目前，昆蟲桿狀病毒（Baculovirus）已被證實可感染哺乳動物細胞，是一種優秀的基因載體，可應用於體內及體外的基因治療。至今，根據桿狀病毒對昆蟲細胞之感染能力所測定出的病毒力價（infectious titer）和multiplicity of infection（MOI），一直都被當作定量病毒劑量的依據。然而，病毒力價和MOI卻不能準確地反映出病毒的轉導能力，造成不同研究之間，基因轉導實驗數據在比較上的困難。為了能快速確實地測定病毒的轉導能力，我們發展一套新的方法「轉導效價分析法（transducing titration）」去定量桿狀病毒的轉導效價（transducing titer）。相較於病毒力價，轉導效價的測定不僅誤差小，所花時間也較短（只需2天）。更重要的是，我們證明有較高轉導效價的昆蟲桿狀病毒，轉導細胞時會有較高的轉導效率以及更長的轉導基因表現時間，這點更加確定了轉導效價能代表病毒的轉導能力。據以上觀點，轉導效價分析法提供了一個簡單、快速、能準確測定桿狀病毒轉導能力的方法，為往後基因轉導相關實驗，甚至病毒的生產及純化等研究，提供一個可靠的定量標準。
在建立了轉導效價分析法之後，為了能依照基因治療相關研究之需求去最佳化病毒的生產及保存程序，我們根據轉導效價的角度對某些影響因素重新進行探討。然而我們發現實驗結果顯示，根據病毒力價和轉導效價進行最佳化所得的條件大致上都相同，這意味著研究者不用再改變以往所用的方法去生產及保存病毒。而在基因轉導時，病毒力價不能準確表示病毒劑量的可能原因，以及與轉導效價之間的關聯，將會在後序進行討論。

Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer (IT) and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol was called “transducing titration” to evaluate the transducing titers (TT) of baculovirus stocks. Comparing to the IT, the determination of TT is more reproducible and faster. More importantly, we demonstrated that baculoviruses with higher TT could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the TT was representative of the baculovirus transducing ability. In this regard, transducing titration provides a simple, fast and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments.
After the development of transducing titration, in order to optimize baculovirus production and storage protocols for gene therapy, several parameters were re-investigated by using TT. We found that these data reveal a general agreement between the optimal conditions based on the IT or TT measurements, and are significant because researchers do not need to alter the production and storage conditions they have been adopting. The reason why IT is at times unreliable to indicate the virus dosage in gene transfer as well as the implications of TT in baculovirus production, storage, and potentially purification processes are discussed.

Abe T, Hemmi H, Miyamoto H, Moriishi K, Tamura S, Takaku H,
Akira S, Matsuura Y. 2005. Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus. J Virol 79: 2847-2858.

Adams JR, Goodwin RH, Wilcox TA. 1977. Electron microscopic investigations on invasion and replication of insect baculoviruses in vivo and in vitro. Biol Cell 28: 261-268.

Airenne KJ, Hiltunen MO, Turunen MP, Turunen AM, Laitinen OH, Kulomaa MS, Yla-Herttuala S. 2000. Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery. Gene Ther 7: 1499-1504.

Blissard GW, Wenz JR. 1992. Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J Virol 66: 6829-6835.

Boublik Y, Di Bonito P, Jones IM. 1995. Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV) for the presentation of foreign proteins on the virus surface. Biotechnology (N Y) 13: 1079-1084.

Boyce FM, Bucher NL. 1996. Baculovirus-mediated gene transfer into mammalian cells. Proc Natl Acad Sci U S A 93: 2348-2352.

Brussaard CP, Marie D, Bratbak G. 2000. Flow cytometric detection of viruses. J Virol Methods 85: 175-182.
Carbonell LF, Klowden MJ, Miller LK. 1985. Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells. J Virol 56: 153-160.

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: 846-854.

Condreay JP, Witherspoon SM, Clay WC, Kost TA. 1999. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proc Natl Acad Sci U S A 96: 127-132.

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.

Duisit G, Saleun S, Douthe S, Barsoum J, Chadeuf G, Moullier P. 1999. Baculovirus vector requires electrostatic interactions including heparan sulfate for efficient gene transfer in mammalian cells. J Gene Med 1: 93-102.

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: 297-307.

Ghosh S, Parvez MK, Banerjee K, Sarin SK, Hasnain SE. 2002. Baculovirus as mammalian cell expression vector for gene therapy: an emerging strategy. Mol Ther 6: 5-11.

Grabherr R, Ernst W, Doblhoff-Dier O, Sara M, Katinger H. 1997. Expression of foreign proteins on the surface of Autographa californica nuclear polyhedrosis virus. Biotechniques 22: 730-735.

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: 291-294.

Hildinger M, Auricchio A, Gao G, Wang L, Chirmule N, Wilson JM. 2001. Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer. J Virol 75: 6199-6203.

Ho YC, Chen HC, Wang KC, Hu YC. 2004. Highly efficient baculovirus-mediated gene transfer into rat chondrocytes. Biotechnol Bioeng 88: 643-651.

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: 860-868.

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: 10099-10103.

Hsu CS, Ho YC, Wang KC, Hu YC. 2004. Investigation of optimal transduction conditions for baculovirus-mediated gene delivery into mammalian cells. Biotechnol Bioeng 88: 42-51.

Hu YC, Bentley WE. 1999. Enhancing yield of infectious Bursal disease virus structural proteins in baculovirus expression systems: focus on media, protease inhibitors, and dissolved oxygen. Biotechnol Prog 15: 1065-1071.

Hu YC, Bentley WE. 2000. A Kinetic and statistical-thermodynamic model for baculovirus infection and virus-like particle assembly in suspended insect cells. Chem Eng Sci 55: 3991-4008.

Hu YC, Bentley WE. 2001. Effect of MOI ratio on the composition and yield of chimeric infectious bursal disease virus-like particles by baculovirus co-infection: deterministic predictions and experimental results. Biotechnol Bioeng 75: 104-119.

Hu YC, Tsai CT, Chang YJ, Huang JH. 2003. Enhancement and prolongation of baculovirus-mediated expression in mammalian cells: focuses on strategic infection and feeding. Biotechnol Prog 19: 373-379.

Jarvis DL, Garcia A, Jr. 1994a. Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein. Virology 205: 300-313.

Jarvis DL, Garcia A, Jr. 1994b. Long-term stability of baculoviruses stored under various conditions. Biotechniques 16: 508-513.

Jorio H, Tran R, Kamen A. 2006a. Stability of serum-free and purified baculovirus stocks under various storage conditions. Biotechnol Prog 22: 319-325.

Jorio H, Tran R, Meghrous J, Bourget L, Kamen A. 2006b. Analysis of baculovirus aggregates using flow cytometry. J Virol Methods 134: 8-14.

King LA, Possee RD. 1992. In The Baculovirus Expression System. Chaman & Hall.

Kost TA, Condreay JP. 2002. Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends Biotechnol 20: 173-180.

Lanier LM, Volkman LE. 1998. Actin binding and nucleation by Autographa california M nucleopolyhedrovirus. Virology 243: 167-177.

Lee SG, Kim S, Robbins PD, Kim BG. 1996. Optimization of environmental factors for the production and handling of recombinant retrovirus. Appl Microbiol Biotechnol 45: 477-483.

Licari P, Bailey JE. 1992. Modeling the population dynamics of baculovirus-infected insect cells: Optimizing infection strategies for enhanced recombinant protein yields. Biotechnol Bioeng 39: 432-441.

Lindley KM, Su JL, Hodges PK, Wisely GB, Bledsoe RK, Condreay JP, Winegar DA, Hutchins JT, Kost TA. 2000. Production of monoclonal antibodies using recombinant baculovirus displaying gp64-fusion proteins. J Immunol Methods 234: 123-135.

Lizee G, Aerts JL, Gonzales MI, Chinnasamy N, Morgan RA, Topalian SL. 2003. Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression. Hum Gene Ther 14: 497-507.

Lo HR, Chao YC. 2004. Rapid titer determination of baculovirus by quantitative real-time polymerase chain reaction. Biotechnol Prog 20: 354-360.

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: 976-988.

Luckow VA, Summers MD. 1988. Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. Virology 167: 56-71.

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: 1986-1991.

Marie D, Brussaard CPD, Thyrhaug R, Bratbak G, Vaulot D. 1999. Enumeration of marine viruses in culture and natural samples by flow cytometry. Appl Environ Microbiol 65: 45-52.

Matilainen H, Rinne J, Gilbert L, Marjomaki V, Reunanen H, Oker-Blom C. 2005. Baculovirus entry into human hepatoma cells. J Virol 79: 15452-15459.

Merrihew RV, Clay WC, Condreay JP, Witherspoon SM, Dallas WS, Kost TA. 2001. Chromosomal integration of transduced recombinant baculovirus DNA in mammalian cells. J Virol 75: 903-909.

Monsma SA, Oomens AG, Blissard GW. 1996. The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. J Virol 70: 4607-4616.

Moreira JL, Alves PM. 1994. Hydrodynamic effects on BHK cells grown as suspended natural aggregate. Biotechnol Bioeng 46: 351-360.

Mottershead D, van der Linden I, von Bonsdorff CH, Keinanen K, Oker-Blom C. 1997. Baculoviral display of the green fluorescent protein and rubella virus envelope proteins. Biochem Biophys Res Commun 238: 717-722.

Mottershead DG, Alfthan K, Ojala K, Takkinen K, Oker-Blom C. 2000. Baculoviral display of functional scFv and synthetic IgG-binding domains. Biochem Biophys Res Commun 275: 84-90.

Nicholson LJ, Philippe M, Paine AJ, Mann DA, Dolphin CT. 2005. RNA interference mediated in human primary cells via recombinant baculoviral vectors. Mol Ther 11: 638-644.

Ojala K, Mottershead DG, Suokko A, Oker-Blom C. 2001. Specific binding of baculoviruses displaying gp64 fusion proteins to mammalian cells. Biochem Biophys Res Commun 284: 777-784.

Olson MR, Axler RP, Hicks RE. 2004. Effects of freezing and storage temperature on MS2 viability. J Virol Methods 122: 147-152.

Oomens AG, Monsma SA, Blissard GW. 1995. The baculovirus GP64 envelope fusion protein: synthesis, oligomerization, and processing. Virology 209: 592-603.

O'Reilly D, Miller L, Luckow V. 1992. Baculovirus expression vector: a laboratory manual. New York: W.H. Freeman and Co.

Ozuer A, Wechuck JB, Russell B, Wolfe D, Goins WF, Glorioso JC, Ataai MM. 2002. Evaluation of infection parameters in the production of replication-defective HSV-1 viral vectors. Biotechnol Prog 18: 476-482.

Pieroni L, Maione D, La Monica N. 2001. In vivo gene transfer in mouse skeletal muscle mediated by baculovirus vectors. Hum Gene Ther 12: 871-881.

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: 743-753.

Pijlman GP, van Schijndel JE, Vlak JM. 2003. Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells. J Gen Virol 84: 2669-2678.

Possee RD. 1997. Baculoviruses as expression vectors. Curr Opin Biotechnol 8: 569-572.

Power JF, Reid S, Radford KM, Greenfield PF, Nielsen LK. 1994. Modeling and optimization of the baculovirus expression vector system in batch suspension-culture. Biotechnol Bioeng 44: 710-719.

Salminen M, Airenne KJ, Rinnankoski R, Reimari J, Valilehto O, Rinne J, Suikkanen S, Kukkonen S, Yla-Herttuala S, Kulomaa MS, Vihinen-Ranta M. 2005. Improvement in nuclear entry and transgene expression of baculoviruses by disintegration of microtubules in human hepatocytes. J Virol 79: 2720-2728.

Sandig V, Hofmann C, Steinert S, Jennings G, Schlag P, Strauss M. 1996. Gene transfer into hepatocytes and human liver tissue by baculovirus vectors. Hum Gene Ther 7: 1937-1945.

Sarkis C, Serguera C, Petres S, Buchet D, Ridet JL, Edelman L, Mallet J. 2000. Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector. Proc Natl Acad Sci U S A 97: 14638-14643.

Shen CF, Meghrous J, Kamen A. 2002. Quantitation of baculovirus particles by flow cytometry. J Virol Methods 105: 321-330.

Shoji I, Aizaki H, Tani H, Ishii K, Chiba T, Saito I, Miyamura T, Matsuura Y. 1997. Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors. J Gen Virol 78 ( Pt 10): 2657-2664.

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: 2156-2165.

Sollerbrant K, Elmen J, Wahlestedt C, Acker J, Leblois-Prehaud H, Latta-Mahieu M, Yeh P, Perricaudet M. 2001. A novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors. J Gen Virol 82: 2051-2060.

Tami C, Farber M, Palma EL, Taboga O. 2000. Presentation of antigenic sites from foot-and-mouth disease virus on the surface of baculovirus and in the membrane of infected cells. Arch Virol 145: 1815-1828.

Tani H, Limn CK, Yap CC, Onishi M, Nozaki M, Nishimune Y, Okahashi N, Kitagawa Y, Watanabe R, Mochizuki R, Moriishi K, Matsuura Y. 2003. In vitro and in vivo gene delivery by recombinant baculoviruses. J Virol 77: 9799-9808.

Taticek RA, Shuler ML. 1997. Effect of elevated oxygen and glutamine levels on foreign protein production at high cell densities using the insect cell-baculovirus expression system. Biotechnol Bioeng 54: 142-152.

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: 1139-1153.

van Loo ND, Fortunati E, Ehlert E, Rabelink M, Grosveld F, Scholte BJ. 2001. Baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids. J Virol 75: 961-970.

Volkman LE, Goldsmith PA. 1983. In vitro study of Autographa californica nuclear polyhedrosis virus interaction with nontarget vertebrate host cells. Appl Environ Microbiol 45: 1085-1093.

Volkman LE, Goldsmith PA, Hess RT, Faulkner P. 1984. Neutralization of budded Autographa californica NPV by a monoclonal antibody: identification of the target antigen. Virology 133: 354-362.

Wang KC, Wu JC, Chung YC, Ho YC, Chang MD, Hu YC. 2005. Baculovirus as a highly efficient gene delivery vector for the expression of hepatitis delta virus antigens in mammalian cells. Biotechnol Bioeng 89: 464-473.

Wang MY, Vakharia V, Bentley WE. 1993. Expression of epoxide hydrolase in insect cells: A focus on the infected cell. Biotechnol Bioeng 42: 240-246.

Wickham TJ, Davis T, Granados RR, Hammer DA, Shuler ML, Wood HA. 1991. Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection. Biotechnol Lett 13: 483-488.

Ye K, Dhiman HK, Suhan J, Schultz JS. 2003. Effect of pH on infectivity and morphology of ecotropic moloney murine leukemia virus. Biotechnol Prog 19: 538-543.

Zeiser A, Elias CB, Voyer R, Jardin B, Kamen AA. 2000. On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process. Biotechnol Prog 16: 803-808.