臺灣博碩士論文加值系統

C型肝炎核心蛋白除了是主要構成病毒顆粒的一部分之外，還藉由與不同細胞因子的交互作用來影響寄主細胞的生長及正常功能。本篇論文主要是利用pQE以及pET兩種質體系統，在大腸桿菌中建立C型肝炎核心蛋白大量表達系統。在所有成功表達的C型肝炎核心蛋白片段中，以移除C端厭水性區域的Core 1-116有最好的表現。本篇中也成功建立起大量純化C型肝炎核心蛋白的方法，並根據西方點墨法的鑑定，分別可以得到大量且高純度的C型肝炎核心蛋白1-191、 1-153以及1-116。更進一步的，我們利用圓二色光譜儀、核磁共振以及分析級超高速離心，來分析C型肝炎核心蛋白的結構特性。根據圓二色光譜的分析結果，C型肝炎核心蛋白1-116在不同濃度尿素濃度中仍可保持有約23% a-helix、 34% b-sheet以及47% random coil穩定的二級結構；相較於低鹽條件下，高鹽條件中，則有28à33%的a-helix的增加；另外，在不同酸鹼值中，C型肝炎核心蛋白1-116則明顯在極酸或極鹼時有不同的二級結構特性，極鹼時會有明顯的random coil比例增加。

The hepatitis c virus core protein is the major component of hepatitis c virus nucleocapsid and also exhibits multiple functions involving in cellular growth, proliferation and other functions. In this study, we successfully established the core protein overexpression system in E. coli. using pQE and pET vector. Our result indicated that the core protein 1-116 without the hydrophobic C-terminal domain is the best expression construct. We succeeded in purifying the large-scale core protein for full length and several C-terminal truncated core proteins including core protein 1-191, core protein 1-153 and core protein 1-116. Furthermore, we characterized the structural properties using CD, NMR, and AUC. In the CD spectra analysis, the core protein 1-116 contains about 23% a-helix, 34% b-sheet and 47% random coil and shows stable structure content during different concentration of urea titration. The ionic strength would cause slightly change, a 28à33% increase of helical content at 0.5 M NaCl, different from that at low salt condition. The pH values shows different pattern in acidic and basic conditions. Besides, NMR and AUC studies indicate that the core protein 1-116 was assembled into an aggregated state at the physiological condition.

[1] Choo, Q., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. and Houghton, M. (1989). Science, 244, 359-362.
[2] Saito, I., Miyammra, T., Ohbayashi, A., Harada, H., Katayama, T., Kikachi, S., Watanabe, Y., Koi, S., Onji, M., and Ohta, Y. (1990). Proc. Natl. Acad. Sci. USA, 87,6547-6549.
[3] Simmonds, P., Alberti, A., Alter, H. J., Bonino, F., Bradley, D. W., Brechot, C., Brouwer, J. T., Chan, S. W., Chayama, K.,and Chen, D. S. (1994). Hepatology, 19,1321-1324.
[4] Simmonds, P., Holmes, E. C., Cha, T. A., Chan, S. W., McOmish, F., Irvine, B., Beall, E., Yap, P. L., Kolberg, J.,and Urdes, M. S. (1993)., J. Gen. Virol., 74,2391-2399.
[5] Bukh, J., Purcell, R. H.,and Miller, R. H. (1994) Proc. Natl. Acad. Sci. USA, 91,8239-8243.
[6] Ohba, K., Mizokami, M., Ohno, T., Suzuki, K., Orito, K., Ina, Y., Lau, J. F. H., and Gojobori, T. (1995). Virus Res., 36,201-204.
[7] He, L. F., Alling, D., Popkin, T., Shapiro, M., Alter, H. J., and Purcell, R. H. (1987). J. Infect. Dis., 156,636-640.
[8] Shimizu, Y. K., Feistone, S. M., Kohara, M., Purcell, R. H., and Yoshikura, H. (1996). Hepatology, 23,205-209.
[9] Kaito, M., Watanabe, S., Tsukiyama-Kohara, K., Yamaguchi, K., Kobayashi, Y., Konishi, M., and Yokoi, M. (1994). J. Gen. Virol., 75,1755-1760.
[10] Choo, Q. L., Richman, K. H., Han, J. H., Berger, K., Lee, C., Dong, C., Gallegos, C., Coit, D., Medina-Selby, A., Barr, P. J., Weiner, A. J., Bradley, D. W., Kuo, G., and Houghton, M. (1991). Proc. Natl. Acad. Sci. USA, 88,2451-2455.
[11] Han, J. H., Shyamala, V., Richman, K. H., Brauer, M. J., Irvine, B., Urdea, M. S.,and Tekamp-Olson, P. (1991). Proc. Natl. Acad. Sci. USA, 88,1711-1715.
[12] Kolykhalov, A. A., Feinstone, S. M., and Rice, C. M. (1996). J Virol., 70,3363-3371.
[13] Tanaka, T., Kato, N., Cho, M. J., Sugiyama, K., and Shimotohno, K. (1996). J. Virol., 70,3307-3312.
[14] Tsukiyama-Kohara, K., Lizuka, N., Kohara, M., and Nomoto, A. (1992). J. Virol., 66,1476-1483.
[15] Jubin, R. (2001). Curr. Opin. Mol. Ther., 3,278-287.
[16] Kolykhalov, A. A., Feinstone, S. M., and Rice, C. M. (1996). J Virol., 70,3363-3371.
[17] Tsukiyama-Kohara, K., Lizuka, N., Kohara, M., and Nomoto, A. (1992). J. Virol., 66,1476-1483.
[18] Tsuchihara, K., Hijikata, M., Fukuda, K., Kuroki, T., Yamamoto, N., and Shimotohno, K. (1999). Virology, 258,100-107.
[20] Ito, T., and Lai, M. M. C. (1997). J Virol., 71,8698-8706.
[21] Kolykhalov, A. A., Mihalik, K., Feinstone, S. M., and Rice, C. M. (2000). J. Virol., 74,2046-2051
[22] Choo, Q. L., Richman, K. H., Han, J. H., Berger, K., Lee, C., Dong, C., Gallegos, C., Coit, D., Medina-Selby, A., Barr, P. J., Weiner, A. J., Bradley, D. W., Kuo, G., and Houghton, M. (1991). Proc. Natl. Acad. Sci. USA, 88,2451-2455.
[23] Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K., and Shimotohno, K. (1993). J. Virol., 67,4665-4675.
[24] Grakoui, A., McCourt, D. W., Wychowski, C., Feistone, S. M., and Rice, C. M. (1993). Proc. Natl. Acad. Sci. USA, 90,10583-10587.
[25] Grakoui, A., Wychowski, C., Lin, C., Feistone, S. M., and Rice, C. M. (1993). J. Virol., 67,1385-1395.
[26] Selby, M. J., Choo, Q. L., Berger, K., Kuo, G., Glazer, E., Eckart, M., Lee, C., Chien, D., Kuo, C., and Houghton, M. (1993). J, Gen. Virol., 74,1103-1113.
[27] Selby, M. J., Choo, Q. L., Berger, K., Kuo, G., Glazer, E., Eckart, M., Lee, C., Chien, D., Kuo, C., and Houghton, M. (1993). J, Gen. Virol., 74,1103-1113.
[28] Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K., and Shimotohno, K. (1993). J. Virol., 67,4665-4675.
[29] Crotta, S., Stilla, A., Wack, A., D’Andrea, A., Nuti, S., and Valiante, N. M. (2002). J. Exp. Med., 195,35-41.
[30] Weiner, A. J., Geysen, H. M., Christopherson, C., Hall, J. E., Mason, T. J., Saracco, G., and Bonino, F. (1992). Proc. Natl. Acad. Sci. USA, 89,3468-3472.
[31] Kato, N., Sekiya, H., Ootsuyama, Y., Nakazawa, T., Hijikata, M., and Shimotohno, K. (1993). J Virol., 67,3923-3930
[32] Koziel, M. J., Dudley, D., Afdhal, N., Choo, Q. L., Houghton, M., Ralstone, R., and Walker, B. D. (1993). J. Virol., 67,7522-7532.
[33] Taniguchi, S., Okamoto, H., Sakamoto, M., Kokima, M., Tsuda, F., Tanaka, T., and Munekata, E. (1993). Virology, 195,297-301.
[34] Large, M. K., Kittlesen, D. J., and Hahn, Y. S. (1999). J. Immunol., 162,931-938.
[35] Moorman, J. P., Joo, M., and Hahn, Y. S. (2001). Arch. Immunol. Ther. Exp., 49,189-194
[36] Grakoui, A., McCourt, D. W., Wychowski, C., Feistone, S. M., and Rice, C. M. (1993). Proc. Natl. Acad. Sci. USA, 90,10583-10587.
[37] Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K., and Shimotohno, K. (1993). J Virol., 67,4665-4675.
[38] Kwong, A. D., Kim, J. L., and Lin, C. (2000). Curr. Top. Microbiol. Immunol., 242,171-196.
[39] De Francesco, R., and Steinkuhler, C. (2000). Curr. Top. Microbiol. Immunol., 242,149-169.
[40] Bartenschlager, R., Ahlborn-Laake, L., Mous, J., and Jacobsen, H. (1993). J Virol., 67,3835-3844.
[41] Grakoui, A., Wychowski, C., Lin, C., Feistone, S. M., and Rice, C. M. (1993). J Virol., 67,1385-1395.
[42] Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K., and Shimotohno, K. (1993). J Virol., 67,4665-4675.
[43] Tomei, L., Failla, C., Santolini, E., De Francesco, R., and La Monica, N. (1993). J. Virol., 67,4017-4026.
[44] Florese, R. H., Nagano-Fujii, M., Iwanaga, Y., Hidajat, R., and Hotta, H. (2002). Virus Res., 90,119-131.
[45] Kaneko, T., Tanji, Y., Satoh, S., Hijitaka, M., Asabe, S., Kimura, K., and Shimotohno, K. (1994). Biophys. Biochem. Res. Commun., 205,320-326.
[46] Enomoto, N., Sakura, I., Asahina, Y., Kurosaki, M., Murakami, T., Yamamoto, C., and Ogura, Y. (1995). N. J. Med., 334,77-81.
[47] Asabe, S., Tanji, Y., Satoh, S., Kaneka, T., Kimura, K., and Shimotohon, K. (1997). J. Virol., 71,790-796.
[48] Behrens, S., Tomei, L., and De Francesco, R. (1996). EMBO J., 15,12-22.
[49] Lohamann, V., Koener, F., Herian, U., and Bartenschlager, R. (1997). J. Virol., 71,8416-8428.
[50] Oh, J. W., Ito, T., and Lai, M, M, C. (1999). J. Virol., 73,7694-7702.
[51] Hagedorn, C. H., van Beers, E. H., and De Staercke, C. (2000). Curr. Top. Microl. Immunol., 242,225-260.
[52] Kao, C. C., Yang, X., Kline, A., Wang, Q. M., Barket, D., and Heinz, B. A. (2000). J. Virol., 72,11121-11128.
[53] Lo, S. Y., Masiarz, F., Hwang, B., Lai, M. M. C, and Ou, J. H. (1995). Virology, 213,455-461.
[54] Hussy, P., Langen, H., Mous, J., and Jaconsen, H. (1996). Virology, 224,93-104.
[55] Yasui, K., Wakita, T., Kyoko, T. K., Funahashi, S. I., Ichikawa, M., Kajita, T., Moradpour, D., Wands, J. R., and Kohara, M. (1998). J. Virol., 72,6048-6055.
[56] Nolandt, O., Kern, V., Muller, H., Praff, E., Theilmann, L., Welker, R., and Krausslich, H. G.. (1997). J. Gen. Virol., 78,1331-1340.
[57] Baumert, T. F., Ito, S., Wong, D. T., and Liang, T. J. (1998). J. Virol., 72,3827-3836.
[58] Kunkel, M., Lorinczi, M., Rijnbrand, R., Lemon, S. M., and Watowich, S. J. (2001). J Virol., 75,2119-2129.
[59] Landford, R. E., Notvall, L., Chavex, D., White, R., Frenzel, G., Simonsen, C., and Kim, J. (1993). Virology, 197,225-235.
[60] Shih, C. M., Lo, S. J., Miyamura, T., Chen, S. Y., and Lee, W. Y. H. (1993). J. Virol., 67,5823-5832.
[61] Suzuki, R., Tamura, K., Li, J., Ishii, K., Matsuura, Y., Miyamura, T., and Suzuki, T. (2001). Virology, 280,301-309.
[62] Lu, W., Strohecker, A., and Ou, J. H. (2001). J. Biol. Chem., 276,47993-47999.
[63] Harada, S., Watanabe, Y., Takeuchi, K., Suzuki, T., Katayama, T., Takebe, Y., and Saito, I. (1991). J Virol., 65,3015-3021.
[64] Shih, C. M., Lo, S. J., Miyamura, T., Chen, S. Y., and Lee, W. Y. H. (1993). J. Virol., 67,5823-5832.
[65] Suzuki, R., Matsuura, Y., Suzuki, T., Ando, A., Chiba, J., Harada, S., and Saito, I. (1995). J. Gen. Virol., 76,53-61.
[66] Shih, C. M., Chen, C. M., Chen, S. Y., and Lee, W. Y. H. (1995). J. Virol., 69,1160-1171.
[67] Santolini, E., Migliaccio, G., and La Monica, N. (1994). J. Virol., 68,3631-3641.
[68] Change, J., Yang, S. H., Hwang, S. B., Hahn, Y. S., and Sung, Y. C. (1998). J. Virol., 72,3060-3065.
[69] Ray, R.B., Lagging, L. M., Meyer, K., and Ray, R. (1996). J. Virol., 70,4438-4443.
[70] Ray, R. B., Lagging ,L. M., Meyer, K., and Ray, R. (1996). J. Virol., 70,4438-4443.
[71] Zhu, N., Khoshnan, A., Schneider, R., Matsumoto, M., Dennert, G., Ware, C., and Lai, M. M. C. (1998). J. Virol. 72,3691-3697.
[72] Marusawa, H., Hijikata, M., Chiba, T.,and Shimotohno, K. (1999). J. Virol., 73,4713-4720.
[73] Ray, R. B., Meyer, K., and Ray, R. (2000). Virology, 271,197-204.
[74] Barba, G., Harper, F., Harada, T., Kohara, M., Goulinet, S., Matsuura, Y., Eder, G., Schaff, Z., Chapman, M.J., Miyamura, T., and Brechot, C. (1997). Proc. Natl. Acad. Sci. USA, 94,1200-1205.
[75] Moriya, K., Fujie, H., Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K., Matsuura, Y., Kimura, S., Miyamura, T., and Koike, K. (1998). Nat. Med., 4,1065-1067.
[76] Kim, D. W., Suzuki, R., Harada, T., Saito, I., and Miyamura, T. (1994). Jpn. J. Med. Sci. Biol., 47,211-220.
[77] Ray, R. B., Lagging, L. M., Meyer, K., Steele, R., and Ray, R. (1995). Virus Res., 37,209-220.
[78] Ray, R. B., Steele, R., Meyer, K., and Ray, R. (1997). J. Biol. Chem., 272,10983-10986.
[79] Shih, C. M., Lo, S. J., Miyamura, T., Chen, S. Y., and Lee, W. Y. H. (1993). J. Virol., 67,5823-5832.
[80] Lu, W., Lo, S. Y., Chen, M., Wu, K. J., Fung, Y. K. T, and Ou, J. H. (1999). Virology, 264,131-141.
[81] Otsuka, M., Kato, N., Lan, K. H., Yoshida, H., Kato, J., Gato, T., Shiraori, Y., and Omata, M. (2000). J. Biol. Chem., 275,34122-34130.
[82] Shrivastava, A., Manna, S. K., Ray, R., and Aggarwal, B. B. (1998). J. Virol., 72,9722-9728.
[83] Bergavist, A., and Rice, C. M. (2001). J. Virol., 75,772-781.
[84] Lee, S., Park, U., and Lee, Y. I. (2001). Virology, 283,167-177.
[85] Yoshida, H., Kato, N., Shiratori, Y., Otsuka, M., Maeda, S., Kato, J.,and Omata, M. (2001). J. Biol. Chem., 276,16399-16405.
[86] Lu, W., Lo, S. Y., Chen, M., Wu, K. J., Fung, Y. K. T, and Ou, J. H. (1999). Virology, 264,131-141.
[87] Otsuka, M., Kato, N., Lan, K. H., Yoshida, H., Kato, J., Gato, T., Shiraori, Y., and Omata, M. (2000). J. Biol. Chem., 275,34122-34130.
[88] Hsieh, T. Y., Matsumoto, M., Chou, H. C., Schneider, R., Hwang, S. B., Lee, A. S., and Lai, M. M. C. (1998). J. Biol. Chem.,28,17651-17659.
[89] Jin, D. Y., Wang, H. L., Zhou, Y., Chun, A. C. S., Kibler, K. V., Hou, Y. D., Kung, H. F., and Jeang, K. T. (2000). EMBO J., 19,729-740.
[90] Aoki, H., Hayashi, J., Moriyama, M., Arakowa, Y., and Hino, O. (2000). J. Virol., 74,1736-1741.
[91] Wang, Z.,and Roeder, R. G.. (1997). Genes Dev., 11,1315-1326.
[92] You, L. R., Chen, C. M., Yeh, T. S., Tsai, T. Y., Mai, R. T., Lin, C. H., and Lee, W. Y. H. (1999). J. Virol., 73,2841-2853.
[93] Nelson, A. C., Viviana, F, Catalina, A., Alexis, M.,Glay, C., MariaChristina de la, R., Armando, R. Santiago, D. C. Victor, T., Mineko, S., Ivon, M, Jose, L. M., Maria M. M. S., Juan, Kouri., and Juan, M. G. (2003). Biophys. Biochem. Res. Commun.,310:48-53.
[94] Nelson, A. R., Julio C. A., Alexis, M., Viviana, F., Ariel, V., Maria, C. R.,and Juan, M. (2001). Biophys. Biochem. Res. Commun., 287:122-125.