臺灣博碩士論文加值系統

　　子宮頸癌，目前高居世界婦女癌症死亡原因的第二位。台灣地區婦女篩檢感染HPV的盛行率為9％。其主要致病原為人類乳突病毒16、18型，病毒的E6、E7為主要致癌基因。E6蛋白質能和細胞內p53蛋白質結合，經由ubiquitin proteolysis system分解p53抑癌蛋白。和癌症發生有密切相關性。
　　HPV16 E6蛋白質分子量大小約為18 kDa，其核酸序列已公布。由於在商業產品上缺乏適當的HPV16 E6抗體。本研究因此利用基因工程技術建構一含E6基因的表現載體，在大腸桿菌中表現E6抗原以免疫兔子製備抗體血清。設計sense-與antisense-兩種序列引子，分別於5’端前包含BamHI及SalI兩限制切點。以含有E6基因的p1436質體為模板，用PCR方式合成含有BamHI及SalI兩限制切點的HPV16 E6基因片段，再以CIP (Calf-Intestinal Phosphatase)將5’-磷酸根去除。接入以BamHI及SalI截切過的商用pET32a載體，此載體上具有thioredoxin及6個histidine等融合蛋白。重組質體轉殖入大腸桿菌BL21。利用限制酵素BamHI及SalI截切及PCR兩種篩選方式，選取有HPV16 E6基因接入的菌株。三次轉殖實驗中有CIP處理的結果共得到30個菌株，有正確接入E6基因的菌株共有2株(第2及6號菌株)，而E6基因發生鹼基錯誤的菌株亦有2株。
　　用IPTG誘發E6融合蛋白的表現後，利用Ni-NTA親和性樹脂，自第2號菌株中純化大量的E6蛋白作為抗原。免疫兔子，得到預期的對HPV16 E6蛋白質的抗體血清，並測定其效價。抗體血清的測試以稀釋1000或3000倍後，經西方墨點法及Cy3-螢光免疫法檢測，結果最少可偵測到1或5 ng的抗原蛋白。本研究所製備的抗體可應用於細胞或組織中觀察E6蛋白質表現。由於子宮頸癌患者血清中通常有HPV16 E6蛋白質抗體的存在，純化的重組E6蛋白質預期可作為抗原，以偵測婦女血清是否有E6蛋白質抗體中的存在。

　　Cervical cancer is the second death cause of all cancers in women. One screening result showed that HPV infection was 9 percent in Taiwan. The major cause of cervical cancer is human papillomavirus (HPVs) type 16 and 18, which consist of oncogenes E5, E6, and E7. E6 protein interacts with tumor suppressor p53 and promotes its degradation through an ubiquitin-dependent pathway. Therefore, E6 protein plays a significant role for malignant transformation.
　　The HPV16E6 protein is about 18 kDa, and has been fully sequenced previously. Because of the absence of commercial available anti-E6 antibodies, it is reasonable to prepare E6 antigen from regular molecular cloning and produce anti-E6 antibodies after immunize E6 in animals to fulfill the purpose. Sense- and antisense- primers containing BamHI and SalI restriction sites in 5’-end and E6 gene containing plasmid-p1436 were used as PCR template. After E6 DNA fragments were obtained from PCR, they were treated by CIP (Calf-Intestinal Phosphatase) to remove 5’-phosphate groups. HPV16 E6 DNA fragment was subcloned into commercially available pET32a expression vector (from Novagen, contain thioredoxin and 6xhis fusion gene sequence). The pET32a-E6 recombinant vector were transformed into BL21 E. coli, and screened for suitable E6 containing candidates in using BamHI and SalI restriction digestion and PCR with E6 primers. Thirty clones were picked up on antibiotic-selective plates from three transformation experiments. Finally, there were 2 candidates (No.2 and No.6) with right base sequences and 2 candidates with wrong base sequences.
　　E6 containing vector in No.2 BL21 E. coli showed most E6 containing fusion protein expression in IPTG- and antibiotic-containing LB medium. Large amount of purified E6 fusion protein from No.2 was collected from Ni-NTA resin elution in this study to serve as antigen in continuous immunization study in rabbits. Expected E6-antiserum was collected from rabbits after several E6 antigen injections into rabbits. The results in from Western blot and Cy3-fluorescent-immunoassay analysis showed that 5 ng and 1 ng E6 antigen could be detected with 1:1000 or 1:3000 dilution of antibodies-containing serum, respectively. From this study, it is expected that E6 antibodies can be used to examine E6 expression in various cells and tissues samples. Since the strong association of HPV16 E6 antibodies in patient’s sera with cervical carcinoma had been reported, it is also expected that E6 fusion protein from this study might contribute to the immunological monitoring of E6-specific antibodies with cervical cancer.

壹、中文摘要………………………………………………………3
貳、英文摘要………………………………………………………4
叁、前言……………………………………………………………5
肆、導論……………………………………………………………5
伍、實驗方法與材料………………………………………………16
陸、結果……………………………………………………………29
柒、討論……………………………………………………………36
捌、參考文獻………………………………………………………43
玖、圖表……………………………………………………………50
拾、附錄……………………………………………………………60

(1998): "Current protocols in cell biology." New York: Wiley.
國家衛生研究院 (2000): 子宮頸癌篩檢及治療共識.
Androphy EJ, Hubbert NL, Schiller JT, Lowy DR (1987): Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines. Embo J 6:989-92.
Band V, Dalal S, Delmolino L, Androphy EJ (1993): Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells. Embo J 12:1847-52.
Barbosa MS, Lowy DR, Schiller JT (1989): Papillomavirus polypeptides E6 and E7 are zinc-binding proteins. J Virol 63:1404-7.
Cain JM, Howett MK (2000): Preventing cervical cancer. Science 288:1753-5.
Chen SL, Han CP, Tsao YP, Lee JW, Yin CS (1993): Identification and typing of human papillomavirus in cervical cancers in Taiwan. Cancer 72:1939-45.
Chen TM, Chen CA, Wu CC, Huang SC, Chang CF, Hsieh CY (1994): The genotypes and prognostic significance of human papillomaviruses in cervical cancer. Int J Cancer 57:181-4.
Choo KB, Shen HD, Leung WY, Lee YN (1988): A distinct difference in the prevalence of papillomavirus infection in cytologically normal and neoplastic cells of the uterine cervix. Zhonghua Yi Xue Za Zhi (Taipei) 42:1-6.
Danos O, Katinka M, Yaniv M (1982): Human papillomavirus 1a complete DNA sequence: a novel type of genome organization among papovaviridae. Embo J 1:231-6.
Durst M, Dzarlieva-Petrusevska RT, Boukamp P, Fusenig NE, Gissmann L (1987): Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene 1:251-6.
Dyson N, Howley PM, Munger K, Harlow E (1989): The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934-7.
Foster SA, Demers GW, Etscheid BG, Galloway DA (1994): The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest. J Virol 68:5698-705.
Guccione E, Massimi P, Bernat A, Banks L (2002): Comparative analysis of the intracellular location of the high- and low-risk human papillomavirus oncoproteins. Virology 293:20-5.
Herrero R, Brinton LA, Reeves WC, Brenes MM, Tenorio F, de Britton RC, Gaitan E, Garcia M, Rawls WE (1990): Sexual behavior, venereal diseases, hygiene practices, and invasive cervical cancer in a high-risk population. Cancer 65:380-6.
Huang H, Li CY, Little JB (1996): Abrogation of P53 function by transfection of HPV16 E6 gene does not enhance resistance of human tumour cells to ionizing radiation. Int J Radiat Biol 70:151-60.
Hubbert NL, Sedman SA, Schiller JT (1992): Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes. J Virol 66:6237-41.
Huibregtse JM, Scheffner M, Beaudenon S, Howley PM (1995): A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci U S A 92:2563-7.
Kanda T, Watanabe S, Zanma S, Sato H, Furuno A, Yoshiike K (1991): Human papillomavirus type 16 E6 proteins with glycine substitution for cysteine in the metal-binding motif. Virology 185:536-43.
Kern SE, Kinzler KW, Bruskin A, Jarosz D, Friedman P, Prives C, Vogelstein B (1991): Identification of p53 as a sequence-specific DNA-binding protein. Science 252:1708-11.
Kim KH, Yoon DJ, Moon YA, Kim YS (1994): Expression and localization of human papillomavirus type 16 E6 and E7 open reading frame proteins in human epidermal keratinocyte. Yonsei Med J 35:1-9.
Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB (1992): Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A 89:7491-5.
Liang XH, Volkmann M, Klein R, Herman B, Lockett SJ (1993): Co-localization of the tumor-suppressor protein p53 and human papillomavirus E6 protein in human cervical carcinoma cell lines. Oncogene 8:2645-52.
Lin D, Shields MT, Ullrich SJ, Appella E, Mercer WE (1992): Growth arrest induced by wild-type p53 protein blocks cells prior to or near the restriction point in late G1 phase. Proc Natl Acad Sci U S A 89:9210-4.
Livingstone LR, White A, Sprouse J, Livanos E, Jacks T, Tlsty TD (1992): Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 70:923-35.
Lunn CA, Kathju S, Wallace BJ, Kushner SR, Pigiet V (1984): Amplification and purification of plasmid-encoded thioredoxin from Escherichia coli K12. J Biol Chem 259:10469-74.
McDougall JK (1994): Immortalization and transformation of human cells by human papillomavirus. Curr Top Microbiol Immunol 186:101-19.
Meschede W, Zumbach K, Braspenning J, Scheffner M, Benitez-Bribiesca L, Luande J, Gissmann L, Pawlita M (1998): Antibodies against early proteins of human papillomaviruses as diagnostic markers for invasive cervical cancer. J Clin Microbiol 36:475-80.
Munger K, Phelps WC, Bubb V, Howley PM, Schlegel R (1989): The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol 63:4417-21.
Nakagawa S, Watanabe S, Yoshikawa H, Taketani Y, Yoshiike K, Kanda T (1995): Mutational analysis of human papillomavirus type 16 E6 protein: transforming function for human cells and degradation of p53 in vitro. Virology 212:535-42.
Oh ST, Kyo S, Laimins LA (2001): Telomerase activation by human papillomavirus type 16 E6 protein: induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites. J Virol 75:5559-66.
Pirisi L, Yasumoto S, Feller M, Doniger J, DiPaolo JA (1987): Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA. J Virol 61:1061-6.
Pisani P, Bray F, Parkin DM (2002): Estimates of the world-wide prevalence of cancer for 25 sites in the adult population. Int J Cancer 97:72-81.
Rapp L, Chen JJ (1998): The papillomavirus E6 proteins. Biochim Biophys Acta 1378:F1-19.
Scheffner M, Munger K, Huibregtse JM, Howley PM (1992): Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins. Embo J 11:2425-31.
Scheffner M, Romanczuk H, Munger K, Huibregtse JM, Mietz JA, Howley PM (1994): Functions of human papillomavirus proteins. Curr Top Microbiol Immunol 186:83-99.
Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM (1990): The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129-36.
Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B, Stremlau A, zur Hausen H (1985): Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314:111-4.
Sedman SA, Barbosa MS, Vass WC, Hubbert NL, Haas JA, Lowy DR, Schiller JT (1991): The full-length E6 protein of human papillomavirus type 16 has transforming and trans-activating activities and cooperates with E7 to immortalize keratinocytes in culture. J Virol 65:4860-6.
Sherman L, Schlegel R (1996): Serum- and calcium-induced differentiation of human keratinocytes is inhibited by the E6 oncoprotein of human papillomavirus type 16. J Virol 70:3269-79.
Smotkin D, Wettstein FO (1986): Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proc Natl Acad Sci U S A 83:4680-4.
Solinas-Toldo S, Durst M, Lichter P (1997): Specific chromosomal imbalances in human papillomavirus-transfected cells during progression toward immortality. Proc Natl Acad Sci U S A 94:3854-9.
Stone KM, Zaidi A, Rosero-Bixby L, Oberle MW, Reynolds G, Larsen S, Nahmias AJ, Lee FK, Schachter J, Guinan ME (1995): Sexual behavior, sexually transmitted diseases, and risk of cervical cancer. Epidemiology 6:409-14.
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990): Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60-89.
Thomas M, Pim D, Banks L (1999): The role of the E6-p53 interaction in the molecular pathogenesis of HPV. Oncogene 18:7690-700.
Tong X, Howley PM (1997): The bovine papillomavirus E6 oncoprotein interacts with paxillin and disrupts the actin cytoskeleton. Proc Natl Acad Sci U S A 94:4412-7.
Um SJ, Lee SY, Kim EJ, Myoung J, Namkoong SE, Park JS (2002): Down-regulation of human papillomavirus E6/E7 oncogene by arsenic trioxide in cervical carcinoma cells. Cancer Lett 181:11-22.
Viscidi RP, Sun Y, Tsuzaki B, Bosch FX, Munoz N, Shah KV (1993): Serologic response in human papillomavirus-associated invasive cervical cancer. Int J Cancer 55:780-4.
Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Munoz N (1999): Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189:12-9.
Werness BA, Levine AJ, Howley PM (1990): Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248:76-9.
White AE, Livanos EM, Tlsty TD (1994): Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins. Genes Dev 8:666-77.
Wlazlo AP, Giles-Davis W, Clements A, Struble G, Marmorstein R, Ertl HC (2001): Generation and characterization of monoclonal antibodies against the E6 and E7 oncoproteins of HPV. Hybridoma 20:257-63.
Yin Y, Tainsky MA, Bischoff FZ, Strong LC, Wahl GM (1992): Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell 70:937-48.
Zhang B, Spandau DF, Roman A (2002): E5 protein of human papillomavirus type 16 protects human foreskin keratinocytes from UV B-irradiation-induced apoptosis. J Virol 76:220-31.
Zivaljevic B, Vlajinac H, Adanja B, Zivaljevic V, Kocev N (2001): Smoking as risk factor for cervical cancer. Neoplasma 48:254-6.
zur Hausen H (1991): Viruses in human cancers. Science 254:1167-73.
zur Hausen H (1996): Papillomavirus infections--a major cause of human cancers. Biochim Biophys Acta 1288:F55-78.
zur Hausen H (2002): Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2:342-50.
zur Hausen H, Meinhof W, Scheiber W, Bornkamm GW (1974): Attempts to detect virus-secific DNA in human tumors. I. Nucleic acid hybridizations with complementary RNA of human wart virus. Int J Cancer 13:650-6.