臺灣博碩士論文加值系統

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，其具有可攜帶大片段外源基因的優點，但已有文獻報導重組桿狀病毒隨著不斷繼代過程中會產生大量的干擾缺損顆粒，造成後代病毒的基因不穩定性並導致重組蛋白的產量減少。目前尚未有研究針對桿狀病毒深入地探討外源片段大小對於重組桿狀病毒基因穩定性的影響，以及其所能夾帶的最大外源片段容量(capacity)。因此本研究的主要目的為探討外源基因片段大小與重組桿狀病毒基因穩定性之間的關聯，並進一步推展至實際應用時，載體中各種不同性質的外源片段對於病毒載體基因穩定性是否造成影響。我們以EGFP (enhanced green fluorescent protein)基因表現匣作為偵測外源片段穩定存在的指標，同時添加不同大小的外源填塞片段 (stuffer)，建構出多株重組桿狀病毒以進行病毒轉導效價及基因DNA定量等分析，深入探討外源片段大小以及病毒代數對於重組桿狀病毒基因穩定性的影響。另外，本實驗室已將長效表現系統Sleeping Beauty (SB)結合桿狀病毒載體應用於癌症的治療且效果顯著，也證實此混成表現系統的應用潛力所在，因此我們在本研究中以SB系統作為基礎平台，插入各種有利於桿狀病毒載體表現的單元因子，在提升SB系統基因表現的同時探討各種因子以及病毒代數對於SB混成重組桿狀病毒基因穩定性的影響。我們發現當外源片段長度增加，第二代病毒仍能維持穩定的外源基因表現，然而隨著病毒代數的提升，其外源片段遺失的現象越明顯，顯示不同大小的外源片段確實會對於重組桿狀病毒的基因穩定性產生影響。另外在混成SB長效表現系統中，將SB100剪切酶表現匣與其轉位子IR/DR置於同一株病毒的建構方式於第二代病毒皆能良好地攜帶外源片段，但隨著病毒連續繼代則明顯較容易產生基因不穩定的現象，我們推測除了外源片段長度的影響之外， CMV啟動子於昆蟲細胞中微量表現的SB100剪切酶也可能致使IR/DR間的egfp表現匣更容易從重組病毒中遭到剃除。這些結果提供實驗室在建構大片段外源基因及SB長效表現系統的重組桿狀病毒時一個基因穩定性的參考。

摘要 I
Abstract II
目錄 III
第一章 文獻回顧 1
1-1 昆蟲桿狀病毒簡介 1
1-2 桿狀病毒/昆蟲細胞蛋白表現系統 2
1-3 Sleeping Beauty (SB)長效表現系統 3
1-4 病毒基因載體之基因穩定性 5
1-5 重組桿狀病毒基因的不穩定性 7
1-5-1 非同源性DNA複製點(non-homologous region ori)對DIP的生成及病毒基因穩定性之影響 7
1-5-2 同源性DNA複製點(homologous regions ori, hr-ori)密度對於基因穩定性之影響 9
1-5-3 利用gp64作為病毒篩選標記(selective marker)對於基因穩定性之影響 10
1-5-4 外源基因片段大小對於基因穩定性之影響 11
1-6 研究動機 12
第二章 實驗材料與方法 16
2-1 聚合酵素連鎖反應(Polymerase Chain Reaction) 16
2-2 DNA純化和回收方法 17
2-2-1 質體純化(Purification of plasmid DNA) 17
2-2-2 瓊脂凝膠萃取DNA片段 (DNA extraction from agarose gel) 18
2-2-3 病毒DNA純化 (Purification of viral DNA) 18
2-2-4 DNA濃度測量 19
2-3 剪切反應、凝膠電泳法、連接反應 19
2-3-1 剪切反應(Digestion) 19
2-3-2 凝膠電泳法(Gel Electrophoresis) 19
2-3-3 接合反應(Ligation) 20
2-4 轉殖作用(Transformation)：化學法大腸桿菌(E. coli)細胞轉殖 20
2-5 細胞培養 21
2-5-1 昆蟲細胞培養 21
2-5-2 哺乳動物細胞培養 21
2-6 建構與放大重組桿狀病毒 21
2-6-1 重組桿狀病毒之Donor plasmid建構 21
2-6-2 重組桿狀病毒之製作與放大 25
2-7 桿狀病毒轉導哺乳動物細胞 25
2-8 轉導效價(transducing titers)的測定與計算 26
2-9 以終點稀釋法滴定病毒力價(infectious titer) 26
2-10 即時偵測同步定量聚合酶連鎖反應分析 27
第三章 結果與討論 37
3-1 探討外源片段長度對於基因穩定性之影響 38
3-2 探討額外表現同源性基因複製點hr1對基因穩定性的影響 40
3-3 探討Sleeping Beauty長效表現系統的建構平台之基因穩定性 43
3-3-1 探討轉位因子IR/DR之基因穩定性 43
3-3-2 置入具功能性外源基因於in cis系統中之基因穩定性 46
3-4 結論 49
第四章 未來展望 64
4-1 外源片段長度對基因穩定性的影響 64
4-2 不同建構系統中基因穩定性的差異 64
4-3 提升大片段外源基因表現匣在重組病毒基因體中的穩定性 66
第五章 參考文獻 67

Adler H, Messerle M, Koszinowski UH. 2001. Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences. J Virol 75(12):5692-6.
Andrew J. Bett LPaFLG. 1993. Packaging capacity and stability of human adenovirus type 5 vectors. J Virol 67(10):10.
Carbonell LF, Klowden MJ, Miller LK. 1985. Baculovirus-Mediated Expression of Bacterial Genes in Dipteran and Mammalian-Cells. J Virol 56(1):153-160.
Chamberlain JS. 2002. Gene therapy of muscular dystrophy. Hum Mol Genet 11(20).
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):846-54.
Dong B, Nakai H, Xiao W. 2010. Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther 18(1):87-92.
Emery DW, Yannaki E, Tubb J, Stamatoyannopoulos G. 2000. A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci U S A 97(16):9150-5.
Felsenfeld MGaG. 2006. Insulators: exploiting transcriptional and epigenetic mechanisms. Nat Rev Genet 7(9):703-13.
Fraefel APdOaC. 2010. Herpes simplex virus type 1 adeno-associated virus hybrid vectors. The Open Virology Journal 4:13.
Goncalves MA, Pau MG, de Vries AA, Valerio D. 2001. Generation of a high-capacity hybrid vector: packaging of recombinant adenoassociated virus replicative intermediates in adenovirus capsids overcomes the limited cloning capacity of adenoassociated virus vectors. Virology 288(2):236-46.
Goncalves MA, van Nierop GP, Tijssen MR, Lefesvre P, Knaan-Shanzer S, van der Velde I, van Bekkum DW, Valerio D, de Vries AA. 2005. Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability. J Virol 79(5):3146-62.
Habib S, Pandey S, Chatterji U, Burma S, Ahmad R, Jain A, Hasnain SE. 1996. Bifunctionality of the AcMNPV homologous region sequence (hr1): enhancer and ori functions have different sequence requirements. DNA Cell Biol 15(9):737-47.
Hitchman RB PR, King LA. 2009. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat Biotechnol 2(1):46-54.
Hu YC. 2005. Baculovirus as a highly efficient expression vector in insect and mammalian cells. Acta Pharmacol Sin 26(4):405-16.
Ikonomou L, Schneider YJ, Agathos SN. 2003. Insect cell culture for industrial production of recombinant proteins. Appl Microbiol Biotechnol 62(1):1-20.
Ivics Z, Hackett PB, Plasterk RH, Izsvak Z. 1997. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91(4):501-10.
Ivics Z, Li MA, Mates L, Boeke JD, Nagy A, Bradley A, Izsvak Z. 2009. Transposon-mediated genome manipulation in vertebrates. Nat Methods 6(6):415-22.
Izsvak Z, Hackett PB, Cooper LJ, Ivics Z. 2010. Translating Sleeping Beauty transposition into cellular therapies: victories and challenges. Bioessays 32(9):756-67.
Izsvak Z, Ivics Z. 2004. Sleeping beauty transposition: biology and applications for molecular therapy. Mol Ther 9(2):147-56.
Jani A LH, Acsadi G, Simoneau M, Huard J, Garnier A, Karpati G, Massie B. 1997. Generation, validation, and large scale production of adenoviral recombinants with large size inserts such as a 6.3 kb human dystrophin cDNA. J Virol Methods 64(2):111-124.
Kaikkonen MU, Raty JK, Airenne KJ, Wirth T, Heikura T, Yla-Herttuala S. 2006. Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo. Gene Ther 13(4):304-12.
Karim Benihoud PYaMP. 1999. Adenovirus vectors for gene delivery.
Kool M, Voncken JW, van Lier FL, Tramper J, Vlak JM. 1991. Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties. Virology 183(2):739-46.
Kost TA, Condreay JP, Jarvis DL. 2005. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol. 23(5):567-575.
Krell PJ. 1996. Passage effect of virus infection in insect cells. Cytotechnology 20:125-137.
Kumar-Singh R. 2008. Barriers for retinal gene therapy: separating fact from fiction. Vision Res 48(16):1671-80.
Liu G, Geurts AM, Yae K, Srinivasan AR, Fahrenkrug SC, Largaespada DA, Takeda J, Horie K, Olson WK, Hackett PB. 2005. Target-site preferences of Sleeping Beauty transposons. J Mol Biol 346(1):161-73.
Logan AC, Nightingale SJ, Haas DL, Cho GJ, Pepper KA, Kohn DB. 2004. Factors influencing the titer and infectivity of lentiviral vectors. Hum Gene Ther 15(10):976-88.
Lung O, Westenberg M, Vlak JM, Zuidema D, Blissard GW. 2002. Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64. J Virol 76(11):5729-5736.
Luo WY, Shih YS, Hung CL, Lo KW, Chiang CS, Lo WH, Huang SF, Wang SC, Yu CF, Chien CH and others. 2011. Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy. Gene Ther.
M. Kool RWGaJMV. 1994. A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus. J Gen Virol 75:3345-3352.
Monsma SA OA, Blissard GW. 1996. The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. J Virol. 70(7):4607-4616.
N Cheshenko1 NK, RC Eisensmith1,2 and VA Krougliak1. 2001. A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus(38kb). Gene Ther(8):846-854.
Pearson M, Bjornson R, Pearson G, Rohrmann G. 1992. The Autographa californica baculovirus genome: evidence for multiple replication origins. Science 257(5075):1382-4.
Pijlman GP. 2003. Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells. J Gen Virol. 84(10):2669-2678.
Pijlman GP, de Vrij J, van den End FJ, Vlak JM, Martens DE. 2004. Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect-cell bioreactors. Biotechnol Bioeng 87(6):743-53.
Pijlman GP, Dortmans JCFM, Vermeesch AMG, Yang K, Martens DE, Goldbach RW, Vlak JM. 2002. Pivotal Role of the Non-hr Origin of DNA Replication in the Genesis of Defective Interfering Baculoviruses. J Virol 76(11):5605-5611.
Pijlman GP, Roode EC, Fan X, Roberts LO, Belsham GJ, Vlak JM, van Oers MM. 2006. Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript. J Biotechnol 123(1):13-21.
Pijlman GP, van den Born E, Martens DE, Vlak JM. 2001. Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells. Virology 283(1):132-8.
Pijlman GP, van Schijndel JE, Vlak JM. 2003a. Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells. J Gen Virol 84(Pt 10):2669-78.
Pijlman GP, Vermeesch AM, Vlak JM. 2003b. Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells. J Invertebr Pathol 84(3):214-9.
Rauschhuber C, Noske N, Ehrhardt A. 2012. New insights into stability of recombinant adenovirus vector genomes in mammalian cells. Eur J Cell Biol 91(1):2-9.
Samulski PEMaRJ. 2000. Adeno-associated virus vectors more pros than cons. MOLECULAR MEDICINE TODAY 6:8.
Smith GA, Enquist LW. 1999. Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus. J Virol 73(8):6405-14.
Smith GA, Enquist LW. 2000. A self-recombining bacterial artificial chromosome and its application for analysis of herpesvirus pathogenesis. Proc Natl Acad Sci U S A 97(9):4873-8.
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.
Theilmann DA, Stewart S. 1992. Tandemly repeated sequence at the 3' end of the IE-2 gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus is an enhancer element. Virology 187(1):97-106.
Tiwari P, Saini S, Upmanyu S, Benjamin B, Tandon R, Saini KS, Sahdev S. 2010. Enhanced expression of recombinant proteins utilizing a modified baculovirus expression vector. Mol Biotechnol 46(1):80-9.
Transfiguracion J, Jaalouk DE, Ghani K, Galipeau J, Kamen A. 2003. Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications. Hum Gene Ther 14(12):1139-53.
Trono D. 2000a. Lentiviral vectors_turning a deadly foe into a therapeutic agent. Gene Therapy 7:3.
Trono D. 2000b. Lentiviral vectors_turning a deadly foe into a therapeutic agent. Gene Ther 7:3.
van Hulten MC, Witteveldt J, Peters S, Kloosterboer N, Tarchini R, Fiers M, Sandbrink H, Lankhorst RK, Vlak JM. 2001. The white spot syndrome virus DNA genome sequence. Virology 286(1):7-22.
van Lier FL, van der Meijs WC, Grobben NG, Olie RA, Vlak JM, Tramper J. 1992. Continuous beta-galactosidase production with a recombinant baculovirus insect-cell system in bioreactors. J Biotechnol 22(3):291-8.
van Lier FL, van Duijnhoven GC, de Vaan MM, Vlak JM, Tramper J. 1994. Continuous beta-galactosidase production in insect cells with a p10 gene based baculovirus vector in a two-stage bioreactor system. Biotechnol Prog 10(1):60-4.
van Oers MM. 2011. Opportunities and challenges for the baculovirus expression system. J Invertebr Pathol 107 Suppl:S3-15.
Vigdal TJ, Kaufman CD, Izsvák Z, Voytas DF, Ivics Z. 2002. Common Physical Properties of DNA Affecting Target Site Selection of Sleeping Beauty and other Tc1/mariner Transposable Elements. J Mol Biol 323(3):441-452.
Volkman LE, Goldsmith PA. 1983. In Vitro Survey of Autographa californica Nuclear Polyhedrosis Virus Interaction with Nontarget Vertebrate Host Cells. Appl Environ Microbiol 45(3):1085-93.
Wagner M, Jonjic S, Koszinowski UH, Messerle M. 1999. Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution. J Virol 73(8):7056-60.
Wickham TJ, Davis T, Granados RR, Hammer DA, Shuler ML, Wood HA. 1991. Baculovirus defective interfering particles are responsible for variations in recombination protein production as a functional of multiplicity of infection. Biotechnol Lett 13:483-488.
Yang F, He J, Lin X, Li Q, Pan D, Zhang X, Xu X. 2001. Complete genome sequence of the shrimp white spot bacilliform virus. J Virol 75(23):11811-20.