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研究生： 張淳欽
Chang, Chun-Chin
論文名稱： 創傷弧菌螢光蛋白基因bfgV的分子研究
MOLECULAR STUDIES OF FLUORESCENT PROTEIN GENE bfgV FROM Vibrio Vulnificus CKM-1
指導教授： 張敏政
Chang, Ming-Chung
學位類別： 博士
Doctor
系所名稱： 醫學院 - 基礎醫學研究所
Institute of Basic Medical Sciences
論文出版年： 2005
畢業學年度： 93
語文別： 中文
論文頁數： 118
中文關鍵詞： 定向演化 、螢光 、短鏈去氫/氧化酵素 、創傷弧菌
外文關鍵詞： Vibrio vulnificus, SDR, fluorescence, directed evolution
相關次數： 點閱：48  下載：0 
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    • 中文摘要

    　　我們由一株非螢光性創傷弧菌CKM-1基因庫內找到一個螢光蛋白基因命名為bfgV，它所轉譯出來的蛋白(BfgV)含有239個氨基酸，經序列分析比對後發現是屬於SDR (short chain dehydrogenase/reductase)家族。將BfgV大量製造並純化後，測其螢光光譜，得知該蛋白主要的激發波峰為352 nm，另有一較小的激發波峰在283 nm; 而其最大的放射波峰出現在456 nm。高效液態層析法(HPLC)與L-glutamic dehydrogenase反應法，證明NADPH與BfgV結合在一起並且與BfgV的螢光有密切的關係，其後的分析結果也顯示，NADPH結合到BfgV後，其內生性螢光會被放大10倍左右。另外，在進行序列分析比對時，我們發現有兩個與bfgV很像的基因分別存在於Vibrio cholera El Tor N16961和 Shewanella oneidensis MR-1，比較這三個基因周邊的基因組態後，我們發現在他們的上游區都緊鄰著一個轉譯調節基因，為了求證bfgV與其上游調節基因(bfgR)的關係，我們設法將bfgR破壞掉，結果發現這種變異株內BfgV蛋白的表現量會增加；而凝膠泳動位移分析(EMSA)也證明純化的BfgR蛋白會與bfgR-bfgV間的啟動子區進行特異性的結合。由這些實驗結果得知bfgV受到上游bfgR的負向調控。另外，我們也用PCR隨機突變以及DNA重排技術對bfgV基因進行定向演化。最後得到一個突變基因D7，此基因產物所表現的螢光指數為野生型的4倍，而其放射波長為440 nm較BfgV的456 nm要短，激發波長則無變化。D7總共有8個氨基酸與BfgV不同，其中7個對螢光增強有實質貢獻，將這些突變標示在BfgV立體分子模型後發現其中有3個突變集中在構成NADPH結合位的loop 4 和loop 6上。因為這些突變非常靠近NADPH，因而推測這個區域在影響BfgV的螢光強度上扮演重要的角色。另外，我們發現不論在有氧或無氧狀況下，D7的大腸桿菌轉型體在蛋白質合成與螢光的出現上幾乎是同步發生，這個特性與綠螢光蛋白(GFP)明顯不同。

    Abstract

    　Blue fluorescent protein BfgV, belonging to the SDR family, was found in non-bioluminescent pathogen Vibrio vulnificus CKM-1. This protein had two excitation peaks at 283 nm and 352 nm respectively, and one emission peak at 456 nm. The results of HPLC analysis and L-glutamic dehydrogenase reaction indicated that BfgV fluoresced through augmenting 10 times the intrinsic fluorescence of NADPH binding on it. Comparison of gene organizations around bfgV and two analogues in Vibrio cholera El Tor N16961 and Shewanella oneidensis MR-1 revealed that each of them had a transcriptional regulatory gene located at the vicinal upstream region. Insertional disruption of the upstream regulatory gene bfgR and electrophoretic mobility shift assay proved that bfgV was negatively regulated by bfgR. In the directed evolution process, wild type bfgV gene was subjected to three cycles of error-prone PCR and one cycle of DNA shuffling. A prominent mutant D7 displayed 4 times the fluorescent intensity of BfgV. The emission peak of D7 was at 440 nm, 16 nm shorter than that of BfgV, but the excitation peaks of these two proteins were the same. D7 possessed eight amino acid substitutions while only seven contributed positive influence on it. After assigning these mutations to the modeled 3D-structure of BfgV, we found three of them appeared at loop 4 and loop 6, which comprised the NADPH binding site. This unusual mutant cluster suggested these regions played a key role on fluorescent intensity of BfgV. In addition, D7 synthesis and fluorescent expression in E. coli transformants were nearly synchronic. This property was quite different from that of GFP.

    目錄 摘要 中文摘要 …………………………………………………………………………… v 英文摘要 …………………………………………………………………………… vi 緒論 ………………………………………………………………………………… 1 材料與方法 ……………………………………………………………………… 6 一、使用之菌株、載體及培養基 ………………………………………………… 6 二、bfgV基因的次選殖 …………………………………………………………… 6 三、利用電穿孔將pFP21送入大腸桿菌BL21(DE3) …………………………… 7 四、觀察菌落螢光 ………………………………………………………………… 8 五、BfgV蛋白的製造與純化 ……………………………………………………… 8 六、探討BfgV螢光蛋白的激發與放射波光譜…………………………………… 9 七、利用膠體過濾法探討BfgV螢光蛋白聚合狀態……………………………… 9 八、利用HPLC和L-Glutamic dehydrogenase分析BfgV蛋白的螢光是否與 NAD(P)H有關 ……………………………………………………………………… 10 九、利用菌落雜交法找出bfgV周邊的DNA片段 ……………………………… 11 十、利用S17-1/pSVI001系統對V. vulnificus CKM-1的bfgR 進行插入性 破壞……………………………………………………………………………… 14 十一、利用電穿孔的方式將載體送入V. vulnificus CKM-1 ………………… 16 十二、利用西方墨點法偵測在CKM-1與URD101菌體內BfgV表現的差異…… 17 十三、BfgR-(His)6融合蛋白的製造與純化 …………………………………… 18 十四、BfgR融合蛋白與bfgV上游的啟動子進行凝膠泳動位移試驗 …………… 20 十五、載體DNA之抽取…………………………………………………………… 21 十六、由電泳膠分離純化DNA片段……………………………………………… 22 十七、接合反應…………………………………………………………………… 22 十八、載體構築…………………………………………………………………… 23 十九、大腸桿菌的轉型…………………………………………………………… 23 二十、SDS-聚丙烯醯胺膠體電泳………………………………………………… 24 二十一、測試BL21轉型體螢光及BfgV蛋白表現情形………………………… 25 二十二、測試螢光指數的線性範圍……………………………………………… 25 二十三、對bfgV進行隨機突變 (error-prone PCR) ………………………… 26 二十四、對bfgV突變基因進行DNA重排技術 (DNA shuffling) …………… 26 二十五、篩選使大腸桿菌轉型體螢光增強的bfgV突變基因 ………………… 27 二十六、利用NADPH滴定法探討BfgV與變種D7螢光蛋白NADPH複合物解離 常數Kd的變化…………………………………………………………… 28 二十七、BfgV蛋白3D-立體結構模擬…………………………………………… 29 二十八、變種D7與野生型GFP發螢光模式比較 ……………………………… 29 二十九、DNA定序 ………………………………………………………………… 30 三十、GenBank 登錄編號………………………………………………………… 30 實驗結果 ………………………………………………………………………… 31 一、bfgV序列比對結果…………………………………………………………… 31 二、BfgV的螢光光譜與聚合度分析……………………………………………… 32 三、BfgV發光原因探討…………………………………………………………… 33 四、BfgV具有放大NADPH內生性螢光的能力…………………………………… 34 五、解析V. vulnificus CKM-1染色體內bfgV的周邊序列與基因組態 …… 34 六、比較bfgV和其類似基因周邊的基因組態 ………………………………… 35 七、製造bfgR變異株 …………………………………………………………… 36 八、比較野生型CKM-1與bfgR變異株URD101內BfgV蛋白表現情形 ……… 36 九、BfgR重組蛋白的製造與純化 ……………………………………………… 37 十、凝膠泳動位移試驗(EMSA)…………………………………………………… 37 十一、以error-prone PCR的方法對野生型的bfgV基因進行第一回合的隨機 突變與篩選………………………………………………………………… 38 十二、以A48為母基因進行第二回合的隨機突變與篩選……………………… 40 十三、以B-系列突變基因為為母基因群進行DNA重排 ……………………… 41 十四、以C-系列突變基因進行第三回合的隨機突變與篩選…………………… 42 十五、DNA定序與分析 …………………………………………………………… 43 十六、野生型BfgV與D7突變蛋白螢光特性比較……………………………… 44 十七、BfgV蛋白分子的立體結構模擬…………………………………………… 45 十八、比較D7與GFP在有氧與無氧狀態下的螢光表現 ……………………… 46 討論 ……………………………………………………………………………… 49 圖目錄 圖一、bfgV的序列分析與其對應蛋白的二級結構預測………………………… 55 圖二、重組載體pFP21構築過程與各成分DNA電泳分析圖…………………… 56 圖三、用SDS-PAGE分析純化過的BfgV蛋白…………………………………… 57 圖四、分析大腸桿菌BL21轉型體表現BfgV時所發出的螢光光譜…………… 58 圖五、BfgV螢光蛋白激發和放射波光譜分析 ………………………………… 59 圖六、利用膠體過濾法做校正曲線求出BfgV的聚合度 ……………………… 60 圖七、用鹼性法萃取純化的BfgV螢光蛋白再經HPLC所得的分析圖………… 61 圖八、BfgV粗蛋白液經過管柱純化後，各收集瓶的分析圖 ………………… 62 圖九、純化的BfgV經GLDH處理後相對螢光的變化…………………………… 63 圖十、純化後的BfgV蛋白溶液與由該蛋白溶液經HPLC分析所得相同濃度的 游離態NADPH的相對螢光強度比較……………………………………… 64 圖十一、菌落雜交試驗所得的輻射曝光顯影圖………………………………… 65 圖十二、單向遠距PCR實驗所得的bfgV上游DNA片段電泳圖……………… 66 圖十三、bfgV及其周邊共4946 bp DNA序列…………………………………… 68 圖十四、bfgV與其類似基因bfgVVc和bfgVSo上下游的基因組態比較………… 69 圖十五、BfgV與另外兩個BfgV類似蛋白氨基酸序列比對 …………………… 70 圖十六、利用定位插入突變法構築bfgR缺失突變株 ………………………… 71 圖十七、用西方墨點法分析BfgR在CKM-1和URD101表現的情形…………… 72 圖十八、BfgR的生產與純化……………………………………………………… 73 圖十九、BfgR融合蛋白與bfgR-bfgV啟動子區的凝膠泳動位移試驗………… 74 圖二十、菌體濃度(OD600)與螢光強度的關係 …………………………………… 75 圖二十一、轉型體BL21/pFP21的生長曲線圖 ………………………………… 76 圖二十二、BL21/pFP21和BL21/p21A48螢光與蛋白表現時程分析圖………… 77 圖二十三、bfgV, A48與A25突變基因的比較 ………………………………… 78 圖二十四、Mg2+和Mn2+對DNaseI切割DNA的影響 ……………………………… 79 圖二十五、DNaseI在含Mg2+ 的buffer內對bfgV DNA片段進行切割 ……… 80 圖二十六、DNA重排實驗 ………………………………………………………… 81 圖二十七、利用定位突變所培育的D7與各突變基因演化相關性 …………… 83 圖二十八、D7變異基因與其直屬變異基因螢光指數變化情形………………… 84 圖二十九、表現野生型bfgV基因與其突變基因D7的大腸桿菌轉型體菌落螢 光表現情形…………………………………………………………… 85 圖三十、比較BfgV和D7蛋白在大腸桿菌表現時螢光光譜的差異…………… 86 圖三十一、利用滴定法求出D7與BfgV 的NADPH複合物的解離常數Kd …… 87 圖三十二、BfgV 與 meso-2,3-butanediol dehydrogenase (m-BD)氨基酸序 列比對………………………………………………………………… 88 圖三十三、BfgV-NADPH複合物部分模擬圖……………………………………… 89 圖三十四、D7的突變點在BfgV分子立體結構上的位置 ……………………… 90 圖三十五、大腸桿菌轉型體表現D7或GFP與其對應螢光的動態變化比較 … 91 圖三十六、BL21/pD719與BL21/pGFP在有氧與無氧條件下培養時螢光表現情形……………………………………………………………………… 92 圖三十七、推測符合人類細胞表現的D7基因序列 …………………………… 93 圖三十八、推測符合斑馬魚細胞表現的D7基因 ……………………………… 94 表目錄 表一、D7與其前代基因所發現的突變位置……………………………………… 82 參考文獻 ………………………………………………………………………… 95 附錄 一、實驗所用的菌株與載體 …………………………………………………… 105 二、載體pET-21b圖譜 ………………………………………………………… 106 三、載體pGFP圖譜……………………………………………………………… 107 四、人類高表現基因密碼頻譜表 ……………………………………………… 108 五、斑馬魚基因密碼頻譜表 …………………………………………………… 109 六、縮寫檢索表 ………………………………………………………………… 110

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