白色來亨雞卵白蛋白基因啟動子之選殖、定序與表現

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Title:	白色來亨雞卵白蛋白基因啟動子之選殖、定序與表現 Cloning, sequencing and expression of ovalbumin gene promoter from White-Leghorn Chicken
Authors:	王君今
Contributors:	嘉南藥理科技大學：生物科技系暨研究所張竣凱蕭振文
Keywords:	表現定序白色來亨雞卵白蛋白啟動子綠色螢光蛋白轉殖 expression sequencing transgenic White Leghorn Chicken ovalbumin promoter GFP
Date:	2011
Issue Date:	2011-10-25 15:29:51 (UTC+8)
Abstract:	以雞做為生物反應器(bioreactor)，生產高價值藥用蛋白質是極具潛力之生物技術。在基因轉殖雞的輸卵管表達外源基因，在雞蛋中可大量生產藥用蛋白質。白色來亨雞(White Leghorn Chicken)之世代間距短，年產約 300顆雞蛋，是做為生物反應器的理想鷄種。因此，本研究之主要目的在選殖白色來亨雞之卵白蛋白(ovalbumin)基因的啟動子(promoter)，構築入綠色螢光蛋白(green fluorescent protein, GFP)報導載體並進行 DNA序列分析，期能供作生產家禽基因轉殖之載體用途。選殖卵白蛋白基因啟動子之引子係參考網路資料庫中雞卵白蛋白基因序列設計，並進行聚合酶連鎖反應 (polymerase chain reaction, PCR)及電泳分析，將 PCR產物回收純化後進行 DNA序列分析確認。本研究分別以 PCR擴增出卵白蛋白基因啟動子轉錄起始點上游 2.8kb及動情素-反應加強子元素區域 (estrogen-responsive enhancer element, ERE) 675bp片段後，並同時將兩個片段(2.8 kb ＋ 675 bp)接合成為 3.5kb之構築體，供測試啟動子調控區對基因表現效率的影響。選殖之構築體經 DNA序列分析確認，構築入含 GFP之報導載體，該質體 DNA經大量純化，進行雞原代管狀腺細胞之基因轉染，經 PCR分析可分別測得 3.5kb卵白蛋白啟動子及 GFP之序列存在，且轉殖細胞在螢光顯微鏡下可觀察到 GFP的表現。此結果顯示，選殖之卵白蛋白啟動子能驅動 GFP基因而表現螢光蛋白。因此，本試驗選殖的白色來亨雞卵白蛋白基因啟動子序列，可發展成為供家禽基因轉殖研究之啓動子來源。 Oviduct-specific expression of exogenous recombinant proteins in transgenic chicken is a promising technology for the large-scale production of therapeutic proteins in eggs. White Leghorn Chicken can produce about 300 egg/year, and has a shorter generation time. Therefore, the objective of this study was to clone and sequencing the ovalbumin gene promoter from White Leghorn Chickens and constructed into the green fluorescent protein (GFP) reporter vector for the study of transgenic chicken. The primers to clone the ovalbumin gene promoter were designed based on the database on the internet. Genomic DNA was purified from blood samples and used as templates for polymerase chain reaction (PCR). The expected PCR products was then purified, and constructed onto DNA sequencing vector. In this work, 2.8 kb upstream of transcription start site and 675 bp of estrogen-responsive enhancer element (ERE) of ovalbumin gene was amplified separately and then ligated to obtain a 3.5 kb construct. After identifying by DNA sequencing, the 3.5 kb construct of ovalbumin gene promoter was sub-cloned into GFP reporter vector to become ova-GFP expression vector. The expression of ova-GFP was confirmed by observed the GFP emitted from chicken tubular gland cells that were transfected with ova-GFP plasmid. The results indicated that the ova-GFP construct can drive GFP expression and could be used as reporter system for the study of transgenic chicken.
Relation:	校內外均一年後公開，學年度：99，74頁
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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