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研究生:程建裕
研究生(外文):Jian-Yu Cheng
論文名稱:純化CCN1重組蛋白並探討其於非小細胞肺癌之角色
論文名稱(外文):Purification of Recombinant CCN1 Protein and Characterization of the Role in Lung Cancers
指導教授:呂世正
指導教授(外文):Shr-Jeng Leu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:150
中文關鍵詞:CCN1重組蛋白桿狀病毒表現系統肺癌
外文關鍵詞:recombinant CCN1 proteinbaculovirus exoression systemlung cancers
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我們利用Bac-to-Bac桿狀病毒表現系統製備了一系列的表現載體,於其N’端放入一段取自蜂毒肽的分泌訊號(insect honeybee melittin secretion signal),可以幫助重組蛋白有效率地分泌至細胞培養液。也更進一步建構一種帶有FLAG(DYKDDDK)及StrepII(WSHPQFEK)雙親和力標籤的新型表現載體,並於其後端加入一段專一性的TEV protease辨識序列(ENLYFGQ)。可以利用SP-Sepharose陽離子交換樹脂及StrepTactin親和力管柱純化重組蛋白,並藉由TEV protease去除前端的親和力標籤。
我們建立了一套低成本、高純度及高產量(cost-effective)的蛋白質純化系統,成功地製造並純化出多種具有生物活性的重組蛋白,其中包含屬於CCN家族成員的CCN1重組蛋白。相較於傳統單一離子層析管柱,這套系統可以不僅可以量產CCN1重組蛋白,同時也將蛋白質純度進一步提升至>99%以上,也成功地利用TEV protease可將N’端的親和力標籤去除。此外,透過多種功能性測試也證實CCN1重組蛋白具有生物活性。
目前CCN1被認為會抑制非小細胞肺癌(NSCLCs)的生長,同時也會降低其腫瘤轉移並增加肺癌病患的存活率。然而,CCN1於非小細胞肺癌上的調控機制目前仍不清楚。實驗結果指出,以CCN1重組蛋白長期處理非小細胞肺癌的三種細胞株:A549、H460及H520,都可以誘導細胞生長中止,我們也將更進一步的去探討CCN1調控非小細胞肺癌的分子機轉。
總結本論文,我們發展並建立了一套低成本、高純度及高產量的蛋白質純化系統,而這套系統將來也可以應用於更多的蛋白質分子上。
We have previously developed a series of expression vectors utilizing the robust Bac-to-Bac baculovirus expression system. In this study, we have advanced to design a new vector that allows expression of a dual-affinity tag, the FLAG and Strep-tag II sequence, followed by a specific TEV protease cleavage site, which are fused to the N-terminus of expressed recombinant proteins. The dual-affinity tag-containing recombinant proteins can be purified by sequential affinity chromatographies including the SP-Sepharose (cationic ion-exchange) and consecutive StrepTactin affinity column, respectively.
We have successfully produced and isolated several human proteins of therapeutic potential, including the matrix-associated CCN1 protein. By such purification setting, the recombinant CCN1 can be obtained with >99% homogeneity as judged by sensitive silver staining and is cost-effective with the comparable low reagent price. In addition, the fused dual-affinity tag sequence can be successfully removed upon TEV protease cleavage. By a variety of test assays, the purified recombinant CCN1 also shows to be bioactive.
Available reports show that CCN1 may play a negative role in human non-small cell lung carcinomas (NSCLCs). However, the molecular mechanism underlying the CCN1-induced tumor growth suppression remains to be clarified. Our data show that treatment of recombinant CCN1 proteins to three representative human NSCLCs, A549, H460, as well as H520, events of the cell cycle arrest are induced. We are examining the cancer cell growth control by recombinant CCN1 in molecular detail.
Taken together, the designed purification scheme provides us a cost-effective strategy to produce many other bioactive human proteins in the near future.
致謝 I
摘要 I
ABSTRACT II
目次 III
圖次 VI
壹、緒論(INTRODUCTION) 1
貳、研究動機與目的 10
參、實驗材料與方法(MATERIALS AND METHODS) 11
肆、實驗結果(RESULTS) 94
伍、實驗討論(DISCUSSION) 100
陸、參考文獻(REFERENCE) 104
柒、實驗圖表(FIGURES AND TABLES) 110
捌、附錄(APPENDIX) 132
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