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研究生:陳一中
研究生(外文):Yi-Jhong Chen
論文名稱:表達重組蛋白GST-hEGF (人表皮生長因子)的酵母菌質體之構築及其在釀酒酵母中的表達
論文名稱(外文):The construction of a yeast plasmid expressing recombinant protein GST-hEGF (human epidermal growth factor) and the protein expression in Saccharomyces cerevisiae
指導教授:蔡榮宗蔡榮宗引用關係
口試委員:陳霈霓傅如輝
口試日期:2020-07-02
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生化微生物免疫研究所
學門:生命科學學門
學類:其他生命科學學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:69
中文關鍵詞:表皮生長因子重組蛋白酵母菌質體釀酒酵母
外文關鍵詞:human epidermal growth factorrecombinant protein GST-hEGFyeast plasmidSaccharomyces cerevisiae
DOI:10.6834/csmu202000086
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EGF (epidermal growth factor;表皮生長因子)是最被早發現的生長因子,功能是促進細胞的生長(cell growth)、增殖(proliferation)和分化 (differentiation)。hEGF (human epidermal growth factor;人表皮生長因子)是由53個胺基酸組成的活性多肽,其分子量為6216道爾頓,分子結構中具有三個二硫鍵,決定著它的生物活性。近代已經進行了許多研究和文獻發表,以探討hEGF在急、慢性傷口癒合、藥物治療和皮膚護理、醫學美容中的潛在應用。hEGF在醫學上已被證實,在燙傷、燒傷、手術傷口、褥瘡、糖尿病足潰瘍、角膜損傷、潰瘍性結腸炎等臨床治療上,取得非常好的療效。隨著醫學美容興起,有關hEGF在皮膚老化的研究方面,也取得不錯的進展;在皮膚護理上的應用與發展是一塊新藍海,它是具有巨大潛力的美容化妝品添加劑。重組DNA技術的出現促進了多種rhEGF (recombinant hEGF;重組人表皮生長因子)的發展及生產,可以用經濟高效的方式來生產rhEGF。本篇研究的主要目的是想建立一個酵母菌的微生物表達系統,可以用來表達及生產GST-hEGF的重組蛋白。未來可以大量生產出具有生物活性的成分,經過萃取、精煉之後,可以應用做成含rhEGF的酒粕面膜;或當作化妝品、保養品的原料,製成有助肌膚保養的醫學美容產品。我的研究是以本實驗室學姐已經構築好了的大腸桿菌質體pGEX-L-EGF-His (含有編碼GST-hEGF融合基因)為基礎,用PCR方式改變限制酶的切位,將切割好的GST-hEGF DNA片段,接入酵母菌的蛋白表達載體pG1-EX1中,以形成新的酵母菌表達質體(命名為pG1-GST-EGF-His)。首先,透過DNA定序分析驗證了它的序列正確性。再將定序分析正確的pG1-GST-EGF-His質體轉形到釀酒酵母中。然後打破酵母菌細胞,將得到的總蛋白藉由Coomassie Blue staining、西方墨點法 (Western Blotting)實驗分析,證實了重組蛋白GST-hEGF的確可以在釀酒酵母中高度表達。
Epidermal growth factor (EGF) is the earliest discovered growth factor and plays an important role in regulating cell growth, proliferation and differentiation. Human epidermal growth factor (hEGF) is an active polypeptide composed of 53 amino acids. Its molecular weight is 6,216 Daltons. There are three disulfide bonds in the molecular structure, which determine its biological activity. In modern times, numerous research studies have been published to explore the potential application of hEGF in acute and chronic wound healing, drug treatment and skin care, and medical aesthetics. hEGF has been proven in medicine, and has achieved very good results in clinical treatment of scalds, burns, surgical wounds, bedsores, diabetic foot ulcers, corneal injuries, and ulcerative colitis. With the rise of medical cosmetology, hEGF has also made good progress in the study of skin aging; the development and application of skin care is a new blue ocean strategy. It is a cosmetic additive with great potential. It has unlimited business opportunities and promising prospects. The emergence of recombinant DNA technology has facilitated the development and production of a great variety of recombinant hEGF (rhEGF) isoforms, and rhEGF can be produced in a cost-effective manner.The main purpose of this study is to establish a yeast microbial expression system that can be used to express and produce recombinant protein GST-hEGF. In the future, biologically active ingredients can be mass-produced. After extraction and refining, they can be used to make rhEGF-containing wine meal masks; or used as raw materials for cosmetics and skin care products to make Cosmeceuticals that can help skin care. My research is based on the E.coli plasmid pGEX-L-EGF-His (which contains genes encoding hEGF and GST) that alumna of the laboratory has constructed. Change the cleavage position of restriction enzyme by PCR. The cut pGEX-L-EGF-His fragment was inserted into the yeast secretion vector pG1-EX1 to form a new yeast expression plasmid (named pG1-GST-EGF-His). First, the accuracy of the new expression plasmid has been verified by DNA sequencing analysis. Then, the correct yeast plasmid expressing recombinant protein GST-hEGF by DNA sequencing analysis was transformed into Saccharomyces cerevisiae, and then the yeast cell was broken. The obtained protein was analyzed by Coomassie Blue staining and Western Blotting experiments, which confirmed that the recombinant protein GST-hEGF can indeed be efficiently expressed in Saccharomyces cerevisiae.
目錄------------------------------------------------------------------------------- 2
中文摘要----------------------------------------------------------------------------3
英文摘要----------------------------------------------------------------------------4
縮寫表------------------------------------------------------------------------------6
緒論--------------------------------------------------------------------------------7
研究背景----------------------------------------------------------------------------7
研究動機---------------------------------------------------------------------------18
實驗材料與方法----------------------------------------------------------------------20
實驗結果---------------------------------------------------------------------------30
討論-------------------------------------------------------------------------------37
參考文獻---------------------------------------------------------------------------39
圖表-------------------------------------------------------------------------------43
附錄-------------------------------------------------------------------------------56
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