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研究生:王權輝
研究生(外文):Chiuan-Huei Wang
論文名稱:Hz-1病毒DNA聚合酶基因表現模式、蛋白質純化與活性分析
論文名稱(外文):Gene expression, protein purification and activity analysis of Hz-1 virus DNA polymerase
指導教授:陳虹樺陳虹樺引用關係
指導教授(外文):Hong-Hwa Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
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:2003
畢業學年度:91
語文別:中文
論文頁數:85
中文關鍵詞:活性分析蛋白質純化DNA聚合脢Hz-1病毒
外文關鍵詞:DNA polymeraseHz-1 virusactivity analysisprotein purificationGene expression
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Hz-1病毒基因組已於2002年定序完成,Hz-1病毒的基因組全長有228,108個鹼基對,計有154個ORF及1個不轉譯的基因PAT-1,經NCBI的BlastP比對後,第131個ORF的比對結果是DNA聚合酶,有趣的是其中的DNA聚合酶所比對到最相似物種為一種耐熱古細菌(Aeropyrum pernix),此種古細菌的最適生存溫度為95°C。
本論文探討Hz-1病毒DNA聚合酶基因表現模式及利用重組病毒技術大量製備Hz-1病毒的DNA聚合酶,並分析酵素活性。
RT-PCR及Northern blot的結果皆顯示,Hz-1病毒的DNA聚合酶從感染後4小時開始轉錄出RNA且一直持續至24小時都可偵測到RNA的表現,因此Hz-1病毒DNA聚合酶屬於早期基因。
首先以Hz-1病毒感染TN368細胞8小時後的RNA進行反轉錄聚合酶連鎖反應,取得Hz-1病毒DNA聚合酶的全長cDNA,全長為3.5 kb,隨後再與傳送載體接合,與線形AcMNPV基因體DNA進行同源重組後轉染至Sf9細胞中,取得重組病毒後,再感染Sf9細胞,最後以His-bind resin純化Hz-1病毒的DNA聚合酶,進行活性分析。
活性分析結果顯示,Hz-1病毒DNA聚合酶於70℃下20分鐘仍有酵素活性。
Genomic sequence of Hz-1 virus has been completely finished in 2002. The Hz-1 virus genome is 228,108 bp containing 154 open reading frames (ORFs) and one untranslated PAT1. The ORF131 is a putative DNA polymerase. Interestingly, the DNA polymerase of Hz-1 virus has a strong similarity to that of Aeropyrum pernix, a strictly aerobic hyperthermophilic crenarchaeon with its optimal growth at 95°C.
DNA polymerase of Hz-1 virus was expressed using recombinant virus and E. coli expression system. Enzymatic activities of the expressed proteins were analyzed. Total RNA was extracted from TN368 cells infected with Hz-1 virus at 8 h post infection (h p.i.). The full-length cDNA of DNA polymerase adding (His)6-tag at both 5’-and 3’-ends were obtained using RT-PCR, and cloned into T-vector. The cDNA fragments was released with Bgl II enzyme and cloned into a transfer vector, pVL1392, and the released products were named as pVL1392-NDPOL and pVL1392-CDPOL for both N’-tag and C’-tag, respectively. These two constructed transfer vectors were mixed well with linear AcMNPV genomic DNA and co-tansfected into Sf9 cells. Recombinant viruses, AcNDPOL and AcCDPOL, were plaque purified and grown to high titers for protein purification. DNA polymerase was purified with His-bind resin and enzymatic activities were analyzed.
Results showed that NDPOL was stable after incubating at 70℃for 20 min, and CDPOL was stable after incubating at 70℃ for 10 min.
第一章、前言………………………………………………………1

第二章、文獻探討…………………………………………………3
1. Hz-1病毒……………………………………………………..3
1.1 Hz-1病毒的發現……………………………………………4
1.2 Hz-1病毒的結構……………………………………………4
1.3 Hz-1病毒的分類地位………………………………………5
1.4 Hz-1病毒的複製及感染期…………………………………5
2. DNA聚合酶…………………………………………………..5
2.1原核生物的DNA聚合酶…………………………………..6
2.2真核生物的DNA聚合酶…………………………………..7
2.3 Hz-1病毒的DNA聚合酶…………………………………..7
2.4 Hz-1病毒DNA聚合酶基因的結構………………………..8
2.5耐熱性DNA聚合酶……………………………………….8

第三章、材料與方法
一、材料
1.細胞株…………………………………………………………10
2.病毒……………………………………………………………10
3.菌種……………………………………………………………11
4.質體……………………………………………………………11
5.軟體……………………………………………………………11
二、方法
1.基因表現模式
1.1感染TN368細胞……………………………………………11
1.2萃取全部RNA………………………………………………12
1.3 Northern blot hybridization
1.3.a RNA探針製備………………………………………….13
1.3.b探針(probe)品質的測定………………………………..14
1.3.c RNA電泳……………………………………………….14
1.3.d 雜合反應……………………………………………….15
1.4選殖Hz-1病毒DNA聚合酶之全長cDNA
1.4.1引子設計……………………………………………….16
1.4.2反轉錄聚合酶連鎖反應………………………………..16
1.4.3接合反應………………………………………………..17
1.4.4細菌轉型反應…………………………………………..17
1.5傳送載體
1.5.1構築含Hz1V-DPOL的傳送載體………………………18
1.5.2大量製備傳送載體……………………………………..19
1.5.3核酸定序……………………………………………….20
1.6轉染傳送載體至Sf9細胞………………………………….20
1.7篩選並放大重組病毒
1.7.1挑選單一病毒斑………………………………………..21
1.7.2放大病毒至24-well……………………………………21
1.7.3放大病毒至6-well……………………………………..22
1.7.4放大病毒至 T25培養瓶………………………………22
1.7.5 TCID50分析…………………………………………….23
1.7.6大量製備病毒液……………………………………….24
2.蛋白質純化
2.1純化Hz-1病毒DNA聚合酶………………………………24
2.2西方氏墨漬法(Western blot)……………………………….25
2.3透析………………………………………………………….27
3.Hz-1病毒DNA聚合酶活性測定
3.1 DNA polymerization assay……………………………...27

第四章、結果
1.基因表現模式
1.3 Hz-1病毒DNA聚合酶基因Blastp比對結果…………….29
1.2 RT-PCR……………………………………………………...29
1.3 Northern blot hybridization…………………………… 30
1.4 Search for exact nearly matches……………………….31
2.蛋白質表現
2.1基因選殖……………………………………………………31
2.2放大重組病毒………………………………………………32
2.3純化His Tag融合蛋白…………………………………….33
2.4西方氏墨漬法……………………………………………….33
3.Hz-1病毒DNA聚合酶活性測定
3.1 Hz1V-DPOL於150 mM分流液之活性分析………………34
3.2 Hz1V-DPOL於125 mM分流液之活性分析………………34

第五章、討論
1. Blastp…………………………………………………………35
2. Hz-1病毒DNA聚合酶基因表現模式………………………37
3. 蛋白質表現
a.昆蟲重組病毒……….………………………………………..38
b.大腸桿菌…………….………………………………………..39
4.蛋白質純化……………………………………………………39
5. DNA聚合酶
(a)Aeropyrum pernix DNA聚合酶的特性……………………..40
(b)昆蟲桿狀病毒的DNA聚合酶………………………………40
(c)Hz-1病毒DNA聚合酶的特性………………………………41
第六章、參考文獻…………………………………………………41
左丹如,2000,Hz-1病毒之感染特性與其胸腺核甘酸合成酶基因的鑑定比對、基因表現和親源分析,國立成功大學生物科技研究所碩士論文。
吳文祥,2001,以含核酸分解酵素、幾丁質分解效素或鹼性脂解酵素基因之重組桿狀病毒開發速效性生物農藥,國立成功大學生物科技研究所碩士論文。
鄭嘉雄,2001,Hz-1病毒基因組之定序與分析,國立成功大學生物科技研究所碩士論文。
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