臺灣博碩士論文加值系統

本研究以胡瓜嵌紋病毒(cucumber mosaic cucumovirus, CMV)及其衛星核酸(satellite RNA)作為模式系統，以探討衛星核酸與輔助病毒外鞘蛋白間交互作用之機制。此為利用衛星核酸作為外源基因載體系統研發成功與否之重要關鍵之一。為求高效率分析核酸與蛋白質分子間之交互作用，本研究研發利用酵素鏈結免疫吸附法分析核酸與蛋白質交互作用之系統（簡稱NB-ELISA），以供更簡便而快速之偵測分析。本實驗利用CMV全長C株系衛星核酸(C-sat)及CMV-NT9株系之外鞘蛋白進行NB-ELISA系統之最適化工作，以獲得二者間反應之最適當條件； 發現其OD405與外鞘蛋白之濃度有線性之正相關，顯示NB-ELISA可應用於核酸與蛋白質交互作用之定量分析；C-sat對於CMV之外鞘蛋白具有專一性交互作用，而與菸草嵌紋病毒外鞘蛋白、小牛血清蛋白、及健康植物萃取蛋白皆無顯著反應；並經由蔗糖密度梯度離心實驗發現C-sat極可能可吸附於CMV病毒顆粒之表面，而不一定需要包被於內部；且C-sat與CMV外鞘蛋白之結合隨時間之增長而有協力作用之現象。為了解外鞘蛋白胺基酸序列中與衛星核酸交互作用之區域，本研究並完成CMV-NT9 及CMV-Gem外鞘蛋白基因之選殖及核④酸序列分析，將CMV-NT9外鞘蛋白之N端（胺基酸位置1-76）、C端（胺基酸位置170-218）、及中段部份（胺基酸位置77-169）分別選殖於pETblue2外源蛋白表現質體中，於大腸桿菌中表現各蛋白片段，結果顯示外鞘蛋白之N端為唯一與衛星核酸結合之區域。為探討衛星核酸中與外鞘蛋白發生交互作用所必需之核酸序列，本研究完成構築C-sat衛星核酸之中段序列刪減突變株及其核④酸序列確認。然而NB-ELISA之結果顯示CMV之外鞘蛋白可與單、雙股之DNA與RNA交互作用，而無顯著之專一性。上述研究結果提供對於衛星核酸胡瓜嵌紋病毒外鞘蛋白生物功能及交互作用更深入之瞭解，以利未來基因載體系統之研發與病毒病害之防治。

The interactions between satellite RNAs (satRNAs) and coat proteins of the helper virus, cucumber mosaic cucumovirus (CMV), were studied to elucidate the corresponding regions in coat proteins and satRNAs responsible for recognition. This work is of vital importance for the successful development of satRNA-based transient expression system. To facilitate efficient detection and analysis of interactions between nucleic acids and protein molecules, a system based on enzyme linked immunosorbent assay (nucleoprotein binding-ELISA, NB-ELISA) was developed and applied to the fast and efficient analysis of interactions between satRNAs and coat proteins of CMV. The satRNA from CMV-C strain (C-sat) and coat proteins of CMV-NT9 strain were used to optimize and standardize the NB-ELISA procedures. A positive, linear correlation was observed between the concentrations of coat proteins of CMV and the observed OD405 value. The C-sat showed binding preference for CMV coat proteins over non-related proteins, such as tobacco mosaic virus coat proteins, bovine serum albumin, and healthy plant extracts. Sucrose density gradient centrifugation assay indicated that the satRNA might attach to the outer surface of the virus particles, besides being encapsidated inside. The result of time course study suggested the binding of satRNAs to coat proteins proceeded in a cooperative manner as a function of time. The coat protein genes of CMV-NT9, CMV-Gem and CMV-M48 strains were cloned and sequenced for further investigation of interactions with satRNAs. The N-, C-terminal and internal fragments (from amino acid number 1 to 76, 176 to 218, and 77 to 169, respectively) of CMV-NT9 coat proteins were subcloned into plasmid pETblue2 for efficient expression in Escherichia coli to study the regions on the coat proteins responsible for the interaction with satRNAs. The N-terminal fragment of coat protein was shown to be the sole required region to recognize satRNAs. Terminal and serial internal deletion mutants of C-satRNA were used to investigate the nucleotide sequences that are required for the proper recognition by the CMV coat proteins. The result indicated that CMV coat proteins interacted with both DNA and RNAs, single- or double-stranded, without evident specificity. These results provided further insight into the biological functions of satRNAs to facilitate the better design of efficient gene-transfer vectors and disease management strategies.

目錄
中文摘要……………………………………………………… 1
英文摘要……………………………………………………… 2
壹、 緒論……………………………………………………. 3
貳、 前言………………………………………………….… 5
一. 胡瓜嵌紋病毒簡介………………………………… 5
二. 胡瓜嵌紋病毒基因體組成………………………… 6
三. 病毒移動…………………………………………….10
四. 外鞘蛋白的功能…………………………………….11
五. 衛星核酸與其基因功能…………………………….12
參、 材料與方法……………………………………………..17
一. 病毒來源…………………………………………….17
二. 病毒的純化………………………………………….17
三. 序列刪減突變株的選殖…………………………….18
四. 生體外轉錄系統合成轉錄子……………………….21
五. 核酸-蛋白結合分析法………………………………22
六. 在大腸桿菌表現系統中誘導鞘蛋白基因大量表現.22
七. 蛋白質的純化……………………………………….24
八. 聚合酵素鏈反應…………………………………….24
九. 定序樣品之製備…………………………………….24
十. 西方墨點分析……………………………………….25
肆、 結果……………………………………………………...24
一. 生體外轉錄產生DIG標定衛星核酸作為探針…….27
二. NB-ELISA反應條件之最適化……………………...27
三. 以NB-ELISA測試衛星核酸對不同蛋白質結合之專一性…………………………………………......……28
四. 完整病毒顆粒與DIG標定衛星核酸鍵結之標準曲線測試…………………………………………………..28
五. 膠體純化之外鞘蛋白與DIG標定衛星核酸鍵結之標準曲線測試…………………………………………..29
六. CMV病毒顆粒在coating buffer與borate buffer下結構變化測試…………………………………………..29
七. CMV病毒顆粒與DIG標定衛星核酸交互作用之時程分析…………………………………………………..30
八. 比較NB-ELISA與北方-西方墨點法兩種方法測試RNA-protein交互作用的結果…………..…………..30
九. 從感病菸草選殖胡瓜嵌紋病毒外鞘蛋白基因…......30
十. 利用聚合酵素鏈反應放大外鞘蛋白各區段……......31
十一. 外鞘蛋白片段選殖到E. coli表現系統pETblue2.31
十二. 外鞘蛋白片段之誘導……………………...……..32
十三. 外鞘蛋白之純化…………………………………..32
十四. CMV不同外鞘蛋白區段與DIG標定衛星核酸交互作用之測定………………………………………..32
十五. 利用限制酵素產生衛星核酸上不同基因段……..33
十六. 衛星核酸序列刪減突變株的篩選………………..33
十七. CMV外鞘蛋白對不同種類DIG標定衛星核酸之專一性測試…………………………………………..33
十八. 在不同種類競爭者存在條件下，以NB-ELISA分析CMV病毒顆粒與衛星核酸之交互作用…….…...34
伍、 討論…………………………………………..………35
一. NB-ELISA方法的建立……………………………...…35
二. 外鞘蛋白上負責與衛星核酸結合區域之探討…..….39
三. 衛星核酸上負責與外鞘蛋白結合序列之探討……….39
陸、 參考文獻………………………………………………42
柒、 附錄……………………………………………………64
捌、 附圖……………………………………………………65

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