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研究生:張智鈞
研究生(外文):Chih-Chun Chang
論文名稱:紅腳蟳(Scyllaolivacea)兩種甲殼類升血糖荷爾蒙異構型特性、重組蛋白表現與功能之研究
論文名稱(外文):Expression, characterization, and function of two structural variants of the crustacean hyperglycemic hormone family in the mud crab Scylla olivacea
指導教授:李奇英
指導教授(外文):Chi-Ying Lee
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:生物學系
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:2008
畢業學年度:96
語文別:中文
論文頁數:119
中文關鍵詞:甲殼類升血糖荷爾蒙圓二色光譜胺化反應
外文關鍵詞:crustacean hyperglycemic hormonecircular dichromaticamidation
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紅腳蟳 (Scylla olivacea) 中,有兩種甲殼類升血糖荷爾蒙 (crustacean hyperglycemic hormone, CHH) 異構物,分別為Sco-CHH 及Sco-CHH-L (CHH-like peptide) 。其特徵為N端前40個殘基完全相同,而C端共32-35個殘基則相異,推測此兩種CHH異構物為RNA替代性剪接 (alternative splicing) 之產物。經生理活性測試得知Sco-CHH 具有升血糖活性,而Sco-CHH-L則無。利用大腸桿菌表現系統,表現出兩種重組蛋白Sco-CHH (rSco-CHH) 及Sco-CHH-L (rSco-CHH-L),經由再折疊 (refold) 後,利用高效能液相色層分析儀 (High Performance Liquid Chromatography, HPLC) 純化,並使用西方點墨法與質譜儀加以確認其重組蛋白。根據所有具升血糖功能之CHH都有C端胺化的現象,因此將rSco-CHH進行C端胺化反應 (amidation) ,並加以純化得到rSco-CHHamide。圓二色 (circular dichroism) 光譜結果顯示,rSco-CHHamide與rSco-CHH-L都是以α-helixes為主的二級結構(分別為47 % 與46 %);藉由蛋白酶切割rSco-CHH與rSco-CHH-L胜肽片段顯示,兩者都具有與其他CHH家族分子相同的雙硫鍵位置。生理功能分析中,注射10或100 pmol的rSco-CHHamides能誘發升血糖反應,而只有rSco-CHH-L能增加鰓上鈉鉀幫浦 (Na+-K+-ATPase) 的活性。在低鹽逆境下Sco-CHH-L基因表現量在第12小時有上升的現象,推測Sco-CHH-L可能與滲透調節有關。由以上結果得知,除了能成功製造出具有生理活性的重組Sco-CHH與Sco-CHH-L外,也發現這兩個CHH異構物有生理功能上的分歧。
Sco-CHH and Sco-CHH-L (CHH-like peptide), two structural variants of the crustacean hyperglycemic hormone family identified in the mud crab (Scylla olivacea), are identical up to the 40th residue, but different from each other in the remaining sequence. In this study, recombinant proteins (rSco-CHH and rSco-CHH-L) were produced by an E. coil expression system, refolded, purified, and confirmed by Western blotting and mass spectrometric analyses. Purified rSco-CHH was C-terminally amidated (rSco-CHHamide) in accordance with its native counterpart. Circular dichromatic spectra of rSco-CHHamide and rSco-CHH-L indicate they are rich in -helixes (47 % and 46 %, respectively); mass spectrometric analyses of peptide fragments of rSco-CHH and rSco-CHH-L reveal a common disulfide bond pattern typical of CHH family peptides. Functionally, rSco-CHHamide at 10 or 100 pmoles/animal elicited significant hyperglycemic responses, whereas rSco-CHH-L had no effect at the same dosage. On the other hand, rSco-CHH-L, but not rSco-CHHamide, dose-dependently (0-400 nM) increased the gill Na+/K+-ATPase activity. Finally, gene expression assays showed that the levels of Sco-CHH-L, but not Sco-CHH, were significantly increased in acclimated animals 12 h after exposing to an osmotic stress. In summary, recombinant Sco-CHH and Sco-CHH-L were successfully produced and characterized. Studies using the recombinant proteins provide evidence revealing that the 2 CHH structural variants diverge functionally.
前言 1
材料與方法 9
結果 26
討論 35
結論 48
參考文獻 49
圖表 64
附件 112

表目次
表一、重組蛋白 rSco-CHH 經胰蛋白酶與 ASP-N 蛋白酶切割後胺基酸片段之質荷比 (m/z) 。 64
表二、重組蛋白 rSco-CHH-L 經胰蛋白酶與 ASP-N 蛋白酶切割後胺基酸片段之質荷比 (m/z) 。 65
圖目次

圖一、包含NdeΙ 與 XhoΙ 切點之Sco-CHH 與Sco-CHH-L 產物。66
圖二、Sco-CHH 或 Sco-CHH-L 成熟肽以及 pET-22 b 質體經限制內切酶 NdeΙ 與 XhoΙ反應切割後產物的瓊脂膠電泳圖。67
圖三、以菌落 PCR確認菌株帶有可以表現 rSco-CHH或 rSco-CHH-L的表現載體。68
圖四、以 IPTG 誘導重組蛋白 rSco-CHH 與表現之蛋白質電泳圖。69
圖五、以 IPTG 誘導重組蛋白 rSco-CHH-L 之表現蛋白質電泳圖。70
圖六、rSco-CHH 與 rSco-CHH-L 之表現蛋白質電泳膠圖。71
圖七、以 Sep-pak C18 管柱初步純化蛋白質 rSco-CHH與 rSco-CHH-L。72
圖八、利用 RP-HPLC 分離未經折疊的 rSco-CHH 之圖譜與蛋白質電泳圖。73
圖九、利用 RP-HPLC 純化 rSco-CHH 之反轉相色層分析圖譜。74
圖十、經由 RP-HPLC 純化的 rSco-CHH 分離液進行 16.5 % Tricine SDS-PAGE 蛋白質電泳分析以及西方墨點呈色。75
圖十一、純化之 rSco-CHH 進行蛋白質分子量分析之質譜圖。76
圖十二、利用 RP-HPLC 純化 rSco-CHH-L 之反轉相色層分析圖譜。77
圖十三、經由 RP-HPLC 純化的 rSco-CHH-L 分離液進行 16.5 % Tricine SDS-PAGE 蛋白質電泳分析以及西方墨點呈色。78
圖十四、純化之 rSco-CHH-L 進行蛋白質分子量分析之質譜圖。79
圖十五、利用 RP-HPLC 純化 rSco-CHHamide 之反轉相色層分析圖譜。80
圖十六、純化之 rSco-CHHamide 的質譜分析圖。81
圖十七、rSco-CHH 與 rSco-CHHamide 圓二色光譜與 α-helix 含量百分比。82
圖十八、rSco-CHH-L 圓二色光譜與 α-helix 含量百分比。83
圖十九、rSco-CHH 經胰蛋白酶 (trypsin) 切割後之膠體過濾色層分析圖譜。84
圖二十、rSco-CHH 經胰蛋白酶 (trypsin) 切割後之反轉相色層分析圖譜。85
圖二十一、rSco-CHH 與 native Sco-CHH 升血糖活性柱狀圖。86
圖二十二、rSco-CHHamide 與 native Sco-CHH 升血糖活性折線圖。87
圖二十三、rSco-CHH-L 升血糖活性柱狀圖。88
圖二十四、native Sco-CHH-L 與 rSco-CHH-L 之反轉相色層分析圖譜。89
圖二十五、Sco-CHH 抗血清進行交叉反應之結果圖。90
圖二十六、Sco-CHH-L 抗血清進行交叉反應之結果圖。91
圖二十七、建立 Sco-CHH 定量之標準曲線。92
圖二十八、建立 Sco-CHH-L 定量之標準曲線。93
圖二十九、血竇腺組織中 Sco-CHH 與 Sco-CHH-L含量。94
圖三十、圍心器官組織中 Sco-CHH 與 Sco-CHH-L 含量。95
圖三十一、rSco-CHH-L 刺激鰓上鈉鉀幫浦活性柱狀圖。96
圖三十二、 rSco-CHHs 刺激鰓上鈉鉀幫浦活性之折線圖。97
圖三十三、紅腳蟳Sco-CHH 熔化曲線分析。98
圖三十四、紅腳蟳Sco-CHH-L 熔化曲線分析。99
圖三十五、紅腳蟳18s rRNA 熔化曲線分析。100
圖三十六、紅腳蟳 18s rRNA、Sco-CHH 與 Sco-CHH-L 在眼柄神經節中專一性引子對之標準曲線圖與其直線方程式以及複製效率。101
圖三十七、紅腳蟳18s rRNA、Sco-CHH 與 Sco-CHH-L 在圍心器官中專一性引子對之標準曲線圖與其直線方程式以及複製效率。102
圖三十八、正常環境中的紅腳蟳在眼柄神經節中,Sco-CHH、Sco-CHH-L 基因相對表現量。103
圖三十九、正常環境中的紅腳蟳在圍心器官中,Sco-CHH、Sco-CHH-L基因相對表現量。104
圖四十、紅腳蟳轉移至不同鹽度環境下,不同時間的眼柄神經節 Sco-CHH 基因相對表現量。105
圖四十一、紅腳蟳轉移至不同鹽度環境下,不同時間的眼柄神經節 Sco-CHH-L 基因相對表現量。106
圖四十二、紅腳蟳轉移至不同鹽度環境下,不同時間的圍心器官Sco-CHH-L 基因相對表現量。107
圖四十三、紅腳蟳轉移至不同鹽度環境下,不同時間的圍心器官 Sco-CHH 基因相對表現量。108
附圖一、rSco-CHH 與 rSco-CHH-L 雙硫鍵位置的確認。109
附圖二、具有升血糖活性的甲殼類升血糖荷爾蒙家族分子胺基酸序列之並列圖。110
附圖三、preproSco-CHH 和 preproSco-CHH-L cDNA 之結構示意圖以及蛋白質表現與相對定量即時 PCR 所使用引子的相對位置。111
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