- 學位論文
雞卵白蛋白啟動子序列之選殖及其功能性測試
Cloning and Fuctional Assessment of the Chicken Ovalbumin Promoter Sequences
摘要
經遺傳選拔之現代萊亨蛋雞，每年產蛋可高達330個，而每顆蛋總蛋白質含量約6.6克，卵黃佔有3.1克，卵白則有3.5克。存在於卵白中之3.5克蛋白質，約有2克是為卵白蛋白（ovalbumin）此種單一蛋白質。而蛋白之生成與分泌係發生於位於輸卵管蛋白分泌部管腔周圍之管狀腺細胞，鑑於此細胞具有大量產生蛋白質之能力，暗示著產蛋雞位於蛋白分泌部的管狀腺細胞具有極大之潛力，成為生產轉基因蛋白質之生物反應器。 本試驗嘗試選殖出卵白蛋白基因之啟動子，並評估利用該啟動子構築轉殖基因之效能。故係萃取產蛋雞血球細胞之基因組DNA （genomic DNA），並利用PCR增殖後以TA cloning方式，擴增並選殖出卵白蛋白啟動子片段，試驗結果自雞基因組DNA擴增出一3.1 kb之DNA片段，而該片段經定序分析後，其5’端序列之1.3 kb為啟動子序列，其3’端1.8 kb之序列則為卵白蛋白之釋泌信息肽（signal peptide）序列，經分析比對後序列無誤。 稍後試驗藉由綠色螢光蛋白此報導基因，針對前述選殖獲得之卵白蛋白啟動子進行效力測試；首先構築一pMAR-OV-GFP質體，令構築之轉殖基因得以利用卵白蛋白啟動子驅動具完整序列之綠色螢光蛋白cDNA。並於質體pOV-GFP中構築一段核基質結合區（matrix attachment regions, MARs），俾謀保護該轉殖基因於轉殖過程中得能減少損害。試驗結果顯示，所構築之質體pMAR-OV-GFP經轉染進入初級培養之管狀腺細胞，經過24-48小時之後，可利用螢光顯微鏡觀察到綠色螢光蛋白之表現，此結果証實，儘管轉染效力不佳，然該選殖而得之卵白蛋白啟動子，仍具啟動GFP cDNA表現之功能。
並列摘要
A modern genetically selected White Leghorn hen lays up to 330 eggs per year and the total proteins contained within each egg has been averaged around 6.6 grams, including 3.5 grams of the egg-white proteins and 3.1grams of the yolk proteins. Of 3.5 grams of the egg-white proteins, 2 grams of them have been identified as the ovalbumin. The fact that egg-white proteins are primarily synthesized and secreted from the tubular gland cells lined on the lumen of the magnum of the oviduct, indicating that these tubular gland cells found in the magnum of laying chicken possess a great potency of serving as the bioreactor for production of transgenic proteins. Attempts of the present studies were made to clone the promoter sequences of ovalbumin gene and to evaluate the potency of using the cloned promoter for construction of the transgene(s). To meet with the described purpose, genomic DNAs extracted from blood cells of the laying chicken hens were subjected to cloning of the ovalbumin promoter sequences by the strategy of PCR-TA cloning techniques that have resulted in a fragment of the genomic DNA lengthen in 3.1 kb was successfully amplified and ligated into the pGlow-TOPO vector. Of the 3.1 kb fragment after sequencing analysis, a 1.3 kb in length of the 5’ end sequences was confirmed to contain the promoter region and the rest fragment lengthen in 1.8 kb of the 3’ end was characterized as the sequences encoding the leading protein. The efficacy of the cloned ovalbumin promoter was further evaluated using the green fluorescence protein (GFP) as a reporter. Of these studies, a transgene named pMAR-OV-GFP was constructed allowing the full length of GFP cDNA sequences driven by ovalbumin promoter and the OV-GFP sequences were equipped with a matrix attachment region (MAR) that will ensure the transgene successfully escaped from damage by those topoisomerases. The expression of transgene of pMAR-OV-GFP was evidenced from the observation of GFP fluorescence detected by fluorescent microscope around 24-48 post the transgene had been transfected into the primary cultured tubular gland cells of oviduct magnum from the laying hen. These results indicate that the cloned ovalbumin promoter does possess potency to drive the GFP cDNA while the efficiency is still far beyond satisfy.