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研究生:呂嘉勳
研究生(外文):Jia-Shiun Leu
論文名稱:核蛋白SP110b特性之研究
論文名稱(外文):Characterization of Nuclear Protein SP110b
指導教授:顏伯勳
指導教授(外文):Bo-Shiun Yan
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
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:42
中文關鍵詞:核蛋白
外文關鍵詞:nuclear protein
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根據統計,三分之一人口曾感染過結核分枝桿菌,但感染者僅有不到十分之一的人會在感染之後的一生中發展成肺結核,這些影響可能是個體間免疫力和基因多樣性而導致; 然而,宿主免疫力和基因之間的關係仍不清楚。先前,在小鼠身上已發現會調控先天免疫的基因區域sst1,並且在此區域找到一個基因Intracellular pathogen resistance 1 (Ipr1),它可能參與先天免疫,細胞凋亡和發病的機制。SP110b是IPR1在人類身上同源性最高的蛋白質 (有41%的相似度),它的基因座落在人類第二條染色體的區域,IPR1和SP110b都是受干擾素誘導的蛋白質,兩者在小鼠和人類的免疫機制上可能有相同的功能。在此論文中,我們利用雙啟動子的慢病毒系統來建構穩定細胞株,以誘導性表現eGFP-SP110b蛋白質,當Doxycycline誘導下,這些細胞會表現eGFP-SP110b。在干擾素活化下, 發現eGFP-SP110b蛋白質的穩定性會提高。除此之外,以免疫共沉澱分析,我們也發現一些蛋白質會和SP110b蛋白形成複合體。下一步,我們將透過MS/MS試著找出在SP110b的蛋白質附合體中的蛋白質來說明它們的功能。在293T和THP1細胞中,我們可觀察到2,5-oligoadenlate synthetase 1 (OAS1)和SP110b的蛋白質交互作用,OAS1參與在RNase L調控的細胞凋亡路徑中。然而,SP110b在OAS1/RNase L路徑的角色仍不清楚。我們的假說推測,在干擾素活化下, SP110b透過OAS1/RNase L的路徑調控細胞凋亡現象。
It is estimated that one-third of people infected with pathogen Mycobacterium tuberculosis (Mtb) and fewer than 10% of them progress to tuberculosis in their lifetime. In a mouse model, intracellular pathogen resistance 1 (Ipr1) gene, which mediates host innate immunity to Mtb infection, has been identified within sst1 (susceptibility to tuberculosis 1) locus. In human, SP110b is the closest homology protein of IPR1 (41% identity), and its gene localizes in region of human chromosome 2. IPR1 and SP110b are both interferon inducible proteins, and they may have the same function involved in immunity in mice and human. In this study, we used dual-promoter lentiviral system to establish the stable cell clones, in which the expression of eGFP-SP110b protein can be induced in presence of doxycycline. The stability of eGFP-SP110b protein is increased after IFN�� treatment. The proteins interacting with SP110b were further characterized by co-immunoprecipating the protein complex interacting with eGFP-SP110b. The data reveal that SP110b protein complex may be more stable in pro-inflammation environment. We will identify the proteins interacting with SP110b by MS/MS analysis. Using the lentiviral expression system, we also demonstrated the OAS1 (2,5- oligoadenylate synthetase 1), which is involved in an RNase L-mediated apoptotic pathway, interacted with SP110b protein in 293T and THP1 cells. However, the role of SP110b in the OAS1/RNase L pathway is still unclear. We hypothesize that SP110b regulates apoptosis through the OAS1/RNase L pathway after IFN treatment.
Contents
Acknowledgement…………………………………………………………………....i
Contents…………………………………………………………………………… 1-3
I Abstract………………………………………………………………………….. 4-6
II Introduction…………………………………………………………………… 7-10
II.01 Background……………………………………………………………..…... 7
II.02 SP110b………………………………………..………………………..……. 8
II.03 2, 5-oligoadenylate synthetase 1 (OAS1)-activated RNase L pathway….. 9
II.4 Future works….…………………………………………………….………. 10
III Materials & Methods....….………………………………………………… 11-17
III.01 Preparation of JM109 competent cells………………………………….. 11
III.02 Construction of lentiviral vectors..……………………………………11-12
III.03 Generation of Lentivirus.…………………………………....................... 12
III.04 Lentiviral transduction………………………………………………. 12-13
III.05 FACS analysis..…………………………………………………………….13
III.06 Magnetic selection..……………………………………………………13-14
III.07 Immunofluoresence assay..……………………………………………….14
III.08 Cell lysis..…………………………………………………………………..15
III.09 Nuclear extraction……………………………………………………….. 15
III.10 Western Blotting..……………………………………………………...15-16
III.11 Co-Immunoprecipitaion…………………………………………………16
III.12 Silver staining………………………………………………………….16-17
IV Results.……………...……………………………………………………….. 18-24
IV.01 Localization of endogenous SP110 proteins in THP1 cells..…………….18
IV.02 FACS analysis of dual-promoter lentiviral system in 293T & THP1 cells..
……...…………………………………………………………………………..18-19
IV.03 Generation of inducible eGFP-SP110b expressing THP1 cells….…..19-20
IV.04 Inducible expression of the eGFP-SP110b protein in stable cell clones, 8E5 and 9F11 ………………………………………………..………………..20-21
IV.05 Sp110b protein complex is more stable in IFN��-activated THP1 cells……
………….…………………………………..…………………………………..21-22
IV.06 Expression of Flag-OAS in 293T & THP1 cells..…..……………….........22
IV.07 Flag-OAS interact with eGFP-Sp110b in the nucleus of 293T cells…….23
IV.08 Flag-OAS is partially co-localized with eGFP-Sp110b after IFN�� treatment.……………...………………………………………………………23-24
V Discussion…..……………………………….…………………………………25-28
V.01 Two inducible stable clones 8E5 and 9F11……………………….…... 25-26
V.02 eGFP-SP110b protein complex is more stable in IFN�� activated cells.26-27
V.03 SP110b may be involved in OAS1/RNase L apoptotic signal……………27
V.04 Perspective ………………………………………………………………….28
VI Figures and Figure Legends………………………………………………...29-39
VII References…………...………………………………………………………40-42
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