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研究生:陳志名
研究生(外文):Chih-Ming Chen
論文名稱:犬之CD150蛋白(犬瘟熱病毒受體)的選殖與表現
論文名稱(外文):Cloning and expression of canine CD150 protein, a canine distemper virus receptor
指導教授:王孟亮王孟亮引用關係
指導教授(外文):Min-Liang Wong
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:90
中文關鍵詞:病毒受體受體犬瘟熱病毒原核表現系統血液凝集素結合性試驗
外文關鍵詞:CD150CD150SLAMcanineRNAcDNAIPTGcanine distemper virushemagglutininpET32denatured formnative form
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CD150又稱signaling lymphocyte activation molecules(SLAM),為大小約70-95 kDa的細胞表面分子。為高度醣化的第一型穿膜蛋白,在蛋白質N端有醣化的V domain 及C2 domain,為 immunoglobulin superfamily特徵。在細胞質內部分C端則有三個以tyrosine(TxYxxV/I/A)為主的構造,會與具有Src-homology 2 ( SH2 ) domain的蛋白質如SLAM-associated protein(SAP)結合。CD150蛋白會持續表現於未成熟胸腺細胞中,並在活化態的B淋巴球、T淋巴球、單核球、樹狀細胞、自然殺手細胞中被誘導表現。功能被認為主要為調控T細胞與B細胞之間交互訊息傳導及增殖。在麻疹病毒研究中,目前已知CD150與CD46為麻疹病毒進入細胞的細胞受體,病毒以其封套醣蛋白血液凝集素( hemagglutinin , H protein )與CD150蛋白的V domain結合,而促使病毒進入。本實驗首先從健康犬隻血液中,萃取出白血球RNA,再進行RT-PCR合成其cDNA,將此cDNA經定序後確定為犬之CD150蛋白(GenBank序號為AF390108),長度為1073 bp。將選殖出的犬CD150蛋白全長與片段cDNA轉接至pET32原核表現系統載體後,於37 ℃利用1 mM 的IPTG誘導,表現並利用親和性樹脂純化出CD150(全長與片段)denatured form重組蛋白。此外,以質譜儀技術進行CD150全長重組蛋白蛋白質鑑定,結果確實為犬之CD150蛋白。最後進行犬CD150蛋白與犬瘟熱病毒H蛋白結合性試驗,在24 ℃環境下,利用0.4 mM 的IPTG誘導表現出犬CD150蛋白N端片段與犬瘟熱病毒H蛋白C端部分native form重組蛋白。將兩蛋白進行結合,發現於非變性膠體蛋白質電泳中有結合現象,但未經西方墨點法確認,因此初步認為此兩種蛋白質之間具有結合性。未來工作將朝向真核表現方向進行,希望藉由真核表現系統解決蛋白質醣基化問題,進一步來探討犬瘟熱病毒血液凝集素與犬CD150蛋白確切結合位置。
CD150 , as known as signaling lymphocyte activation molecules(SLAM), is a cell surface molecule of 70-95 kDa. CD150 is a typeⅠtrans-membrane glycoprotein with N-glycosylation and belongs to the immunoglobulin superfamily with one V domain and one C2 domain. In its cytoplasmic tail are three unique tyrosine-based motifs with the consensus sequence TxYxxV/I/A, which are binding sites for SH2 domain containing proteins such as SLAM-associated protein (SAP). CD150 is constitutively expressed on immature thymocytes, and is induced on activated T cells, B cells, monocytes, dendritic cells, and natural killer cells. The physiological function of CD150 is considered to regulate bidirectional T – B cell stimulation and proliferation. In measles virus research, CD150 and CD46 have been shown to be a cellular receptor for measles virus. Viral envelope glycoprotein, hemagglutinin (H), mediates virus entry into host cell by binding to V domain of CD150. In this work, we extracted the RNA of leukocyte from healthy dog blood, and used RT-PCR to synthetize the cDNA of canine CD150 protein, which’s length is 1073 bp, and confirmed its identity by DNA sequencing (GenBank No. AF390108).The cDNA of canine CD150 was cloned into expression vector pET32 system. We expressed and purified the denatured form canine CD150 of full-length and truncated recombinant proteins, which were induced by IPTG and the temperature is 37 ℃. In addition, we confirmed the recombinant protein identity by LC/MS/MS, and the result is canine CD150 protein. Then, we expressed the native form canine CD150 N-terminal part-length recombinant protein and canine distemper virus hemagglutinin C-terminal part-length recombinant protein, and we expect to study protein-protein interaction between two recombinant proteins
目 次
目次……………………………………………………………………………….……I
圖次…………………………………………………………………………………..Ⅳ
中文摘要……………………………………………………………………………. VI
英文摘要……………………………………………………………………………VII
第一章 前言……………………………………………………….……………….. 1
第二章 文獻探討………………………………………………….……………….. 3
一、 細胞表面蛋白CD150研究……………………………………………...3
1.1 CD150蛋白結構與分佈情形研究…………………………………...3
1.2 CD150生理功能研究……………………………………………...…4
二、 犬瘟熱病毒研究………………………………………………………....5
2.1 犬瘟熱病毒背景與臨床症狀……………………………………..….5
2.2 犬瘟熱病毒病毒特性研究……………………………………….…..7
三、 犬瘟熱病毒封套醣蛋白hemagglutinin之研究……………….………...8
四、 麻疹病毒屬細胞受體CD150研究……………………………………...9
圖表……………………………………………………………………………..12
第三章 材料與方法……………………………………………………………......19
一、 犬隻CD150蛋白選殖………………………………………………….19
1.1 犬隻CD150核酸萃取………………………………………………19
1.2 逆轉錄酶反應……………………………………………………….19
1.3 聚合酵素連鎖反應……………………………………………….…20
1.4 DNA片段純化…………………………………………………...….20
1.5 接合反應……………………………………………………….……21
1.6 勝任細胞的製備…………………………………………….………21
1.7 轉型作用……………………………………………………….……21
1.8 菌株之挑選與質體抽取及確認…………………………………….22
二、 犬隻CD150基因片段載體之建構流程…………...…………………..22
2.1 犬隻CD150基因PCR產物的選殖…………………………………22
2.2 各個CD150基因片段之原核表現質體構築………………………23
三、 犬隻CD150重組蛋白之表現與純化……………………………….....24
3.1 轉型作用…………………………………………………………….24
3.2 菌株之挑選與質體之抽取與確認…………………………….……24
3.3 Isopropyl-β-D-thiogalactopyranoside (IPTG)誘導表現…..……...…25
3.4 蛋白質膠體電泳分析…………………………………..….……..…25
3.5 西方墨點法………………………………………..…………...……26
3.6 蛋白質純化………………………………………..…………..….....28
3.7 Denatured form細胞萃取液之製備………………..…………..…...28
3.8 Denatured form重組蛋白之純化…………………………………...29
四、 犬隻CD150重組蛋白與重組犬瘟熱病毒H蛋白結合性試驗….…....30
4.1 轉型作用及菌株挑選與確認………………………………….…….30
4.2 IPTG誘導表現及native form細胞萃取液之製備…………….……30
4.3 Native form重組蛋白之純化……………………….………….……31
4.4 非變性膠(Native gel)製備及蛋白質電泳………….…………….31
4.5 蛋白質結合性試驗…………………………………….……...……..32
五、 圖表……………………………………………………………………..33
第四章 結果………………………………………………………………………..42
一、 犬隻CD150蛋白基因選殖與定序………………………………...…..42
二、 犬隻CD150蛋白(全長及不同片段)原核表現質體之構築…….……42
三、 犬隻CD150蛋白(全長及不同片段)表現………………...…………..43
四、 利用西方墨點法偵測犬隻CD150(全長及片段)重組蛋白…………..43
五、 犬隻CD150全長及各片段denatured form重組蛋白純化及確認..…..43
六、 以質譜儀技術進行蛋白質鑑定工作…………………………………..44
七、 犬隻CD150 N端重組蛋白與犬瘟熱病毒H蛋白C端重組蛋白native form蛋白表現及純化…………………………………………………..44
八、 犬隻CD150 N端重組蛋白與犬瘟熱病毒H蛋白C端重組蛋白結合性試驗……………………………………………………………………..44
九、 圖表……………………………………………………………………..45
第五章 討論………………………………………………………………………..60
參考文獻……………………………………………………………………………..64
附錄…………………………………………………………………………………..72
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