B型肝炎病毒X基因所產生的具有轉活性HBx蛋白質分子與肝癌形成有密切的關係，且X基因的變異體形成和在癌化過程中扮演著重要角色。因此本研究主要探討在台灣地區肝癌組織中B型肝炎病毒X基因變異體的分子型式。從20個肝癌組織的樣品中抽取核苷酸，並利用專一的寡核苷酸作高溫黏合的聚合酶酵素鏈反應，由不同程度的放大現象顯示X基因嵌入寄主染色體的多寡不同。利用膠上純化所生成的X基因之DNA片段，殖入載體（pUC-T）做轉殖純系化的工作。將8個由肝組織定序出的X基因序列與ayw型的野生型作比較。結果顯示有HBx蛋白質變異體的存在，而變異的區域主要集中在HBx蛋白質分子的胺基酸序列位置第26-45的區段及零星分佈於88、89、116、118、119、127、144等序列點上。同時結果發現8個變異體中有6個序列是相同。序列的分析發現突變區域主要發生於T細胞所辨識的抗原決定基（位置第111-135）及B細胞所辨識的抗原決定基（位置第29-46）和HBx蛋白質的多脯胺酸/絲胺酸區域（PSR）上。這些區域所累積的變異型式，並非來自於亞型的基因多態現象，而可能是因免疫系統的監控篩選下所逃脫出的變異體型式。

Hepatitis B virus (HBV) X gene, encoding a pleotropic transactivator of HBx protein, has been associated with the development of hepatocellular carcinoma (HCC). Molecular information on liver-derived HBV variants isolated from HCC among Taiwanese population was studied. HBV X gene were amplificed from twenty HCC patients in high stringency with specific primers. The results of differential amplification indicated that the variable copy numbers of the HBV X genes were integrated in the genomic DNAs of HCC. The amplified HBV X genes were purified and cloned into pUC-T vector. Sequences of the eight liver-derived X genes were aligned and compared with the wild type, the ayw HBV serotype. Results indicate that variants of the HBx protein were found predominantly within the regions of peptide No. 26-45 in N-terminus, and No. 88, 89, 116, 118, 119, 127, and 144 in position. Sequences from six out of the eight variants were found to be identical. These accumulated sequence mutations among the eight HBx variants were found to coincide with the regions of the B-cell epitopes (No. 29-48), the T-cell epitopes (No. 111-135), and the HBx PSR domain. The observed sequence variation patterns seem to result from accumulation of mutations, rather than, representing subtype-specific polymorphic sites. These mutations of HBx protein may be involved in immunoevasion of HBV.

目錄………………………………………………………………….…1
中文摘要……………………………………………………………...3
英文摘要…………………………………………………………….…4
前言………………………………………………………………….…6
實驗材料及方法……………………………………………………...9
一﹑取得肝炎病人肝腫瘤組織樣本……………..………9
二﹑萃取肝細胞組織的DNA…………………………… …9
三﹑聚合酶酵素鏈反應……………………………………9
四﹑電泳膠的DNA之純化…………………………………10
五﹑重組質體的製備…………………………………….12
六﹑適切化細胞的製備……………………………….…12
七﹑轉化作用………………………………………….…13
八﹑質體的萃取與純化……………………………….…13
九﹑自動定序反應……………………………………….16
十﹑變異體序列分析…………………………………….17
結果…………………………………………………………………..18
討論…………………………………………………………………..23
參考文獻……………………………………………………………..28
圖表………………………………………………………………..…34
附錄…………………………………………………………………..48
Ⅰ.材料配方………………………………………………48
Ⅱ.附圖……………………………………………………52
Ⅲ. B型肝炎病毒ayw亞型核酸序列…………………………......56
Ⅳ. B型肝炎病毒adw亞型核酸序列……………………………....62
Ⅴ.B型肝炎病毒adr亞型核酸序列………………………………...67
個人資料………………………………………………………….….74

1. Bellas RE, FitzGerald MJ, Fausto N, Sonenshein GE. 1997. Inhibition of NF-kappa B activity induces apoptosis in murine hepatocytes. Am. J. Pathol. 151:891-896.
2. Collier L and Oxford J. 1993. Human Virology. Oxford University Press, New York. pp251-267.
3. Chung MK, Yoon H, Min SS, Lee HG, Kim YJ, Lee TG, Lim JS, Kim CM, Park SN. 1999. Induction of cytotoxic T lymphocytes with peptides in vitro: identification of candidate T-cell epitopes in hepatitis B virus X antigen. J. Immunother. 22:279-287.
4. Feitelson MA, Duan LX, Guo J, Sun B, Woo J, Steensma K, Horiike N, Blumberg BS. 1995. X region deletion variants of hepatitis B virus in surface antigen-negative infections and non-A, non-B hepatitis. J. Infect. Dis. 172:713-722.
5. Feitelson MA, Ranganathan PN, Clayton MM, Zhang SM. 1997. Partial characterization of the woodchuck hepatitis virus X antigen in hepatocarcinogenesis. Oncogene 15:327-336.
6. Galibert F, Mandart E, Fitoussi F, Tiollais P, Charnay P. 1979. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature 281:646-650.
7. Gottlob K, Pagano S, Levrero M, Graessmann A. 1998. Hepatitis B virus X protein transcription activation domains are neither required nor sufficient for cell transformation. Cancer Res. 58:3566-3570.
8. Hosono S, Tai PC, Wang W, Ambrose M, Hwang DG, Yuan TT, Peng BH, Yang CS, Lee CS, Shih C. 1995. Core antigen mutations of human hepatitis B virus in hepatomas accumulate in MHC class II-restricted T cell epitopes. Virology 212:151-162.
9. Jung MC, Stemler M, Weimer T, Spengler U, Dohrmann J, Hoffmann R, Eichenlaub D, Eisenburg J, Paumgartner G, Riethmuller G. 1991. Immune response of peripheral blood mononuclear cells to HBx-antigen of hepatitis B virus. Hepatology 13:637-643.
10. Kasai Y, Takeda S, Takagi H. 1996. Pathogenesis of hepatocellular carcinoma: a review from the viewpoint of molecular analysis. Semin. Surg. Oncol. 12:155-156.
11. Kim H, Lee H, Yun Y. 1998. X-gene product of hepatitis B virus induces apoptosis in liver cells. J. Biol. Chem. 273:381-385.
12. Koike K, Moriya K, Iino S, Yotsuyanagi H, Endo Y, Miyamura T, Kurokawa K. 1994. High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice. Hepatology 19:810-819.
13. Koike K, and Takada S. 1995. Biochemistry and functions of hepatitis B virus X protein. Intervirology 38:89-99.
14. Levy JA, Heinz FC, Robert AO. 1994. Virology. Prentic Hall, England. pp143-146.
15. Lin Y, Tang H, Nomura T, Dorjsuren D, Hayashi N, Wei W, Ohta T, Roeder R, Murakami S. 1998. The hepatitis B virus X protein is a co-activator of activated transcription that modulates the transcription machinery and distal binding activators. J. Biol. Chem. 273:27097-27103.
16. Marion PL, Oshiro L, Regnery DC, Scullard GH, Robinson WS. 1980. A virus in Beechey ground squirrels that is related to hepatitis B virus of man. Proc. Natl. Acad. Sci. USA. 77:2941-2945.
17. Mason WS, Seal G, Summers J. 1980. Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. J. Virol. 62:3832-3839.
18. Menzo S, Clementi M, Alfani E, Bagnarelli P, Iacovacci S, Manzin A, Dandri M, Natoli G, Levreo M, Carloni G. 1993. Transactivation of epidermal growth factor receptor gene by the hepatitis B virus X-gene product. Virology 196:878-882.
19. Murakami S, Cheong JH, Kaneko S. 1994. Human hepatitis virus X gene encodes a regulatory domain that represses transactivation of X protein. J. Biol. Chem. 269:15118-15123.
20. Nakatake H, Chisaka O, Yamamoto S, Matsubara K, Koshy R. 1993. Effect of X protein on transactivation of hepatitis B virus promoters and on viral replication. Virology 195:305-314.
21. Park OY, Jin YH, Lee M, Shin HJ, Kim HI, Cho H, Yun CW, Youn JK, Park S. 2000. Characterization and gene cloning of monoclonal antibody specific for the hepatitis B virus X protein. Hybridoma 19:73-80.
22. Passman M, Weinberg M, Kew M, Arbuthnot P. 2000. In situ demonstration of inhibitory effects of hammerhead ribozymes that are targeted to the hepatitis Bx sequence in cultured cells. Biochem. Biophys. Res. Commun. 268:728-733.
23. Petzold DR, Tautz B, Wolf F, Drescher J. 1999. Infection chains and evolution rates of hepatitis B virus in cardiac transplant recipients infected nosocomially. J. Med. Virol. 58:1-10.
24. Renner M, Haniel A, Burgelt E, Hofschneider PH, Koch W. 1995. Transactivating function and expression of the X gene of hepatitis B virus. J. Hepatol . 23:53-65.
25. Seto E, Michell PJ, Yen TSB. 1990. Transactivation by the hepatitis B virus X protein depends on AP-2 and other transcription factors. Nature 344:72-74.
26. Sirma H, Giannini C, Poussin K, Paterlini P, Kremsdorf D, Brechot C. 1999. Hepatitis B virus X mutants, present in hepatocellular carcinoma tissue abrogate both the antiproliferative and transactivation effects of HBx. Oncogene 18:4848-4859.
27. Stemler M, Weimer T, Tu ZX, Wan DF, Levrero M, Jung C, Pape GR, Will H. 1990. Mapping of B-cell epitopes of the human hepatitis B virus X protein. J. Virol. 64:2802-2809.
28. Sung JL, and Chen DS. 1978. Clustering of different subtypes of hepatitis B surface antigen in families of patients with chronic liver diseases. Am. J. Gastroenterol. 69:559-564.
29. Tai PC, Banik D, Lin GI, Pai S, Pai K, Lin MH, Yuoh G, Che S, Hsu SH, Chen TC, Kuo TT, Lee CS, Yang CS, Shih C. 1997. Novel and frequent mutations of hepatitis B virus coincide with a major histocompatibility complex class I-restricted T-cell epitope of the surface antigen. J. Virol. 71:4852-4856.
30. Takada S, Kaneniwa N, Tsuchida N, Koike K. 1997. Cytoplasmic retention of the p53 tumor suppressor gene product is observed in the hepatitis B virus X gene-transfected cells. Oncogene 15: 1895-1901.
31. Tamori A, Nishiguchi S, Kubo S, Koh N, Moriyama Y, Fujimoto S, Takeda T, Shiomi S, Hirohashi K, Kinoshita H, Otani S, Kuroki T. 1999. Possible contribution to hepatocarcinogenesis of X transcript of hepatitis B virus in Japanese patients with hepatitis C virus. Hepatology 29: 1429-1434.
32. Tiollais P, Pourcel C, Dejean A. 1985. The hepatitis B virus. Nature 317: 489-495.
33. Twu JS, Lai MY, Chen DH, Robinson WS. 1993. Activation of protooncogene C-jun by the x protein of hepatitis B virus. Viroloy 192: 346-350.
34. Yeh CT, Shen CH, Tai DI, Chu CM, Liaw YF. 2000. Identification and characterization of a prevalent hepatitis B virus X protein mutant in Taiwanese patients with hepatocellular carcinoma. Oncogene 19: 5213-5220.
35. Yen TSB. 1996. Hepadnaviral X protein: review of recent progress. J. Biomed. Sci. 3: 20-30.
36. Zhang MT, Liu KZ, Jin JH, Ding LM, Zhou SC, Yu QY, Zhang XZ. 1993. Five-year follow-up of immune response to hepatitis B vaccine in juveniles. Chin. Med. J. 106:97-99.
37. Zhang Z, Torii N, Furusaka A, Malayaman N, Hu Z, Liang TJ. 2000. Structural and functional characterization of interaction between hepatitis B virus X protein and the proteasome complex. J. Biol. Chem. 275: 15157-15165.