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研究生:林昱樺
研究生(外文):Lin Yuhua
論文名稱:人類大麻素受體2於桿狀病毒表現系統之建立
論文名稱(外文):Establishment of human Cannabinoid receptor 2 by baculovirus expression system
指導教授:吳美莉吳美莉引用關係
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:食品科學系所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2012
畢業學年度:101
語文別:中文
中文關鍵詞:大麻素受體2競爭性親和力分析G蛋白偶合受體桿狀病毒表現系統
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Cannabinoid receptor 2 (CB2)屬於G蛋白偶合受體(GPCRs)家族並且廣泛存在於免疫系統細胞中。研究證實選擇性的CB2受體激活劑具有止痛和免疫調控功效。因此,CB2受體近來成為藥物開發的重要標的。本實驗中主要的目的為藉由桿狀病毒表現系統建立表面受體平台作為快速篩選大量具有人類CB2受體親和性之生物活性成分。首先以聚合酶鏈鎖反應自人類脾臟cDNA選殖CB2受體基因,將其構築於pFasTBac™載體再轉型至大腸桿菌DH10Bac™進行重組。以抗生素和藍白篩選法篩選出重組成功之bacmids並將其轉染於Sf9昆蟲細胞中進行表現。進一步利用西方墨點法與共軛聚焦顯微鏡確認CB2受體是否可表達於細胞表面,表達成功之CB2受體系統將被用於建立競爭性親和力分析。
摘 要 II
Abstract III
謝 誌 V
目錄 VI
圖表目錄 VIII
第一章 前言 1
第二章 文獻回顧 3
2.1 Endocannabinoid system 3
2.2 內生性配位體 3
2.3 G蛋白偶合受體(G Protein-Coupled Receptors, GPCR) 5
2.4 大麻素受體 6
2.5 Cannabinoid receptor 2 (CB2) 7
2.5.1 CB2受體之表現 7
2.5.2 CB2受體之結構 8
2.5.3 CB2受體之訊息路徑 11
2.6 桿狀病毒表現系統(Baculoviruses expression system) 14
2.6.1 桿狀病毒之分類 14
2.6.2 桿狀病毒之生活史 15
2.6.3 桿狀病毒之基因表現 15
2.6.4 桿狀病毒之構造 17
2.6.5 gp64 17
第三章 材料與方法 19
3. 1 本實驗使用菌株及細胞株種類 19
3.1.1 菌株種類 19
3.1.2 細胞株種類 19
3.1.3 載體 19
3.1.4 儀器設備 20
3.2 建立人類CB2受體蛋白於桿狀病毒/昆蟲細胞之表現系統 21
3.2.1 人類CB2受體蛋白之基因選殖 21
3.2.2 人類CB2受體基因與GP64訊號胜肽於表現載體之構築 28
3.2.3 細胞株之培養 35
3.2.4 昆蟲細胞之感染 38
3.2.5 表現重組蛋白CB2之分析 40
3.2.6 西方墨點法 41
第4章 結果 45
4.1人類CB2受體基因之選殖 45
4.2人類CB2受體基因於昆蟲細胞表現載體之構築 45
4.3人類CB2受體基因於昆蟲細胞表現載體之病毒DNA 46
4.4 人類CB2受體基因於昆蟲細胞之重組蛋白表現分析 46
4.4.1 SDS-PAGE分析之重組蛋白表現 46
4.4.2 西方墨點法分析之重組蛋白表現 46
第五章討論 47
第六章 結論 60
參考文獻 61
作者簡介 72

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