穩定核醣核酸干擾在桿狀病毒表現載體系統之應用

Applications of stable RNA interference in baculovirus expression vector system

林智健(Chih-Chien Lin)

指導教授 ： 黎耀基 ； 徐祖安

國立清華大學/生命科學院/生物科技研究所/博士(2007年)

https://doi.org/10.6843/NTHU.2007.00063

摘要

桿狀病毒表現載體系統（baculovirus expression vector system; BEVS）為使用於大量表現外源蛋白質的表現系統之一。其中最常使用的昆蟲細胞株，如Sf9以及Sf21均由秋行軍蟲（Spodoptera frugiperda; fall armyworm）分離而得。雙股核醣核酸（double-strand RNA; dsRNA）引起的核醣核酸干擾（RNA interference; RNAi）則是在各種真核生物體內皆可以抑制基因的表現。由於以去氧核醣核酸載體（DNA vector）為基礎的穩定核醣核酸干擾未曾使用於秋行軍蟲細胞，所以本研究的第一個部分即是在Sf9細胞中確立以去氧核醣核酸載體為基礎之穩定核醣核酸干擾的新穎方法，而結果顯示此專一性的基因抑制方法，確實可以成功的應用在Sf9細胞中，進而幫助BEVS系統的發展。
在Sf9細胞內，由於桿狀病毒感染（baculovirus infection）或是紫外線照射（UV irradiation）時都會藉由引發Sf-caspase-1活化，進而使細胞進入細胞凋亡的程序。因此本研究的第二部分利用了去氧核醣核酸載體為基礎之穩定核醣核酸干擾方法，在Sf9細胞內持續地抑制Sf-caspase-1蛋白質的表現，希望藉此達到延緩桿狀病毒引起Sf9細胞的細胞凋亡。實驗結果顯示，穩定核醣核酸干擾方法成功地抑制了Sf9細胞中Sf-caspase-1的表現。而穩定抑制Sf-caspase-1的細胞株不但能延緩桿狀病毒與紫外線所引發的細胞凋亡之外，更可以幫助BEVS系統中細胞內及細胞外分泌性的重組蛋白質表現。因此，本策略可以成功地應用於幫助BEVS之中重組蛋白質的生產。

關鍵字

桿狀病毒 ； 雙股核醣核酸 ； 核醣核酸干擾 ； 秋行軍蟲 ； 穩定核醣核酸干擾 ； 細胞凋亡 ； 硫胱胺酸蛋白酶

並列摘要

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.

並列關鍵字

baculovirus ； double-strand RNA ； RNA interference ； fall armyworm (Spodoptera frugiperda) ； stable RNAi ； apoptosis ； caspase

參考文獻

Agrawal N, Malhotra P, Bhatnagar RK. 2004. siRNA-directed silencing of transgene expressed in cultured insect cells. Biochem. Biophys. Res. Commun. 320(2):428-34.

Ahmad M, Srinivasula SM, Wang L, Litwack G, Fernandes-Alnemri T, Alnemri ES. 1997. Spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin FKBP46, is the target of the baculovirus antiapoptotic protein p35. J. Biol. Chem. 272(3):1421-4.

Ames R, Fornwald J, Nuthulaganti P, Trill J, Foley J, Buckley P, Kost T, Wu Z, Romanos M. 2004. BacMam recombinant baculoviruses in G protein-coupled receptor drug discovery. Receptors Channels 10(3-4):99-107.

Berger J, Hauber J, Hauber R, Geiger R, Cullen BR. 1988. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66(1):1-10.

Betenbaugh MJ, Tomiya N, Narang S, Hsu JT, Lee YC. 2004. Biosynthesis of human-type N-glycans in heterologous systems. Curr. Opin. Struct. Biol. 14(5):601-6.