Applications of stable RNA interference in baculovirus expression vector system

• Main Authors: Chih-Chien Lin, 林智健
• Other Authors: Yiu-Kay Lai
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/94321070794589946291

博士 === 國立清華大學 === 生物科技研究所 === 95 === Baculovirus expression vector system (BEVS) is a powerful tool for the expression of heterologous proteins in insect cells. Cell lines derived from Spodoptera frugiperda (fall armyworm), such as Sf9 and Sf21, are the most common insect cell lines used for protein expression. Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. Stable and prolonged silencing has been achieved by DNA vector-based approaches with endogenously expressed dsRNA (including long dsRNA and siRNA) in many species, but never in S. frugiperda cells. To establish a DNA vector-based method for stable RNAi in S. frugiperda cells, in the first part of this study, we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically suppressed the baculovirus-mediated luciferase expression stably. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study. Sf-caspase-1 is the most studied effector caspase of Lepidoptera and its activation may lead cells to apoptosis (programmed cell death) when under ultraviolet (UV) irradiation or baculovirus infection. In the second part of this study, we repressed the expression of Sf-caspase-1 in Sf9 by using constitutive RNAi, and evaluated the effects of stress responses and the production of proteins in recombinant baculovirus infected cells. The Sf-caspase-1-repressed stable cells, Sf9/pIBdsCasp-1 and Sf9/pIBdsCasp-2, showed a significant increase in resistance to UV- and baculovirus-induced apoptosis. These cells produced higher levels of both intracellular (luciferase) and extracellular (secreted alkaline phosphatase; SEAP) recombinant proteins than the parental cells when infected with recombinant baculovirus. Thus, Sf-caspase-1-repressed stable cells have a greater ability to adapt to various culture conditions, and also to provide the benefits of high-level protein production in BEVS.