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研究生:朱慧娟
研究生(外文):Hui-Chuan
論文名稱:探討D型肝炎病毒抗原之啟動子的專一性
論文名稱(外文):Study the promoter specificity of Hepatitis D Virus antigen
指導教授:許國堂
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學分子毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:102
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在自然狀態下,D型肝炎病毒(Hepatitis Delta Virus,簡稱HDV)只存在於共同感染B型肝炎病毒(Hepatitis B Virus,簡稱HBV)的病人中。HDV由一條1.7 kb的環狀單股RNA所構成,而且HDV是最小且環狀的RNA動物病毒。在一般狀態下,其基因體有70%的鹼基對互補可形成雙股RNA的桿狀結構;在HDV的複製週期中,必須利用HBV所產生的蛋白(HBsAg)當莢膜,才能組裝完整的病毒顆粒。
由先前的研究結果顯示,在HDV的cDNA序列中,不論是基因股或是反基因股都有promoter的活性存在,並且證實於反基因股中有一段29個nt的區域(相對於HDV-1.9的第999~1028個nt),具有內生性promoter的活性;至於基因股之promoter活性的探討,則缺乏有利的證據加以證實與定量。根據本實驗室之前的研究指出,HDV的cDNA反基因股970~1070 nt(promoter-8)為HDV之內生性promoter,且其正反向序列皆具有promoter的活性。本篇實驗即以此正反向HDV promoter-8序列進行研究,利用產生的luciferase及Green Fluorescent Protein (GFP)及Hepatitis delta small antigen(SmAg)作為reporter基因,使用RT-PCR的方法比較promoter-8轉錄能力的差異,初步判定當promoter-8表達自己的基因SmAg時,具有選擇性表現,再以Western blotting的方式,同樣得到驗證。最後,利用更靈敏的免疫螢光染色(Fluorescence stain)與GFP做比較,並於顯微鏡下觀察螢光的表現,進而證實promoter-8改變方向後,對表達SmAg之活性亦隨之改變。總結言之,本實驗說明了HDV的cDNA的反基因股上,promoter-8序列確實對其本身的基因,具有選擇性表現,在promoter的研究中可能是新的發現,在未來或許可以利用此promoter序列,對研究HDV RNA複製及轉譯的機制、基因本身可能具有選擇性表現及細胞內可能存在掃瞄基因蛋白等研究,提供一個新的詮釋。
In a natural setting, hepatitis delta virus (HDV) is only found in patients that are also infected with hepatitis B virus (HBV). The genome of HDV is composed of a circular single-stranded RNA molecule of 1.7-kb that is the smallest, and only known circular RNA genome of the aminals RNA virus. Sequence analysis of the HDV genome revealed that as many as 70% of nucleotides have to undergo intramolecular base pairing, thus allowing the RNA to fold into a rod-like structure. HDV RNA genome are assembled using the envelope protein (HBsAg) of HBV. From previously study indicated that HDV cDNA contains endogenous promoters both in genomic and antigenomic strands. It has been shown that the antigenomic cDNA promoter-like sequence of HDV was located to a 29-nucleotide region (corresponding to HDV-1.9 nucleotides 999 ~ 1028). Whereas the promoter activity of the genomic HDV cDNA was still unclear. In ours previously study, we known that anti-genomic HDV的cDNA has an endogenous promoter activity and is located in nucleotide 970 ~ 1070(promoter-8). The activity of this promoter is not restricted by its orientation. In this study, we use luciferase and Green Fluorescent Protein (GFP) as reporter genes that compare with HDV SmAg gene to determine the promoter-8 activity. The results of RT-PCR show this promoter-8 has specific-orientation activity for SmAg. We also use western blotting to comfirm this result. Furthermore, we use Immunofluorescence stain to demonstrate the difference by staining HDV SmAg. Taken together, we propose that promoter-8 has SmAg specific expression activity. These results may have implications for studying HDV RNA replication and transcription mechanisms.
壹、中文摘要 1
貮、英文摘要(Abstract) 3
參、縮寫表(Abbreviation) 5
肆、緖論(Introduction) 6
一、D型肝炎病毒之發現: 6
二、D型肝炎病毒之結構: 7
三、HDV之基因體(genome)構造 8
四、D型肝炎病毒抗原(HDAg) 9
五、HDAg的結構 10
六、HDV RNA的複製模式 13
七、HDV 複製生活史 ( Replication cycle ) 15
八、HDV RNA promoter的特性 18
伍、研究動機(Motivation) 20
陸、實驗材料與方法(Materials and Methods) 22
一、實驗材料(Materials) 22
(一)藥品試劑(Drugs and reagents) 22
(二)細胞株與質體 23
(三)實驗設備 (Instruments) 24
二、實驗方法(Methods) 25
(一)各個質體之構築 25
(二)細胞培養與分盤 37
(三)細胞轉染作用(Transfection) 39
(四)α-amanitin加藥實驗 41
(五)細胞RNA的萃取 41
(六)DNase I digestion處理細胞total RNA 42
(七)cDNA的合成與鏈聚合酵素連鎖反應 43
(八)、自動定序反應的製備(autosequence) 44
(九)西方墨漬法(Western blotting) 45
(十)免疫螢光分析(Immunostaing) 49
柒、結果(Results) 51
一、構築pGL 3-Promoter-8/SmAg質體 52
二、以RT-PCR方式確認只有反向pGL3-Promoter-8/ SmAg質體具有轉錄HDV SmAg序列之功能表現 53
三、以RT-PCR方式確認pGL3-Promoter-8/ luciferase質體,不論Promoter-8方向皆具有轉錄luciferase序列之功能表現 53
四、以co-transfection方式,經RT-PCR確認不同方向的Promoter-8可表現luciferase序列;但只有反向Promoter-8 具SmAg序列之功能表現 55
五、構築pGL3-Promoter-8/ GFP質體 57
六、以co-transfection方式比較正反向pGL3-Promoter-8/ GFP質體及pGL3-Promoter-8/ SmAg質體,其Promoter-8序列之功能表現 58
七、以co-transfection方式,確認正反向pGL3-Promoter-8/ GFP質體及pGL3-Promoter-8/ SmAg質體,其Promoter-8序列在進行轉錄時,對α-amanitin加藥實験之敏感性 59
八、以Western blotting觀察正反向pGL3-Promoter-8 / SmAg質體表現HDV antigen的能力 63
九、以螢光觀察正反向pGL3-Promoter-8 / GFP質體表現GFP之能力 64
十、以免疫螢光染色觀察正反向pGL3-Promoter-8 / SmAg質體表現HDV antigen之能力 65
捌、討論 66
玖、圖表與圖表說明(Figures and Tables) 79
拾、附圖表(Attached graphs and tables) 90
附圖pGL3-Promoter-8/ SmAg、pGL3-Promoter-8/ Luciferase及pGL3-Promoter-8/ GFP質體之primers相對示意圖 90
附表 HDV cDNA、luciferase、GFP及其他primer序列 91
拾壹、參考文獻 92
Beard, M.R., MacNaughton, T.B. and Gowans, E.J. (1996) Identification and characterization of a hepatitis delta virus RNA transcriptional promoter. J Virol, 70, 4986-4995.
Bergmann, K.F. and Gerin, J.L. (1986) Antigens of hepatitis delta virus in the liver and serum of humans and animals. J Infect Dis, 154, 702-706.
Bichko, V., Netter, H.J. and Taylor, J. (1994) Introduction of hepatitis delta virus into animal cell lines via cationic liposomes. J Virol, 68, 5247-5252.
Bichko, V.V., Khudyakov, Y.E. and Taylor, J.M. (1996) A novel form of hepatitis delta antigen. J Virol, 70, 3248-3251.
Bonino, F., Heermann, K.H., Rizzetto, M. and Gerlich, W.H. (1986) Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope. J Virol, 58, 945-950.
Bonino, F., Hoyer, B., Shih, J.W., Rizzetto, M., Purcell, R.H. and Gerin, J.L. (1984) Delta hepatitis agent: structural and antigenic properties of the delta-associated particle. Infect Immun, 43, 1000-1005.
Branch, A.D. and Robertson, H.D. (1984) A replication cycle for viroids and other small infectious RNA''s. Science, 223, 450-455.
Casey, J.L., Brown, T.L., Colan, E.J., Wignall, F.S. and Gerin, J.L. (1993) A genotype of hepatitis D virus that occurs in northern South America. Proc Natl Acad Sci U S A, 90, 9016-9020.
Casey, J.L. and Gerin, J.L. (1995) Hepatitis D virus RNA editing: specific modification of adenosine in the antigenomic RNA. J Virol, 69, 7593-7600.
Chang, M.F., Baker, S.C., Soe, L.H., Kamahora, T., Keck, J.G., Makino, S., Govindarajan, S. and Lai, M.M. (1988) Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity. J Virol, 62, 2403-2410.
Chang, M.F., Chang, S.C., Chang, C.I., Wu, K. and Kang, H.Y. (1992) Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen. J Virol, 66, 6019-6027.
Chang, M.F., Sun, C.Y., Chen, C.J. and Chang, S.C. (1993) Functional motifs of delta antigen essential for RNA binding and replication of hepatitis delta virus. J Virol, 67, 2529-2536.
Chao, M., Hsieh, S.Y. and Taylor, J. (1990) Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication. J Virol, 64, 5066-5069.
Chen, P.J., Chang, F.L., Wang, C.J., Lin, C.J., Sung, S.Y. and Chen, D.S. (1992) Functional study of hepatitis delta virus large antigen in packaging and replication inhibition: role of the amino-terminal leucine zipper. J Virol, 66, 2853-2859.
Chen, P.J., Kalpana, G., Goldberg, J., Mason, W., Werner, B., Gerin, J. and Taylor, J. (1986) Structure and replication of the genome of the hepatitis delta virus. Proc Natl Acad Sci U S A, 83, 8774-8778.
Chou, H.C., Hsieh, T.Y., Sheu, G.T. and Lai, M.M. (1998) Hepatitis delta antigen mediates the nuclear import of hepatitis delta virus RNA. J Virol, 72, 3684-3690.
de Bruin, W., Leenders, W., Kos, T. and Yap, S.H. (1994) In vitro binding properties of the hepatitis delta antigens to the hepatitis B virus envelope proteins: potential significance for the formation of delta particles. Virus Res, 31, 27-37.
Dingle, K., Bichko, V., Zuccola, H., Hogle, J. and Taylor, J. (1998) Initiation of hepatitis delta virus genome replication. J Virol, 72, 4783-4788.
Estell Dumas, Cathy Staedel, Marie Colombat, Sandrine Reigadas, Sandrine Chabas, Therese Astier-Gin, Annie Cahour, Simon Litvak and Michel Ventura. (2003) A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5’ UTR. Nucleic Acids Res, 31, 1275-1281.
Glenn, J.S., Watson, J.A., Havel, C.M. and White, J.M. (1992) Identification of a prenylation site in delta virus large antigen. Science, 256, 1331-1333.
Govindarajan, S., Chin, K.P., Redeker, A.G. and Peters, R.L. (1984) Fulminant B viral hepatitis: role of delta agent. Gastroenterology, 86, 1417-1420.
Gowans, E.J., Baroudy, B.M., Negro, F., Ponzetto, A., Purcell, R.H. and Gerin, J.L. (1988) Evidence for replication of hepatitis delta virus RNA in hepatocyte nuclei after in vivo infection. Virology, 167, 274-278.
Hadler, S.C., Alcala de Monzon, M., Rivero, D., Perez, M., Bracho, A. and Fields, H. (1992) Epidemiology and long-term consequences of hepatitis delta virus infection in the Yucpa Indians of Venezuela. Am J Epidemiol, 136, 1507-1516.
Hoofnagle, J.H. (1989) Type D (delta) hepatitis. Jama, 261, 1321-1325.
Hwang, S.B. and Lai, M.M. (1993a) Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B virus surface antigen. J Virol, 67, 7659-7662.
Hwang, S.B. and Lai, M.M. (1993b) A unique conformation at the carboxyl terminus of the small hepatitis delta antigen revealed by a specific monoclonal antibody. Virology, 193, 924-931.
Imazeki, F., Omata, M. and Ohto, M. (1991) Complete nucleotide sequence of hepatitis delta virus RNA in Japan. Nucleic Acids Res, 19, 5439.
John M. Taylor. (2003) Replication of human hepatitis delta virus: recent developments. TRENDS in Microbiology, 11, 185-190.
Kuo, M.Y., Chao, M. and Taylor, J. (1989) Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen. J Virol, 63, 1945-1950.
Kuo, M.Y., Sharmeen, L., Dinter-Gottlieb, G. and Taylor, J. (1988) Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol, 62, 4439-4444.
Lai, M.M. (1995) The molecular biology of hepatitis delta virus. Annu Rev Biochem, 64, 259-286.
Lazinski, D., Grzadzielska, E. and Das, A. (1989) Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell, 59, 207-218.
Lazinski, D.W. and Taylor, J.M. (1993) Relating structure to function in the hepatitis delta virus antigen. J Virol, 67, 2672-2680.
Lazinski, D.W. and Taylor, J.M. (1994) Expression of hepatitis delta virus RNA deletions: cis and trans requirements for self-cleavage, ligation, and RNA packaging. J Virol, 68, 2879-2888.
Lee, C.Z., Lin, J.H., Chao, M., McKnight, K. and Lai, M.M. (1993) RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication. J Virol, 67, 2221-2227.
Macnaughton, T.B., Beard, M.R., Chao, M., Gowans, E.J. and Lai, M.M. (1993a) Endogenous promoters can direct the transcription of hepatitis delta virus RNA from a recircularized cDNA template. Virology, 196, 629-636.
Macnaughton, T.B., Gowans, E.J., Jilbert, A.R. and Burrell, C.J. (1990) Hepatitis delta virus RNA, protein synthesis and associated cytotoxicity in a stably transfected cell line. Virology, 177, 692-698.
MacNaughton, T.B., Gowans, E.J., McNamara, S.P. and Burrell, C.J. (1991) Hepatitis delta antigen is necessary for access of hepatitis delta virus RNA to the cell transcriptional machinery but is not part of the transcriptional complex. Virology, 184, 387-390.
Macnaughton, T.B., Shi, S.T., Modahl, L.E. and Lai, M.M. (2002) Rolling circle replication of hepatitis delta virus RNA is carried out by two different cellular RNA polymerases. J Virol, 76, 3920-3927.
Macnaughton, T.B., Wang, Y.J. and Lai, M.M. (1993b) Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites. J Virol, 67, 2228-2234.
Makino, S., Chang, M.F., Shieh, C.K., Kamahora, T., Vannier, D.M., Govindarajan, S. and Lai, M.M. (1987) Molecular cloning and sequencing of a human hepatitis delta (delta) virus RNA. Nature, 329, 343-346.
Michael R. Beard, Thomas B. Macnaughton, and Eric J. Gowans* (1996) Identification and characterization of a hepatitis delta virus RNA transcriptional promoter. J Virol, 70, 4986-4995.
Modahl, L.E. and Lai, M.M. (1998) Transcription of hepatitis delta antigen mRNA continues throughout hepatitis delta virus (HDV) replication: a new model of HDV RNA transcription and replication. J Virol, 72, 5449-5456.
Modahl, L.E., Macnaughton, T.B., Zhu, N., Johnson, D.L. and Lai, M.M. (2000) RNA-Dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases. Mol Cell Biol, 20, 6030-6039.
Otto, J.C. and Casey, P.J. (1996) The hepatitis delta virus large antigen is farnesylated both in vitro and in animal cells. J Biol Chem, 271, 4569-4572.
Pohl, C., Baroudy, B.M., Bergmann, K.F., Cote, P.J., Purcell, R.H., Hoofnagle, J. and Gerin, J.L. (1987) A human monoclonal antibody that recognizes viral polypeptides and in vitro translation products of the genome of the hepatitis D virus. J Infect Dis, 156, 622-629.
Polo, J.M., Jeng, K.S., Lim, B., Govindarajan, S., Hofman, F., Sangiorgi, F. and Lai, M.M. (1995) Transgenic mice support replication of hepatitis delta virus RNA in multiple tissues, particularly in skeletal muscle. J Virol, 69, 4880-4887.
Polson, A.G., Bass, B.L. and Casey, J.L. (1996) RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase. Nature, 380, 454-456.
Polson, A.G., Ley, H.L., 3rd, Bass, B.L. and Casey, J.L. (1998) Hepatitis delta virus RNA editing is highly specific for the amber/W site and is suppressed by hepatitis delta antigen. Mol Cell Biol, 18, 1919-1926.
Ponzetto, A., Cote, P.J., Popper, H., Hoyer, B.H., London, W.T., Ford, E.C., Bonino, F., Purcell, R.H. and Gerin, J.L. (1984) Transmission of the hepatitis B virus-associated delta agent to the eastern woodchuck. Proc Natl Acad Sci U S A, 81, 2208-2212.
Ponzetto, A., Negro, F., Popper, H., Bonino, F., Engle, R., Rizzetto, M., Purcell, R.H. and Gerin, J.L. (1988) Serial passage of hepatitis delta virus in chronic hepatitis B virus carrier chimpanzees. Hepatology, 8, 1655-1661.
Rackwitz, H.R., Rohde, W. and Sanger, H.L. (1981) DNA-dependent RNA polymerase II of plant origin transcribes viroid RNA into full-length copies. Nature, 291, 297-301.
Rizzetto, M., Canese, M.G., Arico, S., Crivelli, O., Trepo, C., Bonino, F. and Verme, G. (1977) Immunofluorescence detection of new antigen-antibody system (delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers. Gut, 18, 997-1003.
Rizzetto, M., Canese, M.G., Gerin, J.L., London, W.T., Sly, D.L. and Purcell, R.H. (1980a) Transmission of the hepatitis B virus-associated delta antigen to chimpanzees. J Infect Dis, 141, 590-602.
Rizzetto, M., Hoyer, B., Canese, M.G., Shih, J.W., Purcell, R.H. and Gerin, J.L. (1980b) delta Agent: association of delta antigen with hepatitis B surface antigen and RNA in serum of delta-infected chimpanzees. Proc Natl Acad Sci U S A, 77, 6124-6128.
Roggendorf, M., Pahlke, C., Bohm, B. and Rasshofer, R. (1987) Characterization of proteins associated with hepatitis delta virus. J Gen Virol, 68 ( Pt 11), 2953-2959.
Ryu, W.S., Netter, H.J., Bayer, M. and Taylor, J. (1993) Ribonucleoprotein complexes of hepatitis delta virus. J Virol, 67, 3281-3287.
Sheu, G.T. and Lai, M.M. (2000) Recombinant hepatitis delta antigen from E. coli promotes hepatitis delta virus RNA replication only from the genomic strand but not the antigenomic strand. Virology, 278, 578-586.
Tai, F.P., Chen, P.J., Chang, F.J. and Chen, D.S. (1993) Hepatitis delta virus cDNA monomer can be used in transfection experiments to initiate viral RNA replication. Virology, 197, 137-142.
Wang, H.W., Chen, P.J., Lee, C.Z., Wu, H.L. and Chen, D.S. (1994) Packaging of hepatitis delta virus RNA via the RNA-binding domain of hepatitis delta antigens: different roles for the small and large delta antigens. J Virol, 68, 6363-6371.
Wang, J.G. and Lemon, S.M. (1993) Hepatitis delta virus antigen forms dimers and multimeric complexes in vivo. J Virol, 67, 446-454.
Wang, K.S., Choo, Q.L., Weiner, A.J., Ou, J.H., Najarian, R.C., Thayer, R.M., Mullenbach, G.T., Denniston, K.J., Gerin, J.L. and Houghton, M. (1986) Structure, sequence and expression of the hepatitis delta (delta) viral genome. Nature, 323, 508-514.
Wu, H.N. and Lai, M.M. (1989) Reversible cleavage and ligation of hepatitis delta virus RNA. Science, 243, 652-654.
Wu, J.C., Chiang, T.Y. and Sheen, I.J. (1998) Characterization and phylogenetic analysis of a novel hepatitis D virus strain discovered by restriction fragment length polymorphism analysis. J Gen Virol, 79 ( Pt 5), 1105-1113.
Wu, J.C., Choo, K.B., Chen, C.M., Chen, T.Z., Huo, T.I. and Lee, S.D. (1995) Genotyping of hepatitis D virus by restriction-fragment length polymorphism and relation to outcome of hepatitis D. Lancet, 346, 939-941.
Xia, Y.P. and Lai, M.M. (1992) Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen. J Virol, 66, 6641-6648.
Yamaguchi, Y., Filipovska, J., Yano, K., Furuya, A., Inukai, N., Narita, T., Wada, T., Sugimoto, S., Konarska, M.M. and Handa, H. (2001) Stimulation of RNA polymerase II elongation by hepatitis delta antigen. Science, 293, 124-127.
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