臺灣博碩士論文加值系統

多重抗藥性基因(multidrug resistance，簡稱MDR基因)普遍存在於細菌的基因體中，在鏈黴菌中，抗Ethidium Bromide的MDR基因ebrC，和其抑制者(repressor)基因ebrS形成反向結構，中間包含65個鹼基對大小的片段，含有ebrC和ebrS基因的啟動子(promoter)。在Lee等人(2007)的研究中指出，ebrC和ebrS的啟動子平時不表現，只有當鈣離子出現時才會表現下游的ebrS基因，這非常符合一個好的表現型載體的特色，因為它平時是關閉的，需要表現蛋白時，只要加入鈣離子，它便會被誘發而大量表現蛋白。另外，鈣離子便宜、取得容易，不是合成物或抗生素，是非常理想的天然誘導物。所以這個啟動子非常適合發展成一個蛋白質的表現系統。
本研究是希望借用這套系統，在高拷貝數的質體上，建構出一個可被氯化鈣調控的表現型載體。我把原本包含部份ebrC和ebrS片段的啟動子區域縮小到只包含兩基因之間的DNA序列片段，並在培養基上測試氯化鈣是否能誘發此啟動子的表現，結果顯示當外加的氯化鈣不存在時，ebrC和ebrS方向的啟動子都不會表現，而當氯化鈣存在時ebrC和ebrS方向的啟動子都會表現，但ebrS方向的報告基因表現量比ebrC方向的表現量更強，可能是縮小的啟動子區域往ebrC方向的抑制區域有部分被破壞。我也構築了擁有兩個ebrS方向的啟動子，其表現量比只有一個ebrS或ebrC方向的啟動子都來的更強；所以接下來，我使用生物螢光(luciferase)的活性以及使用抗體偵測有His-tag標記的末端蛋白表現的量，來推算鈣離子對此啟動子產生的誘發活性的效果。這兩部分的初步實驗結果均顯示擁有兩個同方向ebrS啟動子的轉型株在沒有添加氯化鈣的情況下反而會有效佳的表現質，與當初的預期結果不符。我們猜想有可能是內生的EbrS被誘發表現後，回過頭來抑制此啟動子的表現，所以建議後續的實驗要在剔除ebrS的菌株內測試。

Multidrug resistance (MDR) gene is abundant in bacteria genomes. One of Streptomyces MDR gene, ebrC, is able to confer resistance to Ethidium Bromide. ebrC is opposite orientation with ebrS, the repressor gene of ebrC. A 65 bp DNA fragment is in their intergenic region, and it has been identifie as the bidirectional promoter for both genes. Previous studies showed that ebrC and ebrS didn’t express in normal conditions, but expressed when calcium ions were present. These characters are fit to the classic protein expression system, because of no inducer, no induction. The calcium ion is a wonderful inducer that it is cheap, easy to get, and it''s not synthetic compound or antibiotics.
In this study, our purpose is using this ebrC-ebrS promoter system to construct a calcium ions inducible expression vector in Streptomyces. We inserted the shortened promoter, only including intergenic DNA sequence between ebrC and ebrS, into the upstream of a promoter-less kanamycin resistant gene on a high copy number plasmid to test its activity. Our data showed that both of ebrC and ebrS promoters could be induced by calcium ions and there was a stronger inducible activity in the promoter of ebrS than the one of ebrC. It may cause by shortened promoter that the repressor binding sequences of ebrC is damaged. According to the result of paper disc assay, we found that a DNA fragment containing two direct repeats of ebrC-ebrS promoter showed higher expression level than one promoter of each direction. For quantifying the induced promoter activity, we employed a luciferase gene and a his-tag fused TP gene as the measured materials by detecting their enzyme activity or protein amounts. Both parts of primary data showed oppositely expected results that no or fewer proteins were expressed when calcium ions were present, but the proteins expressed well without adding calcium ions. We guess those results may cause by expressed endogenous EbrS proteins which repress this constructed promoter, and we suggest the further experiment should be tested in ebrS deletion strain.

中文摘要 ii
英文摘要 iv
誌謝 vi
目錄 vii
表目錄 viii
圖目錄 ix
第一章 序論 1
第二章 材料與方法 12
第三章 實驗結果 19
第四章 討論 56
參考文獻 64
附錄 66

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