinant Porcine IFNα-IgG Fusion Protein Expressed by Mammalian Cells
| Autor: | Wei-Hsuan Tang, 唐為瑄 |
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| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 99 Mammalian cell expression system can yield properly-folded and post-translationally modified recombinant proteins which benefits greatly the biological activity of proteins. In this study, Chinese hamster ovary (CHO) cells were selected for the expression of recombinant interferon-α (IFNα) proteins with extended serum half-life. Due to the short serum half-life of IFNα, a glycine-serine linker was used to fuse genes of porcine IFNα and the Fc region of porcine IgG antibody for the construction of IFNα with extended serum half-life. The genes were cloned, fused, confirmed with sequence analysis, and subsequently transfected to CHO cells. By ELISA evaluation, PIFNα-Fc recombinant proteins were confirmed as secreted proteins. Secreted proteins were then purified using nickel and Protein G affinity columns. Porcine reproductive and respiratory syndrome virus (PRRSV) was used in assaying the antiviral activity of IFNα with extended half-life. In measuring interferon activity, results showed that 49 nM of IFNα with extended half-life sufficiently displayed antiviral effects against PRRSV. For recombinant protein expression and production of IFNα with extended half-life, 175 cm2 flasks and roller bottles were used. 175 cm2 flasks and roller bottles produced more than 450 μg/ml and about 800 μg/ml of IFNα with extended half-life recombinant proteins, respectively. In the antiviral assay, antiviral effects of IFNα with extended half-life produced in either 175 cm2 flasks or roller bottles were shown to increase over time with longer periods of cell culturing. Furthermore, the antiviral activities of the interferons produced were found to be 691 U ml-1 and 2430 U ml-1, respectively. The antiviral activities of recombinant IFNα proteins with extended half-life produced in roller bottles were significantly higher than those produced in 175 cm2 flasks. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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