臺灣博碩士論文加值系統

　　C型肝炎病毒是引起輸血後之非A非B型肝炎的主要因素，其與肝癌的
發生有密切關聯。C型肝炎病毒為單一正股RNA病毒，長度約9.5kb，負責
轉譯一條3010~3033個胺機酸的多蛋白，經切割後成為10個蛋白，依序為
NH2-C-E1-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH。病毒多蛋白C-NS2區
域是由宿主位於內質網的訊號傳遞蛋白所切割，而NS3-4A，NS4A-4B，
NS4B-5A，NS5A-5B切點是由NS3 N端之類似胰蛋白之絲胺酸蛋白所切
割，其活性中心為Ser1165。NS2-NS3切點則由病毒所轉譯的第二個蛋白
包括大部分NS2區域及完整的NS3蛋白區域之NS2-3蛋白所切割。
NS2-NS3切點若未經切割，則NS3蛋白無法有效作用於其後之切點。因此
C型肝炎病毒多蛋白的加工過程中，NS2/3的水解反應是快速且早期發生的
，然而其作用機轉目前尚不清楚。
本研究論文中利用桿狀病毒及大腸桿菌蛋白質表現系統來表現C型肝
炎病毒NS2-3蛋白最小功能區域的胺基酸片段827-1207，獲得大量
NS2-3蛋白質進一步研究其功能、活性和結構等特性。在桿狀病毒表現系
統方面，目前已製備大量高效價重組病毒液，且重組蛋白質已找得到最佳生
產條件。昆蟲細胞所表現之重組蛋白質經由電泳分析及西方墨點法偵測，
結果發現一25kDa NS2蛋白存在證實此系統所表現之NS2-3蛋白質具有催化
活性，且自切出此25kDa之NS2蛋白。並且利用鎳離子螯合親和性樹脂管柱
進行重組蛋白質的純化。
在大腸桿菌表現系統中，所表現之重組蛋白多以不可溶的型式存在，
這些包涵體可以6M或8M之尿素溶解之，並且經實驗證實其在pH值大於8之
鹼性緩衝液中有較好的溶解度。此外，重組蛋白質所帶有之棕色可能為
NS3金屬結合區域與特殊金屬離子的結合，而重組蛋白質NS2-3經大量誘發
後發現伴隨著一個16kDa蛋白質產生。
綜合以上結果，我們可經由桿狀病毒表現系統得到具有酵素活性之
C型肝炎病毒NS2-3蛋白，並且利用大腸桿菌表現系統表現大量重組蛋白
質NS2-3，NS2及NS3。

Hepatitis C virus (HCV) is a major etiologic agent of
posttransfusion non-A, non-B hepatitis and is also related to
the development of hepatocellular carcinoma. The enveloped
virion contains a single-stranded positive-sense RNA genome of
about 9.5 kb and encodes a polyprotein precursor of 3010 to 3033
amino acids with the following order:
C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The maturation of the
C-NS2 region was mediated by host cell signal peptidases located
in the lumen of the endoplasmic reticulum. A trypsin-like
serine protease with the active site at position Ser-1165
encoded in the N-terminal region of the NS3 gene is responsible
for cleavage at the NS3-4A, NA4A-4B, NS4B-5A, and NS5A-5B
junctions. A second HCV-encoded protease including most of the
NS2 region and the entire NS3 serine protease domain (aa.827 to
1207) is essential for processing at the NS2-3 junction. The
cleavage at the NS2-3 junction is an early and fast event in
proteolytic processing of the HCV precursor polyprotein since
the presence of uncleaved NS2 sequences on the enzyme impeded
NS3-mediated proteolysis at downstream sites in the polyprotein.
However, the molecular mechanism of the proteolytic processing
at the NS2-3 site is poorly understood.
In this study, we used the baculovirus and the E. coli
expression systems to express the HCV NS2-3 protease
(aa827-1207, the minimal region of the polyprotein required for
efficient cleavage at the NS2-3 site) in order to study the
biochemical, enzymatic, and structural properties of the
protein. A large-scale, high-titer recombinant viral stock was
prepared and the conditions for the expression of the
recombinant protein were optimized. The recombinant protein
expressed in insect cells was analyzed by SDS-PAGE followed by
immunoblotting. Immunoactive protein bands of 43kDa NS2-3 and
25 kDa NS2 were obtained indicating that the recombinant protein
might have catalytic activity and the 43kDa NS2-3 was converted
to form 25kDa NS2 by autolysis. The nickel-agarose affinity
column was used to purify the recombinant protein under the
denatured condition.
In the E. coli expression system the target recombinant
proteins were overexpressed as an insoluble form. The inclusion
bodies could be dissolved in 6M or 8M urea and had better
solubility in basic buffer of pH>8. The inclusion bodies had
brown colors indicating that the recombinant protein might bind
with a specific metal ion. A heat shock protein of 16 kDa was
also induced when the recombinant NS2-3 protein was
overexpressed in E. coli .
In conclusion, enzymatically active NS2-3 protease could
be prepared in baculovirus expression system. The large amount
of recombinant NS2-3, NS2 and NS3 proteins could be obtained by
express with E. coli expression system.

目 錄
中文摘要 I
英文摘要 II
誌謝 IV
目錄 V
表目錄 VI
圖目錄 VII
儀器 VIII
藥品 X
縮寫檢索表 XIII
緒論 1
材料與方法 12
HCV NS2-3基因核酸序列分析 12
C型肝炎病毒基因之選殖 16
桿狀病毒表現系統 (Baculovirus expression system) 20
大腸桿菌蛋白質表現系統( E. coli expression system ) 38
結果 46
討論 53
參考文獻 61
表 68
圖 70
附錄 98

Alter, MJ Risk factors for acute non-A, non-B hepatitis in the Untied States and association with hepatitis C virus infection. JAMA 164:2231 (1990)
Amberg SM, Nestoeowicz A, McCourt DW, Rice CM. NS28-3 proteinase-mediated processing in the yellow fever vieus structural region, in vitra and in vivo studies, J. Virol 1994 68:3794-3802
Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H. Kinetic and structural analyses of hepatitis C virus polyprotein processing. Journal of Virology 68:5045-55 (1994)
Bartenschlager R, Lohmann V, Wilkinson T, Koch JO. Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4 and its importance for polyprotein maturation. Journal of Virology 69:7519-28 (1995)
Chen PJ, Lin MH, Tai KF, Liu PC, Lin CJ, Chen DS. The Taiwanese hepatitis C virus genome: sequence determination and mapping the 5* termini of viral genomic and antigenomic RNA. Virology 188:102-113 (1992)
Cuthbert JA. Hepatitis C: Progress and problem. Clinical Microbiology Reviews 7:505-32 (1994)
Dimasi N, Martin F, Volpari C, Brumert; M, Biadsiol G, Altamura S, Cortese R, Francesco RD, Steinkuhler C, Sollazzo M. Chatacterization of engineered hepatitis C virus NS3 protease inhicitors affinity. Selected from human pancreatic secretory trypsin inhibitor and minibody repertoires. J of V 71:7461-69 (1997)
Dubuisson J., and Rice CM. Hepatitis C virus glycoprotein folding: disulfide bond and association with calnexin. Journal of Virology 70:778-786 (1996)
Faila C, Tomei L, De Francesco R. Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. Journal of Virology 68:8147-57 (1994)
Feinstone, S.M. The virology of hepatitis C. Journal of Gastroenterology and Hepatology Suppl. 1:26-28 (1991)
Francesno RD, Urbani A, Nardi MC, Tomei L, Steinkuhler C, Tramontano A. A zinc binding site in viral serine proteinase. Biochemistry 35:13282-7 (1996)
Goodwin, R.H. Replacement of vertevrate serum with lipids and other factors in the culture of invertebrate cells, tissues, parasites, and pathogens. In Vitro Cell. Dev. Biol. 27A, 470A.
Grakoui A, McCourt DW, Wychowski C, Feinstone SM, Rice CM. A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci USA 90:10583-7 (1993)
Han JH., Houghton M. Group specific sequences and conserved secondary structures at the 3* end of HCV genome and its implication for viral replication. Nucl Acids Res 20:3520 (1992)
Henry H.H, Mikhakl D, Harry B.G, Stephen M.F. Characterization of hepatitis C virus structural proteins with a recombinant baculovirus expression system. Hepatology 17(5):763-771 (1993)
Hijikata M, Kato N, Ootsuyama Y, Nakagawa M. Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci USA 88:5547-51 (1991)
Hijikata M, Mizushima H, Akagi T, Mori S, Kakiuchi N, Kato N, Tanaka T, Kimura K, and Shimotohno K. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. Journal of Virology 67:4665-4675 (1993)
Hink, W.F., D.R. Thomsen, D.J.Davidson, A.L. Meyer, F.J. Castellino, Expression of three recombinant proteins using baculovirus vector in 23 insect cell lines. Biotechnol. Prog. 7:9-14 (1991)
Hirowatari Y, Hijikata M, Tanji Y, Nyunoya H, Mizushima H, Kimura K, Tanaka T, Kayo N, and Shimotohno K. Two proteinase activities in HCV polypeptide expressed in insect cells using baculovirus vector. Archives of Virology 133:349-356 (1993)
Honda, M., Kaneko, S., Sakai, A., Unoura, M., Murakami, S., and Kobayashi, K. Degree of diversety of hepatitis C virus quasispecies and progression of liver disease. Hepatology 20:1144-1151 (1994)
Kew MC. Hepatitis C virus and hepatocellular carcinoma. FEMA Microbiology Reviews 14:211 (1994)
Khromykh A.A, Harvey T.J, Abedinia M, Wesatway E.G. Expression and purification of the seven nonstructural proteins of the flavirus Kunjin in the E. coli and the baculovirus expression system. Journal of Virological Methods 61:47-58 (1996)
Kim JL, Morgenstern KA, Lin C, Fox T, Dwyer MD, Landro JA, Chambers SP, Markland W, Lepre CA, O*Malley ET, Harbeson SL, Rice CM, Murcko MA, Caron PR, Thomson JA. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 87:343-355 (1996)
Kitts PA, Possee RD. A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14(5):810-7 (1993)
Knudson, D.L., Tinsley, T.W. Replication of a nuclear polyhedrosis virus in a continuous cell culture of Spodoptera frugiperda; purification , assay of infectivity, and growth characteristics of the virus. Journal of Virology 14:934 (1974)
Koch JO, Bartenschlager R. Determinants of substrate specificity in the NS3 serine proteinase of the hepatitis C virus. Virology 237:78-88 (1997)
Lin C, Pragai BM, Grakoui A, Xu J, Rice CM. Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics. Journal of Virology 68:8147-57 (1994)
Lin R, Lindenbach BD, Prahai BM, McCourt DW, Rice CM. Processing in the hepatitis C virus E2-NS2 region : identification of p7 and two distinct E2-specific products with different C termini. Journal of Virology 68:5063-73 (1994)
Lohmann V, Koch JO, Bartinschlager R. Processing pathway of the hepatitis C virus proteins. Journal of Hepatology 24(Suppl.2): 11-19 (1996)
Love RA, Parge HE, Wickersham JA, Hostomsky Z, Habuka N, Moomaq EW, Adachi T, Hostomska Z. The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site. Cell 87:331-342 (1996)
Matsuura, Y., R.D. Possee, D.H.L. Bishop Expression of the S-coded genes of lymphocytic choriomeningitis arenarivus using a baculovirus vector. Journal of General Virology 67:1515-1529 (1986)
Mink, M., Benichou, S., Madaule, P., Tiollais, P., Prince, A.M., and Inchauspe, G. Characterization and mapping of a B-cell immunogenic domain in hepatitis C virus E2 glycoprotein using a yeast peptide library. Virology 200:246-255 (1994)
Moribe, T., Hahashi, N., Kanazawa, Y., Mita, E., Fusamoto, H., Negi, M., Kaneshige, T., Igimi, H., Kamada, T., and Ushida, K. Hepatitis C viral complexity detected by single-strand conformation polymorphism and response to interferon therapy. Gastroenterology 108:789-795 (1995)
Neddermann P, Tomei L, Steinkuhler C, Gallinari P, Tramontano A, Francesco RD. The nonstructural proteins of the hepatitis C virus: structure and functions. Biol. Chem. 378:469-476 (1997)
Pelham, H. R. B. Speculations on the fuctions of the major heat shock and glucose-requlated protein. Cell 46: 959-961 (1986)
Pieroni L, Santolini E, Fipaldini C, Pacini L, Migliccio G, Monica NL. In vitro study of the NS2-3 protease of hepatitis C virus. Journal of Virology 71: 6373-80 (1997)
Pizzi E, Tramontano A, Tomei L, LaMonica N, Failla C, Sardana M, Wood T, DeFrancesco R. Molecullar model of the specificity pocket of th ehepatitis C virus protease: implications for substrate recognition. Proc. Natl. Acad. Sci. USA 91:888-892 (1994)
Pozzetto B, Bourlet T, Grattard F, Bonnevial L. Structure genomic organization, replication and variability of hepatitis C virus. Nephrology Dialysisi Transplantation 11(suppl 4):2-5 (1996)
Reed K E, Grakoui A, Rice C M. Hepatitis C virus-encoded NS2-3 protease: cleavage-site mutagenesis and requirements for bimolecular cleavage. Journal of Virology 69:4127-36 (1995)
Sali DL, Ingram R, Wendel M, McNemar C, Tsarbopoulos A, Chen JW, Heng Z, Chase R, Risano C, Zhang R, Yao N, Kwong AD, Ramanathan L, Le HV, Weber PC, Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex. Biochemistry 37{10}:3392-3401 (1998)
Santolini E., Migliaccio G., La Monica N. Biosynthesis and biochemical properties of the hepatitis C virus core protein. Journal of Virology 68:3631-41 (1994)
Santolini E, Pancini L, Gipaldini C, Migliaccio G, Monica NL. The NS2 protein of hepatitis C virus is a transmembrane polypeptide. Journal of Virology 69:7461-71 (1995)
Shukla D D, Hoyne P A, Ward C W. Evaluation of complete genome sequences and sequences of individual gene products for the classification of hepatitis C virus. Archives of Virology 140:1747-1761 (1995)
Simmonds P, Holmes EC, Cha T-A, ChanS-W, McOmish F, Irvine B, Beall E, Yap PL, Kolberg J, Ureda MS. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS5 region. Journal of General Virology 74:2391-2399 (1993)
Steinkuhler C, Tomei L, Francessco RD. In vitro acticity of hepatitis C virus protease NS3 purified from recombinant baculovirus-infected Sf9 cells. Journal of Biological Chemistry 271:6367-6373 (1996)
Sudo K, Matsumoto Y, Matsushima M, Fujiwara M, Konno K. Shimotohno K, Shigetas, Yokota T. Novel Hrpatitis C virus Protease inhibitos : Thiazolisine derivatives Biochemical and Biophysical Reserch Communication 238, 643-647 (1997)
van Doorn, L-J. Review: Molecular Biology of the Hepatitis C Virus. Journal of Medical Virology 43:345-356 (1994)
Vishnuvardhan D, Kakiuchi N, Urvil PT, Shimotohno K, Kumar P.K.R, Nishikawa S. Expression of highly active recombinant NS3 protease domain of hepatitis C virus in E. coli. FEBS Letters 402:209-212 (1997)
Wang C, Sarnow P, Siddiwui A. Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. Journal of Virology 67:3338-44 (1993)
Wickham, T.J., T.Davis, R.R. Granades, M.L Shuler and H.A. Wood. Screening of insect cell lines for the profuction of recombinant proteins and infectous virus in the baculovirus expression system. Biotechnol. Prog. 8:391-396 (1992)
Wiskerchen M, Collerr MS. Pestivirus gene expression : protein p80 of bovine viral diarrhea virus is a proteinase involved in ployprotein procedssing. Virology 1991; 184:341-50