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研究生:趙健強
研究生(外文):Chien-Chiang Chao
論文名稱:研究牛痘病毒的寄主範圍基因對於牛痘病毒在HeLa細胞生長之影響
論文名稱(外文):Investigation of poxviral host range genes that are required for vaccinia virus growth in HeLa cells
指導教授:張雯張雯引用關係
指導教授(外文):Wen Chang
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:細胞及分子生物研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
中文關鍵詞:牛痘病毒寄主範圍基因
外文關鍵詞:vaccinia virushost range gene
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前人的實驗結果指出,當牛痘病毒(vaccinia virus ; VV)感染細胞時會使HeLa細胞進行細胞凋亡。並且在此類細胞中,病毒的生命週期也會被終止在中期的基因轉譯,所以我們稱這種現象為「寄主限制」。然而,當cowpox的CP77基因被表現時,則可以抑制細胞進行細胞凋亡並且可以幫助病毒在HeLa細胞中生長。
這篇研究主要的目的是去了解CP77基因在HeLa細胞中所扮演的角色。我們將CP77接到帶有綠色螢光的GFP,以觀察當病毒感染細胞時CP77所在的位置。實驗結果顯示,在病毒感染細胞的早期,GFP-CP77是在細胞的細胞質中,然而到了感染的晚期則會移動到細胞核內。
為了了解CP77是如何在細胞質與細胞核之間穿梭,我做了各種不同突變的CP77然後將之送到HeLa細胞裡,再利用螢光顯微鏡來觀察CP77所在細胞的位置,以推斷CP77哪個區域是具有調節CP77在細胞的移動的功能。就目前的結果顯示,在CP77的胺基酸序列79-275和353~441這兩個區域,似乎對於CP77移動到細胞質是十分重要的。
另一部份,有兩個寄主範圍基因K1L和C7L,已經被證實在某一些細胞中,其功能可以替代CP77,所以我們有興趣想要了解這三個寄主範圍基因是否會活化相同的訊號傳遞路徑,來幫助病毒的生長。以前我們實驗室已經利用酵母菌雙雜交篩選系統,找到五個寄主基因能和CP77有結合作用,根據病毒生長的特性,我們認為這五個基因有可能和K1L、C7L有結合作用,所以我也利用酵母菌雙雜交篩選系統,證實其中的一個寄主基因HMG20A能和K1L和C7L有結合作用,現在我已經將K1L和C7L接到GFP,想要找出這三個寄主範圍基因與HMG20A的結合位置,這些結果應該可以幫助我們了解寄主範圍基因和寄主基因作用的分子機制。
Abstract:
Vaccinia virus infection into Hela was previously shown to trigger apoptosis of the infected cells. In addition, translation of viral intermediate genes was blocked resulting in abortive infection that is commonly referred to as “host restriction”. Expression of a cowpox viral CP77 gene suppressed apoptosis and rescued virus growth in Hela cells. The main focus of my study is to understand the role of CP77 in host restriction. We have fused GFP with CP77 in order to monitor CP77 intracellular location in the infected cells. Expression of GFP-CP77 in the infected cells indicated that early after infection CP77 expresses in the cytoplasm whereas later after infection CP77 was mainly detected in the nuclei of the infected cells.
In order to understand how CP77 is translocated between nucleus and cytoplasm various truncations of CP77 were generated and transfected into Hela cells. Immunofluorescence analyses were performed with these transfected cells to determine which region of CP77 regulates protein movement in cells. The experiments are still in progress and the results indicated that regions, aa 79-275 and aa 353~441 appeared important for cytoplasmic localization of CP77.
Two other viral host range genes K1L and C7L have been shown to substitute the functions of CP77 in other cell types and it will be also interesting to find out whether all three genes activate same signaling pathways in cells for virus rescue. Previously, by two hybrid analyses we have identified 5 cellular cDNAs that bind to CP77. One of the cDNAs, HMG20A, also interacts with K1L and C7L in two hybrid analyses. I have generated GFP-K1L and GFP-C7L constructs and will determine HMG20A-binding domain on CP77 in relation to these viral host range genes. These results should help us to understand molecular mechanism of interactions between viral host range genes and cellular targets.
目次
中文摘要………………………………………….……………...i
英文摘要……………………………………………….………..iii
目次…………………………………………………….………..iv
第壹章 緒論………………………………………..…………...1
第貳章 研究材料與方法…………………………….…………..5
第一節 研究材料
一. 細胞株………………………………………………........5
二. 病毒……………………………..…………………........5
三. reagents used in yeast two hybrid systems….………5
四. 抗體…………………………………………………........7
第二節 研究方法
一. 質體構築……………………………..……………........8
二. 酵母菌雙雜交篩選系統………………………….........12
三. 酵母菌之轉形 (yeast transformation)……………....12
四. Expression of recombinant protein in yeasts………13
五. 西方點墨分析法…………………………..……….......13
六. yeast mating……………………………..………….....14
七. transient assay……………………..………………....15
八. 免疫螢光染色分析法…………………………….........15
第參章 實驗結果與分析…………………………..…………..17
一. Identification of cellular genes that bind to CP77、K1L、C7L, using yeast two-hybrid analysis.…..………………17
二. EGFP-CP77 is expressed in cytoplasm of the infected cells at the early phase and translocated to nucleus at the late phase.……………………………………………………….....18
三. 短期性表現(transient assay)EGFP-CP77原生型和變異型在細胞內座落的位置…………………….........................19
四. CP77之NES移動能力是CRM1-dependent……............25
第肆章 討論………………………………………..….……….29
參考文獻……………………………………………….………..32
附圖1~37………………………………………………………...37
參考文獻
Buller, R. M., and G. J. Palumbo. 1991. Poxvirus pathogenesis. Microbiol. Rev. 55: 80-122.
Chakrabarti, S., K. Brechling, and B. Moss. 1985. Vaccinia virus expression vector: coexpression of b-galactosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol. 5: 3403-3409.
Chen, W., R. Drillien, D. Spehner, and M. L. Buller. 1992. Restricted replication of ectromelia virus in cell culture correlates with mutations in virus-encoded host range gene. Virology 187:433-442
Crameri, A., E. A. Whitehorn, E. Tate, and W. P. C. Stemmer. 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature. Biotechnol. 14: 315-319.
Cressman, D. E., W. J. O’Connor, S. F. Greer, X.-S. Zhu, and J. P.-Y. Ting. 2001. Mechanisms of Nuclear Import and Export That Control the Subcellular Localization of Class II Transactivator. J. Immunol. 167: 3626 - 3634.
Drillien, R., F. Koehren, and A. Kirn. 1981. Host range deletion mutant
of vaccinia virus defective in human cells. Virology 111:488—499.
Drillien, R. D., D. Spehner, and A. Kirn. 1978. Host range restriction
of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff
of protein synthesis. J. Virol. 28: 843—850.
Fenner, F., and J. F. Sambrook. 1966. Conditional lethal mutants of
rabbitpox virus KK. Mutants (p) that fail to multiply in PK-2a cells.
Virology 28: 600—609.
Flach, J., M. Bossie, J. Vogel, A. Corbett, T. Jinks, D. A. Willins, and P. A. Silver. 1994. A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm. Mol. Cell. Biol. 14: 8399-8407.
Gemmell, A., and F. Fenner. 1960. Genetic studies with mammalian poxviruses III White(u) mutants of rabbitpox virus. Virology 11: 219-235.
Geobel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., Winslow, J. P., and Paoletti, E. 1990. Ther complete DNA sequence of vaccinia virus. Virology 179:247-266.
Gillard, S., D. Spehner, R. Drillien, and A. Kirn. 1986. Localization and sequence of a vaccinia virus gene required for multiplication in human cells. Proc Natl. Acad. Sci. USA 83:5573-5577.
Heim, R., D. C. Prasher, and R. Y. Tsien. 1994. Wavelength mutations and posttranslational autoxidation of green fluorescent protein.
Proc Natl. Acad. Sci. USA 91: 12501-12504.
Henderson, B. R. and A. Eleftheriou. 2000. A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals. Experimental Cell Research 256: 213-224.
Hollenberg, S. M., R. Sternglanz, P. F. Cheng, and H. Weintraub. 1995. Identification of a new family of tissue-specific basic helix-loop- helix proteins with a two-hybrid system. Mol. Cell. Biol. 15: 3813-3822.
Hruby, D. E., D. L. Lynn, R. C. Condit, and J. R. Kates. 1980. Cellular differences in the molecular mechanisms of vaccinia virus host range restriction. J. Gen. Virol. 47:485-488.
Ink, B. S., C. S. Gilbert, and G. I. Evan. 1995. Delay of vaccinia virus-induced apoptosis in nonpermissive CHO cells by the coxpox virus CHO hr and adenovirus E1B19K genes. J. Virol. 69:661-668.
Kain, S. R., M. Adams, A. Kondepudi, T. T. Yang, W. W. Ward, and P. Kitts. 1995. The green fluorescent characteristics of the recombinant protein. FEBS Letters. 341: 277-280.
Kitts, P., M. Adams, A. Kondepudi, D. Gallagher, and S. Kain 1995. green fluorescent protein (GFP): A novel reporter gene for monitoring gene expression in living organisms. CLONTECHniques. X(1): 1-3.
Kau T. R. and P. A. Silver. 2003. Nuclear transport as a target for cell growth. DDT. 8: 78-85.
Kotwal, G. J., and B. Moss. 1988. Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition
mutant. Virology 167:524—537.
Marques. S. M. P., J.-L. Veyrune, R. R. Shukla, and A. Kumar. 2003. Restriction of Human immunodeficiency virus type 1 Rev function in murine A9 cells involves the Rev C-terminal domain. J. Virol. 77: 3084-3090.
McClain, M. E. 1965. The host range and plaque morphology of rabbitpox virus (RPu+) and its u mutants on chick fibroblasts PK-2a and L929 cells. Aust. J. Exp. Biol. Med. Sci. 43:31-44.
Moss, B. 1991. Vaccinia virus: a tool for research and vaccine development. Science. 252:1662-1667.
Nishi, K. 1994. Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. J. Biol. Chem. 269: 6320-6324.
Oguiura, N., D. Spehner, and R. Drillien. 1993. Detection of a protein encoded by the vaccinia virus C7L open reading frame and study of its effect on virus multiplication in different cell lines. J. Gen. Virol. 74: 1409-413.
Olson, K. R., J. R. McIntosh and J. B. Olmsted. 1995. Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras. J. Cell Biology. 130: 639-650.
Ossareh-Nazari B., C. Gwizdek and C. Dargemont. 2001. Protein export from the nucleus. Traffic. 2: 684-689.
Perkus, M. E., S. J. Goebel, S. W. Davis, G. P. Johnson, K. Limbach, E. K. Norton, and E. Paoletti. 1990. Vaccina virus host range genes. Virology 179: 276-286.
Ramsey-Ewing, A., and B. Moss. 1995. Restriction of vaccinia virus replication in CHO cells occurs at the stage of viral intermediate protein synthesis. Virology 206: 984-993.
Ramsey-Ewing, A. L., and B. Moss. 1996. Complementation of a vaccinia virus host-range K1L gene deletion by the nonhomologous CP77 gene. Virology 222: 75-86.
Shchelkunov, S. N., P. F. Safronov, A. V. Totmenin, N. A. Petrov, O. I. Ryazankina, V. V. Gutorov, and G. J. Kotwal. 1998. The Genomic Sequence Analysis of the Left and Right Species-Specific Terminal Region of a Cowpox Virus Strain Reveals Unique Sequences and a Cluster of Intact ORFs for Immunomodulatory and Host Range Proteins. Virology 243: 432-460.
Somogyi, P., J. Frazier, and M. A. Skinner. 1993. Fowlpox virus host range restriction: gene expression, DNA replication, and morphogenesis in nonpermissive mammalian cells. Virology. 197:439-444.
Spehner, D., S. Gillard, R. Drillien, and A. Kirn. 1988. A cowpox virus gene required for multiplication in Chinese Hamster ovary cells. J. Virol. 62: 1297-1304.
Sumoy, L., Carim, L., Escarceller, M., Nadal, M., Gratacos, M., Pujana, M. A., Estivill, X., Peral, B. HMG20A and HMG20B map to human chromosomes 15q24 and 19p13.3 and constitute a distinct class of HMG-box genes with unbiquitous expression. 2000. Cytogenet. Cell Genet. 88: 62-67.
Sutter, G., A. Ramsey-Ewing, R. Rosales, and B. Moss. 1994. Stable expression of the vaccinia virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant. J. Virol. 68: 4109-4116.
Tagaya, I., T. Kitamura, and Y. Sano. 1961. A new mutant of dermovaccinia virus. Nature 192: 381-382.
Victoria W. P. and H. M. Michael. 1998. The HIV-1 REV protein. Annu. Rev. Microbiol. 52: 491-532.
Wang, S, and T. Hazelrigg. 1994. Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature. 369: 400-403.
Wolff, B., J. J. Sanglier, and Y. Wang. 1997. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem. Biol. 4. 139-147.
Wyatt, L. S., M. W. Carroll, C. P. Czerny, M. Merchlinsky, J. R. Sisler, and B. Moss. 1998. Marker rescue of the host range restriction defects of modified vaccinia virus Ankara. Virology. 251: 334-342.
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2. 林宏熾 (1997) 〈庇護工場的規劃與功能〉,《就業與訓練》,15(4)期,頁24~31。
3. 林芳玫 (2000) 〈質性研究工作坊系列五--研究者的反省〉,《婦女與兩性研究通訊》,56期,頁17~25。
4. 李永昌 (1999) 〈視覺障礙者也需要支持性就業〉,《特教園丁》,15(2)期,頁24~27。
5. 王麗容 (2002) 〈婦女就業之檢視與政策建議--以再就業為例〉,《就業與訓練》,19(1)期,頁27-32。
6. 洪素英 (1999) 〈身心障礙者就業開發與職務再設計〉,《特教園丁》,14(3)期,頁38~42。
7. 陳秀雅 (1992) 〈按摩業相關問題之探討〉,《特教園丁》,7期,頁45~48。
8. 陳育俊 (1993) 〈德國式殘障庇護工場簡介〉,《就業與訓練》,11(3)期,頁83~89。
9. 萬明美 (1991) 〈視覺障礙者從事按摩業之現況及影響其收入之相關研究〉,《特殊教育學報》,6期,頁1~47。
10. 萬明美,柏廣法 (1999) 〈大學視覺障礙學生畢業後生活狀況之研究〉,《特殊教育研究學刊》,17期,頁107~137。
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12. 趙彥寧 (2000) 〈質性研究工作坊系列四--參與觀察〉,《婦女與兩性研究通訊》,56期,頁9~16。
 
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