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研究生:廖苑君
研究生(外文):Yuan Chun Liao
論文名稱:新穎細胞素-1之鑑定
論文名稱(外文):Identification of Novel Cytokine-1
指導教授:張明熙
指導教授(外文):Ming-shi Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:108
中文關鍵詞:單核球介白素-10介白素-19腫瘤壞死因子-alpha介白素-6新穎細胞素-1
外文關鍵詞:monocyteinterleukin-10interleukin-19TNF-alphainterleukin-6novel cytokine-1
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在人體的免疫反應是由高度複雜的控制因子系統所調節。在這些調節控制因子之中,其中較顯著重要的是抗發炎的細胞素( anti-inflammatory cytokine ) 和特定細胞素的抑制者 ( specific cytokine inhibitor )。免疫系統中,發炎前期細胞素 ( proinflammatory cytokine ) 和抗發炎細胞素 ( anti-inflammatory cytokine ) 存在著一種動態平衡。濃度不足的抗發炎細胞素會導致過度發炎反應,而過多的抗發炎細胞素則會破壞宿主清除外來致病原的機制。
由於人體基因體計劃的進行,興起新穎基因的探索研究。因此,相似IL-10的新穎基因已漸被鑑定出來被稱為IL-10家族 ( IL-10 family )。IL-10家族包括IL-10,IL-19,IL-20,IL-22,AK155和MDA-7。Interleukin-10 ( IL-10 ) 是一種多功能性細胞素,在免疫反應中扮演相當重要的角色。由於IL-10經由降低抗原呈獻 ( antigen presentation )和巨噬細胞 ( macrophage )對刺激的反應來達到抗發炎反應的作用,所以早期IL-10被稱為細胞素合成抑制因子 ( cytokine synthesis inhibitory factor )。
我們自人類腎臟cDNA library分離出與IL-10相似的新穎細胞素-1 ( Novel cytokine-1;NC-1 )。分析人類NC-1基因體結構,發現NC-1基因具5個exon和4個intron。在NC-1 5’端未轉譯區域 ( untranslation region ),exon與intron排列頗多變化。
為了研究此新穎細胞素在人體中所扮演的角色,以及與IL-10功能相關性。我們構築人類NC-1的表現質體,分別在哺乳動物細胞、酵母菌和大腸桿菌表現系統中表達蛋白質,再進行蛋白質純化。將來自酵母菌和大腸桿菌表現系統中純化好的蛋白質處理人類單核球,發現會刺激單核球產生interleukin-6與tumor necrosis factor-a。
另一方面,為了能夠更進一步研究NC-1的生物功能,我們實驗室擬採老鼠作為研究模式。首先,我們在老鼠基因庫 ( database )中找尋到一段與人類NC-1相似的DNA序列。我們再經由5’RACE和3’RACE以找到全長的老鼠NC-1。分析老鼠NC-1的基因體結構 ( genomic structure ),發現老鼠NC-1的intron位置與人類相同。構築老鼠NC-1的表現質體,將老鼠NC-1在大腸桿菌系統中表達蛋白質,進行蛋白質再摺疊 (refolding )與純化。將純化好的老鼠NC-1處理單核球,以ELISA觀察對單核球之影響。發現老鼠NC-1會刺激老鼠單核球產生interleukin-6與tumor necrosis factor-a。此結果與人類NC-1相同。

The human immune response is regulated by a highly complex and intricate network of control elements. Prominent among these regulatory components are the anti-inflammatory cytokines and specific cytokine inhibitors. A dynamic balance exists between proinflammatory cytokines d anti-inflammatory components in human immune system. Inadequate concentrations of anti-inflammatory cytokines result in excess inflammation, yet excess anti-inflammatory cytokine concentrations disrupt clearance mechanisms of microbial pathogens in the host.
The overall sequencing of the human genome gave rise to the discovery of many novel genes. Thus, novel molecules which are homology to the cytokine interleukin-10 ( IL-10 ) have been identified. They were recently combined and referred to as IL-10 family. This family now comprises IL-10, IL-19, IL-20, IL-22, AK155 and melanoma differentiation associated gene 7 ( MDA-7 ). IL-10 has multiple biological activities and plays an import role in the immune system. IL-10 was known to be able to inhibit the inflammatory responses by reducing the antigen presenting capacity of monocytes and the response of macrophage to the stimuli. Therefore, interleukin-10 was originally described as cytokine synthesis inhibitory factor.
We isolated NC-1 ( novel cytokine-1 ) which was homologous to interleukin-10 from human kidney cDNA library. After the analysis of human NC-1 genomic structure, there are five exons and four introns found in the 5’end untranslation region. A lot of variable sorts were existed among these exons and introns
To understand the function of this novel cytokine and the relationship to interleukin-10, we constructed the expression clone of human NC-1 in mammalia cell, Pichia pastoris and E. coli expression system. We purified NC-1 protein from Pichia pastoris and E. coli expression system. Then we treated human monocytes with NC-1 recombinant protein. NC-1 can stimulate human monocytes to produce interleukin-6 and tumor necrosis factor-a.
On the other hand, to study the biological function of NC-1 much more, we processed some experiments using the mice model. First, we isolated a DNA fragment homology to human NC-1 from mouse database. We found the full-length cDNA of mouse NC-1 by 5’ end rapid amplification cDNA end and 3’ end rapid amplification cDNA end. After the analysis of mouse NC-1 genomic structure, we found the location of intron was identical to human NC-1. We constructed the expression clone of mouse NC-1 and expressed mouse NC-1 protein in E. coli expression system. After the refolding and purification of protein, we treated mouse monocytes with NC-1 protein to study the function of mouse NC-1. We also found that mouse NC-1 stimulated monocytes to produce interleukin-6 and tumor necrosis factor-a.

中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
圖目錄 X
表目錄 XII
附錄目錄 XIII
縮寫檢索表 XIV
儀器 XV
第1 章 緒論 1
1-1 免疫反應與細胞素 1
1-2 interleukin-10 ( IL-10 )之介紹 2
1-3 研究動機及內容之簡介 4
第2 章 材料與方法 6
2-1 實驗菌株、質體與培養基配方 6
2-1-1 Host strains and genotypes 6
2-1-2 Vector 6
2-1-3 Growth medium 7
2-2 NC-1基因之來源 10
2-3 NC-1重組基因之構築 10
2-3-1 聚合脢連鎖反應 10
2-3-2 電泳法回收DNA片段 11
2-3-3 TA cloning 11
2-3-4 E. coli Transformation 13
2-3-5 Colony PCR 14
2-3-6 限制脢處理 15
2-3-7 PCR產物純化法  15
2-3-8 接合反應 16
2-3-9 小量質體的抽取 17
2-4 Ligation independent cloning 17
2-5 Pichia pastoris EasyComp transformation 18
2-6 人類NC-1_pCEP4重組蛋白質的製備 19
2-6-1 蛋白質之表達 19
2-6-2 偵測蛋白質表達情形 21
2-7 人類NC-1_pPICZaA重組蛋白質的製備 24
2-8 人類 NC-1_pET 30 EK/LIC重組蛋白質的製備 26
2-9 人類 NC-1重組蛋白質之功能分析 27
2-9-1 人類單核球 ( monocyte ) 之純化 27
2-9-2 處理單核球 ( monocyte ) NC-1重組蛋白質 28
2-9-3 Enzyme linked immunoabsorbent assay ( ELISA ) 28
2-10 5’ Rapid amplification of cDNA ends ( 5’RACE ) 29
2-11 3’ Rapid amplification of cDNA ends ( 3’RACE ) 30
2-12 北方轉漬法 31
2-13 老鼠NC-1重組蛋白質的製備 31
2-14 老鼠NC-1重組蛋白質之功能分析 33
2-14-1老鼠單核球 ( monocyte ) 之純化 33
2-14-2處理單核球 ( monocyte ) NC-1重組蛋白質 33
2-14-3 Enzyme linked immunoabsorbent assay ( ELISA ) 33
第3章 35
3-1 人類NC-1與interleukin-10之胺基酸序列比對 35
3-2 人類NC-1之基因體結構 35
3-3人類NC-1之5’端未轉譯區域 35
3-4人類NC-1重組基因的製備 36
3-5人類NC-1重組蛋白質的製備 37
3-6人類NC-1重組蛋白質之功能分析 39
3-7全長( full-length )老鼠NC-1 cDNA 40
3-8北方轉漬之結果 42
3-9老鼠NC-1與人類NC-1之胺基酸序列比對 42
3-10老鼠NC-1之基因體結構 43
3-11老鼠NC-1之重組基因的製備 43
3-12老鼠NC-1重組蛋白質的製備 43
3-13老鼠NC-1重組蛋白質之功能分析 44
第4章 討論 45
參考文獻 50
圖 56
表 81
附錄 83
自述

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