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研究生:顏郡誼
研究生(外文):Yen, Chun-Yi
論文名稱:利用乳蛋白基因啟動子於豬乳腺上皮細胞中表現並分泌外源蛋白質
論文名稱(外文):Expression and secretion of exogenous proteins directed by the promoter of milk protein gene in porcine mammary epithelial cells
指導教授:林志生林志生引用關係
指導教授(外文):Lin, Chih-Sheng
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:98
中文關鍵詞:信號胜&信號胜&信號胜&信號胜&信號胜&信號胜&
外文關鍵詞:recombinant proteinmammary epithelial cellssignal peptide
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&;#63965;用 DNA 重組技術生產重組蛋白質已發展超過 30 &;#63886;之久。至今,超過百種醫藥用重組蛋白質已被美國食品與藥物管&;#63972;局(Food and Drug Administration, FDA)核准使用於醫&;#63937;上。早期用&;#63789;大&;#63870;生產重組蛋白質的生物宿主以微生物為主,&;#63925;如細菌或酵母菌,但使用微生物系統生產蛋白質常有蛋白質轉譯後修飾(post-modification)功能&;#63847;完全的問題,此可能影響蛋白質產物的穩定性與生物功能性。因此,目前用&;#63789;生產重組蛋白質之生物宿主,仍以哺乳&;#63952;動物細胞為主。&;#64022;乳腺上皮細胞 SI-PMEC 是一自&;#63848;乳&;#64022;乳腺組織所分&;#63978;並篩選出&;#63789;的細胞株,此細胞株已持續培養超過 8 個月(約 70 代)。SI-PMEC細胞生長在 Matrigel 上,並給予&;#63848;乳激素(prolactin),細胞會分化形成三維的&;#63952;乳腺網&;#63994;結構,並高&;#64001;表現乳蛋白基因。將帶有&;#63805;螢光蛋白與螢光&;#37238;表現載體轉殖至 SI-PMEC細胞中,可測得顯著地這&;#63864;個報導基因的表現;&;#63745;進一步,SI-PMEC 細胞已成功地被用
&;#63789;產生重組抗凝血水蛭素(hirudin)。
由於α-乳白蛋白和β-&;#63769;蛋白是哺乳動物乳中主要的蛋白質。因此,在我們的研究中,假設此&;#63864;種蛋白質基因的啟動子(promoter)在 SI-PMEC 細胞中有較強的表現能&;#63882;,因此&;#63965;用這&;#63864;個蛋白質基因的啟動子與其信號胜&;#32957;(signal peptide)序&;#63900;&;#63789;建構表現載體,製造出能分&;#63848;至細胞外的重組蛋白質,即報導螢光&;#37238;。本實驗結果顯示:(1) 轉殖有表現基因載體的 SI-PMEC 細胞,培養於第一型膠原蛋白與 Matrigel 基質上,細胞呈分化外貌&;#63994;,且可高&;#64001;表現報導蛋白質;(2) firefly 螢光&;#37238;一旦被分&;#63848;至 SI-PMEC 細胞外,其活性很快地衰減,因此&;#63847;適用於細胞分&;#63848;性質的報導蛋白質,反之 Gaussia 螢光&;#37238;被分&;#63848;至細胞外時,其活性穩定;(3) 沒有構築乳蛋白基因的信號胜&;#32957;序&;#63900;時,螢光&;#37238;在細胞內的表現&;#63870;較有信號胜&;#32957;序&;#63900;時為高;(4) 以 α-乳白蛋白和 β-&;#63769;蛋白的信號胜&;#32957;序&;#63900;置換Gaussia 螢光&;#37238;本身所帶有的信號胜&;#32957;序&;#63900;,其分&;#63848;螢光&;#37238;的效&;#63841;&;#64009;低;(5)
α-乳白蛋白基因啟動子在 SI-PMEC 細胞中有顯著較高的表現與分&;#63848;螢光&;#37238;的效&;#63841;;(6) 在&;#63745;換培養基處&;#63972;下,轉殖有表現載體的分化 SI-PMEC 細胞可持續的表現與分&;#63848;螢光&;#37238;。
本研究提供一個可生產與分&;#63848;重組蛋白質的乳腺細胞培養系統,儘管本系統完備之前還有很多條件須探討和最佳化,但此一系統用於生產醫用蛋白質可能具有產物純化程序簡單與因可&;#63898;續操作而產能高的優勢,值得後人&;#63851;考&;#63965;用之。
Using recombinant DNA technology to produce recombinant proteins have developed over 30 years. Until today, over hundred therapeutic recombinant proteins have been approved by Food and Drug Administration (FDA) and used in medical purposes. In the early days, the microorganisms, e.g., bacteria and yeast, were the major host cells to produce recombinant proteins. However, the major limitation of the microorganisms is possibly lack of post-translation modification that may affect the stability and activity of produced proteins. For the reason, the therapeutic recombinant proteins are mainly produced by mammalian cells so far. Spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) was isolated from mammary gland of a lactating Landrace pig and maintained for more than 8 months (70 passages). When SI-PMEC cells grew on Matrigel and stimulated by prolactin, the cells could differentiate and form three-dimensional mammary gland-like structure and strongly express the gene of milk proteins. The SI-PMEC cells transfected with the express vector encoding the enhanced green fluorescence protein (EGFP) and luciferase were approved they could highly express both reporter genes. Furthermore, the cells have been used to produce recombinant anticoagulant hirudin successfully.
Based on the knowledge of α-lactalbumin and β-casein is the major proteins in mammal milk, we suppose that the gene promoter of α-lactalbumin and β-casein can strongly express in mammary epithelial cells. Therefore, the sequences of promoter and signal peptide of both genes were constructed into expression vectors that were used to produce secreted recombinant proteins, firefly and Gaussia luciferase, in this study. Our results showed that: (1) the expression vectors transfected SI-PMEC cells cultured on the dish with type-I collagen or Matrigel as substratum could differentiate and strongly express the exogenous proteins; (2) while firefly luciferase was secreted to the culture medium, the luciferase activity decreased quickly; on the contrary, the activity of Gaussia luciferase secreted to the culture medium was stable; (3) the expressed luciferase of SI-PMEC cells transfected with a express vector without signal peptide sequence is higher than that with the signal peptide sequence of milk protein genes; (4) changing the native signal peptide of reporter Gaussia luciferase by the signal peptide sequences of α-lactalbumin and ??casein genes showed a reduced efficiency of produced luciferase secretion; (5) using the α-lactalbumin promoter to drive Gaussia luciferase expression, the culturing SI-PMEC cells had the highest expression of luciferase in the cultured medium; (6) the express vector transfected and differentiated SI-PMECs could continuously express and secrete the luciferase when treating the cultures with medium change.
Our studies provide a culture system of mammary epithelial cells to produce and secrete recombinant proteins. Even though there are many conditions that have to be discussed and optimized in this system but also many advantages that are worthy to utilize, such as collecting the secreted recombinant proteins easily, simplifying the procedure of product purification, and possibly a high productivity in a continuous culture system.
Acknowledgement………………………………………………………iii
Chinese Abstract .........................................v
English Abstract .......................................vii
Abbreviation .......................................... ix
Content ..................................................x
List of Figures........................................ xiii
List of Tables ..........................................xv
I. Literature review …………………………………………......1
1-1. Recombinant protein.................................. 1
1-1-1. Development of recombinant protein………………………1
1-1-2. Use microorganisms to express recombinant proteins…2
1-1-3. Use mammalian cells to express recombinant proteins.3
1-2. Mammary epithelial cells..............................4
1-2-1. Mammary gland………………………………………………….4
1-2-2. Mammary epithelial cells…………………………………..6
1-2-3. The effects of lactogenic hormones to mammary epithelial cells………... 7
1-3. Signal peptide........................................8
1-3-1. Concepts of protein secretion…………………………….8
1-3-2. Concepts of signal peptide……………………………….10
1-3-3. The applications of signal peptides……………………11
II. Research Purpose and Strategy……………………………….21
2-1. The porcine mammary epithelial cells as new host to produce recombinant protein...............................21
2-2. Using the promoters of α-lactalbumin and β-casein to drive recombinant proteins expression in PMECs………………21
2-3. Producing the secretory proteins by the signal peptide...................................................22
2-4. The research strategy and flowchat in this experiment22
III. Materials and Methods…………………………………………24
3-1. Cell culture.........................................24
3-2. Plasmid construction.................................24
3-2-1. pCMV-AL-EGFP……………………………………………….24
3-2-2. pCMV-BL-EGFP……………………………………………...25
3-2-3. pαLa-EGFP………………………………………………….25
3-2-4. pαLa-AL-EGFP....................................26
3-2-5. pCMV-Luc……………………………………............26
3-2-6. pCMV-AL-Luc……………………………...............26
3-2-7. pCMV-BL-Luc……………………………….............27
3-2-8. pαLa-Luc……………………………………............27
3-2-9. pαLa-AL-Luc……………………………….............27
3-2-10. pCMV-GLuc………………………………................28
3-2-11. pCMV-AL-Gluc……………………………...............28
3-2-12. pCMV-BL-Gluc……………………………...............28
3-2-13. pαLa-GLuc………………………………………………....29
3-2-14. pGB568-Gluc…………………………………………………29
3-3. Transfection of cells and selection for stable cell populations .............................................30
3-4. Confocal scanning microscope .......................30
3-5. Firefly Luciferase assay .......................... 31
3-6. Gaussia Luciferase assay............................31
IV. Results………………………………………………………....33
4-1. Plasmid construction................................33
4-1-1. pCMV-AL-EGFP……………………………………………..33
4-1-2. pCMV-BL-EGFP……………………………………………..35
4-1-3. pαLa-EGFP………………………………………………….37
4-1-4. pαLa-AL-EGFP....................................39
4-1-5. pCMV-Luc…………………………………………........41
4-1-6. pCMV-AL-Luc……………………………………………….43
4-1-7. pCMV-BL-Luc.....................................45
4-1-8. pαLa-Luc…………………………………………………..47
4-1-9. pαLa-AL-Luc……………………………………………….49
4-1-10. pCMV-GLuc…………………………………………………..51
4-1-11. pCMV-AL-Gluc……………………………………………….53
4-1-12. pCMV-BL-Gluc……………………………………………….55
4-1-13. pαLa-GLuc…………………………………………………..57
4-1-14. pGB568-Gluc…………………………………………………59
4-2. Enhanced green fluorescence protein expression in PMECs cultured on Petri dish………….....................61
4-3. Enhanced green fluorecence protein expression in PMECs cultured on Matrigel . .................................63
4-4. Enhanced green fluorecence protein expression driven by CMV or α-lactalbumin promoter in PMECs ...............66
4-5. Signal peptides affect the intracellular amount of luciferase in PMECs .....................................68
4-6. The luciferase expression driven by α-lactalbumin promoter and cultured on the type-I collagen gel of different concentrations ................................70
4-7. Firefly luciferase activity decreased quickly in cultured medium………………..............................72
4-8. Gaussia luciferase expressed stably and continuously in cultured medium.......................................74
4-9. Alteration the signal peptide and comparison the Gaussia luciferase expression in the cultured medium.....76
4-10. Gaussia luciferase expression on different substrata in stably transfected PMECs ............................78
4-11. Gaussia luciferase measurements in PMECs/pCMV-GLuc with different cell number on Matrigel..................80
4-12. Gaussia luciferase expression and secretion continuously in cultured medium.........................82
V. Discussion……………………………………….............86
5-1. The signal peptides derived from mammalian proteins might not be the most efficient to transport proteins in mammalian cell..........................................86
5-2. The modulation of signal peptide might affect the secretory efficiency....................................86
5-3. Prolactin affects the gene expression of milk proteins in PMECs................................................88
5-4. The different substrata affect the cells differentiation and protein production…................88
VI. Conclusions …………………………………………………. 90
VII. References ……………………………………………………92
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