Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52975
標題: 利用乳腺表現型基因轉殖動物系統產製具生物活性之人類第八凝血因子B-區刪除重組蛋白
Production of Biologically Active B-Domain Deleted Human Factor VIII in the Milk of Transgenic Animals Driven by Mammary-Specific Expression Cassettes
作者: 陳全木
關鍵字: 畜牧獸醫類
技術發展
摘要: 血友病(Hemophilia)為遺傳性凝血功能異常之疾病,患者中約有80%為A 型血友病(Hemophilia A),人類第八凝血因子(human factor VIII; hFVIII)係體內凝血過程不可或缺之重要成分,人體一旦缺乏或減少第八凝血因子,將會引發不同程度之凝血功能問題,而導致所謂的A 型血友病。目前血友病患係以血漿製劑或由正常人血液中純化之hFVIII 蛋白進行針劑補充療法,然而因費用昂貴,以及無法避免血源病毒感染(如HBV 或HIV)之潛在危險,因此利用生物科技發展出以非血源性的hFVIII 醫藥蛋白生產平台更顯迫切。又根據資料統計,A 型血友病好發於東方人,因此在東方國家第八凝血因子蛋白的短缺是醫療衛生上的一大問題,所以大量製造重組FVIII 醫藥蛋白必為當務之急。本研究室的先前研究成果以利用基因轉殖小鼠模式,成功在乳腺組織生產人類第八凝血因子,並於乳汁中檢測出含有7.0–50.2ug/ml 的FVIII 重組蛋白,其濃度為正常血液含量的35-200 倍之多,並且具有13.4U/ml 的凝血活性。在此一基礎下,本計畫將進一步以基因轉殖猪為動物模式(目前亦產製出No. 235 與No. 335 兩個品系),應用基因工程技術改造全長的FVIII 基因為B-區刪除的FVIII 基因(B-domain deleted FVIII; FVIII△B),探討其提升重組蛋白產製的效果,三年期計畫之重點研究規劃,包括:第一年度將建構三種不同的FVIII轉殖基因,分別為全長FVIII cDNA、B-區刪除FVIII cDNA (FVIII△B)、及FVIII△B 加上His-Tag 等形式,並於基因轉殖小鼠系統中進行重組蛋白質之生產效能與純化效率分析;第二年度將針對已產製的FVIII△B 轉殖基因猪,建立不同的系譜族群,進行染色體基因定位與基因表現分析,並於猪乳樣中分析重組蛋白之含量;第三年則著重於基因轉殖猪乳之蛋白質體分析、FVIII△B 重組蛋白之純化與活性測定、並以A 型血友病基因刪除鼠為動物模式,進行活體之凝血功能確效分析。評估於基因轉殖乳汁中大量生產並純化人類第八凝血因子之可行性,以期降低昂貴FVIII 醫藥蛋白之生產成本,未來可開發成應用於A 型血友病治療之新式醫藥蛋白生產來源。
Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency orabnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with wholeplasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and thecost of highly purified FVIII are the major factors that restrict prophylaxis in hemophiliatherapy. One of the challenges created by the biotechnology revolution is the development ofmethods for the economical production of highly purified proteins in large scales. Recentdevelopments indicate that manipulating milk composition using transgenesis has focusedmainly on the mammary gland as a bioreactor to produce pharmaceuticals. In our pilot study,a fusion gene containing bovine -lactalbumin and human FVIII full-length cDNA (7.2-kb)was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. Totalnumber of 9 (4 females/ 5 males) mice produced were confirmed to be successfully integratedand stably germ-line transmitted with the LA-hFVIII fusion gene. Western-blot analysisagainst milk samples have further shown that the recombinant hFVIII was secreted into themilk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2 g/ml, over35- to 200-fold higher than that in normal human plasma. Up to 13.4 U/ml of rFVIII wasdetected in an assay in which rFVIII restored normal clotting activity to FVIII-deficienthuman plasma. In this 3 years proposal, to improve the large-scale production of rFVIIIpharmaceutical protein from transgenic milks, we will attempt to generate B-domain FVIII(FVIII △ B) gene constructs and mammary gland-specific expression in the milks oftransgenic pigs during lactation. A fast protein liquid chromatography (FPLC) will be appliedto large-scale purify recombinant FVIII△B protein and the in vivo clotting functional assaywill also be validated in FVIII-knockout mice. Whenever this project has been successfullydone, it could provide a novel strategy to large-scale production of a potential rFVIIIpharmaceutical drug use in Hemophilia A therapy.
URI: http://hdl.handle.net/11455/52975
其他識別: NSC100-2313-B005-028-MY3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=2325446&plan_no=NSC100-2313-B005-028-MY3&plan_year=100&projkey=PD10007-0303&target=plan&highStr=*&check=0&pnchDesc=%E5%88%A9%E7%94%A8%E4%B9%B3%E8%85%BA%E8%A1%A8%E7%8F%BE%E5%9E%8B%E5%9F%BA%E5%9B%A0%E8%BD%89%E6%AE%96%E5%8B%95%E7%89%A9%E7%B3%BB%E7%B5%B1%E7%94%A2%E8%A3%BD%E5%85%B7%E7%94%9F%E7%89%A9%E6%B4%BB%E6%80%A7%E4%B9%8B%E4%BA%BA%E9%A1%9E%E7%AC%AC%E5%85%AB%E5%87%9D%E8%A1%80%E5%9B%A0%E5%AD%90B-%E5%8D%80%E5%88%AA%E9%99%A4%E9%87%8D%E7%B5%84%E8%9B%8B%E7%99%BD
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