丙型肝炎病毒被膜蛋白的表达、纯化与应用研究
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摘要
丙型肝炎病毒(hepatitis C virus, HCV)感染可导致慢性肝炎、肝硬化以至肝癌，是威胁人类健康的重大病原体。研制抗HCV疫苗是当前重要而且紧迫的任务。多种证据表明，HCV被膜蛋白E1(aa 192-383)和E2(384-746)在HCV感染靶细胞的过程中可能发挥了重要而且关键的作用，因此成为HCV疫苗研制的主要靶分子。基于被膜蛋白的HCV疫苗研究急需解决抗原检测和疫苗接种后的免疫反应评价的问题，需要首先得到大量高纯度的被膜蛋白抗原及其相应抗体或抗血清。为此，在大肠杆菌系统中进行了1b亚型HCV被膜蛋白表达和纯化的研究。
采用6xHis融合表达体系，在大肠杆菌中实现了E2 aa 385-565、aa 450-565、aa 567-730和aa 385-730四个片段的高表达，并获得了高度纯化的产物。在免疫印迹检测中，上述片段与丙型肝炎病人血清有特异性反应，表明大肠杆菌系统表达的E2蛋白携带有HCV E2特异的、不依赖于糖化和立体构象的抗原决定簇。利用aa 385-730片段，建立了病人血清中抗E2抗体的ELISA检测体系，该体系在56%的丙型肝炎病人血清内检出了抗E2抗体，并且发现抗E2抗体的存在与HCV病毒血症存在正相关。利用aa 385-565片段，建立了E2疫苗免疫后抗体反应的ELISA评价体系，并已成功用于重组痘苗病毒疫苗和DNA疫苗的研究。利用aa 450-565和aa 385-730两片段免疫家兔，获得了可识别大肠杆菌和哺乳动物细胞表达之E2蛋白的多抗血清，并且在某些亚型之间具有一定程度的交叉反应性。
采用6xHis融合表达体系，在大肠杆菌中实现了E1 aa 192-340和aa 192-326两个片段的高表达，并获得了高度纯化的产物。利用aa 192-326免疫家兔，获得了可识别大肠杆菌和哺乳动物细胞表达之E1蛋白的多抗血清，表明大肠杆菌系统表达的E1蛋白携带有HCV E1特异的、不依赖于糖化和立体构象的抗原决定簇。
通过以上工作，得到了大量、高纯的被膜蛋白抗原和被膜蛋白特异的抗血清，建立了被膜蛋白抗原、抗体的检测体系。同时，在表达被膜蛋白的过程中，对E1和E2在大肠杆菌中的表达规律作了一些有益的探索。
Hepatitis C virus (HCV) infection could lead to chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma, constituting a major threat to human health. There is a pressing need to develop effective anti-HCV vaccines. Different lines of evidence suggest that the envelope proteins of HCV, E1 (aa 192-383) and E2 (384-746), may play important and vital roles in its infection of target cells, thus becoming the main targets of HCV vaccine research. Research on envelope protein-based HCV vaccine demands a prompt solution to the problems of detection of antigen and evaluation of post-vaccination immune responses, which requires large amounts of highly purified envelope proteins and corresponding antibody or antiserum. Therefore, the expression and purification of subtype 1b envelope proteins by using the Escherichia coli system were studied.
Four E2 fragments, aa 385-565, aa 450-565, aa 567-730 and aa 385-730, were expressed as 6xHis fusion proteins in E. coli to a high level and purified to high purity. In immuno-blotting, these fragments reacted specifically with hepatitis C patients' sera, suggesting that E. coli-derived E2 proteins carried HCV E2-specific, glycosylation-and-conformation-independent epitopes. ELISA system for the detection of anti-E2 antibodies in patients' sera was established using aa 385-730 fragment, which detected anti-E2 in 56% of hepatitis C patients and demonstrated a correlation between anti-E2 and HCV viraemia. By using aa 385-365 fragment, an ELISA system for the evaluation of post-E2-vaccination humoral immune responses was also established, and was successfully applied to recombinant vaccinia virus- and DNA-based vaccine research. By immunizing rabbits with aa 450-565 or aa 385-730 fragment, polyclonal sera were obtained that were able to recognize E2 proteins expressed in both E. coli and mammalian cells, and cross-react between certain HCV subtypes.
Two E1 fragments, aa 192-340 and aa 192-326, were expressed as 6xHis fusion proteins in E. coli to a high level and purified to high purity. By immunizing rabbits with aa 192-326 fragment, polyclonal sera were obtained that were able to recognize E1 proteins expressed in both E. coli and mammalian cells, suggesting that E. coli-derived E2 proteins carried HCV E1-specific, glycosylation-and-conformation-independent epitopes.
This work produced large amounts of high-purity envelope protein antigens and envelope-specific antisera, and established detection systems for envelope antigens as well as antibodies. Meanwhile, in the process of expressing envelope proteins, some useful insights into the mechanisms governing HCV envelope protein expression in E. coli were obtained.
采用6xHis融合表达体系，在大肠杆菌中实现了E2 aa 385-565、aa 450-565、aa 567-730和aa 385-730四个片段的高表达，并获得了高度纯化的产物。在免疫印迹检测中，上述片段与丙型肝炎病人血清有特异性反应，表明大肠杆菌系统表达的E2蛋白携带有HCV E2特异的、不依赖于糖化和立体构象的抗原决定簇。利用aa 385-730片段，建立了病人血清中抗E2抗体的ELISA检测体系，该体系在56%的丙型肝炎病人血清内检出了抗E2抗体，并且发现抗E2抗体的存在与HCV病毒血症存在正相关。利用aa 385-565片段，建立了E2疫苗免疫后抗体反应的ELISA评价体系，并已成功用于重组痘苗病毒疫苗和DNA疫苗的研究。利用aa 450-565和aa 385-730两片段免疫家兔，获得了可识别大肠杆菌和哺乳动物细胞表达之E2蛋白的多抗血清，并且在某些亚型之间具有一定程度的交叉反应性。
采用6xHis融合表达体系，在大肠杆菌中实现了E1 aa 192-340和aa 192-326两个片段的高表达，并获得了高度纯化的产物。利用aa 192-326免疫家兔，获得了可识别大肠杆菌和哺乳动物细胞表达之E1蛋白的多抗血清，表明大肠杆菌系统表达的E1蛋白携带有HCV E1特异的、不依赖于糖化和立体构象的抗原决定簇。
通过以上工作，得到了大量、高纯的被膜蛋白抗原和被膜蛋白特异的抗血清，建立了被膜蛋白抗原、抗体的检测体系。同时，在表达被膜蛋白的过程中，对E1和E2在大肠杆菌中的表达规律作了一些有益的探索。
Hepatitis C virus (HCV) infection could lead to chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma, constituting a major threat to human health. There is a pressing need to develop effective anti-HCV vaccines. Different lines of evidence suggest that the envelope proteins of HCV, E1 (aa 192-383) and E2 (384-746), may play important and vital roles in its infection of target cells, thus becoming the main targets of HCV vaccine research. Research on envelope protein-based HCV vaccine demands a prompt solution to the problems of detection of antigen and evaluation of post-vaccination immune responses, which requires large amounts of highly purified envelope proteins and corresponding antibody or antiserum. Therefore, the expression and purification of subtype 1b envelope proteins by using the Escherichia coli system were studied.
Four E2 fragments, aa 385-565, aa 450-565, aa 567-730 and aa 385-730, were expressed as 6xHis fusion proteins in E. coli to a high level and purified to high purity. In immuno-blotting, these fragments reacted specifically with hepatitis C patients' sera, suggesting that E. coli-derived E2 proteins carried HCV E2-specific, glycosylation-and-conformation-independent epitopes. ELISA system for the detection of anti-E2 antibodies in patients' sera was established using aa 385-730 fragment, which detected anti-E2 in 56% of hepatitis C patients and demonstrated a correlation between anti-E2 and HCV viraemia. By using aa 385-365 fragment, an ELISA system for the evaluation of post-E2-vaccination humoral immune responses was also established, and was successfully applied to recombinant vaccinia virus- and DNA-based vaccine research. By immunizing rabbits with aa 450-565 or aa 385-730 fragment, polyclonal sera were obtained that were able to recognize E2 proteins expressed in both E. coli and mammalian cells, and cross-react between certain HCV subtypes.
Two E1 fragments, aa 192-340 and aa 192-326, were expressed as 6xHis fusion proteins in E. coli to a high level and purified to high purity. By immunizing rabbits with aa 192-326 fragment, polyclonal sera were obtained that were able to recognize E1 proteins expressed in both E. coli and mammalian cells, suggesting that E. coli-derived E2 proteins carried HCV E1-specific, glycosylation-and-conformation-independent epitopes.
This work produced large amounts of high-purity envelope protein antigens and envelope-specific antisera, and established detection systems for envelope antigens as well as antibodies. Meanwhile, in the process of expressing envelope proteins, some useful insights into the mechanisms governing HCV envelope protein expression in E. coli were obtained.
引文
1. Choo QL, Kuo G, Weiner AJ, Bradley RD, and Houghton M: Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-362.
2. Seeff LB: Natural history of hepatitis C. Am J Med 1999;107:10S-15S.
3. Report of a WHO consultation organized in collaboration with the viral hepatitis prevention board, Antwerp, Belgium: Global surveillance and control of hepatitis C. J Viral Hepatitis 1999;6:35-47.
4. Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA, and Summers MD (editors): Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses. Vienna & New York: Springer-Verlag 1995, pp. 424-426.
5. Neddermann P, Tomei L, Steinkühler C, Gallinari P, Tramontano A, and De Francesco R: The nonstructural proteins of the hepatitis C virus: structure and functions. Biol Chem 1997;378:469-476.
6. Suzuki R, Suzuki T, Ishii K, Matsuura Y, Miyamura T: Processing and functions of hepatitis C virus proteins. Intervirology 1999;42:145-152.
7. Xu Z, Choi J, Yen TSB, Lu W, Strohecker A, Govindarajan S, Chien D, Selby MJ, and Ou J: Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO J 2001;20:3840-3848.
8. Smith DB and Simmonds P: Review: molecular epidemiology of hepatitis C virus. J Gastroenterol Hepatol 1997;12:522-527.
9. Grakoui A, Wychowski C, Lin C, Feinstone SM, and Rice CM: Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 1993;67: 1385-1395.
10. Selby MJ, Choo QL, Berger K, Kuo G, Glazer E, Eckart M, Lee C, Chien D, Kuo C, and Houghton M: Expression, identification and subcellular localization of the proteins encoded by the hepatitis C. J Gen Virol 1993;74:1103-1113.
11. Rosa D, Campagnoli S, Moretto C, Guenzi E, Cousens L, Chin M, Dong C, Weiner AJ, Lau JYN, Choo QL, Chien D, Pileri P, Houghton M, and Abrignani S: A quantative test to estimate neutralizing antibodies to the hepatitis C virus: Cytofluorimetric assessment of envelop glycoprotein 2 binding to target cells. Proc Natl Acad Sci USA 1996;93:1759-1763.
12. Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, Weiner AJ, Houghton M, Rosa D, Grandi G, and Abrignani S: Binding of hepatitis C virus to CD81. Science 1998;282:938-941.
13. Flint M, Maidens C, Loomis-Price LD, Shotton C, Dubuisson J, Monk P, Higginbottom A, Levy S, and McKeating JA: Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81. J Virol 1999;73:6235-6244.
14. Kronenberger B, Ruster B, Elez R, Weber S, Piiper A, Lee JH, Roth WK, and Zeuzem S: Interferon alfa down-regulates CD81 in patients with chronic hepatitis C. Hepatology 2001;33:1518-1526.
Hamaia S, Li C, and Allain JP: The dynamics of hepatitis C virus binding to platelets
15. and 2 mononuclear cell lines. Blood 2001;98:2293-2300.
16. Wunschmann S, Medh JD, Klinzmann D, Schmidt WN, and Stapleton JT: Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor. J Virol 2000;74:10055-10062.
17. Itakura J, Nagayama K, Enomoto N, Sakamoto N, Tazawa J, Izumi N, Marumo F, and Sato C: CD81 nucleotide mutation in hepatocellular carcinoma and lack of CD81 polymorphism in patients at stages of hepatitis C virus infection. J Med Virol 2001;63:22-28.
18. Tseng CT, Miskovsky E, Houghton M, and Klimpel GR: Characterization of liver T-cell receptor gammadelta T cells obtained from individuals chronically infected with hepatitis C virus (HCV): evidence for these T cells playing a role in the liver pathology associated with HCV infections. Hepatology 2001;33:1312-1320.
19. Wack A, Soldaini E, Tseng C, Nuti S, Klimpel G, and Abrignani S: Binding of the hepatitis C virus envelope protein E2 to CD81 provides a co-stimulatory signal for human T cells. Eur J Immunol 2001;31:166-175.
20. Lagging LM, Meyer K, Owens RJ, and Ray R: Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus. J Virol 1998;72:3539-3546.
21. Meyer K, Basu A, and Ray R: Functional features of hepatitis C virus glycoproteins for pseudotype virus entry into mammalian cells. Virology 2000;276:214-226.
22. Matsuura Y, Tani H, Suzuki K, Kimura-Someya T, Suzuki R, Aizaki H, Ishii K, Moriishi K, Robison CS, Whitt MA, and Miyamura T: Characterization of pseudotype VSV possessing HCV envelope proteins. Virology 2001;286:263-275.
23. Flint M, Thomas JM, Maidens CM, Shotton C, Levy S, Barclay WS, McKeating JA: Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol 1999;735:6782-6790.
24. Takikawa S, Ishii K, Aizaki H, Suzuki T, Asakura H, Matsuura Y, Miyamura T: Cell fusion activity of hepatitis C virus envelope proteins. J Virol 2000;745:5066-5074.
25. Choo QL, Kuo G, Ralston R, Weiner AJ, Chien DY, Van Nest G, Han J, Berger K, Thudium K, Kuo C, Kansopon J, McFarland J, Tabrizi A, Ching K, Moss B, Cummins LB, Houghton M, and Muchmore M: Vaccination of chimpazees against infection by the hepatitis C virus. Proc Natl Acad Sci USA 1994;91:1294-1298.
26. Abrignani S and Rosa D: Perspectives for a hepatitis C virus vaccine. Clin Diagn Virol 1998;10:181-185.
27. Farci P, Shimoda A, Wong D, Cabezon T, De Gioannis D, Strazzera A, Shimizu Y, Shapiro M, Alter HJ, and Purcell RH: Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Natl Acad Sci USA 1996;93:15394-15399.
28. Esumi M, Rikihisa T, Nishimura S, Goto J, Mizuno K, Zhou YH, and Shikata T: Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees. Arch Virol 1999;144:973-980.
Goto J, Nishimura S, Esumi M, Makizumi K, Rikihisa T, Nishihara T, Mizuno K, Zhou Y, Shikata T, Fujiyama S, and Tomita K: Prevention of hepatitis C virus
29. infection in a chimpanzee by vaccination and epitope mapping of antiserum directed against hypervariable region 1. Hepatol Res 2001;19:270-283.
30. Vrielink H, Reesink HW, van den Burg PJM, Zaaijer HL, Cuypers HTM, Lelie PN, and van der Poel CL: Performance of three generations of anti-hepatitis C virus enzyme-linked immunosorbent assays in donors and patients. Transfusion 1997;37:845-849.
31. Fried MW: Diagnostic testing for hepatitis C: practical considerations. Am J Med 1999:107:31S-35S.
32. Leon P, Lopez JA, Elola C, Domingo CJ, and Echevarria JM: Detection of antibody to hepatitis C virus E2 recombinant antigen among samples indeterminate for anti-HCV after wide serological testing and correlation with viremia. The Spanish Study Group for Blood Donors at Risk of Transmission of HCV. Vox Sang 1996;70:213-216.
33. Da-Silva-Cardoso M, Sturm D, Koerner K, Dengler T, Kerowgan M, and Kubanek B: Anti-HCV envelope prevalence in blood donors from Baden-Wurttemberg. Ann Hematol 1997;74:135-137.
34. Chien DY, Arcangel P, Selby AM, Coit D, Baumeister M, Nguyen S, Nascimento CG, Gyenes A, Kuo G, and Valenzuela P: Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection. J Clin Microbiol. 1999;37:1393-1397.
35. Lesniewski R, Okasinski G, Carrick R, Van-Sant C, Desai S, Johnson R, Scheffel J, Moore B, Mushahwar I: Antibody to hepatitis C virus second envelope (HCV-E2) glycoprotein: a new marker of HCV infection closely associated with viremia. J Med Virol 1995;45:415-422.
36. Fong TL, Lee SR, Briggs WK, Valinluck B, Govindarajan S, Hoffman A, Jaczko B, Redeker AG: Clinical significance of hepatitis C viral RNA status and its correlation to antibodies to structural HCV antigens in anti-HCV reactive patients with normal liver tests. J Med Virol 1996;49:253-258.
37. Fournillier-Jacob A, Lunel F, Cahour A, Cresta P, Frangeul L, Perrin M, Girard M, Wychowski C: Antibody responses to hepatitis C envelope proteins in patients with acute or chronic hepatitis C. J Med Virol 1996;50:159-167.
38. Yuki N, Hayashi N, Kasahara A, Hagiwara H, Mita E, Ohkawa K, Katayama-K, Fusamoto H, Kamada T: Quantitative analysis of antibody to hepatitis C virus envelope 2 glycoprotein in patients with chronic hepatitis C virus infection. Hepatology 1996;23:947-952.
39. Psichogiou M, Katsoulidou A, Vaindirli E, Francis B, Lee SR, Hatzakis A: Immunologic events during the incubation period of hepatitis C virus infection: the role of antibodies to E2 glycoprotein. Multicentre Hemodialysis Cohort Study on Viral Hepatitis. Transfusion 1997;37:858-862.
40. Kobayashi M, Tanaka E, Matsumoto A, Ichijo T, Kiyosawa K: Antibody response to E2/NS1 hepatitis C virus protein in patients with acute hepatitis C. J Gastroenterol Hepatol 1997;12:73-76.
Grellier L, Brown D, Power J, Dusheiko G: Absence of anti-envelope antibodies and clearance of hepatitis C virus in a cohort of Irish women infected in 1977. J Viral
41. Hepat 1997;4:379-381.
42. Ishii K, Rosa D, Watanabe Y, Katayama T, Harada H, Wyatt C, Kiyosawa K, Aizaki H, Matsuura Y, Houghton M, Abrignani S, Miyamura T: High titers of antibodies inhibiting the binding of envelope to human cells correlate with natural resolution of chronic hepatitis C. Hepatology 1998;28:1117-1120.
43. Zibert A, Kraas W, Ross RS, Meisel H, Lechner S, Jung G, Roggendorf M: Immunodominant B-cell domains of hepatitis C virus envelope proteins E1 and E2 identified during early and late time points of infection. J Hepatol 1999;30:177-184.
44. Prince AM, Brotman B, Lee DH, Ren L, Moore BS, Scheffel JW: Significance of the anti-E2 response in self-limited and chronic hepatitis C virus infections in chimpanzees and in humans. J Infect Dis 1999;180:987-991.
45. Gane EJ, Maertens G, Ducatteeuw A, Qian KP, Lau JY, Jones H, Davies E, Naoumov NV, Williams R: Antibodies to hepatitis C virus envelope proteins correlate with hepatitis C viraemia after liver transplantation. Transplantation 1999;67:78-84.
46. Leroux-Roels G, Esquivel CA, DeLeys R, Stuyver L, Elewaut A, Philippe J, Desombere I, Paradijs J, and Maertens G. Lymphoproliferative responses to hepatitis C virus core, E1, E2, and NS3 in patients with chronic hepatitis C infection treated with interferon alfa. Hepatology 1996;23:8-16.
47. L?hr, HF, Schmitz D, Arenz M, Weyer S, Gerken G, and zum Büschenfelde KM: The viral clearance in interferon-treated chronic hepatitis C is associated with increased cytotoxic T cell frequencies. J Hepatol 1999;31:407-415.
48. Depraetere S, Van Kerschaever E, Van Vlierberghe H, Elewaut A, Brouwer JT, Niesters HG, Schalm SW, Maertens G, and Leroux-Roels G: Long term response to interferon treatment in chronic hepatitis C patients is associated with a significant reduction in anti-E1 envelope antibody titers. J Med Virol 2000;60:126-132.
49. Taylor DR, Shi ST, Romano PR, Barber GN, and Lai MMC: Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 1999;285:107-110.
50. Taylor DR, Tian B, Romano PR, Hinnebusch AG, Lai MM, and Mathews MB: Hepatitis C virus envelope protein E2 does not inhibit PKR by simple competition with autophosphorylation sites in the RNA-binding domain. J Virol 2001;75:1265-1273.
51. Sarrazin C, Kornetzky I, Ruster B, Lee JH, Kronenberger B, Bruch K, Roth WK, and Zeuzem S: Mutations within the E2 and NS5A protein in patients infected with hepatitis C virus type 3a and correlation with treatment response. Hepatology 2000;31:1360-1370.
52. Berg T, Mas Marques A, Hohne M, Wiedenmann B, Hopf U, and Schreier E: Mutations in the E2-PePHD and NS5A region of hepatitis C virus type 1 and the dynamics of hepatitis C viremia decline during interferon alfa treatment. Hepatology 2000;32:1386-1395.
Chayama K, Suzuki F, Tsubota A, Kobayashi M, Arase Y, Saitoh S, Suzuki Y, Murashima N, Ikeda K, Takahashi N, Kinoshita M, and Kumada H: Association of amino acid sequence in the PKR-eIF2 phosphorylation homology domain and
53. response to interferon therapy. Hepatology 2000;32:1138-44
54. Gerotto M, Dal Pero F, Pontisso P, Noventa F, Gatta A, and Alberti A: Two PKR inhibitor HCV proteins correlate with early but not sustained response to interferon. Gastroenterology 2000;119:1649-1655.
55. Puig-Basagoiti F, Saiz JC, Forns X, Ampurdanes S, Gimenez-Barcons M, Franco S, Sanchez-Fueyo A, Costa J, Sanchez-Tapias JM, and Rodes J: Influence of the genetic heterogeneity of the ISDR and PePHD regions of hepatitis C virus on the response to interferon therapy in chronic hepatitis C. J Med Virol 2001;65:35-44.
56. Lo S, and Lin HH: Variations within hepatitis C virus E2 protein and response to interferon treatment. Virus Res 2001;75:107-112.
57. Yi M, Kaneko S, Yu DY, Murakami S: Hepatitis C virus envelope proteins bind lactoferrin. J Virol 1997;71:5997-6002.
58. Inudoh M, Nyunoya H, Tanaka T, Hijikata M, Kato N, and Shimotohno K: Antigenicity of hepatitis C virus envelope proteins expressed in Chinese hamster ovary cells. Vaccine 1996;14:1590-1596.
59. Ralston R, Thudium K, Berger K, Kuo C, Gervase B, Hall J, Selby M, Kuo G, Houghton M, and Choo QL: Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. J Virol 1993;67:6753-6761.
60. Choo QL, Kuo G, Ralston R, Weiner AJ, Chien DY, Van Nest G, Han J, Berger K, Thudium K, Kuo C, Kansopon J, McFarland J, Tabrizi A, Ching K, Moss B, Cummins LB, Houghton M, and Muchmore M: Vaccination of chimpazees against infection by the hepatitis C virus. Proc Natl Acad Sci USA 1994;91;1294-1298.
61. Lee KJ, Suh YA, Cho YG, Cho YS, Ha GW, Chung KH, Hwang JH, Yun YD, Lee DS, Kim CM, and Sung YC: Hepatitis C virus E2 protein purified from mammalian cells is frequently recognized by E2-specific antibodies in patient sera. J Biol Chem 1997;272:30040-30046.
62. Seong YR, Lee CH, and Im DS: Characterization of the structural proteins of hepatitis C virus expressed by an adenovirus recombinant. Virus Res 1998;55:177-185.
63. Mita E, Hayashi N, Ueda K, Kasahara A, Fusamoto H, Takamizawa A, Matsubara K, Okayama H, and Kamada T: Expression of MBP-HCV NS1/E2 fusion protein in E. coli and detection of anti-NS1/E2 antibody in type C chronic liver disease. Biochem Biophys Res Commun 1992;183:925-930.
64. Hüssy P, Faust H, Wagner JC, Schmid G, Mous J, and Jacobsen H: Evaluation of hepatitis C virus envelope proteins expressed in E. coli and insect cells for use as tools for antibody screening. J Hepatology 1997;26:1179-1186.
65. Due?as-Carrera S, Vi?a A, Garay HE, Reyes O, Alvarez-Lajonchere L, Guerra I, González LJ, and Morales J: Immunological evaluation of Escherichia coli-derived hepatitis C virus second envelope protein (E2) variants. J Pept Res 2001;58:221-228.
66. Chien DY, Choo QL, Ralston R, Spaete R, Tong M, Houghton M, and Kuo G: Persistence of HCV despite antibodies to both putative envelope glycoproteins [letter] Lancet 1993;342:933.
Mustilli AC, Izzo E, Houghton M, Galeotti CL: Comparison of secretion of a
67. hepatitis C virus glycoprotein in Saccharomyces cerevisiae and Kluyveromyces lactis. Res Microbiol 1999;150;179-187.
68. Nishihara T, Nozaki C, Nakatake H, Hoshiko K, Esumi M, Hayashi N, Hino K, Hamada F, Mizuno K, Shikata T: Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells. Gene 1993;129:207-214.
69. Hüssy P, Schmid G, Mous J, Jacobsen H: Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells. Virus Res 1996;45:45-57.
70. Ciccaglione AR, Marcantonio C, Equestre M, Jones IM, and Rapicetta M: Secretion and purification of HCV E1 protein forms as glutathione-S-transferase fusion in the baculovirus insect cell system. Virus Res 1998;55:157-165.
71. Li Y, Li G, Kong Y, Wang Y, Wang Y, and Wen Y: Expression of structural proteins of hepatitis C virus (HCV) in mammalian cells. Science in China (Series C) 1998;41:47-55.
72. Wang Y, Okamoto H, Tsuda F, Nagayama R, Tao QM, and Mishiro S: Prevalence, genotypes, and an isolate (HC-C2) of hepatitis C virus in Chinese patients with liver disease. J Med Virol 1993;40:254-260.
73. Yokosuka O, Ito Y, Imazeki F, Ohto M, and Omata M: Detection of antibody to hepatitis C E2/NS1 protein in patients with type C hepatitis. Biochem Biophys Res Commun. 1992;189:565-571.
74. Cerino A, Bissolati M, Cividini A, Nicosia A, Esumi M, Hayashi N, Mizuno K, Slobbe R, Oudshoorn P, Silini E, Asti M and Mondelli MU: Antibody responses to the hepatitis C virus E2 protein: Relationship to viraemia and prevalence in anti-hcv seronegative subjects. J Med Virol 1997;51:1-5.
75. Yan BS, Liao LY, Leou K, Chang YC, and Syu WJ: Truncating the putative memberane association region circumvents the difficulty of expressing hepatitis C virus protein E1 in Escherichia coli. J Virol Methods 1994;49:343-352.
76. Ciccaglione AR, Marcantonio C, Costantino A, Equestre M, Geraci A, and Rapicetta M: Expression and membrane association of hepatitis C virus Envelope 1 protein. Virus Genes 2000;21:223-226.
77. Yang L, Zhu LX, Wang Y, and Li GD: Fusion expression, immunogenicity and applications of C-terminally truncated HCV core proteins. Acta Biochim Biophys Sin 1999;31:61-66.
78. Amersham pharmacia biotech: Glutathione Sepharose 4 Fast Flow - Instructions. Edition AB.
79. Craig Ta, Lutz WH, and Kumar R: Association of prokaryotic and eukaryotic chaperone proteins with the human 1alpha,25-dihydroxyvitamin D(3) receptor. Biochem Biophys Res Commun 1999;260:446-452.
80. Rohman M, and Harrison-Lavoie KJ: Separation of copurifying GroEL from glutathione-S-transferase fusion proteins. Protein Expr Purif 2000;20:45-47.
Zhu J, Kong YY, Liu J, Zhang ZC, Wang Y, and Li GD: Secretory expression of different C-ternimal truncated HCV E1 proteins in mammalian cells and characterization of the expressed products. Acta Biochim Biophys Sin
81. 2001;33:634-640
2. Seeff LB: Natural history of hepatitis C. Am J Med 1999;107:10S-15S.
3. Report of a WHO consultation organized in collaboration with the viral hepatitis prevention board, Antwerp, Belgium: Global surveillance and control of hepatitis C. J Viral Hepatitis 1999;6:35-47.
4. Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA, and Summers MD (editors): Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses. Vienna & New York: Springer-Verlag 1995, pp. 424-426.
5. Neddermann P, Tomei L, Steinkühler C, Gallinari P, Tramontano A, and De Francesco R: The nonstructural proteins of the hepatitis C virus: structure and functions. Biol Chem 1997;378:469-476.
6. Suzuki R, Suzuki T, Ishii K, Matsuura Y, Miyamura T: Processing and functions of hepatitis C virus proteins. Intervirology 1999;42:145-152.
7. Xu Z, Choi J, Yen TSB, Lu W, Strohecker A, Govindarajan S, Chien D, Selby MJ, and Ou J: Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO J 2001;20:3840-3848.
8. Smith DB and Simmonds P: Review: molecular epidemiology of hepatitis C virus. J Gastroenterol Hepatol 1997;12:522-527.
9. Grakoui A, Wychowski C, Lin C, Feinstone SM, and Rice CM: Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 1993;67: 1385-1395.
10. Selby MJ, Choo QL, Berger K, Kuo G, Glazer E, Eckart M, Lee C, Chien D, Kuo C, and Houghton M: Expression, identification and subcellular localization of the proteins encoded by the hepatitis C. J Gen Virol 1993;74:1103-1113.
11. Rosa D, Campagnoli S, Moretto C, Guenzi E, Cousens L, Chin M, Dong C, Weiner AJ, Lau JYN, Choo QL, Chien D, Pileri P, Houghton M, and Abrignani S: A quantative test to estimate neutralizing antibodies to the hepatitis C virus: Cytofluorimetric assessment of envelop glycoprotein 2 binding to target cells. Proc Natl Acad Sci USA 1996;93:1759-1763.
12. Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, Weiner AJ, Houghton M, Rosa D, Grandi G, and Abrignani S: Binding of hepatitis C virus to CD81. Science 1998;282:938-941.
13. Flint M, Maidens C, Loomis-Price LD, Shotton C, Dubuisson J, Monk P, Higginbottom A, Levy S, and McKeating JA: Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81. J Virol 1999;73:6235-6244.
14. Kronenberger B, Ruster B, Elez R, Weber S, Piiper A, Lee JH, Roth WK, and Zeuzem S: Interferon alfa down-regulates CD81 in patients with chronic hepatitis C. Hepatology 2001;33:1518-1526.
Hamaia S, Li C, and Allain JP: The dynamics of hepatitis C virus binding to platelets
15. and 2 mononuclear cell lines. Blood 2001;98:2293-2300.
16. Wunschmann S, Medh JD, Klinzmann D, Schmidt WN, and Stapleton JT: Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor. J Virol 2000;74:10055-10062.
17. Itakura J, Nagayama K, Enomoto N, Sakamoto N, Tazawa J, Izumi N, Marumo F, and Sato C: CD81 nucleotide mutation in hepatocellular carcinoma and lack of CD81 polymorphism in patients at stages of hepatitis C virus infection. J Med Virol 2001;63:22-28.
18. Tseng CT, Miskovsky E, Houghton M, and Klimpel GR: Characterization of liver T-cell receptor gammadelta T cells obtained from individuals chronically infected with hepatitis C virus (HCV): evidence for these T cells playing a role in the liver pathology associated with HCV infections. Hepatology 2001;33:1312-1320.
19. Wack A, Soldaini E, Tseng C, Nuti S, Klimpel G, and Abrignani S: Binding of the hepatitis C virus envelope protein E2 to CD81 provides a co-stimulatory signal for human T cells. Eur J Immunol 2001;31:166-175.
20. Lagging LM, Meyer K, Owens RJ, and Ray R: Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus. J Virol 1998;72:3539-3546.
21. Meyer K, Basu A, and Ray R: Functional features of hepatitis C virus glycoproteins for pseudotype virus entry into mammalian cells. Virology 2000;276:214-226.
22. Matsuura Y, Tani H, Suzuki K, Kimura-Someya T, Suzuki R, Aizaki H, Ishii K, Moriishi K, Robison CS, Whitt MA, and Miyamura T: Characterization of pseudotype VSV possessing HCV envelope proteins. Virology 2001;286:263-275.
23. Flint M, Thomas JM, Maidens CM, Shotton C, Levy S, Barclay WS, McKeating JA: Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol 1999;735:6782-6790.
24. Takikawa S, Ishii K, Aizaki H, Suzuki T, Asakura H, Matsuura Y, Miyamura T: Cell fusion activity of hepatitis C virus envelope proteins. J Virol 2000;745:5066-5074.
25. Choo QL, Kuo G, Ralston R, Weiner AJ, Chien DY, Van Nest G, Han J, Berger K, Thudium K, Kuo C, Kansopon J, McFarland J, Tabrizi A, Ching K, Moss B, Cummins LB, Houghton M, and Muchmore M: Vaccination of chimpazees against infection by the hepatitis C virus. Proc Natl Acad Sci USA 1994;91:1294-1298.
26. Abrignani S and Rosa D: Perspectives for a hepatitis C virus vaccine. Clin Diagn Virol 1998;10:181-185.
27. Farci P, Shimoda A, Wong D, Cabezon T, De Gioannis D, Strazzera A, Shimizu Y, Shapiro M, Alter HJ, and Purcell RH: Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Natl Acad Sci USA 1996;93:15394-15399.
28. Esumi M, Rikihisa T, Nishimura S, Goto J, Mizuno K, Zhou YH, and Shikata T: Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees. Arch Virol 1999;144:973-980.
Goto J, Nishimura S, Esumi M, Makizumi K, Rikihisa T, Nishihara T, Mizuno K, Zhou Y, Shikata T, Fujiyama S, and Tomita K: Prevention of hepatitis C virus
29. infection in a chimpanzee by vaccination and epitope mapping of antiserum directed against hypervariable region 1. Hepatol Res 2001;19:270-283.
30. Vrielink H, Reesink HW, van den Burg PJM, Zaaijer HL, Cuypers HTM, Lelie PN, and van der Poel CL: Performance of three generations of anti-hepatitis C virus enzyme-linked immunosorbent assays in donors and patients. Transfusion 1997;37:845-849.
31. Fried MW: Diagnostic testing for hepatitis C: practical considerations. Am J Med 1999:107:31S-35S.
32. Leon P, Lopez JA, Elola C, Domingo CJ, and Echevarria JM: Detection of antibody to hepatitis C virus E2 recombinant antigen among samples indeterminate for anti-HCV after wide serological testing and correlation with viremia. The Spanish Study Group for Blood Donors at Risk of Transmission of HCV. Vox Sang 1996;70:213-216.
33. Da-Silva-Cardoso M, Sturm D, Koerner K, Dengler T, Kerowgan M, and Kubanek B: Anti-HCV envelope prevalence in blood donors from Baden-Wurttemberg. Ann Hematol 1997;74:135-137.
34. Chien DY, Arcangel P, Selby AM, Coit D, Baumeister M, Nguyen S, Nascimento CG, Gyenes A, Kuo G, and Valenzuela P: Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection. J Clin Microbiol. 1999;37:1393-1397.
35. Lesniewski R, Okasinski G, Carrick R, Van-Sant C, Desai S, Johnson R, Scheffel J, Moore B, Mushahwar I: Antibody to hepatitis C virus second envelope (HCV-E2) glycoprotein: a new marker of HCV infection closely associated with viremia. J Med Virol 1995;45:415-422.
36. Fong TL, Lee SR, Briggs WK, Valinluck B, Govindarajan S, Hoffman A, Jaczko B, Redeker AG: Clinical significance of hepatitis C viral RNA status and its correlation to antibodies to structural HCV antigens in anti-HCV reactive patients with normal liver tests. J Med Virol 1996;49:253-258.
37. Fournillier-Jacob A, Lunel F, Cahour A, Cresta P, Frangeul L, Perrin M, Girard M, Wychowski C: Antibody responses to hepatitis C envelope proteins in patients with acute or chronic hepatitis C. J Med Virol 1996;50:159-167.
38. Yuki N, Hayashi N, Kasahara A, Hagiwara H, Mita E, Ohkawa K, Katayama-K, Fusamoto H, Kamada T: Quantitative analysis of antibody to hepatitis C virus envelope 2 glycoprotein in patients with chronic hepatitis C virus infection. Hepatology 1996;23:947-952.
39. Psichogiou M, Katsoulidou A, Vaindirli E, Francis B, Lee SR, Hatzakis A: Immunologic events during the incubation period of hepatitis C virus infection: the role of antibodies to E2 glycoprotein. Multicentre Hemodialysis Cohort Study on Viral Hepatitis. Transfusion 1997;37:858-862.
40. Kobayashi M, Tanaka E, Matsumoto A, Ichijo T, Kiyosawa K: Antibody response to E2/NS1 hepatitis C virus protein in patients with acute hepatitis C. J Gastroenterol Hepatol 1997;12:73-76.
Grellier L, Brown D, Power J, Dusheiko G: Absence of anti-envelope antibodies and clearance of hepatitis C virus in a cohort of Irish women infected in 1977. J Viral
41. Hepat 1997;4:379-381.
42. Ishii K, Rosa D, Watanabe Y, Katayama T, Harada H, Wyatt C, Kiyosawa K, Aizaki H, Matsuura Y, Houghton M, Abrignani S, Miyamura T: High titers of antibodies inhibiting the binding of envelope to human cells correlate with natural resolution of chronic hepatitis C. Hepatology 1998;28:1117-1120.
43. Zibert A, Kraas W, Ross RS, Meisel H, Lechner S, Jung G, Roggendorf M: Immunodominant B-cell domains of hepatitis C virus envelope proteins E1 and E2 identified during early and late time points of infection. J Hepatol 1999;30:177-184.
44. Prince AM, Brotman B, Lee DH, Ren L, Moore BS, Scheffel JW: Significance of the anti-E2 response in self-limited and chronic hepatitis C virus infections in chimpanzees and in humans. J Infect Dis 1999;180:987-991.
45. Gane EJ, Maertens G, Ducatteeuw A, Qian KP, Lau JY, Jones H, Davies E, Naoumov NV, Williams R: Antibodies to hepatitis C virus envelope proteins correlate with hepatitis C viraemia after liver transplantation. Transplantation 1999;67:78-84.
46. Leroux-Roels G, Esquivel CA, DeLeys R, Stuyver L, Elewaut A, Philippe J, Desombere I, Paradijs J, and Maertens G. Lymphoproliferative responses to hepatitis C virus core, E1, E2, and NS3 in patients with chronic hepatitis C infection treated with interferon alfa. Hepatology 1996;23:8-16.
47. L?hr, HF, Schmitz D, Arenz M, Weyer S, Gerken G, and zum Büschenfelde KM: The viral clearance in interferon-treated chronic hepatitis C is associated with increased cytotoxic T cell frequencies. J Hepatol 1999;31:407-415.
48. Depraetere S, Van Kerschaever E, Van Vlierberghe H, Elewaut A, Brouwer JT, Niesters HG, Schalm SW, Maertens G, and Leroux-Roels G: Long term response to interferon treatment in chronic hepatitis C patients is associated with a significant reduction in anti-E1 envelope antibody titers. J Med Virol 2000;60:126-132.
49. Taylor DR, Shi ST, Romano PR, Barber GN, and Lai MMC: Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 1999;285:107-110.
50. Taylor DR, Tian B, Romano PR, Hinnebusch AG, Lai MM, and Mathews MB: Hepatitis C virus envelope protein E2 does not inhibit PKR by simple competition with autophosphorylation sites in the RNA-binding domain. J Virol 2001;75:1265-1273.
51. Sarrazin C, Kornetzky I, Ruster B, Lee JH, Kronenberger B, Bruch K, Roth WK, and Zeuzem S: Mutations within the E2 and NS5A protein in patients infected with hepatitis C virus type 3a and correlation with treatment response. Hepatology 2000;31:1360-1370.
52. Berg T, Mas Marques A, Hohne M, Wiedenmann B, Hopf U, and Schreier E: Mutations in the E2-PePHD and NS5A region of hepatitis C virus type 1 and the dynamics of hepatitis C viremia decline during interferon alfa treatment. Hepatology 2000;32:1386-1395.
Chayama K, Suzuki F, Tsubota A, Kobayashi M, Arase Y, Saitoh S, Suzuki Y, Murashima N, Ikeda K, Takahashi N, Kinoshita M, and Kumada H: Association of amino acid sequence in the PKR-eIF2 phosphorylation homology domain and
53. response to interferon therapy. Hepatology 2000;32:1138-44
54. Gerotto M, Dal Pero F, Pontisso P, Noventa F, Gatta A, and Alberti A: Two PKR inhibitor HCV proteins correlate with early but not sustained response to interferon. Gastroenterology 2000;119:1649-1655.
55. Puig-Basagoiti F, Saiz JC, Forns X, Ampurdanes S, Gimenez-Barcons M, Franco S, Sanchez-Fueyo A, Costa J, Sanchez-Tapias JM, and Rodes J: Influence of the genetic heterogeneity of the ISDR and PePHD regions of hepatitis C virus on the response to interferon therapy in chronic hepatitis C. J Med Virol 2001;65:35-44.
56. Lo S, and Lin HH: Variations within hepatitis C virus E2 protein and response to interferon treatment. Virus Res 2001;75:107-112.
57. Yi M, Kaneko S, Yu DY, Murakami S: Hepatitis C virus envelope proteins bind lactoferrin. J Virol 1997;71:5997-6002.
58. Inudoh M, Nyunoya H, Tanaka T, Hijikata M, Kato N, and Shimotohno K: Antigenicity of hepatitis C virus envelope proteins expressed in Chinese hamster ovary cells. Vaccine 1996;14:1590-1596.
59. Ralston R, Thudium K, Berger K, Kuo C, Gervase B, Hall J, Selby M, Kuo G, Houghton M, and Choo QL: Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. J Virol 1993;67:6753-6761.
60. Choo QL, Kuo G, Ralston R, Weiner AJ, Chien DY, Van Nest G, Han J, Berger K, Thudium K, Kuo C, Kansopon J, McFarland J, Tabrizi A, Ching K, Moss B, Cummins LB, Houghton M, and Muchmore M: Vaccination of chimpazees against infection by the hepatitis C virus. Proc Natl Acad Sci USA 1994;91;1294-1298.
61. Lee KJ, Suh YA, Cho YG, Cho YS, Ha GW, Chung KH, Hwang JH, Yun YD, Lee DS, Kim CM, and Sung YC: Hepatitis C virus E2 protein purified from mammalian cells is frequently recognized by E2-specific antibodies in patient sera. J Biol Chem 1997;272:30040-30046.
62. Seong YR, Lee CH, and Im DS: Characterization of the structural proteins of hepatitis C virus expressed by an adenovirus recombinant. Virus Res 1998;55:177-185.
63. Mita E, Hayashi N, Ueda K, Kasahara A, Fusamoto H, Takamizawa A, Matsubara K, Okayama H, and Kamada T: Expression of MBP-HCV NS1/E2 fusion protein in E. coli and detection of anti-NS1/E2 antibody in type C chronic liver disease. Biochem Biophys Res Commun 1992;183:925-930.
64. Hüssy P, Faust H, Wagner JC, Schmid G, Mous J, and Jacobsen H: Evaluation of hepatitis C virus envelope proteins expressed in E. coli and insect cells for use as tools for antibody screening. J Hepatology 1997;26:1179-1186.
65. Due?as-Carrera S, Vi?a A, Garay HE, Reyes O, Alvarez-Lajonchere L, Guerra I, González LJ, and Morales J: Immunological evaluation of Escherichia coli-derived hepatitis C virus second envelope protein (E2) variants. J Pept Res 2001;58:221-228.
66. Chien DY, Choo QL, Ralston R, Spaete R, Tong M, Houghton M, and Kuo G: Persistence of HCV despite antibodies to both putative envelope glycoproteins [letter] Lancet 1993;342:933.
Mustilli AC, Izzo E, Houghton M, Galeotti CL: Comparison of secretion of a
67. hepatitis C virus glycoprotein in Saccharomyces cerevisiae and Kluyveromyces lactis. Res Microbiol 1999;150;179-187.
68. Nishihara T, Nozaki C, Nakatake H, Hoshiko K, Esumi M, Hayashi N, Hino K, Hamada F, Mizuno K, Shikata T: Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells. Gene 1993;129:207-214.
69. Hüssy P, Schmid G, Mous J, Jacobsen H: Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells. Virus Res 1996;45:45-57.
70. Ciccaglione AR, Marcantonio C, Equestre M, Jones IM, and Rapicetta M: Secretion and purification of HCV E1 protein forms as glutathione-S-transferase fusion in the baculovirus insect cell system. Virus Res 1998;55:157-165.
71. Li Y, Li G, Kong Y, Wang Y, Wang Y, and Wen Y: Expression of structural proteins of hepatitis C virus (HCV) in mammalian cells. Science in China (Series C) 1998;41:47-55.
72. Wang Y, Okamoto H, Tsuda F, Nagayama R, Tao QM, and Mishiro S: Prevalence, genotypes, and an isolate (HC-C2) of hepatitis C virus in Chinese patients with liver disease. J Med Virol 1993;40:254-260.
73. Yokosuka O, Ito Y, Imazeki F, Ohto M, and Omata M: Detection of antibody to hepatitis C E2/NS1 protein in patients with type C hepatitis. Biochem Biophys Res Commun. 1992;189:565-571.
74. Cerino A, Bissolati M, Cividini A, Nicosia A, Esumi M, Hayashi N, Mizuno K, Slobbe R, Oudshoorn P, Silini E, Asti M and Mondelli MU: Antibody responses to the hepatitis C virus E2 protein: Relationship to viraemia and prevalence in anti-hcv seronegative subjects. J Med Virol 1997;51:1-5.
75. Yan BS, Liao LY, Leou K, Chang YC, and Syu WJ: Truncating the putative memberane association region circumvents the difficulty of expressing hepatitis C virus protein E1 in Escherichia coli. J Virol Methods 1994;49:343-352.
76. Ciccaglione AR, Marcantonio C, Costantino A, Equestre M, Geraci A, and Rapicetta M: Expression and membrane association of hepatitis C virus Envelope 1 protein. Virus Genes 2000;21:223-226.
77. Yang L, Zhu LX, Wang Y, and Li GD: Fusion expression, immunogenicity and applications of C-terminally truncated HCV core proteins. Acta Biochim Biophys Sin 1999;31:61-66.
78. Amersham pharmacia biotech: Glutathione Sepharose 4 Fast Flow - Instructions. Edition AB.
79. Craig Ta, Lutz WH, and Kumar R: Association of prokaryotic and eukaryotic chaperone proteins with the human 1alpha,25-dihydroxyvitamin D(3) receptor. Biochem Biophys Res Commun 1999;260:446-452.
80. Rohman M, and Harrison-Lavoie KJ: Separation of copurifying GroEL from glutathione-S-transferase fusion proteins. Protein Expr Purif 2000;20:45-47.
Zhu J, Kong YY, Liu J, Zhang ZC, Wang Y, and Li GD: Secretory expression of different C-ternimal truncated HCV E1 proteins in mammalian cells and characterization of the expressed products. Acta Biochim Biophys Sin
81. 2001;33:634-640