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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 張世宗 | |
dc.contributor.author | I-Hsun Wei | en |
dc.contributor.author | 魏義峋 | zh_TW |
dc.date.accessioned | 2021-06-15T01:21:08Z | - |
dc.date.available | 2009-08-20 | |
dc.date.copyright | 2009-08-20 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-25 | |
dc.identifier.citation | Beninga J, Rock KL, Goldberg AL (1998) Interferon-gamma can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase. J Biol Chem 273: 18734-18742
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PLoS ONE 3: e3658 Fruci D, Giacomini P, Nicotra MR, Forloni M, Fraioli R, Saveanu L, van Endert P, Natali PG (2008) Altered expression of endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 in transformed non-lymphoid human tissues. J Cell Physiol 216: 742-749 Fruci D, Niedermann G, Butler RH, van Endert PM (2001) Efficient MHC class I-independent amino-terminal trimming of epitope precursor peptides in the endoplasmic reticulum. Immunity 15: 467-476 Fukasawa KM, Fukasawa K, Kanai M, Fujii S, Harada M (1996) Molecular cloning and expression of rat liver aminopeptidase B. J Biol Chem 271: 30731-30735 Funk CD, Radmark O, Fu JY, Matsumoto T, Jornvall H, Shimizu T, Samuelsson B (1987) Molecular cloning and amino acid sequence of leukotriene A4 hydrolase. Proc Natl Acad Sci U S A 84: 6677-6681 Goldberg AL, Rock KL (1992) Proteolysis, proteasomes and antigen presentation. 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FEBS Lett 354: 1-6 Komlosh A, Momburg F, Weinschenk T, Emmerich N, Schild H, Nadav E, Shaked I, Reiss Y (2001) A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex class I antigenic peptides. J Biol Chem 276: 30050-30056 Lobigs M, Chelvanayagam G, Mullbacher A (2000) Proteolytic processing of peptides in the lumen of the endoplasmic reticulum for antigen presentation by major histocompatibility class I. Eur J Immunol 30: 1496-1506 Mehta AM, Jordanova ES, Corver WE, van Wezel T, Uh HW, Kenter GG, Jan Fleuren G (2009) Single nucleotide polymorphisms in antigen processing machinery component ERAP1 significantly associate with clinical outcome in cervical carcinoma. Genes Chromosomes Cancer 48: 410-418 Miyashita H, Yamazaki T, Akada T, Niizeki O, Ogawa M, Nishikawa S, Sato Y (2002) A mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase is expressed in endothelial cells and plays an important role in angiogenesis. Blood 99: 3241-3249 Mo XY, Cascio P, Lemerise K, Goldberg AL, Rock K (1999) Distinct proteolytic processes generate the C and N termini of MHC class I-binding peptides. J Immunol 163: 5851-5859 Rammensee HG, Friede T, Stevanoviic S (1995) MHC ligands and peptide motifs: first listing. Immunogenetics 41: 178-228 Reits E, Neijssen J, Herberts C, Benckhuijsen W, Janssen L, Drijfhout JW, Neefjes J (2004) A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation. Immunity 20: 495-506 Rock KL, York IA, Goldberg AL (2004) Post-proteasomal antigen processing for major histocompatibility complex class I presentation. Nat Immunol 5: 670-677 Rogi T, Tsujimoto M, Nakazato H, Mizutani S, Tomoda Y (1996) Human placental leucine aminopeptidase/oxytocinase. A new member of type II membrane-spanning zinc metallopeptidase family. J Biol Chem 271: 56-61 Roitt IM, Brostoff J, Male DK (2001) Immunology, Ed 6th. Mosby, Edinburgh ; New York Saric T, Chang SC, Hattori A, York IA, Markant S, Rock KL, Tsujimoto M, Goldberg AL (2002) An IFN-gamma-induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I-presented peptides. Nat Immunol 3: 1169-1176 Saveanu L, Carroll O, Hassainya Y, van Endert P (2005) Complexity, contradictions, and conundrums: studying post-proteasomal proteolysis in HLA class I antigen presentation. Immunol Rev 207: 42-59 Saveanu L, Carroll O, Lindo V, Del Val M, Lopez D, Lepelletier Y, Greer F, Schomburg L, Fruci D, Niedermann G, van Endert PM (2005) Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum. Nat Immunol 6: 689-697 Saveanu L, Fruci D, van Endert P (2002) Beyond the proteasome: trimming, degradation and generation of MHC class I ligands by auxiliary proteases. Mol Immunol 39: 203-215 Schomburg L, Kollmus H, Friedrichsen S, Bauer K (2000) Molecular characterization of a puromycin-insensitive leucyl-specific aminopeptidase, PILS-AP. Eur J Biochem 267: 3198-3207 Serwold T, Gonzalez F, Kim J, Jacob R, Shastri N (2002) ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum. Nature 419: 480-483 Snyder HL, Yewdell JW, Bennink JR (1994) Trimming of antigenic peptides in an early secretory compartment. J Exp Med 180: 2389-2394 Stoltze L, Schirle M, Schwarz G, Schroter C, Thompson MW, Hersh LB, Kalbacher H, Stevanovic S, Rammensee HG, Schild H (2000) Two new proteases in the MHC class I processing pathway. Nat Immunol 1: 413-418 Strehl B, Seifert U, Kruger E, Heink S, Kuckelkorn U, Kloetzel PM (2005) Interferon-gamma, the functional plasticity of the ubiquitin-proteasome system, and MHC class I antigen processing. Immunol Rev 207: 19-30 Tanioka T, Hattori A, Masuda S, Nomura Y, Nakayama H, Mizutani S, Tsujimoto M (2003) Human leukocyte-derived arginine aminopeptidase. The third member of the oxytocinase subfamily of aminopeptidases. J Biol Chem 278: 32275-32283 Tanioka T, Hattori A, Mizutani S, Tsujimoto M (2005) Regulation of the human leukocyte-derived arginine aminopeptidase/endoplasmic reticulum-aminopeptidase 2 gene by interferon-gamma. FEBS J 272: 916-928 Taylor A (1993) Aminopeptidases: structure and function. FASEB J 7: 290-298 Tsujimoto M, Hattori A (2005) The oxytocinase subfamily of M1 aminopeptidases. Biochim Biophys Acta 1751: 9-18 van Endert PM, Tampe R, Meyer TH, Tisch R, Bach JF, McDevitt HO (1994) A sequential model for peptide binding and transport by the transporters associated with antigen processing. Immunity 1: 491-500 Yamamoto N, Nakayama J, Yamakawa-Kobayashi K, Hamaguchi H, Miyazaki R, Arinami T (2002) Identification of 33 polymorphisms in the adipocyte-derived leucine aminopeptidase (ALAP) gene and possible association with hypertension. Hum Mutat 19: 251-257 York IA, Chang SC, Saric T, Keys JA, Favreau JM, Goldberg AL, Rock KL (2002) The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8-9 residues. Nat Immunol 3: 1177-1184 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42729 | - |
dc.description.abstract | 在高等的脊椎動物中,大部分的抗原胜肽是由泛素-蛋白解體途徑所產生之胜肽衍生而來。26S蛋白解體系統將病毒蛋白質降解為2-25個胺基酸之胜肽後,有少部分的胜肽會被運輸至內質網中,經ERAP1 (endoplasmic reticulum aminopeptidase 1) 和ERAP2 (endoplasmic reticulum aminopeptidase 2) 處理為成熟之抗原胜肽,進而與第一類主要組織相容性複合體 (major histocompatibility complex class I) 分子結合而呈現於細胞表面,此即為免疫系統的監視機制 (immunosurveillance)。由於ERAP1與ERAP2具有不同的受質專一性,因此,本論文欲探討造成這個差異的可能原因。
為了進一步分析ERAP1與ERAP2之特性,首先必須表現及純化全長之ERAP1及ERAP2蛋白質。然而,先前的研究結果指出,若藉由大腸桿菌表現系統大量表現,容易形成降解片段,甚至累積於不溶體中的情形。因此本論文嘗試利用桿狀病毒表現系統,表現並純化全長之ERAP1及ERAP2。然而在此系統中,仍無法順利表現出具有胺肽酶活性之ERAP2。為了探討是否是由於ERAP2之5’ 及3’ 序列的影響,造成其基因表現不易,本論文成功建構了數個5’ 或3’ 端截短之ERAP2載體以及chimera融合基因載體,並將其表現於大腸桿菌表現系統、哺乳類動物細胞表現系統以及桿狀病毒表現系統。結果顯示,N端及C端截短之ERAP2蛋白質雖可經由大腸桿菌表現,但卻是以不溶體之形式存在,因此本論文亦建立並探討了純化不溶體之最適條件與方法。除此之外,雖然ERAP1可成功地表現於293T細胞中,但是卻無法順利將帶有Kozak序列之ERAP2、3’ 截短之ERAP2或ERAP1與ERAP2的chimera基因轉染至HeLa或是293T細胞中。同樣地,chimera之重組蛋白質亦無法正常表現於桿狀病毒表現系統中。綜合以上結果,推測ERAP2之5’ 極可能具有特殊之序列或形成複雜的RNA二級結構,而造成其表現上的不易。針對ERAP1與ERAP2之RNA二級結構進行比較分析後發現,相較於ERAP1,ERAP2之RNA結構的確較為複雜,因此除了不同表現系統中密碼子使用 (codon usage) 的問題外,RNA的二級結構亦可能是造成表現困難的原因之一。 | zh_TW |
dc.description.abstract | In higher vertebrates, most of the antigenic peptides are derived from peptides generated during protein degradation by the ubiquitin-proteasome pathway. The 26S proteasome is able to degrade viral proteins to peptides of about 2~25 residues long. However, a small fraction of peptides escapes complete degradation and is transported into the endoplasmic reticulum, where they are processed by ERAP1 (endoplasmic reticulum aminopeptidase 1) and ERAP2 (endoplasmic reticulum aminopeptidase 2) into mature antigenic peptides. These mature antigens then bind to major histocompatibility complex (MHC) class I molecules and are presented to the cell surface. This process is known as immunosurveillance. Since ERAP1 and ERAP2 possess substrate specificity, our primary interest is to investigate further the probable reasons behind this difference.
To further analyze the unique properties of ERAP1 and ERAP2, we first need to express and purify recombinant ERAP1 and ERAP2 proteins. Previous studies have shown that if E. coli expression system is used, it is easy to observe proteins as degraded fragments or even in the form of inclusion bodies. Therefore, we tried expressing full-length ERAP1 and ERAP2 using the baculovirus protein expression system. However, in this alternate expression system, we still failed to express ERAP2 with aminopeptidase activity. In order to determine whether the 5’-end and 3’-end sequence of ERAP2 affect its ability to express, we successfully generated several 5’- or 3’-truncated ERAP2 constructs as well as chimera constructs and expressed them in E. coli expression system, mammalian expression system and the baculovirus expression system. The results have shown that, although N-terminal and C-terminal truncated ERAP2 proteins can be expression in E. coli, they accumulated as inclusion bodies. Consequently, we have set up the optimal condtions for purifying inclusion bodies. Interestingly, we have found that ERAP1 can be expressed in 293T cells while Kozak-ERAP2, C-terminal truncated ERAP2 and chimera fusion proteins cannot. Chimera fusion proteins also cannot be successfully expressed using baculovirus expression system. From the above mentioned results we have come to suspect ERAP2’s unique 5’ sequence or complex RNA secondary structure may have an effect on its expression. It is therefore not surprising when we found the RNA structure of ERAP2 is much more complicated than that of ERAP1 after comparing and analyzing the RNA secondary structures of both proteins. Thus, it is safe to conclude that other than codon usage in different expression systems, the RNA secondary structure of ERAP2 is the most likely factor contributing to expression difficulties. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:21:08Z (GMT). No. of bitstreams: 1 ntu-98-R96B47219-1.pdf: 2989745 bytes, checksum: 97ec3023f11e7f707a70c0e08c124827 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 中文摘要 i
英文摘要 ii 縮寫表 iv 第一章 緒論 1 1.1抗原呈現與免疫反應 1 1.1.1主要組織相容性複合體 (major histocompatibility complex, MHC) 1 1.1.2蛋白解體與第一類MHC抗原呈現 2 1.1.3 TAP與第一類MHC抗原呈現 2 1.2 內質網胺肽酶 (endoplasmic reticulum aminopeptidase) 3 1.2.1胺肽酶之識別 4 1.2.2 ERAP1之發現 5 1.2.3 ERAP1之受質專一性 6 1.2.4 ERAP2之發現與受質專一性 6 1.2.5 ERAP1與ERAP2之生理角色 7 1.3 ERAP1之作用機制 8 1.4研究動機 9 第二章 材料與方法 11 2.1實驗材料 11 2.1.1動物細胞 11 2.1.2昆蟲細胞 11 2.1.3大腸桿菌菌株 11 2.2各基因表現載體之建構 12 2.2.1真核表現系統載體 12 2.2.2原核表現系統載體 13 2.2.3核酸引子設計 14 2.2.4聚合酶鏈鎖反應 14 2.2.5限制酶切反應 (restriction enzyme digestion) 16 2.2.6鹼性磷酸酶去磷酸化反應 17 2.2.7接合反應 (ligation) 17 2.3原核宿主細胞表現系統 18 2.3.1化學法勝任細胞製備 18 2.3.2大腸桿菌細胞轉形 19 2.3.3重組蛋白質誘導表現 20 2.4重組蛋白質之純化方法 21 2.4.1 6xHis-tag重組蛋白質親和性層析法 21 2.4.2 離子交換法 22 2.4.3不溶體之純化 23 2.5真核宿主細胞表現系統 26 2.5.1動物細胞培養 26 2.5.2昆蟲細胞培養 28 2.5.3真核細胞之基因轉染 29 2.5.4細胞冷凍保存 33 2.5.5細胞解凍 34 2.6分子生物學相關實驗 35 2.6.1小量質體DNA製備 35 2.6.2大量質體DNA製備 36 2.6.3洋菜膠體電泳 37 2.6.4核酸定量 38 2.6.5核酸純化 (gel extraction) 39 2.7蛋白質相關實驗 40 2.7.1蛋白質定量法 40 2.7.2蛋白質電泳檢定 41 2.7.3蛋白質電泳膠片染色法 44 2.7.4蛋白質轉印法 45 2.7.5酵素免疫染色 46 2.7.6酵素免疫染色退染 (stripping) 48 2.8 ERAP酵素活性分析 48 2.9桿狀病毒表現系統 49 2.9.1重組質體之建構 50 2.9.2質體轉形 51 2.9.3重組Bacmid之純化 51 2.9.4 PCR檢驗法 52 2.9.5重組Bacmid之轉染 53 2.9.6病毒效價檢驗 55 2.9.7重組蛋白之表現 56 2.9.8重組蛋白之純化及分析 56 第三章 結果 58 3.1 ERAP1與ERAP2表現載體之建構與表現 58 3.1.1 ERAP1及ERAP2表現載體之建構 58 3.1.2 ERAP1及ERAP2於哺乳類動物細胞系統之表現 62 3.1.3 ERAP1及ERAP2之桿狀病毒表現載體之建構 71 3.1.4 ERAP1及ERAP2於桿狀病毒表現系統之表現 73 3.2截短之ERAP2表現載體之建構、表現與純化 78 3.2.1 N端截短ERAP2表現載體之建構 78 3.2.2 N端截短之ERAP2蛋白質於大腸桿菌表現系統之表現與純化 79 3.2.3 N端截短之ERAP2蛋白質不溶體之純化 86 3.2.4 C端截短之ERAP2表現載體之建構與表現 92 3.3 Chimera表現載體之建構與表現 96 3.3.1 Chimera之桿狀病毒表現載體之建構與表現 96 3.3.2 Chimera之哺乳類動物表現載體之建構與表現 100 第四章 討論 103 4.1 ERAP1、ERAP2與chimera之表現探討 103 4.1.1 ERAP1、ERAP2及chimera於動物細胞表現系統之表現探討 103 4.1.2 ERAP1、ERAP2及chimera於桿狀病毒表現系統之表現探討 104 4.1.3 截短之ERAP2於大腸桿菌表現系統之表現探討 105 4.2 ERAP2之N端序列或二級結構可能造成表現困難 105 第五章 未來展望 112 參考文獻 113 | |
dc.language.iso | zh-TW | |
dc.title | 內質網胺肽酶II之蛋白質表現研究與探討 | zh_TW |
dc.title | Studies on the protein expression of endoplasmic reticulum aminopeptidase 2 | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 莊榮輝,陳威戎,張麗冠 | |
dc.subject.keyword | ERAP1,ERAP2,第一類主要組織相容性複合體,不溶體, | zh_TW |
dc.subject.keyword | ERAP1,ERAP2,MHC class I,inclusion bodies, | en |
dc.relation.page | 116 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-27 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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