請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38585
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳培哲(Pei-Jer Chen) | |
dc.contributor.author | Jyun-Yuan Huang | en |
dc.contributor.author | 黃鈞源 | zh_TW |
dc.date.accessioned | 2021-06-13T16:38:15Z | - |
dc.date.available | 2005-07-11 | |
dc.date.copyright | 2005-07-11 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-05 | |
dc.identifier.citation | Acs, G., Sells, M.A., Purcell, R.H., Price, P., Engle, R., Shapiro, M., Popper, H. (1987). 'Hepatitis B virus produced by transfected Hep G2 cells causes hepatitis in chimpanzees.' Proc. Natl. Acad. Sci. U.S.A. 84(13): p4641-4644.
Albin, C., Robinson ,W.S. (1980). 'Protein kinase activity in hepatitis B virus.' J. Virol. 34(1): p297-302. Anderson, N. L. (2002). 'The Human Plasma Proteome: History, Character, and Diagnostic Prospects.' Mol. Cell Proteomics 1(11): p845-867. Beasley, R. (1988). 'Hepatitis B virus: the major etiology of hepatocellular carcinoma.' Cancer 61(10): p1942-1956. Bechtel, J. T., Winant ,R.C., Ganem ,D. (2005). 'Host and viral proteins in the virion of Kaposi's sarcoma-associated herpesvirus.' J. Virol. 79(8): p4952-4964. Beck, J.,Nassal, M., (2003). 'Efficient Hsp90-independent in vitro activation by Hsc70 and Hsp40 of duck hepatitis B virus reverse transcriptase, an assumed Hsp90 client protein.' J. Biol. Chem. 278(38): p36128-36138. Bourbonnais, R., Guevin ,R.M., Delvin ,E.E. (1975). 'Australia antigen 'ad' and 'ay' subtypes. Purification and partial characterization.' Vox Sang. 29(4): p269-279. Cantin , R., Methot ,S., Tremblay ,M.J. (2005). 'Plunder and stowaways: incorporation of cellular proteins by enveloped viruses.' J. Virol. 79(11): p6577-6587. Cho, G., Park ,S.G., Jung ,G. (2000). 'Localization of HSP90 Binding Sites in the Human Hepatitis B Virus Polymerase.' Biochem. Biophys. Res. Commun. 269(1): p191-196. Crowther, R. A., Kiselev, N.A., Bottcher, B., Berriman, J.A., Borisova, G.P., Ose, V., Pumpens, P., (1994). 'Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy.' Cell 77(6): p943-950. Dane,D. S., Cameron,C.H., Briggs ,M. (1970). 'Virus- like particles in serum of patients with Australia-antigen-associated hepatitis.' Lancet 1(7649): p695-698. Daub,H., Blencke, S., Habenberger, P., Kurtenbach, A., Dennenmoser, J., Wissing, J., Ullrich, A., Cotten, M. (2002). 'Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein.' J. Virol. 76(16): p8124-8137. Effersoe,P. (1951). 'Polyfractionation of the serum proteins by means of salting-out with ammonium sulphate and quantitative determination of the fractions by means of spectrophotometry.' Scand. J. Clin. Lab. Invest. 3(1): p6-13. Fauvel,M. L., Babiuk,E.T.,Sheaff,L.S. (1975). 'Preparation od a dane core (hepatitis B )antigen from human plasma.' Can. J. Microbiol. 21(6): p905-910. Feitelson,M. A., Marion ,P.L., Robinson ,W.S. (1982). 'Core particles of hepatitis B virus and ground squirrel hepatitis virus. II. Characterization of the protein kinase reaction associated with ground squirrel hepatitis virus and hepatitis B virus.' J. Virol. 43(2): p741-748. Franke,E. K., Yuan ,H.E., Luban ,J. (1994). 'Specific incorporation of cyclophilin A into HIV-1 virions.' Nature. 372(6504): p359-362. Gerlich,W. H., Goldmann ,U., Muller ,R., Stibbe ,W., Wolff ,W. (1982). 'Specificity and localization of the hepatitis B virus-associated protein kinase.' J. Virol. 42(3): p761-766. Gurer,C., Cimarelli ,A., Luban ,J. (2002). 'Specific incorporation of heat shock protein 70 family members into primate lentiviral virions.' J. Virol. 76(9): p4666-4670. Hatton,T., Zhou ,S., Standring ,D.N. (1992). 'RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication.' J. Virol. 66(9): p5232-5241. Heermann, K. H., Gerlich ,W.H., Chudy ,M., Schaefer ,S., Thomssen ,R. (1999). 'Quantitative detection of hepatitis B virus DNA in two international reference plasma preparations. Eurohep Pathobiology Group.' J. Clin. Microbiol. 37(1): p68-73. Heermann, K. H., Goldmann, U., Schwartz, W., Seyffarth, T., Baumgarten,H.,Gerlich,W.H. (1984). 'Large surface protein of hepatitis B virus containing the pre-s sequence.' J. Virol. 52(2): p396-402. Heermann,K. H., Kruse,F. , Seifer, M. ,Gerlich,W. H. (1987). 'Immunogenicity of the gene S and Pre-S domains in hepatitis B virions and HBsAg filaments.' Intervirology 28(1): p14-25. Hilleman,M. R., McAleer ,W.J., Buynak ,E.B., McLean ,A.A. (1983). 'The preparation and safety of hepatitis B vaccine.' J .Infection. 7(Suppl 1): p3-8. Hu,J., Anselmo ,D. (2000). 'In Vitro Reconstitution of a Functional Duck Hepatitis B Virus Reverse Transcriptase: Posttranslational Activation by Hsp90.' J. Virol. 74(24): p11447-11455. Hu,J., Flores ,D., Toft ,D., Wang ,X., Nguyen ,D. (2004). 'Requirement of Heat Shock Protein 90 for Human Hepatitis B Virus Reverse Transcriptase Function.' J. Virol. 78(23): p13122-13131. Hu,J., Seeger,C. (1996). 'Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.' Proc. Natl. Acad. Sci. USA 93(3): p1060-1064. Hu,J., Toft, D. O. ,Seeger,C. (1997). 'Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids.' EMBO J. 16(1): p59-68. Huovila ,A. P., Eder ,A.M., Fuller ,S.D. (1992). 'Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment.' J .Cell .Biol 118(6): p1305-1320. Kann, M., Gerlich ,W.H. (1994). 'Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus.' J. Virol. 68(12): p7993-8000. Kann, M., Sodeik, B., Vlachou ,A., Gerlich ,W.H., Helenius ,A. (1999). 'Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex.' J. Cell. Biol. 145(1): p45-55. Kann, M., Thomssen ,R., Kochel ,H.G., Gerlich ,W.H. (1993). 'Characterization of the endogenous protein kinase activity of the hepatitis B virus.' Arch. Virol. Suppl. 8: p53-62. Kao, S., Khan ,M.A., Miyagi ,E., Plishka ,R., Buckler-White ,A., Strebel ,K. (2003). 'The human immunodeficiency virus type 1 Vif protein reduces intracellular expression and inhibits packaging of APOBEC3G (CEM15), a cellular inhibitor of virus infectivity.' J. Virol. 77(21): p11398-11407. Kau, J. H., Ting ,L.P. (1998). 'Phosphorylation of the core protein of hepatitis B virus by a 46-kilodalton serine kinase.' J. Virol. 72(5): p3796-3803. Kenney,J. M., von Bonsdorff, C.H., Nassal, M., Fuller, S.D., (1995). 'Evolutionary conservation in the hepatitis B virus core structure: comparison of human and duck cores.' Structure 3(10): p1009-1119. Krone, B., Lenz,A., Heermann ,K.H., Seifer ,M., Lu ,X.Y., Gerlich ,W.H. (1990). 'Interaction between hepatitis B surface proteins and monomeric human serum albumin.' Hepatology. 11(6): p1050-1056. Lanford, R. E., Notvall ,L. (1990). 'Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity.' Virology 176(1): p222-233. Lin, J. Y., Hsieh ,Y.S., Chu ,S.C. (1987). 'The isolation and purification of pre-S2 containing hepatitis B virus surface antigen by chemical affinity chromatography.' Appl. Biochem .Biotechnol. 15(Appl. Biochem. Biotechnol.): p255-263. Mangeat, B., Turelli ,P., Caron ,G., Friedli ,M., Perrin ,L., Trono ,D. (2003). 'Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts.' Nature. 424(6944): p99-103. Marion, P. L., Oshiro ,L.S., Regnery ,D.C., Scullard ,G.H., Robinson ,W.S. (1980). ' A virus in Beechey ground squirrels that is related to hepatitis B virus of humans.' Proc. Natl. Acad. Sci. USA 77(5): p2941-2945. Mason , W. S., Seal ,G., Summers ,J. (1980). 'Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus.' J. Virol. 36(3): p829-836. Miller, R. H., Marion ,P.L., Robinson ,W.S. (1984). 'Hepatitis B viral DNA-RNA hybrid molecules in particles from infected liver are converted to viral DNA molecules during an endogenous DNA polymerase reaction.' Virology 139(1): p64-72. Misumi, S., Fuchigami ,T., Takamune ,N., Takahashi ,I., Takama ,M., Shoji ,S. (2002). 'Three isoforms of cyclophilin A associated with human immunodeficiency virus type 1 were found by proteomics by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry.' J. Virol. 76(19): p10000-10008. MR, H. (2001). 'Overview of the pathogenesis, prophylaxis and therapeusis of viral hepatitis B, with focus on reduction to practical applications.' Vaccine 19: 1837-1848. Murakami, S. (2001). 'Hepatitis B virus X protein: a multifunctional viral regulator.' J.Gastroenterol.36: p651-60. Nassal , M., Schaller ,H. (1993). 'Hepatitis B virus replication.' Trends.Microbiol.1: p221-228. Nassal , M., Schaller,H. (1996). 'Hepatitis B virus replication--an update.' J.Viral.Hepat.3(5): p217-226. Onodera, S., Ohori ,H., Yamaki ,M., Ishida ,N. (1982). 'Electron microscopy of human hepatitis B virus cores by negative staining- carbon film technique.' J.Med.Virol.10(2): p147-155. Ott, D. E. (2002). 'Potential roles of cellular proteins in HIV-1.' Rev.Med.Virol.12(6): p359-374. Ou, J. H., Laub ,O., Rutter ,W.J. (1986). 'Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen.' Proc. Natl. Acad. Sci. U.S.A. 83(6): 1578-1582. Parekh, S., Zoulim ,F., Ahn ,S.H., Tsai ,A., Li ,J., Kawai ,S., Khan ,N., Trepo ,C., Wands ,J., Tong ,S. (2003). 'Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants.' J.Virol.77(12): p6601-6612. Park, J. H., Choi ,E.A., Cho ,E.W., Lee ,Y.J., Park ,J.M., Na ,S.Y., Kim ,K.L. (2000). 'Detection of cellular receptors specific for the hepatitis B virus preS surface protein on cell lines of extrahepatic origin.' Biochem. Biophys. Res. Commun. 277(1): p246-254. Park, S. G., Jung ,G. (2001). 'Human Hepatitis B Virus Polymerase Interacts with the Molecular Chaperonin Hsp60.' J. Virol. 75(15): p6962-6968. Park, S. G., Kyung Rho ,J., Jung ,G. (2002). 'Hsp90 makes the human HBV Pol competent for in vitro priming rather than maintaining the human HBV Pol/pregenomic RNA complex.' Arch. Biochem. Biophys. 401(1): p99-107. Parkin, D. M., Pisani, P., Ferlay,J. (1999). 'Estimates of the worldwide incidence of 25 major cancers in 1990.' Int. J. Cancer 80(6): 827-841. Petit, M. A., Capel ,F., Pillot ,J. (1985). 'Demonstration of a firm association between hepatitis B surface antigen proteins bearing polymerized human albumin binding sites and core-specific determinants in serum hepatitis B viral particles.' Mol. Immunol. 22(11): p1279-1287. Pollack, J. R., Ganem,D. (1993). 'An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation.' J. Virol. 67(6): p3254-3263. Pugh, J., Zweidler A, Summers J (1989). 'Characterization of the major duck hepatitis B virus core particle protein.' J. Virol. 63(3): p1371-1376. Rall, L. B., Standring ,D.N., Laub ,O., Rutter ,W.J. (1983). 'Transcription of hepatitis B virus by RNA polymerase II.' Mol. Cell .Biol. 3(10): p1766-73. Roossinck , M. J., Siddiqui ,A. (1987). 'In vivo phosphorylation and protein analysis of hepatitis B virus core antigen.' J. Virol. 61(4): p955-61. Ryu, C. J., Cho ,D.Y., Gripon ,P., Kim ,H.S., Guguen-Guillouzo ,C., Hong ,H.J. (2000). 'An 80-kilodalton protein that binds to the pre-S1 domain of hepatitis B virus.' J.Virol. 74(1): p110-116. Schagen, F. H., Rademaker ,H.J., Rabelink ,M.J., van Ormondt ,H., Fallaux ,F.J., van der Eb ,A.J., Hoeben ,R.C. (2000). 'Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield.' Gene Ther. 7(18): p1570-1574. Schlicht , H., Bartenschlager ,R., Schaller ,H. (1989). 'The duck hepatitis B virus core protein contains a highly phosphorylated C terminus that is essential for replication but not for RNA packaging.' J. Virol. 63(7): p2995-3000. Schmitt , S., Glebe ,D., Tolle ,T.K., Lochnit ,G., Linder ,D., Geyer ,R., Gerlich ,W.H. (2004). 'Structure of pre-S2 N- and O-linked glycans in surface proteins from different genotypes of hepatitis B virus.' J. Gen. Virol. 85(7): p2045-2053. Seeger, C., Mason,W.S. (2000). 'Hepatitis b virus biology.' Microbiol. Mol. Biol. Rev. 64(1): p51-68. Sen, A. (1981). 'Purified low-molecular-weight protein kinase from murine sarcoma virus particles catalyzes tyrosine phosphorylation endogenously but phosphorylates cellular proteins at serine.' J. Virol. 39(2): p612-624. Sheehy, A. M., Gaddis ,N.C., Choi ,J.D., Malim ,M.H. (2002). 'Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein.' Nature. 418(6898): p646-650. Sprengel, R., Kaleta, E. F. ,Will ,H. (1988). 'Isolation and characterization of a hepatitis B virus endemic in herons.' J. Virol. 62(10): p932-937. Stannard , L. M., Hodgkiss, M. (1979). 'Morphological Irregularities in Dane Particles Cores.' J. Gen. Virol. 45(2): p509-514. Stibbe , W., Gerlich ,W.H. (1982). 'Variable protein composition of hepatitis B surface antigen from different donors.' Virology 123(2): p436-442. Summers , J., Mason ,W.S. (1982). 'Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate.' Cell 29(2): p403-415. Summers , J., Smolec, J. and Snyder,R. (1978). 'A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks.' Proc. Natl. Acad. Sci. USA 75(9): p4533?537. Svarovskaia, E. S., Xu ,H., Mbisa ,J.L., Barr ,R., Gorelick ,R.J., Ono ,A., Freed ,E.O., Hu ,W.S., Pathak ,V.K. (2004). 'Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs.' J. Biol. Chem. 279(34): p35822-35828. Thali , M., Bukovsky ,A., Kondo ,E., Rosenwirth ,B., Walsh ,C.T., Sodroski ,J., Gottlinger ,H.G. (1994). 'Functional association of cyclophilin A with HIV-1 virions.' Nature.372(6504): p363-365. Trepanier , P., Payment ,P., Trudel ,M. (1981). 'Concentration of human respiratory syncytial virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration.' J. Virol. Methods 3(4): p201-211. Tuttleman , J. S., Pourcel ,C., Summers ,J. (1986). 'Formation of the pool of covalently closed circular viral DNA in hepadnavirus- infected cells.' Cell 47(3): p451-460. Varnum , S. M., Streblow ,D.N., Monroe ,M.E., Smith ,P., Auberry ,K.J., Pasa-Tolic ,L., Wang ,D., Camp ,D.G. 2nd, Rodland ,K., Wiley ,S., Britt ,W., Shenk ,T., Smith ,R.D., Nelson ,J.A. (2001). 'Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome.' J. Virol. 78(20): p10960-10966. Wang, G. H., Zoulim, F.,Leber, E. H. , Kitson,J., Seeger,C. (1994). 'Role of RNA in enzymatic activity of the reverse transcriptase of hepatitis B viruses.' J. Virol. 68(12): p8437-8442. Wang, X., Grammatikakis, N., Hu, J., (2002). 'Role of p50/CDC37 in hepadnavirus assembly and replication.' J. Biol. Chem. 277(27): p24361-24367. Wei , Y., Tavis ,J.E., Ganem ,D. (1996). 'Relationship between viral DNA synthesis and virion envelopment in hepatitis B viruses.' J. Virol. 70(9): p6455-6458. Wen, L. T., Henneberger ,M., Nguyen ,N., McPherson ,R.A. (1994). 'Quantitation of hepatitis B virus DNA in serum by ammonium sulfate precipitation and molecular hybridization.' J .Clin. Lab. Anal. 8(1): p44-50. Will , H., Reiser, W., Weimer ,T., Pfaff ,E., Buscher ,M., Sprengel ,R., Cattaneo ,R.,Schaller ,H. (1987). 'Replication strategy of human hepatitis B virus.' J. Virol. 61(3): p904-911. Wingfield , P. T., Stahl ,S.J., Williams ,R.W., Steven ,A.C. (1995). 'Hepatitis core antigen produced in Escherichia coli: subunit composition, conformational analysis, and in vitro capsid assembly.' Biochemistry 34(15): p4919-4932. Wu , T. T., Coates ,L., Aldrich ,C.E., Summers ,J., Mason ,W.S. (1990). 'In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway.' Virology 175(1): p255-261. Wynne, S. A., Crowther, R.A., Leslie, A.G., (1999). 'The crystal structure of the human hepatitis B virus capsid.' Mol.Cell 3(6): p771-780. Yaginuma,K., Shirakata, Y., Kobayashi, M., Koike, K., (1987). 'Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA.' Proc.Natl. Acad.Sci.U.S.A. 84(9): p2678-2682. Yeh , C. T., Ou ,J.H. (1991). 'Phosphorylation of hepatitis B virus precore and core proteins.' J. Virol. 65(5): p2327-2331. Yeh , S. H., Tsai ,C.Y., Kao ,J.H., Liu ,C.J., Kuo ,T.J., Lin ,M.W., Huang ,W.L., Lu ,S.F., Jih ,J., Chen ,D.S., Chen ,P.J. (2004). 'Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis.' J .Hepatol. 41(4): p659-666. Zhou, S., Standring, D.N., (1992). 'Hepatitis B virus capsid particles are assembled from core-protein dimer precursors.' Proc. Natl. Acad. Sci. U.S.A. 89(21): p10046-10050. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38585 | - |
dc.description.abstract | 人類B型肝炎病毒是帶有外套膜的DNA病毒,內有一個二十面體的核心顆粒。在受到病毒感染的肝細胞中,核心顆粒的組裝是在細胞質中發生,組裝的過程中伴隨著包裹前基因體RNA與病毒DNA聚合酶的結合複合體和一些細胞因子像是熱休克蛋白與會磷酸化核心蛋白的磷酸激酶。磷酸激酶的活性在其他種類的肝炎病毒中也有被發現,並有一些研究鑑定出具有此活性之蛋白質。而熱休克蛋白像是Hsp90、Hsp70、p23都有報導說其對於病毒進行protein priming與病毒顆粒組裝是必需的。這些細胞蛋白質似乎在HBV生活史中,都扮演很重要的角色。因此我們很有興趣去找尋在病毒顆粒中,被特定包裹於其中的細胞蛋白質。
為了能取得大量的病毒顆粒,於是我們選擇使用HBV感染者的血清來當做純化病毒的來源。利用不同濃度硫酸銨沉澱與sucrose cushion來去除血清中大量的血清蛋白像是白蛋白(albumin)。再接著使用蔗糖濃度梯度與氯化銫密度梯度,成功的分離出高純度的病毒顆粒,並同時將表面抗原顆粒與鄧氏顆粒分離出來。我們再利用電子顯微鏡和南方墨點法確認病毒顆粒的結構與鄧氏顆粒內的DNA基因體。最後,我們建立了一套能夠從血清中獲得高純度的病毒顆粒純化方法。 | zh_TW |
dc.description.abstract | The hepatitis B virus (HBV) is an enveloped DNA virus with an icosahedral nucleocapsid. In an HBV infected cell, assembly of nucleocapsid occurs in the cytosol and is accompanied by packaging of the viral pregenomic RNA bound to the viral polymerase protein together with cellular protein such as chaperons and protein kinases phosphorylating core protein. This kinase activity is also conserved in other members of hepadnaviridae and plays an important role in viral life cycle. Besides, chaperons like hsp90,hsp70 ,p23 are required for protein priming and encapsidation .Therefore , we are interested in finding out specific cellular proteins incorporated into HBV virions.
In order to obtain enough quantity of hepatitis B virus, we chose serum from HBV-infected patients as materials for virus purification. Ammonium sulfate precipitation and sucrose cushion were used to remove high abundant serum proteins, like albumins. After sucrose gradient and cesium chloride gradient, Dane particles and empty particles were obtained with high purity. Then we utilized electron microscopy and Southern-blotting to confirm the structure of particles and virus genome inside Dane particles. Finally, we set up a system for virus purification from serum with high purity. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T16:38:15Z (GMT). No. of bitstreams: 1 ntu-94-R92445108-1.pdf: 1623979 bytes, checksum: 966b217f45147c023f3b72a0d6a6166f (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 中文摘要……………………………………………………………… .Ⅱ
英文摘要……………………………………………………………… .Ⅲ 目錄…………………………………………………………………… .Ⅳ 表次……………………………………………………………………. Ⅴ 圖次……………………………………………………………………. Ⅵ 壹、序論 B型肝炎病毒簡介…………………………………… .1 B型肝炎病毒的基因與蛋白質……………………… .2 B型肝炎病毒結構…………………………………… .3 B型肝炎病毒的生活史………………………………..4 細胞蛋白與病毒蛋白之交互作用…………………….5 研究目的……………………………………………… 9 貳、材料與方法 大腸桿菌之轉形(Transformation)………………… 10 HBV copy number 標準品之製備………………….. 10 HBV病毒顆粒之純化………………………………… 11 HBV病毒DNA之萃取……………………………….....12 定量即時聚合酶連鎖反應…………………………… 13 TCA(Trichloroacetic acid)沉澱法……………… 14 南方墨點法(Southern blotting)………………… 14 西方墨點法(Western blotting)…………………… 16 銀染法(Silver stain)……………………………… 17 穿透式電子顯微鏡及負染法………………………….17 參、結果 (一) 所需病毒之來源……………………………………………18 (二)硫酸銨沉澱病毒顆粒……………………………………….18 (三)蔗糖濃度梯度分離不同大小的病毒顆粒………………….19 (四)氯化銫密度梯度分離鄧氏顆粒與次病毒顆粒…………….20 (五)證明鄧氏顆粒存在………………………………………….21 肆、討論……………………………………………………………….23 附表…………………………………………………………………….27 附圖…………………………………………………………………… 30 伍、參考文獻………………………………………………………….44 表次 表一、不同硫酸銨濃度中,表面抗原與DNA的含量………28 表二、病毒純化之回收率……………………………………29 圖次 圖一、B型肝炎病毒顆粒種類及結構…………………………………31 圖二、B型肝炎病毒生活史……………………………………………32 圖三、肝炎病毒核心顆粒組裝之假設模型……………………………33 圖四、不同的硫酸銨濃度沉澱血清內的蛋白…………………………34 圖五、用不連續蔗糖濃度梯度純化HBV病毒顆粒…………………..35 圖六、用氯化銫密度梯度分離出次病毒顆粒…………………………36 圖七、氯化銫fraction中的蛋白質分析(Ⅰ)……………………… 37 圖八、用氯化銫密度梯度分離出鄧氏顆粒……………………………38 圖九、氯化銫fraction中的蛋白質分析 (Ⅱ)…………………… .39 圖十、氯化銫fraction中的蛋白質分析 (Ⅲ)………………………40 圖十一、濃縮後的病毒液與病毒蛋白分析……………………………41 圖十二、電子顯微鏡觀察結果…………………………………………42 圖十三、南方墨點法偵測病毒基因體…………………………………43 | |
dc.language.iso | zh-TW | |
dc.title | 大量B型肝炎病毒顆粒純化方法之發展 | zh_TW |
dc.title | Purification of HBV Particles from Patients' Serum | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 周綠蘋(Lu-Ping CHOW),董馨蓮(Shin-Lian Doong),許萬枝(Wan-Jr Syu) | |
dc.subject.keyword | B型肝炎病毒,純化,病毒顆粒, | zh_TW |
dc.subject.keyword | Hepatitis B virus,purification,virus particle, | en |
dc.relation.page | 58 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-05 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-94-1.pdf 目前未授權公開取用 | 1.59 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。