重組桿狀病毒表現系統目前已被廣泛應用在蛋白生產、疫苗開發及基因治療上。在實際應用時，桿狀病毒的劑量將依據其病毒效價而定，然而目前常見的病毒效價定量方法為TCID50，此法雖然成本低廉，但仍具有耗時、不易判讀等缺點；因此，如何開發一套快速且準確之病毒效價定量方法是十分重要的議題。經由G418長期篩選下，本研究成功建立病毒效價測定專用之Sf-9ET細胞株，攜帶有polyhedrin啟動子-綠色螢光蛋白 (polyhedron promoter-EGFP) 表現匣，故只有在桿狀病毒進入細胞後，才能驅動病毒的polyhedrin啟動子 (晚期啟動子) 表現綠色螢光蛋白。利用Sf-9ET細胞株進行病毒效價測定時，我們發現在感染後7天 (7 dpi, days post-infection) 所觀察到的螢光表現和11 dpi 觀察細胞的毒理反應，兩者計算所得到的病毒效價值相近似。接著，我們利用能夠表現紅色螢光蛋白的桿狀病毒 (Bac-Polh-DsRed) 感染Sf-9ET細胞，結果顯示紅、綠螢光的表現完全同步，而各自所計算的效價值也相同，證明此Sf-9ET細胞株的準確度。我們也同時針對多種不同的桿狀病毒株進行病毒效價的分析，其定量結果與傳統方法測得的結果皆相近，證實此細胞之泛用性。最後，我們證實Sf-9ET細胞在繼代培養至P50時仍保有病毒效價測定的靈敏性。綜觀以上，本研究所建立的Sf-9ET細胞株可大幅縮短病毒效價的測定時間並且有效降低判讀時的困難度，同時兼具準確度、泛用性以及穩定性等優點，為桿狀病毒載體的應用領域提供了一項便捷的量測工具。

Recombinant baculovirus expression system has been widely used in protein production, gene therapy and vaccine development. In applications, the dose of baculovirus is determined by virus titers. However, the common method for baculovirus titration TCID50 which cost is lower than other method, but this is a time-consuming and difficult to be determined. Thus, we would like to develop a fast and accurate method for baculovirus titration. We successfully establish a Sf-9ET cell lines for baculovirus titration via G418 screening. Sf-9ET cell lines contain the polyhedrin promoter-green fluorescent protein expression cassette. While Sf-9ET cell lines infected by baculovirus, baculovirus will drive the polyhedrin promoter and express green fluorescent protein. We using Sf-9ET for baculovirus titration , we found that the fluorescence observed at 7 dpi (days post-infection) and cell cytopathic effect observed at 11 dpi, both obtained similar baculovirus titer respectively. Then, we use the baculovirus Bac-Polh-DsRed which express red fluorescent protein in infected Sf-9ET cells, the results show the red and green fluorescence observed at same time, we obtained same titer of the baculovirus. Thus, Sf-9ET cell line is accurate for baculovirus titration. Several type of baculovirus titer obtained by Sf-9ET which are similar to original baculovirus titration method. Sf-9ET cell line is universal for all baculovirus for titration. Finally, we demonstrate that Sf-9ET cells were subcultured to P50 still retains the sensitivity of titration. In summary, this study established Sf-9ET cell line can significantly shorten the time for titration and easy to determine. Sf-9ET cell line is accurate and stable.

第一章 文獻回顧 1
1-1 昆蟲桿狀病毒 1
1-1-1 昆蟲桿狀病毒簡介 1
1-1-2 桿狀病毒/昆蟲細胞蛋白表現系統 2
1-1-3 昆蟲桿狀病毒/哺乳動物細胞表現系統 2
1-2 昆蟲桿狀病毒效價定量 3
1-3 研究動機 5
第二章 實驗材料與方法 9
2-1 質體的製備純化與定量 9
2-1-1 轉殖作用：化學法大腸桿菌(E. coli)細胞轉殖 9
2-1-2 質體純化 (Purification of plasmid DNA) 9
2-1-3 DNA定量 10
2-2 DNA片段放大與純化 11
2-2-1 聚合酵素連鎖反應 (Polymerase Chain Reaction) 11
2-2-2 瓊脂凝膠萃取DNA片段 (DNA extraction from agarose gel) 12
2-3 剪切反應、凝膠電泳法、連接反應 12
2-3-1 剪切反應 (Digestion) 12
2-3-2 凝膠電泳法 (Gel Electrophoresis) 13
2-3-3 Klenow Fragment 13
2-3-4 接合反應 (Ligation) 13
2-4 細胞培養 14
2-4-1 昆蟲細胞培養與繼代 14
2-5 Sf-9 ET (Easy Titer) 細胞株之製備 14
2-5-1 pBac-Sf9ET 質體之建構 14
2-5-2 Sf-9 細胞之質體轉染 16
2-5-3 Sf-9ET 細胞之篩選 16
2-6 重組桿狀病毒的製備 17
2-6-1 pBacPolh-DsRed建構 17
2-6-2 質體之轉置反應(transposition)(Bac-to-Bac system) 17
2-6-3 重組Bacmid之純化 17
2-6-4 重組Bacmid之轉染反應 (transfection) (製備P0病毒) 18
2-6-5 製作P1重組桿狀病毒 18
2-6-6 製作P2重組桿狀病毒 18
2-6-7 桿狀病毒效價定量 19
第三章 結果與討論 21
3-1 Sf-9ET 細胞之建構 21
3-2 Sf-9ET 細胞之改良 22
3-3 Sf-9ET 觀察螢光時間與病毒效價之關係 23
3-4 Sf-9ET靈敏性之探討 24
3-5 Sf-9ET泛用性之探討 25
3-6 Sf-9ET穩定性之探討 26
第四章 未來展望 38
第五章 參考文獻 39

Airenne KJ， Hu YC， Kost TA， Smith RH， Kotin RM， Ono C， Matsuura Y， Wang S， Yla-Herttuala S. 2013. Baculovirus: an insect-derived vector for diverse gene transfer applications. Mol Ther 21(4):739-49.
Bilello JP， Cable EE， Myers RL， Isom HC. 2003. Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes. Gene Ther 10(9):733-49.
Boyce FM， Bucher NLR. 1996. Baculovirus-mediated gene transfer into mammalian cells. Proc Natl Acad Sci U S A 93(6):2348-2352.
Chang CQ， Ding Z， Hung YS， Fung PCW. 2008. Fast network component analysis (FastNCA) for gene regulatory network reconstruction from microarray data. Bioinformatics 24(11):1349-1358.
Chen CY， Wu HH， Chen CP， Chern SR， Hwang SM， Huang SF， Lo WH， Chen GY， Hu YC. 2011. Biosafety assessment of human mesenchymal stem cells engineered by hybrid baculovirus vectors. Mol Pharm 8(5):1505-14.
Cheshenko N， Krougliak N， Eisensmith RC， Krougliak VA. 2001. A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Ther 8(11):846-854.
Dee KU， Shuler ML. 1997. A mathematical model of the trafficking of acid-dependent enveloped viruses: application to the binding， uptake， and nuclear accumulation of baculovirus. Biotechnol Bioeng 54:468-490.
Dwarakanath RS， Clark CL， McElroy AK， Spector DH. 2001. The use of recombinant baculoviruses for sustained expression of human cytomegalovirus immediate early proteins in fibroblasts. Virology 284(2):297-307.
Hara K， Funakoshi M， Tsuda K， Kawarabata T. 1994. Plaque assay for Spodoptera exigua and Autographa californica nuclear polyhedrosis viruses in a newly established cell line of the beet armyworm， Spodoptera exigua. Acta Virol 38(5):291-4.
Hitchman RB PR， King LA. 2009. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat Biotechnol 2(1):46-54.
Ho YC， Chung YC， Hwang SM， Wang KC， Hu YC. 2005. Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells. J Gene Med 7(7):860-8.
Ho YC， Lee HP， Hwang SM， Lo WH， Chen HC， Chung CK， Hu YC. 2006. Baculovirus transduction of human mesenchymal stem cell-derived progenitor cells: variation of transgene expression with cellular differentiation states. Gene Ther 13(20):1471-1479.
Hofmann C， Sandig V， Jennings G， Rudolph M， Schlag P， Strauss M. 1995. Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci U S A 92(22):10099-103.
Hopkins R， Esposito D. 2009. A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line. Biotechniques 47(3):785-8.
Hu YC. 2008. Baculoviral vectors for gene delivery: a review. Curr Gene Ther 8(1):54-65.
Kost TA， Condreay JP. 2002. Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends Biotechnol 20(4):173-80.
Kost TA， Condreay JP， Jarvis DL. 2005a. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol. 23(5):567-575.
Kost TA， Condreay JP， Jarvis DL. 2005b. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology 23(5):567-575.
Leisy DJ， Lewis TD， Leong JA， Rohrmann GF. 2003. Transduction of cultured fish cells with recombinant baculoviruses. J Gen Virol 84(Pt 5):1173-8.
Lo HR， Chao YC. 2004. Rapid titer determination of baculovirus by quantitative real-time polymerase chain reaction. Biotechnol Prog 20(1):354-60.
Ma L， Tamarina N， Wang Y， Kuznetsov A， Patel N， Kending C， Hering BJ， Philipson LH. 2000. Baculovirus-mediated gene transfer into pancreatic islet cells. Diabetes 49(12):1986-91.
O'Reilly D， Miller L， Luckow V. 1992. Baculovirus expression vector: a laboratory manual. Baculovirus expression vector. New York: W.H. Freeman and Co.
Roldao A， Oliveira R， Carrondo MJ， Alves PM. 2009. Error assessment in recombinant baculovirus titration: evaluation of different methods. J Virol Methods 159(1):69-80.
Shen CF， Meghrous J， Kamen A. 2002. Quantitation of baculovirus particles by flow cytometry. J Virol Methods 105(2):321-30.
Smith GE， Summers MD， Fraser MJ. 1983. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol 3(12):2156-65.