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研究生:張繼中
研究生(外文):Ji-Chung Chang
論文名稱:轉殖水稻生產豬乳鐵蛋白之研究:同質品系篩選、蛋白質純化及生物活性分析
論文名稱(外文):Production of porcine lactoferrin in transgenic rice: Selection of homozygous lines, protein purification and biological activity assay
指導教授:陳良築
指導教授(外文):Liang-Jwu Chen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:69
中文關鍵詞:乳鐵蛋白純化同質
外文關鍵詞:porcinelactoferrinpurificationhomozygous
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乳鐵蛋白為哺乳動物乳汁中的一種多功能的醣蛋白,其具有結合鐵離子、抑菌、增進免疫力和消除自由基的能力。本研究以轉入豬乳鐵蛋白基因之水稻為材料進行分析。由於轉入豬乳鐵蛋白基因之轉殖水稻第一代不是同質品系,基因仍會分離;因此,本研究利用PCR和西方墨點法來篩選轉殖水稻,以獲得轉殖水稻同質品系。目前在水稻胚特有基因起動子-721LF轉殖水稻獲得10個轉殖水稻同質品系,而利用CaMV35S起動子之35LF轉殖水稻獲得2 個轉殖水稻同質品系,其中721LF轉殖水稻已栽培至第六代,而35LF轉殖水稻則栽培至第五代,南方墨漬法分析發現這些同質品系的豬乳鐵蛋白基因分別嵌入水稻染色體的不同位置。由西方墨漬法的結果顯示不同起動子之轉殖水稻所表現的豬乳鐵蛋白含量的確有差異,而來自不同世代之相同轉殖水稻所表現的豬乳鐵蛋白含量則沒有差異。由研究結果初步推測在721LF轉殖水稻糙米、米糠中之豬乳鐵蛋白佔總蛋白質之百分比分別為0.04%、0.06%,而在35LF轉殖水稻之糙米、米糠、精白米中之豬乳鐵蛋白佔總蛋白質之百分比分別為0.07%、0.04%、0.06%。利用組織免疫墨漬法(Tissue immunoblots) 來分析在不同起動子所起動的豬乳鐵蛋白在水稻種子組織的位置,結果顯示在705LF、721LF、731LF轉殖水稻主要表現在種子胚和糊粉層之中,而在持續表現起動子轉殖水稻種子中則整粒都有表現。利用Heparin-sepharose純化轉殖水稻同質品系中之豬乳鐵蛋白,結果發現豬乳鐵蛋白會在0.6 M NaCl和0.8 M NaCl淋洗出來,同時亦發現由水稻所表現的豬乳鐵蛋白能和鐵離子結合及抑制大腸桿菌DH5α生長的功能。此外,本研究的結果顯示轉殖水稻的農藝性狀不會因為有外來的基因嵌入而有明顯的改變。
Lactoferrin is a multifunctional glycoprotein, found mostly in milk and involved in iron absorption, antibacterial activity, immune response and free-radical scavenging. The analysis of lactoferrin gene expression in transgenic rice was performed in this study. Because the porcine lactoferrin gene was segregated in the first generation (T1), the homozygous lines were screened with the medium containing hygromycin, and further detected by PCR and Western blot assay. In the present, we obtained ten homozygous lines of Ose721 promoter and two of CaMV35S promoter. The homozugous line of Ose721 promoter was cultivated to the sixth (T6) generation, whereas the homozygous line of CaMV35S promoter was cultivated to fifth (T5) generation. The amount of porcine lactoferrin detected in brown rice and bran of transgenic rice of Ose721 promoter were 0.04% and 0.06% of total proteins, whereas the amount in brown rice, bran and milled rice of transgenic rice of CaMV35S promoter were 0.07%, 0.04% and 0.06% of total proteins respectively. Results of tissue immunoblots showed that porcine lactoferrin gene driven by Ose705, Ose721and Ose731 promoters, porcine lactoferrin was detected in embryo and aleurone cells, whereas the gene driven by actin and CaMV35S promoters, porcine lactoferrin was observed in whole seeds. Porcine lactoferrin purified by heparin-sepharose column from transgenic homozygous line was eluted with 0.6M and 0.8M NaCl, while the iron absorption and the growth inhibition of E. Coli-DH5α was observed in this study. Besides, the alternations of agronomic trait of transgenic rice were not significant due to the insertion of foreign gene.
ABSTRACT 1
中文摘要 2
壹、前言 3
貳、前人研究 5
一、利用植物生產外源蛋白之研究現況 5
1. 起動子之選擇 6
2. 外源蛋白之表現 8
3. 轉殖植物的同質品系篩選 9
二、關於乳鐵蛋白的研究 11
三、轉殖植物表現乳鐵蛋白的研究 13
參、材料與方法 15
(一)材料 15
(二)方法 15
1. 篩選轉殖水稻同質品系 15
2. 少量質體DNA之萃取 16
3. DNA 片段之純化回收 16
4. 水稻total DNA之抽取 18
5. 轉殖植物之確認 18
6. 雜交探針之製備 19
7. 南方墨點分析 19
8. 自動放射顯影 20
9. 利用不同蛋白萃取液分析水稻中的蛋白質 20
10. 蛋白質膠體電泳分析 21
11. 西方墨點分析 21
12. 豬乳鐵蛋白於轉殖水稻中之表現分析 22
13. 轉殖水稻種子中豬乳鐵蛋白的含量 22
14. 豬乳鐵蛋白在轉殖水稻組織中之位置 23
15. 以Heparin-sepharose管柱純化豬乳鐵蛋白 23
16. 抑菌活性分析 25
17. 鐵離子結合分析 25
18. 轉殖水稻的農藝性狀調查 25
肆、結果 26
一、 同質轉殖水稻品系之篩選 26
1. 利用含抗生素的培養基篩選 26
2. 聚合酵素鏈鎖反應分析 27
3. 同質轉殖水稻品系之確認分析:西方墨點法 27
4. 同質轉殖水稻品系之南方墨點分析 27
二、 轉殖水稻生產豬乳鐵蛋白之純化 28
1. 不同蛋白質萃取液之應用 28
2. 利用硫銨沈澱法部分純化豬乳鐵蛋白 29
3. 利用heparin-sepharose 管柱純化豬乳鐵蛋白 29
三、轉殖水稻種子中豬乳鐵蛋白之表現 29
1. 水稻種子之糙米、米糠及精白米之蛋白質種類之分布 30
2. 豬乳鐵蛋白在721LF轉殖水稻種子之含量百分比 30
3. 豬乳鐵蛋白在35LF轉殖水稻種子之含量百分比 30
4. 不同轉殖水稻植株之豬乳鐵蛋白表現 31
5. 轉殖水稻不同世代之豬乳鐵蛋白表現 31
6. 利用組織免疫墨漬法分析豬乳鐵蛋白在轉殖水稻種子中的分布 32
四、 豬乳鐵蛋白之活性分析 32
1. 豬乳鐵蛋白與鐵離子結合分析 32
2. 抑菌活性分析 33
五、轉殖水稻之農藝性狀調查 33
伍、討論 35
一、轉殖水稻同質品系之篩選 35
二、水稻生產豬乳鐵蛋白之純化 36
三、轉殖水稻種子中豬乳鐵蛋白之表現 37
四、豬乳鐵蛋白之活性分析 40
1. 豬乳鐵蛋白與鐵離子結合分析 40
2. 抑菌活性分析 40
五、轉殖水稻之農藝性狀調查 41
陸、參考文獻 42
圖表 49
陸、參考文獻
王簾讀 (1997) 水稻胚油體膜蛋白基因與似LEA蛋白基因之分離與分析,中興大學分子生物學研究所碩士論文。
李寛志 (2000) 轉基因水稻生產D-hydantoinase之研究,中興大學分子生物學研究所碩士論文。
涂倉榮 (2000) 水稻胚Ose705及Ose719基因起動子之分析,中興大學分子生物學研究所碩士論文。
陳鵬文 (1997) 水稻胚發育時期特有表現基因之分離與分析,中興大學植物學研究所博士論文。
陳瑞斌 (2001) 豬乳鐵蛋白基因在轉殖水稻表現之研究,中興大學分子生物學研究所碩士論文。
蔡靜宜 (1999) 以水稻轉殖系統表現豬乳鐵蛋白基因之研究,中興大學分子生物學研究所碩士論文。
羅慧如 (1998) 水稻胚發育時特有表現基因Ose731之選殖與分析及Ose705 Ose712、Ose730之原位雜交分析,中興大學分子生物學研究所碩士論文。
Allen, G. C., Hall, G., Michalowski, S., Newman, W., Spiker, S., Weissinger, A. K. and Thompson, W. F. (1996) High-level transgene expression in plants cells: effects of a strong scaffold assachment region from tobacco . Plant Cell. 8:899-913.
Allen, G. C., Spiker, S. and Thompson, W. F. (2000) Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Mol. Biol. 43:361-376.
Aguilera, O., Ostolaza, H., Quiros, L. H. and Fierro, J. F. (1999) Permeabilizing action of antimircrobial lactoferrin-derived peptide on bacterial and artificial membranes. FEBS Lett. 462:273-277.
Arakawa, T., Chong, DK., Slattery, CW., Langridge, WH. (1999) Improvements in human health through production of human milk proteins in transgenic food plants. Adv Exp Med Biol. 464:149-159.
Brock J. (1995) Lactoferrin: a multifunctional immunoregulatory protein? Immunol. Today 16(9):417-419.
Chen, P. W., Fang, L. W., Lin, J. W., Tsay, H. S., Wu, H. K. and Chen, L. J. (1997) Isolation of clones for genes that are specifically expressed in the rice embryo. Bot. Bull. Acad. Sin. 38:13-20.
Cheng, X., Sardana, R., Kaplan, H. and Altosaar, I. (1998) Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer. Proc. Natl. Acad. Sci. USA 95:2767-2772.
Chong, D. K.X. and Langridge, W. H. R. (2000) Expression of full-length bioactive antimicrobial human lactoferrin in potato plants.Transgenic Res. 9:71-78.
Chu, M. R., Wang S. R., Weng C. N. and Pursel V. G. (1993) Isolation and characterization of porcine milk lactoferrin. Am. J. Vet. Res. 54(7):1154-1159.
Darkakaki, G., Christou, P. and Stoger, E. (2000) Constitutive expression of soybean ferritin cDNA in transgenic wheat and rice results in increased iron levels in vegetative tissues but not in seeds. Transgenic res. 9:445-452.
DeZoeten, GA., Penswick, JR., Horisberger, MA., Ahl, P., Schultze, M. and Hohn, T. (1989) The expression, localization, and effect of a human interferon in plants. Virology. 172(1):213-222.
Fischer, R., Drossard, J., Commandeur, U., Schillberg, S., Emans, N. (1999) Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants. Biotechol Appl Biochem. 30(Pr2): 101-8.
Fleet, J. C. (1995) A New Role for Lactoferrin:DNA Binding and Transcription Activation. Nutrition Reviews 53(8):226-231.
Gaikwad, A., Tewari, K. K., Kumar D., Chen W., and Mukherjee, S. K.(1999) Isolation and characterisation of the cDNA encoding a glycosylated accessory protein of pea chloroplast DNA polymerase. Nucleic Acids Res. 27(15):3120-3129.
Goddijn, O. J. M. and Pen, J. (1995) Plant as bioreactors. Trends Biotechnol. 13:379-387.
Goto, F., Yoshihara, T., Shigemoto, N., Toki, S. and Takaiwa, F. (1999) Iron fortification of rice seed by the soybean ferritin gene. Natrue Biotech. 17:282-286.
Groenink, J., Walgreen-Weterings, E., Hof, W. V., Veerman, E. C. I. and Amerongen, A. V. N. (1999) Cationic amphipathic peptides , derived from bovine and human lactoferrins ,with antimicrobial activity against oral pathogens. FEMS Microbiol. Lett. 179:217-222.
Hashizume, S., Kuroda, K., and Murakami, H. (1983) Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium. Biochim. Biophys.Acta. 763:377-382.
Houdebine, LM. Transgenic animal bioreactors. (2000) Transgenic Res. 9(4-5):301-304.
Ikeda, M., Nozaki A., Sugiyama, K., Tanaka, T., Naganuma, A., Tanaka, K., Sekihara, H., Shimotohno, K., Saito, M. and Kato, N. (2000) Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells. Virus Res. 66:51-63.
Iyer, S. and Lonnerdal, B. (1993) Lactoferrin, lactoferrin receptors and iron metabolism. Eur. J. Clin. Nutr. 47:232-241.
Janne, J., Hyttinen, JM., Peura, T., Tolvanen, M., Alhonen, L., Sinervirta, R., Halmekyto M. (1994) Transgenic bioreactors. Int J Biochem. 26(7):859-70.
Janne, J., Alhonen, L., Hyttinen, JM., Peura, T., Tolvanen, M., Korhonen, VP. (1994) Transgenic bioreactors. Biotechol Annu Rev. 4:55-74.
Jin, Y. Z., Bannai, S., Dacheux, F., Dacheux, J. L. and Okamura, N. (1997) Direct evidence for the secretion of lactoferrin and its binding to sperm in the porcine epididymis. Mol. Reprod. Dev. 47:490-496.
Kalla, R., Shimamoto, K., Potter, R., Nielsen, P. S., Linnestad, C. and Olsen, O. A. (1994) The promoter of the barley aleurone-specific gene encoding a putative 7 kDa lipid transfer protein confers aleurone cell-specific expression in transgenic rice. Plant J. 6(6): 849-860.
Kamachi, K., Yamaya, T., Hayakawa, T., Mae, T. and Ojima, K. (1992) Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves. Plant Physiol. 99:1481-1486.
Levay, P. F., and Viljoen, M. (1995) Lactoferrin: a general review. Haematologica 80:252-267.
Mitra, A., and Zhang, Z. (1994) Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial protein(s). Plant Physiol. 106:977-981.
Muttler, I. J. (1987) A simple and rapid method for minipreparation of DNA from tissue cultured plant cells. Plant Mol. Biol. Reporter 5:346-349.
Normile, D. (2000) Agricultural biotechnology. Monsanto donates its share of golden rice. Science. 289:843-845.
Peeters, K., De Wilde, C., De Jaeger, G., Angenon, G., and Depicker, A. (2001) Production of antibodies and antibody fragmints in plants. Vaccine. 19(17-19):2756-2761.
Prakash J. (1996) Rice bran proteins: properties and food uses. Critical Reviews in Food Sci .and Nutr. 36(6):537-552.
Rao, K. V., Rathore, K. S., Hodges, T. K., Fu, X., Stoger, E., Sudhakar, D., Williams, S., Christou, P., Bharathi, M., Bown, D. P., Powell, K. S., Spence, J., Gatehouse, A. M. R. and Gatehouse J. A. (1998) Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper. Plant J. 15(4):469-477.
Salmon, V., Legrand, D., Slomianny, M., Yazidi, I. E., Spik, G., Gruber, V., Bourant, P., Olagnier, B., Mison, D., Theisen, M. and Merot, B. (1998) Production of human lactoferrin in transgenic tobacco. Plants. Protein Express Purif. 13:127-135.
Schibli, D. J., Hwang, P. M. and Vogel, H. J. (1999) The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles. FEBS Lett. 446:213-217.
Sorrentino, S., D’Alessandro, A. M., Maras, B., Di Ciccio, L., D’Andrea, G., De Prisco, R., Bossa, F., Libonati, M. and Oratore, A. (1999) Purification of a 76-kDa iron —binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin. Biochimica. et. Biophysica. Acta. 1430:103-110.
Spik, G., Cheron, A., Montreuil, J. and Dolby, J. (1978) Bacteriostasis of a milk-sensitive strain of Escherichia coli by immunoglobulins and iron-binding protein in association. Immunology 35:663-671.
Stoger, E., Vaquero, C., Torres, E., Sack, M., Nicholson, L., Drossard, J., Williams, S., Keen, D., Perrin, Y., Christou, P., and Fischer, R. (2000) Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol. 42(4):583-590.
Thanavala, Y., Yang, Y., Lyons, P., Mason, HS. and Arntzen, C. (1995) Immunogenicity of transgenic plant-derived Hepatitis B surface antigen. Proc Natl Acad Sci USA. 92:3358-3361.
Torres, E., Vaquero, C., Nicholson, L., Sack, M., Stoger, E., Drossard, J., Christou, P., Fischer, R., and Perrin, Y. (1999) Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic Res. 8(6):441-449.
Tuccari, G., Giuffre, G., Crisafulli, C., and Barresi G.(1999) Immunohistochemical detection of lactoferrin in human astrocytomas and multiforme glioblastomas. Eur. J. Histochem. 43(4):317-322.
Udaka, J., Koga, T., Tsuji, H., Kimoto, M. and Takumi, K. (2000) Efficient extraction and some properties of storage proteins (prolamin and glutelin) in ancient rice cultivars. J. Nutr. Sci. Vitaminol.46:84-90.
Van Berkel, P. H. C., Geerts, M. E. J., Van Veen, H. A., Kooiman, P. M., Pieper, F. R., De Boer, H. A. and Nuijens, J. H. (1995) Glycosylated and unglycosylated human lactoferrin both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis. Biochem. J. 312(1):107-114.
Verpoorte, R., Heijden, R. V. D. and Memelink, J. (2000) Engineering the plant cell factory for secondary metabolite production. Transgenic res. 9:323-343.
Weinberg, E. D. (1993) The development of awareness of iron-withholding defense. Perspect. Bio. Med. 36:215-221.
Wilde, C. D., Houdt, H. V., Buck, S. D., Angenon, G., Jaeger, G. D. and Depicker, A. (2000) Plants as bioreactors for protein production: avoiding the problem of transgene silencing. Plant Mol. Biol. 43:347-359.
Wilmink, A., Ven, B. C. E. V. D. and Dons, J. J. M. (1995) Activity of constitutive promoters in various species from liliaceae. Plant Mol. Biol. 28:949-955.
Ye, X., Al-Babili, S., Kioti, A., Zhang, J., Lucca, P., Beyer, P. and Potrykus, I. (2000) Engineering the provitamin A(beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science. 287:303-305.
Ye, X. Y., Wang, H. X., Liu, F. and Ng, T. B. (2000) Ribonuclease, cell-free translation—inhibitory and superoxide radical scavenging activities of the iron —binding protein lactoferrin from bovine milk. J. Biochem. Cell. Biol. 32:235-241.
Ye, Z. H. and Varner J. E. (1991) Tissue-specific expression of cell wall proteins in developing soybean tissues. Plant Cell. 3:23-37.
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1. 6.林資敏, 2000. “從商品概念出發掌握虛擬商機”. 商業現代化37期.
2. 9.吳政達, 1996. “3D/VR在網路之運用與探討”:虛擬購物中心雛形建置. 光碟月刊, pp39-42.
3. 14.程治, 1996. “VRML─在WWW上製作虛擬實境的應用”. 電腦與通訊46期, pp23-32.
4. 18.鄭文俊, 1999. “資訊產品螢幕圖像操作介面設計思索與實務”. 大葉學報, 第八卷, 第一期, pp111-12.
5. 李雅玲、陳月枝、林國信(1993).癌症兒童家庭主要照顧者對支持系統的感受.護理雜誌季刊,40(3),59-69。
6. 林麗嬋、歐美、吳肖琪(1997).長期照顧中主要照顧者之家庭功能、社會支持與情緒.護理研究,5(1),77-86。
7. 高迪理(民80).社會支持體系概念之架構之探討.社區發展季刊,54,
8. 高淑芳、盧孳豔、葉淑惠、劉雪娥(1999).探討家庭功能、社會支持與社區殘病老人照顧者負荷之關係.護理研究,7(2),172-181。
9. 陳金彌、毛新春、陳月枝、黃棣棟(2000).母親面對惡性腦瘤病童接受癌症療程的經驗.護理研究,8(3),338-347。
10. 陳秀勤、王秀紅(1997).血液腫瘤住院患者的希望、社會支持與人口學特性之相關性探討.護理研究,5(6),487-497。
11. 許淑蓮、黃秀梨(1986).Mishel疾病不確定感量表之中文版測試.護理研究,4(1),59-68。
12. 葉美玲、陳興夏(1998).測量工具之翻譯與效度.長庚護理,9(1),64-68。
13. 穆佩芬、馬鳳岐、顧小明、許洪坤、黃碧桃(2000).有罹患癌病兒童之家庭壓力:對母親之衝擊.護理研究,8(5),568-577。
14. 盧成皆、李瑛慈(1995).醫學研究常見之統計分析謬誤.中國統計學報,33(2),275-285。
 
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