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研究生:邱千綾
研究生(外文):Chien-Ling, Chiu
論文名稱:以細菌表現人類乳突病毒16型E6蛋白質及免疫血清之製備
論文名稱(外文):The Expression of Human Papillomavirus Type 16 Protein in Bacteria and the Production of Anti-E6 Serum
指導教授:黃海美
指導教授(外文):Hai-Mei, Huang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:73
中文關鍵詞:人類乳突病毒第16型E6蛋白質多株抗體
外文關鍵詞:HPV16E6 proteinpolyclone antiserum
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  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

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