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研究生:陳俐君
研究生(外文):Li-chun Chen
論文名稱:人類DC2穿膜基因及其產物之分子特性分析
論文名稱(外文):Molecular Characterization of A Novel Transmembrane Gene DC2
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:2004
畢業學年度:92
語文別:中文
論文頁數:78
中文關鍵詞:未知功能基因穿膜蛋白
外文關鍵詞:novel transmembrane genetransmembrane proteinunknown gene
相關次數:
  • 被引用被引用:2
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hsDC2基因位在染色體4q25位置上,此基因的蛋白質產物含有149個胺基酸,大約為16.8 kDa。生物資訊軟體預測hsDC2可能是一個位在內質網上的穿膜蛋白,含有三個穿過膜的helices,為一未知功能的基因。根據RT-PCR及RNA dot blot實驗的結果顯示hsDC2在許多組織中皆會表現,如腦組織、脊髓、腦幹、腎臟、肌肉、心臟、肝臟、脾臟、肺臟、腸胃等,不過似乎在癌組織中mRNA表現量較多。以即時定量PCR分析癌細胞株發現hsDC2可能與癌細胞株之分化程度有關,在分化程度較好的鼻咽癌、肝癌及以溫度誘導分化的骨母細胞株中mRNA表現量較多。我們也利用生物資訊資料庫對hsDC2進行分析、預測得到許多有助益的資訊。為了進一步研究hsDC2蛋白質的功能,我們製作能辨識hsDC2蛋白質之多株抗體,希望能應用於分析蛋白質表現及其他功能特性分析的實驗中,但hsDC2的生物功能仍有待進一步的研究加以釐清。
The hsDC2, an unknown gene, was located on chromosome 4q25. Its genetic protein product contains 149 amino acids with the molecular weight of 16.8 kDa approximately. Predicted by bioinformatics, hsDC2 might be a transmembrane protein with three transmembrane helices on the endoplasmic reticulum. Based on the results of reverse transcription-polymerase chain reaction, it revealed that hsDC2 was expressed in many tissues. It was showed that there were more mRNA expression in the cancer tissue. Using real-time quantitative PCR to analyze cancer cell lines, we found that hsDC2 might be related to differentiation status of cells. Among the well-differentiated cells such as nasopharyngeal carcinoma cell line NPC TW01, hepatoma cell line Hep3B and temperature-induced differentiated human fetal osteoblast cell line (hFOB), there were more hsDC2 mRNA expression. We also obtained some useful information from bioinformatics databases. To further elucidate the biological functions of the gene, glutathion S-transferase-hsDC2 fusion protein was used to generate anti-hsDC2 polyclonal antibody. However, we still need further research to clarify the biological function of hsDC2.
目錄
中文摘要--------------------------------------------------1
英文摘要--------------------------------------------------2
壹、緒論--------------------------------------------------3
貳、實驗目的---------------------------------------------13
參、實驗材料與方法---------------------------------------14
一、生物資訊軟體分析-------------------------------------14
二、細胞培養(cell culture) ------------------------------14
三、臨床檢體---------------------------------------------15
四、反轉錄聚合酵素連鎖反應-------------------------------16
五、質體重組DNA建構--------------------------------------20
六、蛋白質表現及純化-------------------------------------25
七、hsDC2多株抗體製備------------------------------------31
八、以ELISA的方式測定抗體力價----------------------------31
九、RNA dot blot-----------------------------------------32
十、Real-Time quantitative PCR---------------------------34
肆、結果與討論-------------------------------------------37
一、生物資訊預測結果-------------------------------------37
二、hsDC2在各癌細胞株、臨床組織mRNA的表現----------------55
三、DC2在成熟小鼠各組織mRNA的表現------------------------61
四、質體重組DNA建構--------------------------------------63
五、表現GST-hsDC2融合蛋白--------------------------------66
六、anti-hsDC2抗體的製備---------------------------------70
伍、結論-------------------------------------------------72
陸、參考資料---------------------------------------------74
圖表附錄
圖一、Ct值的確定------------------------------------------7
圖二、hsDC2核苷酸序列及胺基酸序列------------------------38
圖三、hsDC2在染色體的位置--------------------------------39
圖四、GeneCard資料庫預測的hsDC2表現情形------------------41
圖五、SOURCE資料庫預測hsDC2在組織中表現量最多的前十名結果-42
圖六、hsDC2蛋白結構預測圖--------------------------------44
圖七、hsDC2 之親疏水圖譜---------------------------------47
圖八、ELM motif search預測hsDC2可能含有的功能性位置------48
圖九、hsDC2胺基酸序列與OST3利用T-Coffee做alignment比對結果--53
圖十、hsDC2胺基酸序列與OST6利用T-Coffee做alignment比對結果-54
圖十一、利用RT-PCR檢測hsDC2 mRNA的表現情形--------------56
圖十二、利用real-time PCR分析癌細胞株hsDC2 mRNA的表現情形--58
圖十三、利用RT-PCR檢測小鼠正常組織中DC2 mRNA的表現情形---61
圖十四、以RNA Dot blot觀察在小鼠正常組織中DC2的表現情形--62
圖十五、論文中所用的蛋白質表現載體pGEX-6p-1圖譜----------64
圖十六、建構蛋白質表現載體pGEX-6p-1/hsDC2 a1~31及a55~113---65
圖十七、分段表現hsDC2蛋白質的不同片段 a1~31及a55~113------67
圖十八、小量之粗製表現蛋白-------------------------------68
圖十九、利用GST親合性管柱純化系統純化GST-hsDC2融合蛋白結果-69
圖二十、利用ELISA的方式測定hsDC2的抗體力價---------------71
表一、PCR primer序列-------------------------------------18
表二、PCR反應條件----------------------------------------19
表三、Real-time quantitative PCR primer序列--------------36
表四、ELM motif search預測hsDC2的功能性位置結果----------49
參考資料

ELM http://elm.eu.org/

Ensembl Gene Report http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000138804

Ensembl Protein Report http://www.ensembl.org/Homo_sapiens/protview?peptide=ENSP00000340993&db=core

Ensembl Exon Report http://www.ensembl.org/Homo_sapiens/exonview?transcript=ENST00000344885&db=core

GeneCards http://genome-www.stanford.edu/cgi-bin/genecards/carddisp?DC2

NCBI Map Viewer http://www.ncbi.nih.gov/mapview/

ProtScale http://tw.expasy.ch/cgi-bin/protscale.pl

PSORT II http://psort.ims.u-tokyo.ac.jp/cgi-bin/runpsort.pl

SMART (Simple Modular Architecture Research Tool) http://smart.embl-heidelberg.de/

SOSUI http://sosui.proteome.bio.tuat.ac.jp/sosui_submit.html

SOURCE http://genome-www5.stanford.edu/cgi-bin/source/sourceResult

T-COFFEE server http://www.ch.embnet.org/software/TCoffee.html

TMHMM http://www.cbs.dtu.dk/services/TMHMM/

UCSC Genome Bioinformatics http://genome.cse.ucsc.edu/

UCSC Human Genome Browser http://genome.cse.ucsc.edu/cgi-bin/hgGateway


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