請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32452
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王重雄(Chung-Hsiung Wang) | |
dc.contributor.author | Kuang-Hung Lin | en |
dc.contributor.author | 林光宏 | zh_TW |
dc.date.accessioned | 2021-06-13T03:50:12Z | - |
dc.date.available | 2016-07-24 | |
dc.date.copyright | 2006-07-28 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-25 | |
dc.identifier.citation | 趙榮台。2002。邁向黑角舞蛾的綜合防治。農政與農情。122期,81-84頁。
潘秀雯。2005。黑角舞蛾核多角體病毒融合蛋白基因之選殖及其在吉普賽舞蛾細胞之表現。碩士論文。國立臺灣大學昆蟲學研究所。臺北。中華民國。 蕭剛柔。1992。中國森林昆蟲。中國林業出版社。 1090-1092頁。 Blissard, G. W., and G. F. Rohrmann. 1989. Location, sequence, transcriptional mapping, and temporal expression of the gp64 envelope glycoprotein gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 170: 537-555. Blissard, G. W., and J. R. Wenz. 1992. Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J. Virol. 66: 6829-6835. Blissard, G., B. Black, N. Crook, B. A. Keddie, R. Possee, G. Rohrmann, D. Theilmann, and L. Volkman. 2000. Family Baculoviridae: taxonomic structure of the family. pp. 195-220. In: M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle, and R. B. Wickner, eds. Virus Taxonomy: Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego. Bonning, B. C., and B. D. Hammock. 1996. Development of recombinant baculoviruses for insect control. Annu. Rev. Entomol. 41: 191-210. Bradford, M. B., G. W. Blissard, and G. F. Rohrmann. 1990. Characterization of the infection cycle of the Orgyia pseudosugata multicapsid nuclear polyhedrosis virus in Lyman-tria dispar cells. J. Gen. Virol. 71: 284-286. Bulach, D. M., C. A. Kumar, A. Zaia, B. Liang, and D. E. Trible. 1999. Group II nucleopolyhedrovirus subgroups revealed by phylogenetic analysis of polyhedrin and DNA polymerase gene sequences. J. Invertebr. Pathol. 73: 59-73. Carrel, A. 1912. On the permant life of tissue outside the organism. J. Exp. Med. 15:516-528. Chao, J. T., P. W. Schaefer, Y. B. Fan, and S. S. Lu. 1996. Host plants and infestation of casuarina moth Lymantria xylina (Lepidoptera: Lymantriidae) in Taiwan. Taiwan J. Forest Sci. 11: 23-28. Disney, J. E., and W. J. McCarthy. 1985. A modified technique for the improved characterization of lepidopteran chromosomes from cells in culture. In Vitro Cell. Dev. Biol. 21: 563-568. Ennis T. J., and S. S. Sohi. 1976. Chromosomal characterisation of five lepidopteran cell lines of Malacosoma disstria (Lasiocampidae) and Christoneura fumiferana (Tortricidae). Can. J. Genet. Cytol. 18: 471–477. Federici, B. A. 1997. Baculovirus pathogenesis. pp. 33-56. In: L. K. Miller, ed. The Baculoviruses. Plenum Press, New York. Friesen, P. D., and L. K. Miller. 2001. Insect viruses. pp. 443-472. In: D. M. Fuchs, M. S. Woods, and R. F. Weaver. 1983. Viral transcription during Autographa californica nuclear polyhedrosis virus infection: a novel RNA polymerase induced in infected Spodoptera frugiperda cells. J. Virol. 48: 641-646. Garner, K. J. and J. M. Slavicek. 1996. Identification and characterisation of a RAPD-PCR marker for distinguishing Asian and North American gypsy moths. Insect Molecular Biology 5:81-91. Goding, J. W. 1980. Antibody production by hybridomas. J. Immunol. Methods 39 : 285 308. Goldsby, R. A., and E. Zipper. 1969. The isolation and replica plating of mammalian cell colonies. Exp. Cell Res. 54: 271-274 Goldschmidt, R. 1915. Some experiments on spermatogenesis in vitro. Proc. Natl. Acad. Sci. 2: 220-222. Goodman, C. L., and A. H. McIntosh. 1994. Production of baculoviruses for insect control using cell culture. pp. 33–56. In: K. Maramorosch, A. H. McIntosh, eds. Insect cell biotechnology. CRC Press, Boca Raton, FL. Goodwin R. H., G. J. Tompkins, P. McCawley. 1978. Gypsy moth cell lines divergent in viral susceptibility. In Vitro 14: 485-493. Grace, T. D. C. 1962. Establishment of the four strains of cell from insect tissue grown in vitro. Nature 195: 788-789. Grace, T. D. C. 1968, The Development of Clones from Lines of Antheraea eucalypti Cells Grown in Vitro. Exp. Cell Res. 52: 451-458. Granados, R. R. and K. A. Williams. 1986. In vivo infection amd replication of baculovirus. pp.89-108.In: R. R. Granados, and B. A. Federici, eds. The biology of baculovirus. CRC Press. Boca Raton. Greene, A. E., J. Charney, W. W. Nichols, and L. L. Coriell. 1972. Species identity of insect cell lines. In Vitro 7: 313-322. Harrison, R. G. 1907. Obervation on the living developing nerve fiber. Proc. Soc. Exp. Biol. Med. 4:140-143. Heim, R., A. B. Cubitt, and R.Y. Tsien, 1995. Improved green fluorescence. Nature 373,663-664. Herniou, E. A., T. Luque, X. Chen, J. M. Vlak, D. Winstanley, J. S. Cory, and D. R. O’Reilly. 2001. Use of whole-genome sequence data to infer baculovirus phylogeny. J. Virol. 75: 8117-8126. Herniou, E. A., J. A. Olszewski, D. J. S. Cory, and R. O’Reilly. 2003. The genome sequence and evolution of baculovirus. Annu. Rev. Entomol. 48: 211-234. Herniou, E. A., J. A. Olszewski, D. R. O’Reilly, and J. S. Cory. 2004. Ancient coevolution of baculoviruses and their insect host. J. Virol. 78: 3244-3251. Hofmann C., V. Sandig, G. Jennings, M. Rudolph, P. Schlag, and M. Strauss. 1995. Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc. Natl. Acad. Sci. U. S. A. 92: 10099-103. Holtke, H. J., G. Sanger, C. KesSLer, and G. Schmitz. 1992. Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acid: a fast and simple protocol and it`s application. Biotechniques 12: 104-113. IJkel, W. F. J., E. A. Van Strien, J. G. Heldens, R. Broer, D. Zuidema, R. W. Goldbach, and J. M. Valk. 1999. Sequence and organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome. J. Gen. Virol. 80: 3289-3304. IJkel, W. F. J., M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, and D. Zuidema. 2000. A novel baculovirus envelope fusion protein with a proprotein convertase cleavage site. Virology 275: 30-41. Kariuki, C. W., A. H. McIntosh, and C. L Goodman,. 2000. In vitro host range studies with a new baculovirus isolate from the diamondback moth Plutella xylostella (L.) (Plutellidae: Lepidoptera). In Vitro Cell. Dev. Biol. 36A: 271–276. Kawai, Y., and J. Mitsuhashi. 1997. An insect cell line descrimination method by RAPD-PCR. In Vitro Cell. Dev. Biol. 33: 512-515. Kuzio, J., M. N. Pearson, S. H. Harwood, C. J. Funk, J. T. Evans, J. M. Slavicek, and G. F. Rohrmann. 1999. Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar. Virology 253: 17-34. Liebhold, A. M., K. G. Gottschalk, R. Muzika, M. E. Montgomery, R. Young, K. O’Day, and B. Kelly. 1995. Suitability of North American tree species to the gypsy moth: a summary of field and laboratory tests. USDA General Technical Report NE-211, USDA Forest Service, Delaware, Ohio, USA. Liebhold, A. M., H.A. Halverson, and G.A. Elmes. 1992. Gypsy moth invasion of North America: a quantitative analysis. J. Biogeogra. 19: 1-8. Luckow, V. A. 1993. Baculovirus systems for the expression of human gene product. Curr. Opin. Biotechnol. 4: 564-572. Lynn, D. E., E. M. Dougherty, J. T. McClintock, and M. Loeb 1988. Development of cell lines from various tissues of Lepidoptera. pp.239–242. In: Y. Kuroda, E. Kurstak, K. Maramorosch, eds. Invertebrate and Fish Tissue Culture. Japan Scientific Societies Press. Tokyo. Lynn, D. E. 1991. Establishing invertebrate cells in culture: Continued need for new insect cell lines. pp. 1–6. In: M. J. Fraser, Jr., ed. Proceedings of the 8th International Conference on Invertebrate and Fish Tissue Culture. Tissue Culture Association, Columbia, MD. Lynn, D. E. 1999. Development of insect cell lines: Virus susceptibility and applicability to prawn cell culture. Methods in Cell Science 21: 173-181. Macpherson, I., and L. Montagnier. 1964. Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23: 291-294. Mairella, B. I., D. Shauger, and D. Harano. 1988. Large-scale insect cell culture for recombinant proteins production. Bio Technol. 6: 1406-1410. Matsumura, S. 1933. Lymantridae of Japan-Empire. Ins. Matsum. 7: 111-152. McIntosh, A. H. and C. Pechtoris. 1974. Insect Cells:Colony Formation and Cloning in Agar Medium. In Vitro 10:1-5. McIntosh, A. H., P. D. Christian, and J. J. Grasela. 1999. The establishment of Heliothine cell lines and their susceptibility to two baculovirus. In Vitro Cell. Dev. Biol. 35: 94-97. McIntosh, A. H., J. J. Grasela, and R. L. Matteri. 1996. Identification of insect cell lines by DNA amplification fingerprinting (DAF). Insect Mol. Biol. 5: 187-195. McKenna, K. A., H. Hong, E. vanNunen, and R. R. Granados. 1998. Establishment of new Trichoplusia ni cell lines in serum-free medium for baculovirus and recombinant protein production. J. Invertebr. Pathol. 71: 82–90. Miltenburger, H. G., W. L.Naser, J. P. Harvey. 1984. The cellular substrate: a very important requirement for baculovirus in vitro replication. Z. Naturforsch. 39: 993–1002. Mitsuhashi, J. 1990. Comparison of zymograms of some insect cell lines by means of APIZYM system. Appl. Entomol. Zool. 25: 535. Monsma, S. A., A. G. P. Oomens, and G. W. Blissard. 1996. The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. J. Virol. 70: 4607-4616. Murhammer, D. W. 1996. Production of viral insecticides in cell culture and their use for pest control. Appl. Biochem. Biotechnol. 59: 199–220. Nakajima, S. and T. Miyake. 1976. Effective Colony Formation in Drosophila Cell Lines using Conditioned Medium. pp.279- 287. In:E. Kurstak, and K. Maramorosch. eds. Invertebrate Tissue Culture:Applications in Medicine, Biology and Agriculture. Academic Press, New York. Nichols, W. W., C. Bradt, and W. Bowne. 1971. Cytogenetic studies on cells in culture from the class insecta. Curr. Top. Microbiol. Immunol. 55: 61-69. Olsen, G. J., and C. R. Woese. 1993. Ribosomal RNA: A key to phylogeny. FASEB J. 7: 113-123. O’Reilly, D. R., L. K. Miller, and V. A. Luckow. 1992. Virus structure and the infection process. pp. 4-6. In: Baculovirus Expression Vector. W. H. Freeman and Company, New York. Pearson, M. N., C. Groten, and G. F. Rohrmann. 2000. Identification of the Lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae. J. Virol. 74: 6126-6131. Pearson, M. N., R. L. Q. Russell, and G. F. Rohrmann. 2001. Characterization of a baculovirus-encoded protein that is associated with infected-cell membranes and budded virions. Virology 291: 22-31. Pijlman, G. P., E. V. D. Born, D. E. Marten, and J. M. Vlak. 2001. Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells. Virology 283: 132-138. Prasher, D. C. 1995. Using GFP to see the light. Trends Genet. 11: 320-323. Puck, T. T. and P. I. Marcus. 1955. A rapid method for viable cell titration and clone production with HeLa cells in tissue culture: The use of X-irradiated cells to supply conditioning factors. Proc. Nat. Acad. Sci. U.S.A. 41: 432-437. Puck, T. T. and H. W. Fisher. 1956. Genetics of somatic mammalian cells. I. Demonstration of the existence of mutants with different growth requirements in a human cancer cell strain (HeLa). J. Exp. Med. 104: 427-434. Puck, T. T., P. I. Marcus, and S. J. Cierciura. 1956. Clonal growth of mammalian cells in vitro: Growth characteristics of colonies from single HeLa cells with and without a “Feeder layer”. J. Exp. Med., 103: 273-284. Rochford, R. 1984. Establishmnet of a cell line from embryos of the cabbage looper Trichoplusia ni (Hübner). In Vitro 20: 823-825. Sanford, K. K., W. R. Earle, and G. D. Likely. 1948. The growth in vitro of single isolated tissue. J. Nat. Cancer Inst 9:229-246. Shimomura, O., F. H. Johnson, and Y. Saiga. 1962. Extraction, purification, and properties of Aequorin, a bioluminescent protein from luminous Hydromedusan, Aequorea. J. Cell Comp. Physiol. 59: 223-239. Shouche Y. S., M. S. Patole, U. Pant, S. Paranjpe, and K. Banerjee. 1999. Authentication of two cell lines developed from the larval and pupal ovaries of Spodoptera litura by RNA based methods. In Vitro Cell Dev Biol (Animal) 35: 244-245. Smith, G. E., M. D. Summers, and M. J. Fraser. 1983. Production of human β-interferon in insect cells infect with a baculovirus expression vector. Mol. Cell Biol. 3: 2156-2165. Suitor, E. C., L. L. Chang, and H. H. Liu. 1966. Establishment and characterization of a clone from Grace’s in vitro cultured mosquite (Aedes aegyptic L.) Cells. Expt. Cell Res. 44:572-578. Tabachnick, W. J., and D. L. Knudson. 1980. Characterization of invertebrate cell lines Ⅱ. Isozyme analysis employing starch gel electrophoresis. In Vitro 16: 392-398. Terry R. G., R. S. Nowak, and R. J. Tausch. 2000. Genetic variation in chloroplast and nuclear ribosomal DNA in Utah juniper (Juniperus osteosperma, Cupressaceae): evidence for interspecific gene flow. Am. J. Bot. 87: 250-258. Trager, W. 1935. Cultivation of the virus of grasserie in silkworm tissue cultures. J. Exp. Med. 61: 501-513. Trouvelot, E. L. 1867. The American Silk Worm. Am. Nat., 1:30-38. Vaughn J. L., R. H. Goodwin, G. J. Tompkins, P. McCawley. 1977. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13: 213–217. Volkman, L. E., and M. D. Summers. 1976. Comparative studies with clones derived from a cabbage looper ovarian cell line, TN-368. pp.289-296. In:E. Kurstak, and K. Maramorosch, eds. Invertebrate Tissus Culture:Applications in Medicine, Biology and Agriculture. Academic Press. New York.): Volkman, L. E., P. A. Goldsmith, R. T. Hess, and P. Faulkner. 1984. Neutralization of budded Autographa californica NPV by a monoclonal antibody: identification of the target antigen. Virology 133: 354-362. Wallner, W. E. 1996. Invasion of the tree snatchers. American Nurseryman March: 28-30. Wang, C. H., C. M. Chou, H. C. Lin, S. L. Kau, G. H. Kou, and C. F. Lo. 1996. Continuous cell line from pupal ovary of Perina nuda (Lepidoptera: Lymantriidae)that is permissive to nuclear polyhedrosis virus from P. nuda. J. Invertebr. Pathol. 67: 199-204. Wang, M.-Y., Y. Y. Kuo, M. S. Lee, S. R. Doong, J.Y. Ho, and L. H. Lee. 2000. Self-assembly of the infectious bursal disease virus capsid protein, rVP2, expressed in Insect cells and purification of immunogenic chimeric rVP2H particles by immobilized metal-ion affinity chromatography. Biotech. Bioengi. 67: 104-111. Westenberg, M., H. Wang, W. F. J. IJkel, R. W. Goldbach, J. M. Vlak, and D. Zuidema. 2002. Furin is involved in baculovirus envelope fusion protein activation. J. Virol. 76: 178-184. White T. J., T. Bruns, S. Lee., and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications 38: 315-322. Whitford, M., S. Stewart, J. Kuzio, and P. Faulkner. 1989. Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus. J. Virol. 63: 1393-1399. Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18: 6531-6535. Wood, H. A., and R. R. Granados. 1991. Genetically engineered baculoviruses as agents for pest control. Annu. Rev. Microbiol. 45: 67-87. Wu, C. Y., and C. H. Wang. 2005. Characterization and polyhedron gene cloning of Lymantria xylina multiple nucleopolyhedrovirus. J. Invertebr. Pathol. 88: 238-246. Yu, C. C., H. H. Kao, C. H. Wang, J. T. Chao, and S. S. Lu. 1997. Characterization of Lymantria xylina nucloepolyhedrovirus (LyxyNPV) and the establishment of in vitro multiplication of LyxyNPV. Chinese J. Entomol. 17: 11-22. (In Chinese) Zanotto, P. M., B. D. Kessing, and J. E. Maruniak. 1993. Phylogenetic interrelationships among baculoviruses: evolutionary rates and host associations. J. Invertebr. Pathol. 62: 147-164. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32452 | - |
dc.description.abstract | 利用次選殖(sub-clone)技術從舞毒蛾 IPLB-LD652Y 母細胞株選殖出 IPLB-LD652Y-5、IPLB-LD652Y-7、IPLB-LD652Y-b、IPLB-LD652Y-d、IPLB-LD652Y-f、IPLB-LD652Y-g、及IPLB-LD652Y-h 共 7 株次選殖株。此 7 株次選殖株與母株在形態上皆可以區分為四型:圓形、鱗狀、梭狀及多形態細胞,其中以圓形細胞所佔之比例為最高。黑角舞蛾核多角體病毒(LyxyNPV)感受性試驗結果,顯示對照組 SL-7B 細胞株對各株供試病毒(Ly5、LyxyExp-DsRed、LyxyExp-EGFP) 均無感受性;而陽性對照組 NTU-LY 系列細胞株則對各株供試病毒均呈現高感受性。至於 IPLB-LD652Y 系列細胞株對 Ly5 皆具高感受性,但對 LyxyExp-DsRed 則均呈現低感受性;對 LyxyExp-EGFP 之感受性除 IPLB-LD652Y-h 較低外,其餘各株均呈現高感受性。依產 Ly5 胞外病毒能力,選出產量高的 3 株次選殖株(IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f)與其母株作進一步細胞特性之探討。選出之 3 株次選殖細胞株之生長速率均顯著高於母株,而 IPLB-LD652Y-f 又顯著高於 IPLB-LD652Y-5。染色體數分析結果顯示這 4 株細胞株染色體數分布皆很相近,其平均值介於 78.02 至 87.28。同功異構酶分析顯示 IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f 皆有相同的 EST Esterase (EST) 、 Malate dehydrogenase ( MDH ) 及 Lactate dehydrogenase ( LDH ) 電泳圖譜,且明顯不同於 NTU-LY1 的電泳圖譜。ITS (Internal transcribed spacer) 序列分析顯示 IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f 細胞株之 ITS 片段序列並無任何差異。感染 LyxyExp-EGFP 後之 EGFP 相對螢光分析顯示出4 株細胞株 (IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f) 之 EGFP 相對螢光值,隨著感染時間的增加會有明顯的改變。綜合以上結果可篩選出對 LyxyMNPV 感受性、產量最高及生長速率快之IPLB-LD652Y-7細胞株。 | zh_TW |
dc.description.abstract | Seven new cell lines, IPLB-LD652Y-5, IPLB-LD652Y-7, IPLB-LD652Y-b, IPLB-LD652Y-d, IPLB-LD652Y-f, and IPLB-LD652Y-h, have been sub-cloned from the parental cell line, IPLB-LD652Y. There were four kinds of cell shape within these cell lines including round, squamous, spindle, and polymorphous shape. Among them, the round shape cells were the most preponderant. According to the virus infection results, SL-7B cannot been infected with Ly5, LyxyExp-DsRed, and LyxyExp-EGFP while the NTU-LY cell lines had high susceptibility with the same viruses infection. Moreover, the IPLB-LD652Y and subcloned cell lines showed similar results as the NTU-LY cell lines, but them showed lower susceptibility when infected with LyxyExp-DsRed, and only IPLB-LD652Y-h had lower susceptibility when infected with LyxyExp-EGFP. According to the results, we chose three sub-clonal cell lines with high production of Ly5 virus, IPLB-LD652Y-5, IPLB-LD652Y-7, and IPLB-LD652Y-f for the further comparison with the parental cell line. We can observe that the chosen cell lines have higher growth rate than the parental cell line, and the growth rate of IPLB-LD652Y-f were higher than that of IPLB-LD652Y-5.The distribution of chromosome numbers of these four cell lines were very close, between 78.02 to 87.28. In the result of isozyme, whether on EST Esterase (EST)、 Malate dehydrogenase ( MDH ) or Lactate dehydrogenase ( LDH ), the electrophoresis patterns of the chose cell lines were all the same and significant different from that of the NTU-LY1. The ITS (Internal transcribed spacer) sequences of the four cell lines, and they showed no significant difference. In the relative fluorescent unit (RFU) comparison, significant curve growth and decline with the increasing with the time post LyxyExp-EGFP inoculation were observed. Integrating the results, the IPLB-LD652Y-7 could be sifted as the highest virus susceptibility, production, and speedy growth cell line when infected with LyxyMNPV. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T03:50:12Z (GMT). No. of bitstreams: 1 ntu-95-P93632001-1.pdf: 1186909 bytes, checksum: efab442b509bd56f3ad38a84fc383be9 (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | i
目錄 中文摘要………………………………………………………………………………1 英文摘要………………………………………………………………………………2 壹、緒言………………………………………………………………………………3 貳、往昔研究…………………………………………………………………………5 一、舞毒蛾與木毒蛾背景簡介…………………………………………………5 二、核多角體病毒簡介……………………………………………………6 三、昆蟲細胞培養之發展…………………………………………………9 四、細胞株之選殖…………………………………………………………11 五、細胞株之特性與鑑定…………………………………………………11 六、螢光蛋白基因的發現與應用…………………………………………13 參、材料與方法………………………………………………………………………15 一、供試細胞株…………………………………………………………………15 二、供試病毒株…………………………………………………………………15 三、母株之次選殖………………………………………………………………15 四、細胞之冰凍保存與解凍培養………………………………………………16 五、胞外病毒效價測定…………………………………………………………16 六、次選殖細胞株形態及病毒感受性之比較…………………………………17 1. 形態上之比較 ……………………………………………………………18 2. 對供試病毒之感受性測試 ………………………………………………18 七、細胞株特性之建立…………………………………………………………18 1. 生長速率測定 ……………………………………………………………18 2. 染色體數目分析 …………………………………………………………19 3. 同功異構酶之分析 ………………………………………………………19 4. DNA 分子標示鑑定法 …………………………………………………20 八、EGFP 相對螢光值測量……………………………………………………22 ii 1. 感染細胞之蛋白質取樣 …………………………………………………22 2. 蛋白質定量 ………………………………………………………………23 3. 螢光值測量 ………………………………………………………………23 肆、結果………………………………………………………………………………24 一 、次選殖 (sub-clone) 細胞株的建立……………………………………24 二、次選殖細胞株形態及病毒感受性之比較…………………………………24 1. 形態上之比較 ……………………………………………………………24 2. 對各種供試病毒之感受性 ………………………………………………24 三、選殖細胞株之特性…………………………………………………………26 1. 細胞株生長速率之比較…………………………………………………26 2. 染色體數目分析…………………………………………………………26 3. 同功異構酶之分析………………………………………………………26 4. ITS 序列分析 ……………………………………………………………27 四、EGFP 相對螢光分析………………………………………………………27 伍、討論………………………………………………………………………………28 陸、參考文獻…………………………………………………………………………33 柒、圖表………………………………………………………………………………43 捌、附錄………………………………………………………………………………66 附錄一、同功異構酶電泳與染色……………………………………………………66 附錄二、實驗所使用之引子對及其序列……………………………………………70 iii 表次 表一 IPLB-LD652Y 細胞株系母株與次選殖株之不同細胞形態比例…………43 表二 IPLB-LD652Y 細胞株系各細胞形態大小 ……………………………… 44 表三 IPLB-LD652Y 細胞株系、SL-7B 細胞及 NTU-LY4S 在感染 Ly5 後第 7 天之感染細胞比率………………………………………………………. 45 表四 IPLB-LD652Y 細胞株系、SL-7B 細胞及 NTU-LY4S 在感染 Ly5 後第 14 天之感染細胞比率………………………………………………………46 表五IPLB-LD652Y 細胞株系、SL-7B 細胞及 NTU-LY4S 在感染 LyxyExp-DsRed 後第 3、7、10 及 14 天之發螢光細胞比率…………… 47 表六IPLB-LD652Y 細胞株系、SL-7B 細胞及 NTU-LY4S 在感染 LyxyExp-EGFP 後第 3、7、10 及 14 天之發螢光細胞比率…………48 表七 0%、4%、8% 及 6% 血清濃度下培養 IPLB-LD652Y、IPLB-LD652Y-5、 IPLB-LD652Y-7 及 IPLB-LD652Y-f 細胞株之細胞生長所需倍增時 間…………………………………………………………………………… 49 表八 ITS rDNA 序列相似度………………………………………………………50 iv 圖次 圖一 核醣體 DNA 之結構及各引子與 r DNA 之關係 ………………………51 圖二 IPLB-LD652Y 細胞株系細胞形態…………………………………………52 圖三 IPLB-LD652Y 細胞株感染Ly5 後第 7 天之感染形成之細胞病變…….54 圖四 IPLB-LD652Y 細胞株系細胞感染Ly5 後7 天之胞外病毒效價…………55 圖五 IPLB-LD652Y 細胞株系細胞感染Ly5 後7 天之核多角體產量 ………56 圖六 IPLB-LD652Y 細胞在感染Lyxy Exp-DsRed 後第 3、7、10 及 14 天之 發螢光細胞………………………………………………………………… 57 圖七 IPLB-LD652Y 細胞在感染Lyxy Exp-EGFP 後第 3、7、10 及 14 天之 發螢光細胞………………………………………………………………… 58 圖八 不同血清濃度下培養 IPLB-LD652Y(a)、IPLB-LD652Y-5(b)、 IPLB-LD652Y-7(c) 及 IPLB-LD652Y-f (d) 細胞株之細胞生長曲線…59 圖九 8 % 血清濃度條件下培養 IPLB-LD652Y 、IPLB-LD652Y-5 、 IPLB-LD652Y-7 及 IPLB-LD652Y-f 細胞株之細胞生長曲線及倍增時間 之比較 ………………………………………………………………………60 圖十 IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f 細 胞株之染色體數分布圖…………………………………………………… 61 圖十一 IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7、IPLB-LD652Y-f 及 LY1 細胞株之同功異構酶電泳圖譜 ………………………………… 62 圖十二 IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7 及IPLB-LD652Y-f 細胞株之 ITS 片段序列比較 ………………………………………… 64 圖十三 IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f 細胞株在感染 Lyxy Exp-EGFP 後第 48、96、144、192 及 240 小 時之EGFP 相對螢光值………………………………………………… 65 | |
dc.language.iso | zh-TW | |
dc.title | 木毒蛾核多角體病毒(LyxyMNPV)量產用之舞毒蛾細胞(IPLB-LD652Y)次選殖株系篩選 | zh_TW |
dc.title | Screening of sub-clonal cell lines from IPLB-LD652Y for the mass production of Lymantria xylina multiple nucleopolyhedrovirus(LyxyMNPV) | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳文哲,陳秋男,吳宗遠,蔡恕人 | |
dc.subject.keyword | 舞毒蛾細胞株,木毒蛾核多角體病毒,次選殖, | zh_TW |
dc.subject.keyword | IPLB-LD652Y cell line,LyxyMNPV,sub-clone, | en |
dc.relation.page | 70 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-26 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 昆蟲學研究所 | zh_TW |
顯示於系所單位: | 昆蟲學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-95-1.pdf 目前未授權公開取用 | 1.16 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。