Development of Bicistronic Expression Vector using Internal Ribosome Entry Sites (IRESes) and 2A-like sequences from the Genome of Perina nuda virus (PnV) in Mammalian cells
| Autor: | Yi-Hsi Chen, 陳以希 |
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| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 2A is an oligopeptide sequence mediating a ribosome “skipping” effect, producing two non-overlapping proteins. This sequence can be facilitated to connect sequences encoding several proteins into a single open reading frame. 2A (identified in picornaviruses) and 2A-like sequences (found in other mammalian and insect viruses) have become widely used in biotechnology and biomedicine. In previous studies, perina nuda picorna-like virus (PnV) derived 2A-like sequences including 2AI and 2AII were successfully used in the baculovirus expression vector system. However, whether these sequences can be applied in mammalian system is unclear. In this study, we examined the translation activity of 2A-like sequences derived from PnV in different mammalian cells and compared their activity with other approaches, like internal ribosome entry site (IRES) and DNA fusion that used in bi-cistronic vectors to express two genes simultaneously. We found that a donut-shaped fluorescence distribution pattern in mammalian cells transient transfected with plasmids carrying either the red fluorescent protein (DsRed) or secretory alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP) reporter genes joined by an in-frame fusion linker. In contrast, the fluorescence distribution pattern was homogenous in cells transfected with expression vectors in which the downstream EGFP gene is produced independently by way of PnV 2A-like sequences as well as IRESes. Based on these observations, we suggest the self-cleavage 2A-like sequences from PnV, an insect virus, can also function in mammalian cells. Furthermore, we also employed the SEFP reporter gene (a EGFP and SEAP fusion gene) to quantify the activity the PnV 2AI and PnV 2AII in different mammalian cells. Our results indicated PnV 2AI was not only more effective than PnV 2AII but also more stable than IRESes. In the future, we suppose that virus-derived 2A-like sequence in a bi-cistronic expression vector might be rapidly screened and quantified. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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