臺灣博碩士論文加值系統

MRF4是肌肉特異性轉錄因子之一，參與肌肉芽細胞的分化及增生。先前的研究發現斑馬魚MRF4基因有不同的兩型，即MRF4a與MRF4b，而且與其他物種的肌肉轉錄因子群（MRFs）的三位成員–Myf5、MyoD及Myogenin一樣，在蛋白質結構上具有一共同的保守區域bHLH domain；近年來有研究發現另一個具功能性的蛋白序列其位於bHLH domain之後，經電腦預測為一α-helix的結構，特此命名為「Helix 3 domain」，且證實此段被預測為螺旋結構的蛋白序列於骨骼肌生成上，具有促進內生性肌肉專一性調控基因的轉錄。由於MRFs家族的羧端內「Helix 3 domain」序列具高度保留性，因此本研究選擇在斑馬魚MRFb的羧端內「Helix 3 domain」 （胺基酸序列210至224）以site-directed mutagenesis PCR製造MRF4b胺基酸序列219位置Serine點突變成Proline，來測定「Helix 3 domain」突變對於二級結構所造成的影響；同時也利用酵母菌單雜合分析（yeast one-hybrid system）試圖探討「Helix 3 domain」在MRFs家族的轉錄活性上，所扮演的角色。首先利用E.coli BL21-CodonPlus（DE3）-RIL 菌株製造MRF4a羧端蛋白、MRF4b羧端突變與未突變的重組蛋白，由圓二色旋光儀（circular dichroism）的光譜分析其二級結構，結果指出此蛋白功能區存在有「β-sheet」。此外，酵母菌單雜合分析試驗中觀測宿主酵母菌GAL1-HIS3報導基因的表現情形，也發現以MRF4b全長蛋白為activation domain之報導基因，宿主酵母菌生長情況較好；故推論具有「Helix 3 domain」的MRF4b蛋白活性較高。

MRF4 belongs to the family of myogenic regulatory factors（MRFs）and participates in the differentiation and proliferation of myoblasts. Previous studies in Zebrafish indicated the existence of two different types of MRF4 gene, namely MRF4a and MRF4b. This situation is the same as the three other members of MRF family: Myf5, MyoD, Myogenin of which they share a conserved bHLH domain. Other studies indicated another functional protein sequence is present at the carboxyl end following the bHLH domain. It is predicted to be an amiphipathic α-helix, and is referred to as「Helix 3 domain」. It was also verified that this α-helix is capable of promoting expression of endogenous muscle gene during skeletal myogenesis. Because「Helix 3 domain」of carboxy-terminal region of MRFs family exhibits highly conserved sequence﹐I substituted amino acid 219 for proline in the carboxyl terminal sequence of MRF4b by site-directed mutagenesis PCR to determine any change in the secondary structure. I also utilized yeast one-hybrid analysis to investigate the role of「Helix 3 domain」in the transcription activity of MRFs. I used E.coli BL21-CodonPlus (DE3) RIL strain to produce MRF4b C-terminal protein, MRF4b C-terminal protein and MRF4b C-terminal mutant protein. I then compared the difference of secondary structures of these three different recombinant proteins by circular dichroism. The results indicated「Helix 3 domain」contains a「β-sheet」. In addition, yeast one-hybrid system utilizes GAL1-HIS3 reporter gene to observe the transcription activity of MRFs in the yeast host cell. I found MRF4b is the best of all three MRFs. I infer that MRF4b with「Helix 3 domain」has a higher activity than the other two forms of MRF.

第一章 前言 ------------------------------------------- 1
第二章 材料與方法 --------------------------------- 12
第一節 菌種、質體與抗體................................................................12
第二節 ZMRF4a與ZMRF4b羧端蛋白質表現質體構築...............13
2.2.1 聚合酶連鎖反應（Polymerase Chain Reaction﹐PCR）
...........................................................................................13
2.2.2 膠體電泳………………………………………………....14
2.2.3 DNA純化………………………………………………...14
2.2.4 ZMRF4a與ZMRF4b羧端基因之次選殖（TA cloning）
.…………………………………………………………..15
2.2.5 ZMRF4a與ZMRF4b羧端基因之選殖…………………...16
2.2.6 勝任細胞（competent cells）之製備…………………….....17
2.2.7 細胞轉形（transformation）………………………………..18
2.2.8 質體的小量製備..................................................................18
2.2.9 DNA定序……………………………………………….....20
2.2.10 胺基酸演化變異樹狀圖分析……………………………..20

第三節 ZMRF4a與ZMRF4b羧端重組蛋白的誘發表現及純化...20
2.3.1 蛋白質誘生時間之檢測…………………………………20
2.3.2 SDS-PAGE蛋白質電泳………………………………….21
2.3.3 菌體超音波震碎…………………………………………22
2.3.4 His6-tag ZMRF4a與His6-tag ZMRF4b融合蛋白之大量 純…………………………………………………………23
2.3.5 His6-tag ZMRF4a與His6-tag ZMRF4b融合蛋白與Thrombin蛋白水解酶作用……....……………………....24
2.3.6 離子交換層析（anion exchanger chromatography）…...…25
2.3.7 西方墨點法………………………………………………26
2.3.8 Bradford 蛋白定量………..……………………………..27
2.3.9 圓二色分光光譜之分析（Circular Dichroism spectroscopy）.....................................................................27

第四節 ZMRF4b羧端突變蛋白質表現質體之構築與重組蛋白的誘發表現及純化…………………………………………..28
第五節 酵母菌單雜合分析（yeast one-hybrid system）…………..30
2.5.1 酵母菌單雜合分析載體之構築…………………………30
2.5.2 酵母菌勝任細胞的製備…………………………………31
2.5.3 酵母菌的細胞轉形（transformations）…………………...31
2.5.4 Culture assay ………………………………………….....33

第三章 結果與討論 --------------------------------- 34
第一節 斑馬魚MRF4之分子結構與其他物種肌肉調控蛋白之胺基酸序列比對……………………………………………..34

第二節 大腸桿菌蛋白表現型及酵母菌單雜合分析型載體的構築…………………………………………………………..36

3.2.1 目標基因之擴增…………………………………………36
3.2.2 大腸桿菌蛋白表現型載體之構築……………………...36
3.2.3 酵母菌單雜合分析載體的構築………………………...37

第三節 融合蛋白之表現分析……………………………………39
第四節 蛋白之純化分析…………………………………………40
第五節 二級結構預測……………………………………………43
第六節 酵母菌單雜合分析（yeast one-hybrid system）............44
第七節 圓二色分光光譜分析（Circular Dichroism spectroscopy）
……………………………………………………………47

參考文獻 ------------------------------------------------ 50
附錄 ------------------------------------------------------ 76
圖表目錄

圖一､ MRFs功能區之結構分析。 --------------------------------------- 56
圖二､pET28c（＋）之map。 ------------------------------------------- 58

圖三､ 重疊延伸法產生MRF4b羧端突變片段。 ---------------------- 59

圖四､pGBDU-C1之map。 ---------------------------------------------- 60

圖五､斑馬魚MRF4與其他物種MRF4胺基酸序列之比對圖。---- 61

圖六､ZMRF4a､ZMRF4b與其各羧端DNA利用PCR反應，經2％

agarose gel分析之結果。 ----------------------------------------- 63

圖七､His6-tag ZMRF4a羧端重組蛋白經0.5％乳糖誘生表現之10％

Tricine SDS-PAGE。 ----------------------------------------------- 64

圖八､His6-tag ZMRF4b羧端重組蛋白經0.5％乳糖誘生表現之10％

Tricine SDS-PAGE。 ----------------------------------------------- 65

圖九､以10％ Tricine SDS-PAGE分析His6-tag ZMRF4a羧端重組蛋

白的表現。 ----------------------------------------------------------- 66

圖十､以10％Tricine SDS-PAGE及Western blotting分析His6-tag

ZMRF4b羧端與ZMRF4b羧端重組蛋白的表現。-------------- 67

圖十一､ZMRF4b羧端蛋白經FPLC MonoQ管柱層析。------------- 68

圖十二､ZMRF4b羧端突變蛋白經FPLC MonoQ管柱層析。------- 69

圖十三､NNPREDICT程式預測ZMRF4b羧端與其突變蛋白二級結

構之結果。 --------------------------------------------------------- 70

圖十四､比較ZMRF4b羧端與其突變蛋白之CD光譜。------------- 73

表一､ PCR所需之引子。 ------------------------------------------------- 74

表二､ 酵母菌單雜合分析之試驗結果。 -------------------------------- 75

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