臺灣博碩士論文加值系統

在本文裡, 我們研究了20隻突變株在E.coli thioredoxin 蛋白兩個主要位置產生置換所帶來的影響, 這些胺基酸的置換主要是在可能產生蛋白質交互作用的疏水性區域。我們利用點突變法在位置G33及G74 改成其他不同類型的胺基酸, 例如serine, alanine, lysine…等, 製造20種不同單一及雙重的thioredoxin 突變株。 這20種不同的突變株我們拿來做噬菌體的感染測試, 我們發現在G74位置以其他胺基酸取代在37℃可支持T3/T7噬菌體的生長, 然而其他剩下的G33及G74突變株在37℃及在42℃下則不同程度地抑制T3/T7 噬菌體的生長。對於T7噬菌體, 在G74位置以serine 或threonine 和G33位置以alanine或histidine取代在37℃可支持T7噬菌體的生長, 然而剩下的G33及G74突變株在37℃及在42℃下也抑制了T7 噬菌體的生長, 但程度上沒像抑制T3/T7那麼嚴重。至於f1及M13噬菌體, 我們看出G74位置對噬菌體f1及M13的影響似乎比G33位置來得大, 至於thioredoxin 與線形噬菌體蛋白的詳細作用機制, 目前仍然不太清楚。但我們可以確定thioredoxin 與線形噬菌體蛋白的作用區域仍是座落於由胺基酸殘基32-35,72-76, and 91-94所組成的疏水平面上。

In this study, we report the effects of twenty different substitutions in E.coli thioredoxin on two major regions of the protein: the G33 site and G74 site where are the hydrophobic areas implicated in protein/protein interactions. There single and double mutants are produced by site-directed mutagenesis. The twenty different mutants have been tested in vivo with bacterial phages. Substitutions of glycine74 by some support phage T3/T7 growth at 37 ℃ whereas the rest substitutions prevented phage T3/T7 growth to different degrees. Substitution of glycine74 by threonine and substitution of glycine33 by alanine or histidine support phage T7 growth at 37 ℃. However, the rest mutants prevented phage T7 growth. As to f1 and M13 phages, glycine74 is more important than glycine33 when E.coli thioredoxin interacts with the protein of filamentous phage. We know little about the mechanism of thioredoxin with filamentous gene I protein. But we are sured that the interaction areas are still located on the hydrophobic surface of E.coli thioredoxin, including amino acid residues 32-35,72-76, and 91-94.

壹、緒論-------------------------------------------------------------------------1
ㄧ、E.coli Thioredoxin-------------------------------------------------------1
二、大腸桿菌Thioredoxin與噬菌體感染作用--------------------------2
1.T7與T3/T7噬菌體-------------------------------------------------------2
2.線形噬菌體-----------------------------------------------------------------3
三、 E.coli thioredoxin 突變株的誕生與影響--------------------------5
四、.研究目的---------------------------------------------------------6
貳、材料與方法-----------------------------------------------------------------7
一、實驗材料-------------------------------------------------------------------7
1.菌株---------------------------------------------------------------------------7
2.噬菌體------------------------------------------------------------------------8
3.質體---------------------------------------------------------------------------9
4.引子--------------------------------------------------------------------------11
5.酵素--------------------------------------------------------------------------12
6.培養基-----------------------------------------------------------------------12
7.蛋白質電泳溶液-----------------------------------------------------------13
8.DNA電泳溶液-------------------------------------------------------------14
9.DNA定序材料-------------------------------------------------------------15
10.試劑組(Kits) 及 Markers-----------------------------------------------15
11.儀器-------------------------------------------------------------------------16
12.溶液-------------------------------------------------------------------------17
二、實驗方法--------------------------------------------------------------------18
1.質體的萃取----------------------------------------------------------------18
2.電泳-------------------------------------------------------------------------18
3. DNA自動定序電泳-----------------------------------------------------19
4.定點突變-------------------------------------------------------------------20
5.E.coli thioredoxin 蛋白表現--------------------------------------------22
6.噬菌體感染作用----------------------------------------------------------23
參、實驗結果--------------------------------------------------------------------27
ㄧ、利用PCR方法製作Thioredoxin 突變基因------------------------27
二、質體的建構---------------------------------------------------------------27
三、DNA定序結果-----------------------------------------------------------27
四、 Thioredoxin 突變株蛋白質的表現-----------------------------------28
五、比較不同菌株的Thioredoxin蛋白的表現量-----------------------29
六、噬菌体感染Thioredoxin 突變株之E.O.P.值表---------------------29
肆、討論---------------------------------------------------------------------------53
伍、結論---------------------------------------------------------------------------58
陸、未來展望---------------------------------------------------------------------58
柒、參考文獻---------------------------------------------------------------------59
附圖一-----------------------------------------------------------------------------62
附圖二-----------------------------------------------------------------------------63
附圖三-----------------------------------------------------------------------------64
附圖四-----------------------------------------------------------------------------65
附圖五-----------------------------------------------------------------------------66
附圖六-----------------------------------------------------------------------------67
附圖七-----------------------------------------------------------------------------68
附圖八-----------------------------------------------------------------------------70
附圖九-----------------------------------------------------------------------------71
附圖十-----------------------------------------------------------------------------72
附圖十一--------------------------------------------------------------------------73
附圖十二--------------------------------------------------------------------------74
附圖十三--------------------------------------------------------------------------75
附圖十四--------------------------------------------------------------------------76
附圖十五--------------------------------------------------------------------------77
附圖十六--------------------------------------------------------------------------78
附圖十七--------------------------------------------------------------------------79
附圖十八--------------------------------------------------------------------------80
附圖十九--------------------------------------------------------------------------81
附圖二十--------------------------------------------------------------------------82
附圖二十一-----------------------------------------------------------------------83
附圖二十二-----------------------------------------------------------------------84
附圖二十三-----------------------------------------------------------------------85
附圖二十四-----------------------------------------------------------------------86
附圖二十五-----------------------------------------------------------------------87
附圖二十六-----------------------------------------------------------------------88
附圖二十七-----------------------------------------------------------------------89
附圖二十八-----------------------------------------------------------------------90
附圖二十九-----------------------------------------------------------------------91
附圖三十--------------------------------------------------------------------------92
附圖三十一-----------------------------------------------------------------------93
附圖三十二-----------------------------------------------------------------------94
附圖三十三-----------------------------------------------------------------------95
附圖三十四-----------------------------------------------------------------------96
附圖三十五-----------------------------------------------------------------------97
附圖三十六-----------------------------------------------------------------------98
附圖三十七-----------------------------------------------------------------------99
附圖三十八---------------------------------------------------------------------100
附圖三十九---------------------------------------------------------------------101
附圖四十------------------------------------------------------------------------102
附圖四十一---------------------------------------------------------------------103
附圖四十二---------------------------------------------------------------------104
附圖四十三---------------------------------------------------------------------105
附表一---------------------------------------------------------------------------106
附表二---------------------------------------------------------------------------107

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