跳到主要內容

臺灣博碩士論文加值系統

(44.201.97.0) 您好!臺灣時間:2024/04/24 11:55
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:沈家漢
研究生(外文):Gia-Han Shen
論文名稱:製備具有凝血酶切點及活化型血小板結合能力之重組鏈激酶融合蛋白
論文名稱(外文):Preparation of Recombinant Streptokinase Fusion Proteins with Thrombin Cutting Site and Specific Affinity to the Activated Platelets
指導教授:施桂月
指導教授(外文):Guey-Yueh Shi
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:114
中文關鍵詞:鏈激酶凝血酶切點活化型血小板凝血酶
外文關鍵詞:StreptokinaseThrombin cutting siteActivated plateletsThrombin
相關次數:
  • 被引用被引用:0
  • 點閱點閱:177
  • 評分評分:
  • 下載下載:11
  • 收藏至我的研究室書目清單書目收藏:0
鏈激酶(streptokinase, SK),是由β-溶血性鏈球菌所分泌的菌體外蛋白質,具有414個胺基酸的單一多胜肽。雖然鏈激酶自己不具有酵素活性,但是當鏈激酶和人類血纖維蛋白溶酶原(human plasminogen, HPlg)或人類血纖維蛋白溶酶(human plasmin, HPlm)形成活化複合體,就能將人類血纖維蛋白溶酶原活化成人類血纖維蛋白溶酶。因此,臨床上鏈激酶常被用來當做血栓溶解劑,治療心血管方面的疾病,如急性心肌梗塞。
鏈激酶是一種血栓溶解劑,但是對於血栓並無專一性。它除了會將新血栓溶解外,也會水解血纖維蛋白原(fibrinogen)及人體恆定存在的舊血栓(haemostatic plugs),因而產生出血的副作用。為了改進鏈激酶在血栓治療上的安全性,我們構築並測試重組鏈激酶蛋白是否能專一性的在新血栓上活化人類血纖維蛋白溶酶原。
這些新穎的鏈激酶重組蛋白是在SK(16-378)的NH2端分別接上RGDS或血纖維蛋白 COOH端γ-chain部分的胜肽,並且在鏈激酶蛋白和胜肽間設計凝血酶切點(thrombin cutting site, T)。這些鏈激酶重組蛋白在in vitro下具有較低活化人類血纖維蛋白溶酶原的能力,且對新血栓上的活化型血小板具有較高的親合力。此外,當這些鏈激酶重組蛋白和凝血酶(thrombin)反應後,鏈激酶活化人類血纖維蛋白溶酶原的能力會上升。
總結以上,我們證明這些鏈激酶重組蛋白對活化型血小板有較高的親合力,且能被新血栓附近的凝血酶誘導出活化人類血纖維蛋白溶酶原的能力。這些鏈激酶重組蛋白比原型鏈激酶對新血栓更具有專一性。
Streptokinase (SK), a secretory protein of -hemolytic streptococcus, is consisted of single polypeptide of 414 amino acid residues. Although SK by itself possesses no intrinsic catalytic activity, the complex of SK and human plasminogen (HPlg) or plasmin (HPlm) becomes a plasminogen activator that is able to catalyze the conversion of HPlg to HPlm. Therefore, SK has been used as a thrombolytic agent in treatment of thromboembolic blockages including acute myocardial infarction.
SK was considered as a thrombolytic agent with not blood clot specificity. It could induce systemic hydrolysis of fibrinogen and haemostatic plugs as well, leading to bleeding side effect episodes. To improve the safety of SK as a thrombolytic agent, a recombinant SK that can specifically activate plasminogen in the fresh blood clot was constructed and tested.
The novel recombinant SKs are consisted of RGDS and/or partial fibrinogen γ-chain at the N-terminal of SK (16-378). In the recombinant SK we also designed a thrombin specific cutting site between fusion peptide and SK. The recombinant SKs have lower plasminogen activator activity in vitro. On the other hands, the recombinant SKs have higher affinity for activated platelet that is abundant in the fresh blood clot. In addition, the specific plasminogen activator activity increased, when the recombinant SK was pretreated with thrombin.
In conclusion, we demonstrated the recombinant SK has the potential to bind to activated platelet and be activated by thrombin that may exist in the fresh blood clot. The recombinant SKs could induce fresh blood clot more specifically than native SK.
中 文 摘 要 I
英 文 摘 要 II
致 謝 IV
目 錄 V
表 目 錄 XIII
圖 目 錄 XIII
附 錄 XIII
縮 寫 檢 索 表 IX
緒 論 1
藥 品 及 材 料 5
儀 器 9
方 法 11
一、DNA基本技術操作 11
1-1 小量質體DNA的抽取 11
1-2 聚合酶連鎖反應 13
1-3 瓊脂膠電泳分析 14
1-4 以電泳法回收DNA 15
1-5 限制酶切割 16
1-6 去磷酸根 17
1-7 接合反應 17
1-8 形質轉移 18
1-9 定序DNA 20
1-10 長期保存菌種 20
二、構築鏈激酶重組基因 22
2-1 載體的製備 22
2-2 PCR產物的製備 22
2-3 構築鏈激酶重組基因 23
2-4 選殖鏈激酶重組基因 23
三、表現鏈激酶重組蛋白 25
四、純化鏈激酶重組蛋白 27
4-1 準備High-Q膠體管柱 27
4-2 準備鏈激酶重組蛋白樣品 28
4-3 純化鏈激酶重組蛋白 29
五. 純化原型鏈激酶蛋白 30
5-1 準備Blue-Sepharose 6B膠體管柱 30
5-2 準備原型鏈激酶蛋白樣品 31
5-3 純化原型鏈激酶蛋白 32
5-4 第二次純化原型鏈激酶蛋白 33
六、蛋白質基本技術操作 34
6-1 蛋白濃度測定 34
6-2 SDS-PAGE製備 35
6-3 SDS-PAGE電泳分析 37
6-4 PVDF membrane轉印分析 39
6-5 蛋白HN2端定序前處理 40
七、鏈激酶比活性測定 41
八、血小板吸附試驗 43
8-1 製備血小板懸浮液 43
8-2 血小板吸附試驗 45
九、血小板凝集試驗 47
9-1 製備血小板懸浮液 47
9-2 血小板凝集試驗 49
十、利用凝血酶和鏈激酶作用後活化HPlg試驗 51
10-1 凝血酶和鏈激酶作用 51
10-2 利用凝血酶和鏈激酶作用後活化HPlg試驗 52
十一、利用凝血酶和鏈激酶作用後溶解血漿凝塊試驗 53
11-1 將血纖維蛋白原接上FITC 53
11-2 形成血漿凝塊 54
11-3 凝血酶和鏈激酶作用 55
11-4 利用凝血酶和鏈激酶作用後溶解血漿凝塊試驗 56
結 果 57
一、鏈激酶重組基因的構築與選殖 57
(1)基因的構築 57
(2)基因的選殖 58
二、鏈激酶重組蛋白表現、純化及蛋白HN2端定序分析 58
(1)蛋白表現 58
(2)蛋白純化 58
(3)蛋白NH2端定序分析 59
三、鏈激酶重組蛋白活化HPlg的能力測試 60
(1)比活性 60
四、鏈激酶重組蛋白的功能分析 61
(1)血小板吸附試驗 61
(2)血小板凝集試驗 62
(3)利用凝血酶和鏈激酶作用後活化HPlg試驗 63
(4)利用凝血酶和鏈激酶作用後溶解血漿凝塊試驗 64
討 論 66
總結 71
參 考 文 獻 72
表 76
圖 79
附 錄 112
自 述 114
Albelda SM. Role of integrins and other cell adhesion molecules in tumor progression and metastasis. Lab Invest. 1993 Jan;68(1):4-17. Review.
Bellavite P, Andrioli G, Guzzo P, Arigliano P, Chirumbolo S, Manzato F, Santonastaso C.A colorimetric method for the measurement of platelets adhesion in microtiter plates. Anal Biochem. 1994 Feb 1;216(2):444-50.
Booyse FM, Osikowicz G, Feder S, Scheinbuks J. Isolation and characterization of a urokinase-type plasminogen activator (Mr = 54,000) from cultured human endothelial cells indistinguishable from urinary urokinase. J Biol Chem. 1984 Jun 10; 259(11):7198-205.
Braunwald E, Knatterud GL, Passamani E, Robertson TL, Solomon R. Update from the Thrombolysis in Myocardial Infarction Trial. J Am Coll Cardiol. 1987 Oct; 10(4):970.
Chesebro JH, Knatterud G, Roberts R, Borer J, Cohen LS, Dalen J, DAge HT, Francis CK, Hillis D, Ludbrook P, et al. Thrombolysis in Myocardial Infarction (TIMI) Trial, Phase I: A comparison between intravenous tissue plasminogen activator and intravenous streptokinase. Clinical findings through hospital discharge. Circulation. 1987 Jul; 76(1):142-54.
Colman RW. Receptors that activate platelets.Proc Soc Exp Biol Med. 1991 Jul;197 (3):242-8. Review
Eliceiri BP, Cheresh DA. Adhesion events in angiogenesis.Curr Opin Cell Biol. 2001 Oct;13(5):563-8. Review.
Farrell DH, Thiagarajan P, Chung DW, Davie EW. Role of fibrinogen alpha and gamma chain sites in platelets aggregation.Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10729-32.
Francis CW, Markham RE Jr, Barlow GH, Florack TM, Dobrzynski DM, Marder VJ. Thrombin activity of fibrin thrombi and soluble plasmic derivatives. J Lab Clin Med. 1983 Aug;102(2):220-30.
Ginsberg MH, Loftus JC, Plow EF. Cytoadhesins, integrins, and platelets.Thromb Haemost. 1988 Feb 25;59(1):1-6. Review.
Gold HK, Leinbach RC, Garabedian HD, Yasuda T, Johns JA, Grossbard EB, Palacios I, Collen D. Acute coronary reocclusion after thrombolysis with recombinant human tissue-type plasminogen activator: prevention by a maintenance infusion. Circulation. 1986 Feb;73(2):347-52.
Hawiger J, Timmons S. Binding of fibrinogen and von Willebrand factor to platelets glycoprotein IIb-IIIa complex.Methods Enzymol. 1992;215:228-43.
Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J Biol Chem. 1982 Mar 25;257(6):2912-9.
Korninger C, Collen D. Studies on the specific fibrinolytic effect of human extrinsic (tissue-type) plasminogen activator in humanblood and in various animal species in vitro. Thromb Haemost. 1981 Aug 28; 46(2):561-5.
Levin EG, Loskutoff DJ. Cultured bovine endothelial cells prAuce both urokinase and tissue-type plasminogen activators. J Cell Biol. 1982 Sep; 94(3):631-6.
Marder VJ, Sherry S. Thrombolytic therapy: current status (1). N Engl J Med. 1988 Jun 9; 318(23):1512-20.
Marder VJ, Sherry S. Thrombolytic therapy: current status (2). N Engl J Med. 1988 Jun 16; 318(24):1585-95.
McClintock DK, Bell PH. The mechanism of activation of human plasminogen by streptokinase. Biochem Biophys Res Commun. 1971 May 7; 43(3):694-702.
McEver RP. Adhesive interactions of leukocytes, platelets, and the vessel wall during hemostasis and inflammation.Thromb Haemost. 2001 Sep;86(3):746-56. Review.
Mohri H, Ohkubo T. Effects of hybrid peptide analogs to receptor recognition domains on alpha- and gamma-chains of human fibrinogen on fibrinogen binding to platelets. Thromb Haemost. 1993 May 3; 69(5):490-5.
Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding to integrins.J Biol Chem. 2000 Jul 21;275(29):21785-8. Review.
Rao AK, Pratt C, Berke A, Jaffe A, Ockene I, Schreiber TL, Bell WR, Knatterud G, Robertson TL, Terrin ML. Thrombolysis in Myocardial Infarction (TIMI) Trial--phase I: hemorrhagic manifestations and changes in plasma fibrinogen and the fibrinolytic system in patients treated with recombinant tissue plasminogen activator and streptokinase. J Am Coll Cardiol. 1988 Jan; 11(1):1-11.
Reddy KN, Markus G. Mechanism of activation of human plasminogen by streptokinase. Presence of active center in streptokinase-plasminogen complex. J Biol Chem. 1972 Mar 25; 247(6):1683-91.
Reddy KN, Markus G. Esterase activities in the zymogen moiety of the streptokinase-plasminogen complex. J Biol Chem. 1974 Aug 10; 249(15):4851-7.
Savage B, Bottini E, Ruggeri ZM. Interaction of integrin alpha IIb beta 3 with multiple fibrinogen domains during platelets adhesion.J Biol Chem. 1995 Dec 1;270(48):28812-7.
Savage B, Cattaneo M, Ruggeri ZM. Mechanisms of platelets aggregation.Curr Opin Hematol. 2001 Sep;8(5):270-6. Review
Schick LA, Castellino FJ. Interaction of streptokinase and rabbit plasminogen. Biochemistry. 1973 Oct 23;12(22):4315-21.
Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelets paradigm. Blood. 1998 Apr 15;91(8):2645-57. Review.
Steiner B, Cousot D, Trzeciak A, Gillessen D, Hadvary P. Ca2+-dependent binding of a synthetic Arg-Gly-Asp (RGD) peptide to a single site on the purified platelets glycoprotein IIb-IIIa complex. J Biol Chem. 1989 Aug 5; 264(22):13102-8.
van der Flier A, Sonnenberg A. Function and interactions of integrins.Cell Tissue Res. 2001 Sep;305(3):285-98. Review
White HD, Rivers JT, Maslowski AH, Ormiston JA, Takayama M, Hart HH, Sharpe DN, Whitlock RM, Norris RM. Effect of intravenous streptokinase as compared with that of tissue plasminogen activator on left ventricular function after first myocardial infarction. N Engl J Med. 1989 Mar 30; 320(13):817-21.
Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, Jordan RE. Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets. Blood. 1996 Aug 1;88(3):907-14.
Yusuf S, Collins R, Peto R, Furberg C, Stampfer MJ, Goldhaber SZ, Hennekens CH. Intravenous and intracoronary fibrinolytic therapy in acute myocardial infarction: overview of results on mortality, reinfarction and side-effects from 33 randomized controlled trials. Eur Heart J. 1985 Jul;6(7):556-85.
Yusuf S. Interventions that potentially limit myocardial infarct size: overview of clinical trials. Am J Cardiol. 1987 Jul 15;60(2):11A-17A. Review.
Yusuf S. An overview of the clinical trials of agents (other than beta-blockers) that potentially limit myocardial infarct size. J Cardiovasc Pharmacol. 1988;12 Suppl 1:S48-55. Review.
Yusuf S, Wittes J, Friedman L. Overview of results of randomized clinical trials in heart disease. I. Treatments following myocardial infarction.JAMA. 1988 Oct 14;260(14):2088-93. Review.
Young KC, Shi GY, Chang YF, Chang BI, Chang LC, Lai MD, Chuang WJ, Wu HL.Interaction of streptokinase and plasminogen. Studied with truncated streptokinase peptides. J Biol Chem. 1995 Dec 8; 270(49):29601-6.
石燕萍. (1997)製備具有活化型血小板結合能力之重組鏈激酶融合蛋白. 國立成功大學醫學院生物化學研究所. 碩士論文
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top