臺灣博碩士論文加值系統

Part I: Studies of the Purine Nucleoside Phosphorylase Gene from Grouper Iridovirus
中文摘要
嘌呤核苷磷酸酶(purine nucleoside phosphorylase, PNP)為嘌呤再利用途徑(purine salvage pathway)中之關鍵酵素，以可逆的方式將嘌呤(或去氧嘌呤)核苷[(2’-deoxy)ribonucleoside]分解為鹼基(free base)與嘌呤(或去氧嘌呤)-1-磷酸[(2’-deoxy)ribose 1-phosphate]。石斑魚虹彩病毒(grouper iridovirus, GIV)基因體研究中，首次發現此病毒攜帶有PNP基因，將其命名為GIV PNP基因。此基因可轉錄出一段由285個胺基酸組成的開放讀架，分子量為30,332 dalton的蛋白質，其胺基酸序列與人類及其他哺乳類的 PNP有近50%的相似性。北方與西方轉漬雜合反應的結果證實，受GIV感染的石斑魚腎臟 (GK) 細胞在病毒感染後6小時即可見到PNP的表現，其表現的程度隨時間增加而加強。免疫細胞化學反應 (immunocytochemistry)的結果顯示GIV PNP位於GIV感染細胞之細胞質中。此外，在PNP與綠螢光蛋白(EGFP)表現載體轉殖的GK與HeLa細胞之細胞質中，偵測到PNP與EGFP的複合蛋白質 (PNP-EGFP)表現有綠色螢光。以大腸桿菌系統表現的GIV PNP，以親合力層析法(affinity chromatography)純化PNP重組蛋白質。由雙圓極化光吸收光譜技術(circular dichroism spectroscopy)顯示此重組蛋白質之二級結構主要由-螺旋(- helix)所組成。高壓液態色層分析(HPLC) ，發現此重組蛋白質具有功能性，可磷酸化嘌呤類核醣核苷(purine ribonucleosides)，可以鳥糞嘌呤核苷(guanosine)，肌嘌呤核苷(inosine)及腺嘌呤核苷(adenosine)為受質。帶有GIV PNP基因的HeLa細胞經過 fludarabine phosphate （一種腺嘌呤核苷之類似物）處理後，未攜有GIV PNP基因之細胞株存活率較低。依據此實驗結果，推估GIV PNP基因可做為基因-引導酵素前驅藥物治療癌症的方法(gene-directed enzyme prodrug therapy)之一。

Part II: Studies of the Deoxyuridine Triphosphatase Gene from Grouper Iridovirus
中文摘要
石斑魚虹彩病毒(grouper iridovirus, GIV)基因體研究中，發現病毒開放讀碼區(open reading frame, ORF) 028L之核苷酸序列與小鼠的去氧尿嘧啶三磷酸酶(deoxyuridine triphosphatase, dUTPase)基因具有66%的相同。dUTPase將去氧尿嘧啶三磷酸(dUTP)分解為去氧尿嘧啶單磷酸(dUMP) 與焦磷酸鹽(pyrophosphate)。本實驗證實ORF 028L為dUTPase基因，其可轉譯出由159個胺基酸組成，分子量為17,483 dalton的蛋白質，此蛋白質胺基酸序列中含有五個具序列保守性的模組(motif)。北方轉漬雜合反應顯示，受GIV感染的石斑魚腎臟 (GK) 細胞在病毒感染後6小時即可見到二個RNA的訊號。若先以複製抑制劑或轉錄抑制劑處理細胞後，再感染GIV，發現GIV dUTPase為後前期(delayed-early)表現的基因。西方轉漬雜合反應顯示， GK細胞在GIV感染後偵測出二個蛋白質，分別為18 kDa及36 kDa，推測dUTPase在GIV感染的細胞中形成同質雙體(homodimer) ，此型態亦可在非還原狀態下的GIV dUTPase重組蛋白質發現。免疫細胞化學反應 (immunocytochemistry)顯示，GIV dUTPase位於GIV感染細胞之細胞質中。利用大腸桿菌系統來表現GIV dUTPase基因，並利用親合力層析法(affinity chromatography)純化dUTPase重組蛋白質，證實此重組蛋白質具有酵素活性，可分解dUTP。

Part I: Studies of the Purine Nucleoside Phosphorylase Gene from Grouper Iridovirus
英文摘要
Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway. It catalyses the reversible phosphorolysis of purine (2'-deoxy)ribonucleosides to free bases and (2'-deoxy)ribose 1-phosphates. A novel piscine viral PNP gene from grouper iridovirus (GIV), a causative agent of an epizootic fish disease, is reported. The putative GIV PNP gene encodes a protein of 285 amino acids with a predicted molecular mass of 30,332 Da. A high similarity between this GIV PNP protein and human PNP was found. GIV PNP expression analyzed by Northern and Western blot was increased after 6 h post-infection in grouper kidney (GK) cells. GIV PNP was localized in the cytoplasm of GIV-infected host cells by immunocytochemistry. The PNP–EGFP fusion protein was observed in the cytoplasm of transfected GK and HeLa cells. GIV PNP gene was expressed in Escherichia coli and purified by affinity chromatography. Results obtained from HPLC analysis indicates the recombinant GIV PNP protein catalyzes the reversible phosphorolysis of purine nucleosides, such as guanosine, inosine and adenosine. A predominantly -helix was found in the recombinant protein structure verified by circular dichroism spectroscopy. A GIV PNP gene transfected HeLa cells showed a lower survival rate compared to mock-transfected cells after treatment with fludarabine phosphate, the analog of adenosine. These results suggested a potential application of GIV PNP gene in the field of gene-directed enzyme prodrug therapy.

Part II: Studies of the Deoxyuridine Triphosphatase Gene from Grouper Iridovirus
英文摘要
Analysis of the complete genome of grouper iridovirus (GIV), an open reading frame (028L) was shown to have a 66% nucleotide sequence identity to a mouse deoxyuridine triphosphatase (dUTPase). dUTPase catalyzes the pyrophosphate cleavage of dUTP into dUMP. Here, ORF 028L was identified as a GIV dUTPase gene. GIV dUTPase gene encodes a protein of 159 amino acids with a predicted molecular mass of 17,483 dalton and has five conserved motifs. Northern blot hybridization analysis of GIV infected grouper kidney (GK) cells revealed two RNAs appearance in cells from 6 hours post-infection (h p.i.). In the presence of replication or translation inhibitors, GIV dUTPase is a delayed-early gene. Results from Western blot analysis showed two proteins, 18 kDa and 36 kDa were detected, suggesting a homodimeric form of dUTPase existed in GIV infected cells. The homodimeric dUTPase was further confirmed by the purified recombinant protein in the non-reducing condition. Immunocytochemistry study indicated GIV dUTPase was localized in the cytoplasm of GIV infected GK cells. The GIV dUTPase expressed from E. coli was found active and had a catalytic ability on dUTP.

Part I: Studies of the Purine Nucleoside Phosphorylase Gene from Grouper Iridovirus
頁
1. INTRODUCTION 1
2. MATERIALS AND METHODS 5
2.1 Virus and cells 5
2.2 PCR amplification and plasmid construction 5
2.3 Sequence and phylogenetic analysis 6
2.4 Recombinant GIV PNP expression in E. coli and its purification 6
2.5 N-terminal sequencing 7
2.6 Circular dichroism (CD) measurement 7
2.7 Preparation of anti-GIV PNP antibody 7
2.8 Northern blot hybridization 8
2.9 Western blot hybridization 8
2.10 Immunocytochemistry 9
2.11 EGFP fusion protein expression in cells 9
2.12 Enzymatic activity analysis 9
2.13 Coupled in vitro transcription/translation reaction 10
2.14 Prodrug treatment and WST-1 assay 10
2.15 Construction of GIV PNP structure model 11
3. RESULTS 12
3.1 Detection of the ORF encoding PNP in a partial genome of GIV 12
3.2 Recombinant GIV PNP expression in E. coli and its purification 19
3.3 Retention of structure confirmed by CD spectroscopy 21
3.4 Expression of GIV PNP in GIV-infected cells 23
3.5 Subcellular localization of GIV PNP 26
3.6 Enzymatic assay of GIV PNP 28
3.7 Expression of recombinant GIV PNP in a cell-free system 31
3.8 Effects of fludarabine-P on GIV PNP expressed HeLa cells 33
4. DISCUSSION 37
5. CONCLUSION 43
6. REFERENCES 44

Part II: Studies of the Deoxyuridine Triphosphatase Gene from Grouper Iridovirus
頁
1. INTRODUCTION 55
2. MATERIALS AND METHODS 58
2.1 Virus and cells 58
2.2 PCR amplification and plasmid construction 58
2.3 Sequence, phylogeny and structure analyses 58
2.4 Recombinant GIV dUTPase expression in E. coli and its purification 59
2.5 Preparation of anti-GIV dUTPase antibody 59
2.6 Northern blot hybridization 60
2.7 Western blot hybridization 60
2.8 Immunocytochemistry 61
2.9 Enzymatic activity analysis 61
2.10 Construction of GIV dUTPase structure model 62
3. RESULTS 63
3.1 Sequence analysis of GIV dUTPase 63
3.2 Recombinant GIV dUTPase expression in E. coli and its purification 67
3.3 Expression of GIV dUTPase gene in GIV infected cells 69
3.4 Subcellular localization of GIV dUTPase 72
3.5 Enzymatic assay of GIV dUTPase 74
3.6 Structure remodeling of GIV dUTPase protein 74
4. DISCUSSION 78
5. CONCLUSION 82
6. REFERENCES 83

Part I:Studies of the Purine Nucleoside Phosphorylase Gene from Grouper Iridovirus

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Part II: Studies of the Deoxyuridine Triphosphatase Gene from Grouper Iridovirus

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