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研究生:陳世昌
研究生(外文):Chen Shue-Chan
論文名稱:以基因重組技術於哺乳類細胞和酵母菌生產第一型人類環氧酶(前列腺素H合成酶)
論文名稱(外文):Expression of Recombinant Human COX-1(Prostaglandin H Synthase 1)in Mammalian Cells and Yeast
指導教授:楊美桂楊美桂引用關係盧志峰盧志峰引用關係王進賢
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
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:2007
畢業學年度:96
語文別:中文
論文頁數:59
中文關鍵詞:前列腺素環氧酶蛋白質表現系統
外文關鍵詞:CyclooxygenaseK. lactisNT-2293TPGHS1CMV
相關次數:
  • 被引用被引用:0
  • 點閱點閱:248
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  • 下載下載:78
  • 收藏至我的研究室書目清單書目收藏:0
前列腺素(Prostaglandins, PGs)屬於脂質性媒介物(lipid mediators),其生成涉及人類正常生理的維持和炎性反應的發生。環氧酶(Cyclooxygenase, COX;前列腺素H合成酶Prostaglandin H Synthase, PGHS)是合成前列腺素的關鍵酵素,有兩個異構酶(isozymes):COX-1和COX-2。COX-2是屬於誘發性酵素(inducible enzyme),其生成與發炎反應有關。相反的,COX-1是屬於經常性酵素(housekeeping enzyme),與維持正常的生理作用有關。在本研究中,我們利用基因工程技術重組COX-1基因使其表現六個組胺酸(6×histidines)標記於人類COX-1(hCOX-1)羧基端的融合蛋白質(fusion protein)的基因,再經由哺乳類細胞NT-2、293T和酵母菌K. lactis來生產重組的第一型人類環氧酶(hCOX-1-His);羧基端附加6×His則有利於重組hCOX-1酵素的純化。結果發現:在NT-2細胞內,重組的hCOX-1-His基因並沒有表現;在293T細胞內,重組的hCOX-1-His基因有表現但是產量少;在酵母菌細胞內,重組的hCOX-1-His基因有較好的表現。我們利用溶壁酶(lyticase)溶解酵母菌細胞壁後,再利用0.2% 的Triton X-100 萃取脂溶性的hCOX-1-His酵素,大約有40%以上的hCOX-1-His酵素可以順利被萃取出。結果顯示維持少量的前列腺素可能是一般哺乳類細胞生存所必須,過量生產前列腺素可能對NT-2和293T細胞有毒害,因此細胞本身可能會自行調節hCOX-1的產量,以維持穩定量的前列腺素。若要在哺乳類細胞中量產第一型人類環氧酶,可能需要更進一步研究其控制機轉。在酵母菌中,hCOX-1-His屬於外來蛋白質,在合適的啟動子誘導下,便可以量產。雖然文獻報告指出hCOX-1可在昆蟲病毒表現系統 (baculovirus expression system)和牛痘病毒載體(vaccinia vector)轉染COS-7細胞表現系統來生產。 但是兩系統的流程複雜且成本相對昻貴,同時存在病毒污染的危險性。本研究結果顯示可利用簡單的轉染或轉形方式,在哺乳類細胞或酵母菌生產hCOX-1酵素,提供研究之用。
The prostaglandins are lipid mediators, which are made by the bifunctional enzyme, Cyclooxygenase(COX), containing both cyclooxygenase and peroxidase activities. Prostaglandins are involved in physiological functions such as protection of the stomach mucosa, aggregation of platelets and regulation of kidney function...etc. They also have pathological functions such as induction of inflammation, fever and pain...etc. There are two isoforms of cyclooxygenase: Cyclooxygenase-1(COX-1)and Cyclooxygenase-2(COX-2). The COX-1 is a housekeeping or constitutive enzyme making prostaglandins which are important for maintaining physiological functions. In this study, a recombinant human COX-1(hCOX-1; human prostaglandin H synthase 1, PGHS1)was expressed in yeast, K. lactis, and 293T mammalian cells, but not in NT-2 cells. In the yeast system, the full-length cDNA for hCOX-1 was placed under the control of the LAC4 promoter and was integrated into the yeast genome by homologous recombination. The hCOX-1 enzyme was extracted from the transformed yeast by a solution containing 0.2% of Triton X-100, and the efficacy of hCOX-1 enzyme extraction was over 40% as estimated by Western blotting. In the mammalian cell system, the full-length cDNA for hCOX-1 was under the control of CMV ( cytomegalovirus) promoter from the mammalian expression vector, pcDNA3.1, and was transiently transfected into both NT-2 and 293T cells. The recombinant hCOX-1 enzyme expressed in both yeast and mammalian systems contains six-histidine tag (His-tag) at the carboxyl terminus, and was identified by Western blotting using both anti-COX-1 and anti-His antibodies. Although no recombinant hCOX-1 proteins were identified in NT-2 cells, and only small amounts of the recombinant hCOX-1 were detected in the transfected 293T cells, a larger quantity of recombinant proteins was found in the yeast expression system. Previously, it was reported that hCOX-1 successfully expressed in both the BVES (baculovirus expression system)and in the VV (vaccinia vector transfected)COS-7 cells. However, both processes were rather complicated and involving the use of viral vectors which carry potential hazards of environmental contamination. The data presented here demonstrate that it is possible to produce hCOX-1 enzyme for biochemical, biophysical, and pharmacological investigations using both the yeast K. lactis and 293T mammalian cell expression systems circumventing the risk of possible viral contamination. The low expression of recombinant hCOX-1 in transfected 293T cells is possibly caused by cellular auto-regulation mechanism to avoid toxicity of prostaglandin over-production. Further investigation will warrant the production of recombinant hCOX-1 in the mammalian cells via simple transfection.
第一章、文獻回顧………………………………………………1.

第二章、實驗材料………………………………………………9.

第三章、實驗方法………………………………………………11.

第四章、結果……………………………………………………25.

第五章、討論……………………………………………………29.

第六章、結論……………………………………………………33.

第七章、參考文獻………………………………………………56.
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