臺灣博碩士論文加值系統

地衣芽孢桿菌 (Bacillus licheniformis) 為革蘭氏陽性(gram-positive) 、兼性厭氧 (facultative anaerobe) 之產孢桿菌，廣泛的存在於自然界的土壤及榖類中。由於其生長容易、具有分泌胞外蛋白質之能力，且同時為GRAS (generally recognized as safe) 級宿主，能安全而廣泛的被使用在酵素工業及生產蛋白質上，故被認為是遺傳工程上為具有發展潛力之宿主。
本研究建立了一個簡單又快速的地衣芽孢桿菌電轉形方法，在培養勝任細胞的培養基組成中添加1％ 甘胺酸，可於電阻200 Ω、電容25 μF、電場強度為10 kv cm-1下，得到最佳轉型效率1.72 × 103 transformants μg DNA-1。此外，鹼性彈性蛋白酶基因以及增強型的綠螢光蛋白基因於本宿主中皆適合作為報導基因。比較各種表現元件活性，結果以本實驗室所設計構築的σA型人工合成啟動子配合最適SD 序列的pOA-系列載體有最強活性表現。
Ling Zhi-8是由靈芝 (Ganoderma lucidum) 菌絲體 (mycelia) 中純化而來，為具有免疫調節功能之小分子蛋白質，有作為生物反應調節劑 (biological response modifier) 或免疫調節試劑 （immunomodul- ating agent） 之潛力。本實驗室先前以人工合成方式設計、合成重組靈芝免疫調節蛋白，包括N端帶有或不帶有來自鹼性彈性蛋白酶之訊息胜肽，及C端帶有6個組胺酸 (histidine) 以利純化。本研究中，將其表現於地衣芽孢桿菌及枯草桿菌蛋白酶缺損株 (Bacillus subtilis WB800) 中。結果顯示，N端不帶有訊息胜肽之rLZ-8，亦可分泌表現至胞外，且表現量更甚於N端帶有訊息胜肽者。而小鼠脾臟初代細胞增生能力結果則顯示，來自B. subtilis WB800之rLZ-8無法增生或抑制BALB/c小鼠脾臟初代細胞；然而來自地衣芽孢桿菌之重組靈芝免疫調節蛋白，具有抑制BALB/c小鼠脾臟初代細胞增生之能力。

Bacillus licheniformis is a gram-positive, facultative anaerobic, endospore-forming bacterium and widely distributed in the environment. Due to the characteristics of B. licheniformis such as: easy to growth, efficient secretion ability and considered GRAS (generally recognized as safe) by FDA (US Food and Drug Administration). B. licheniformis is a potential strain for secretion and production of heterologous proteins.
In this study, a simple and rapid electroporation procedure of this strain was established. The effect of electrical parameters and medium composition on electroporation efficiency was determined. Highest transformation efficiency (1.72 × 103 transformants μg DNA-1) was obtained when glycine-treated B. licheniformis were subjected to one pulse with 2.0 kv and 200 Ω with plasmid DNA.
The suitability of subtilisin YaB and GFP+ as reporters and function analysis of various expression signals in B. licheniformis was examined. Results suggest that Subtilisin YaB and GFP+ is suitable as reporters and expression signal containing artificial σA type promoter and optimized SD sequence pOA achieved highest expression level in B. licheniformis.
LZ-8, a fungal immunomodulatory protein (FIP) from Ganoderma lucidium, is considered as potential biological response modifier or immunomodulating agent. In our previously study, recombinant LZ-8 was designed and constructed with or without signal peptide in N-terminus and six histidine tag in C-terminus. The rLZ-8 was expressed in B. licheniformis and B. subtilis WB800 by pOA. The rLZ-8 without signal peptide could be directly secreted into supernatant of those hosts. Expression level of rLZ-8 was higher in B. licheniformis. The cell proliferation assay showed that rLZ-8 from B. subtilis WB800 did not significantly affect the proliferation of splenocytes from BALB/c mice. However, rLZ-8 from B. licheniformis inhibited the proliferation of spleen cells from the mice.

中文摘要…………………………………………………………................. i

Abstract……………………………………………………………………… ii

壹、前言……………………………………………………………………. 1
一、 芽孢桿菌屬 (Bacillus spp.)………………………………………..... 1
（一） 地衣芽孢桿菌之簡介……………………………………............... 1
（二） 枯草桿菌之簡介……………………………................................... 12
二、 靈芝免疫調節蛋白（Ling Zhi-8）…………………………………. 18
（一） 靈芝 (Ganoderma lucidum) 之簡介………………………………. 18
（二） 靈芝免疫調節蛋白 (Ling Zhi-8) 之發現………………………… 21
（三） 真菌免疫調節蛋白
(fungal immunomodulatory protein family, FIP family)..................... 21
（四） 真菌免疫調節蛋白之結構 24
三、 實驗目的……………………………………………………………... 26
四、 實驗策略…………………….…………….…………….…………… 27
貳、材料與方法…………………………………..….…………………….. 28
一、 菌種與質體………………………………..……….………………… 28
二、 藥品與試劑………………………………….………….……………. 32
三、 宿主特性分析……..………………………………............................. 32
四、 質體DNA抽取法……..…………...…….………………………….. 32
五、 電轉形法…………………………………………………………….. 33
六、 不同報導基因於地衣芽孢桿菌之適用性評估 .……………............ 35
七、 不同表現訊號於地衣芽孢桿菌之適用性評估……………............... 36
八、 DNA分子之電泳、剪切、回收及黏合…………………..………… 36
九、 重組靈芝免疫調節蛋白表現載體之構築………………................... 38
十、 重組之靈芝免疫調節蛋白於不同宿主中之分泌表現與偵測……... 38
十一、 不同來源的重組靈芝免疫調節蛋白之純化…....………………... 39
十二、 重組靈芝免疫調節蛋白對於老鼠脾臟細胞增生能力之評估....... 40
參、結果與討論..………………....………………....………...…………… 41
一、 宿主特性分析結果………....………...……………............................ 41
二、 地衣芽孢桿菌電轉形方法之建立………....………………............... 41
三、 不同報導基因於地衣芽孢桿菌之適用性評估……………............... 45
四、 不同表現訊號於地衣芽孢桿菌之適用性評估……………............... 45
五、 重組靈芝免疫調節蛋白表現載體之構築………....………………... 48
六、 重組靈芝免疫調節蛋白於地衣芽孢桿菌中之表現情形…............... 52
七、 重組靈芝免疫調節蛋白於B. subtilis WB800中之表現情形............ 52
八、 不同來源的重組靈芝免疫調節蛋白之純化……........…................... 56
九、 不同來源的重組靈芝免疫調節蛋白對於老鼠脾臟初代細胞增生
能力之評估…………………………………………………………
56
肆、結論........….....……........…......……........…..........….....……............... 59
伍、參考文獻….....……........…......……........…..........….....……................. 60

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