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研究生:陳桂秋
研究生(外文):Kuei-Chiu Chen
論文名稱:人類基因ROGDI蛋白質多株抗體製備及初步特性分析
論文名稱(外文):Cloning, Expression and Initial Characterization of a Novel Human Gene ROGDI
指導教授:卓忠隆
指導教授(外文):Chung-Lung Cho
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:95
語文別:中文
論文頁數:88
中文關鍵詞:肝纖維化抗體製備未知基因
外文關鍵詞:polyclonal antibodyliver fibrosisROGDI
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ROGDI是一個尚不知功能的基因,依據NCBI基因資料庫所示,其位於人類染色體16p13.3上,遺傳編碼區為864 bp,轉譯出蛋白質有287個胺基酸,是由NEDO人類基因序列計畫中從人類腎上皮細胞中選殖出。其相關序列(RefSeq NM_024589)命名為Homo sapiens rogdi homolog (Drosophila), ROGDI。生物資訊預測其可能為一個親水性的蛋白質,表現於細胞質中,並含有leucine zipper domain的結構。之前本實驗室已選殖出此基因,為了近一步探討此基因及其產物之功能角色,將此基因的遺傳編碼區轉殖pGEX-6p及pET-28a質體入E. coli BL21 (DE3)中表現蛋白,並純化以製備多株抗體。北方點墨法分析人類正常器官中此基因的表現量,發現並非於所有器官中均有表現。從RT-PCR與西方點墨法的結果中,發現此基因在多數的癌細胞株均有表現,但是於正常細胞株中表現量較少。在免疫細胞化學染色與表現GFP螢光融合蛋白實驗發現此基因於細胞核與質中均有表現,然而於細胞核的表現量比細胞質多。此外,在以TAA誘發小鼠肝纖維化的早期,此基因有明顯的表現量增加。因此,推測此基因可能在癌症與肝纖維化的過程中扮演重要角色。
ROGDI is a novel gene which has unknown function. According to GenBank, the gene is located on chromosome 16p13.3 and the size of coding region is 864 bp which encodes 287 amino acids. It was a novel gene isolated from primary human renal epithelial cells in NEDO human cDNA sequencing project (AK026039). The definition of its reference sequence (RefSeq NM_024589) described as Homo sapiens rogdi homolog (Drosophila), ROGDI. By bioinformatic analysis, the gene was predicted as a hydrophilic protein with leucine zipper domain and located in cytoplasm. A partial cDNA of this gene was cloned in our laboratory. For further study of the biological function of the gene, the coding region of this gene was cloned into pGEX-6p and pET-28a vector and expressed in E. coli BL21 (DE3). The fusion protein was partially purified for preparation of polyclonal antibody. Northern blot analysis revealed that the gene was not expressed in all tissues. From the results of RT-PCR and western blot analysis, it can be concluded that the products, both mRNA and protein, of this gene were found in many cancer cell lines, but protein level expression of the gene was much less in normal cell lines. By
immunocytochemistry analysis and subcellular localization analysis of GFP-tagged ROGDI, the gene was expressed both in the nucleus and cytoplasm, but expressed more in the nucleus than cytoplasm. In addition, ROGDI was up-regulated in early stages of liver fibrosis of TAA-treated mouse livers. This novel gene may play roles in tumorigenesis and liver fibrosis.
中文摘要
英文摘要
目錄
縮寫詞彙
前言 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒1
ROGDI﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒1
肝纖維化 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒3
實驗目的 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒6
材料與方法 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒7
生物資訊軟體分析 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒7
Total RNA的萃取﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒7
反轉錄 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒8
聚合酵素連鎖反應 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒9
建構蛋白質表達質體﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒10
轉形作用﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒11
製備小量質體﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒11
表現小量蛋白質﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒12
表現與萃取大量蛋白質﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒13
純化GST-ROGDI及His-ROGDI融合蛋白 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒14
SDS-聚丙烯醯胺膠體電泳 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒15
Coomassie Blue染色﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒16
膠內蛋白質分解﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒16
基質輔助雷射脫附游離/飛行時間質譜分析 ﹒﹒﹒﹒﹒﹒﹒﹒17
ROGDI多株抗體製備及純化 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒18
北方點墨法﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒19
蛋白質的萃取與定量﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒20
西方點墨法﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒21
細胞培養﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒22
細胞轉染﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒23
以抗原競爭抗體做抗體專一性分析﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒24
以RNA干擾作用做抗體專一性分析﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 25
免疫細胞化學染色﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒26
細胞內定位分析﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒27
細胞群落形成分析﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒27
誘發小鼠肝纖維化﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒27
結果與討論﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒29
生物資訊軟體分析ROGDI胺基酸序列 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒29
GST-ROGDI與His-ROGDI融合蛋白的表現﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒29
MALDI-TOF分析並確認抗原 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒31
GST-ROGDI與His-ROGDI融合蛋白的純化﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒33
多株抗體anti-ROGDI的製備與純化 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒33
多株抗體anti-ROGDI的確認 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒34
ROGDI的mRNA在人類正常器官之表現量 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒37
ROGDI在細胞株的mRNA及蛋白質表現量 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒38
ROGDI在細胞內所表現的位置 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒39
ROGDI蛋白質增量表現對HEK293T細胞生長的影響﹒﹒﹒﹒﹒﹒40
ROGDI在小鼠肝纖維化過程的蛋白質表現量 ﹒﹒﹒﹒﹒﹒﹒﹒41
結論﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒43
參考文獻﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒45
圖表﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒48
表一、PCR引子的序列、PCR的條件與產物大小﹒﹒﹒﹒﹒﹒﹒48
圖一、小鼠與果蠅同源基因與人類胺基酸比對﹒﹒﹒﹒﹒﹒﹒49
圖二、Pole Bioinformatique Lyonnais以GOR4模式預測ROGDI的蛋白質二級結構﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒50
圖三、Pole Bioinformatique Lyonnais以HNN模式預測ROGDI的蛋白質二級結構﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒51
圖四、Jpred與SCRATCH預測ROGDI的蛋白質二級結構 ﹒﹒﹒﹒52
圖五、ProtScale與PSORT II預測ROGDI的性質與細胞內表現位置﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒53
圖六、實驗室先前大量GST-ROGDI融合蛋白表現模式的純化 ﹒54
圖七、MALDI-TOF偵測並確認抗原ROGDI﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒55
圖八、大量蛋白表現後GST-ROGDI及His-ROGDI融合蛋白的純 化﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒56
圖九、以西方點墨法確認抗體與質體轉譯出的抗原之專一性﹒57
圖十、以免疫競爭確認抗體與質體轉譯出的抗原之專一性﹒﹒58
圖十一、以RNAi確認抗體與內生性抗原之專一性﹒﹒﹒﹒﹒﹒59
圖十二、ROGDI的mRNA在人類正常器官中的表現量 ﹒﹒﹒﹒﹒60
圖十三、ROGDI的mRNA在各個細胞株內的表現量 ﹒﹒﹒﹒﹒﹒61
圖十四、ROGDI蛋白質在各個細胞株內的表現量 ﹒﹒﹒﹒﹒﹒62
圖十五、免疫細胞化學染色偵測ROGDI在細胞中的表現位置 ﹒63
圖十六、雷射共軛焦顯微鏡偵測GFP-ROGDI融合蛋白在細胞中的表現位置﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒65
圖十七、ROGDI蛋白質增量表現對HEK293T細胞生長的影響﹒﹒67
圖十八、ROGDI蛋白質在小鼠肝纖維化過程的表現量 ﹒﹒﹒﹒68
附錄﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒69
附錄一、在NCBI登錄的ROGDI基本資料 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒69
附錄二、ROGDI的遺傳編碼區與胺基酸序列 ﹒﹒﹒﹒﹒﹒﹒﹒70
附錄三、AmiGO對小鼠與果蠅同源基因的功能性預測 ﹒﹒﹒﹒71
附錄四、果蠅的rogdi基因突變後的結果 ﹒﹒﹒﹒﹒﹒﹒﹒﹒72
附錄五、肝臟受損後,肝臟細胞的改變﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒73
附錄六、各種因素均導致肝星狀細胞活化,進而形成肝纖維化74
附錄七、肝星狀細胞活化後所產生的生物機制﹒﹒﹒﹒﹒﹒﹒75
Andrade ZA. Regression of hepatic fibrosis. Rev Soc Bras Med Trop 2005; 38:514-520.
Bataller R and Brenner DA. Liver fibrosis. J Clin Invest 2005; 115:209-218.
Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 2000; 275:2247–2250.
Geerts A. History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin Liver Dis 2002; 21:311–335.
Halliwell B and Chirico S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 1993; 57:715-725.
Hellerbrand C, Jobin C, iimuro Y, Licato L, Sartor RB, Brenner DA. Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor. Hepatology 1998; 27:1285-1295.
Lee HS, Miau LH, Chen CH, Chiou LL, Huang GT, Yang PM, Sheu JC. Differential role of p38 in IL-1alpha induction of MMP-9 and MMP-13 in an established liver myofibroblast cell line. J Biomed Sci 2003; 10:757-765.
McCord JM. The evolution of free radicals and oxidative stress. Am J Med 2000; 108:652-659.
Olaso E, Salado C, Egilegor E, Gutierrez V, Santisteban A, Bru PS, Friedman SL, Vanaclocha FV. Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis. Hepatology 2003; 37:674-685.
Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, et al. Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat Genet 2004; 36:40-45.
Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Wakeield L, Roberts AB, Sporn MB, Thorgeirsson SS. Hepatic expression of mature transforming growth factor ß1 in transgenic mice results in multiple tissue lesions. PNAS 1995; 92:2572-2576.
Schwabe RF, Schnabl B, Kweon YO, Brenner DA. CD40 activates NF-kappa B and c-Jun N-terminal kinase and enhances chemokine secretion on activated human hepatic stellate cells. J Immunol 2001; 166: 6812-6819.
Svegliati-Baroni G, Saccomanno S, van Goor H, Jansen P, Benedetti A, Moshage H. Involvement of reactive oxygen species and nitric oxide radicals in activation and proliferation of rat hepatic stellate cells. Liver 2001; 21:1-12.
Xu L, Hui AY, Albanis E, Arthur MJ, O’Byrne SM, Blaner WS, Mukherjee P, Friedman SL, Eng FJ. Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 2005; 54:142-151.
FlyBase http://flybase.bio.indiana.edu/
Gene Ontology http://www.geneontology.org/
Jpred http://www.compbio.dundee.ac.uk/~www-jpred/
MASCOT Peptide Mass Fingerprint http://www.matrixscience.com
BLAST 2 SEQUENCES http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi
Pole Bioinformatique Lyonnais http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sspred.html
ProtScale http://www.expasy.org/tools/protscale.html
PSORT II http://psort.hgc.jp/form2.html
SCRATCH http://www.igb.uci.edu/tools/scratch/
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