臺灣博碩士論文加值系統

resistn是一種分泌性的蛋白質，屬於FIZZ蛋白質家族成員之一，主要由脂肪細胞所分泌，且其分泌量之多寡與肥胖和第二型糖尿病有關聯性，在老鼠身上已經證實。為了明瞭此蛋白質與細胞之間的交互作用及完整的訊號傳遞模式，則必須使用大量的resistin作為研究的材料，因而選擇利用桿狀病毒表現系統(BEVS)表現resistin分泌性蛋白質，其優點在於蛋白質的產量大且具有類似哺乳類動物細胞蛋白質後修飾作用的功能，讓所生產的蛋白質具有活性。
本實驗利用time course的方法於48、60及72小時去尋找此蛋白質最佳表現情況，及品質最佳的時間點，結果發現於60及72小時的蛋白質表現量比48小時來得多。
為了找出較佳的打破細胞的方法，藉由加入不同的試劑如：Guanidine-HCl buffer、Urea buffer 、Tris-HCl buffer及NP-40 lysis buffer的條件下，評估其對蛋白質之製備是否會影響，結果則發現添加Guanidine-HCl buffer能收穫較多的蛋白質。
為了模擬於昆蟲細胞培養液中，是否能純化所要的Histidine其標定的蛋白質，則將培養液調成pH 6.3及pH 8.0的情況之下，對利用His-bind resin純化蛋白質效率的影響，結果發現無法純化出所要的蛋白質。
於此論文，我們獲得表現Resistin的重組桿狀病毒，而重組蛋白質乃存在於細胞中，而不是分泌到培養液中。此可能是由於Resistin的signal peptide 於昆蟲中沒有功能。因此我們未來將用昆蟲細胞的signal peptide協助Resistin的分泌。

Resistin is a member of FIZZ protein family and mainly secreted by adipocyte. The quantity of secreted resistin is related to obesity and typeII D.M., which was proved on mouse model. In order to understand the interaction of protein and cells and their signal transduction pathway, we need to produce recombinant resistin in large scale. Therefore, we choose the Baculovirus Expression System (BEVS) to produce recombinant resistin protein. The advantage of Baculovirus Expression System is that (1) it can produce recombinatn protein largely, and (2) there is post-translation modification available in insect cells, similar to that in mammalian cells. Thus the recombinant proteins, produced in insect cells, is likely to be active.
In the experiment of baculovirus expression system, time course has been run for the optimal peroid for the maximum expression. The cells were harvested at 48, 60 and 72 hour post transfection and the quantity of recombinant preoteins were analyzed. We found that higher amount of recombinat proteins were observed at 60 and 72 hour post transfection, compare to the protein produced 48 hour.
While we tried to harvest and analyze the proteins, different lysis reagents were added, such as guanidine-HCl buffer, urea buffer, Tris-HCl buffer and NP-40 lysis buffer. We evaluated the efficiency of these buffer, and found that more recombinant protein were harvested by using Guanidine-HCl buffer.
In order to know whether we can purifiy the his-tagged protein, proteins have been purified by His-bind resin in the Grace medium at pH 6.3 and 8.0. The recombinant protein can not be purified under this condition.
In this thesis, we obtained the recombinat baculovirus, expressing the recombinant resistin in the cells, but not in the culture medium. This may due to the ill-function of the signal peptide, therefore, we would like to use signal peptide of insect cells for carrying the secretion of resistin in the future.

目錄
中文摘要 Ⅰ
英文摘要 Ⅲ
目錄 Ⅴ
圖目錄 Ⅷ
表目錄 Ⅹ
縮寫檢索表 ⅩⅠ
一、緒論 1
二、實驗材料 8
2.1儀器 8
2.2藥品 8
三、 實驗方法 12
3.1構築質體 12
3.1-1 連鎖聚合反應 12
3.1-2 DNA接合反應 13
3.1-3 形質轉移 14
3.1-4 小量抽取細菌質體 15
3.1-5 利用限制酵素切DNA 16
3.1-6 跑DNA電泳 16
3.1-7中量培養細菌質體 17
3.1-8中量抽取細菌質體 18
3.1-9 電泳法回收DNA片段 19
3.2 桿狀病毒表現系統 20
3.2-1昆蟲細胞之介紹 20
3.2-1.1 配製昆蟲細胞培養液 20
3.2-2昆蟲細胞培養 21
3.2-3共轉染作用 21
3.2-4噬菌斑測試 22
3.2-5蛋白質的表現及製備 23
3.2-6 跑SDS-PAGE蛋白質電泳 24
3.2-7西方點墨法 26
3.2-8蛋白質純化 27
四、結果 30
五、討論 33
六、參考文獻 36
圖 44
表 54
附錄 55
自述 62

六、參考文獻
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