跳到主要內容

臺灣博碩士論文加值系統

(44.200.171.74) 您好!臺灣時間:2022/08/12 19:57
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:黃建豪
研究生(外文):Chain-Hao Huang
論文名稱:基因轉殖植物生產人類血清蛋白
論文名稱(外文):Producing human serum albumin in transgenic plants
指導教授:林國知
指導教授(外文):Kuo-Chih Lin
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
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:86
中文關鍵詞:人類血清蛋白基因轉殖植物阿拉伯芥
外文關鍵詞:human serum albumintransgnic plantsarabidopsis
相關次數:
  • 被引用被引用:3
  • 點閱點閱:538
  • 評分評分:
  • 下載下載:96
  • 收藏至我的研究室書目清單書目收藏:1
中文摘要
本研究的目的係將人類血清蛋白(human serum albumin, HSA)轉殖於阿拉伯芥中表現。近二十年來,由於生物技術及基因轉殖技術的蓬勃發展,使許多科學家將具有醫療用途的蛋白質表現於植物體中。而從植物生產醫藥用品不僅生產成本相對地較低,最重要的可減少使用從人體或動物細胞萃取醫藥用品受到未知病毒污染的潛在危險性。吾人為了使人類血清蛋白能夠表現於阿拉伯芥細胞內、細胞外,及可為人體腸道細胞吸收,分別構築三種表現載體:pHSA-1、pHSA-2及pHSA-3。pHSA-1含prepro HSA基因;pHSA-2則含成熟之HSA,並在其N-端接上一段signal peptide以利分泌於胞外;pHSA-3除了成熟之HSA外,亦於N-端接上霍亂毒素B次單元(CTB)及signal peptide,而所接上之CTB可幫助HSA於腸道細胞之吸收。我們分別將pHSA-1、pHSA-2及pHSA-3轉形入農桿菌品系LBA4404,然後將LBA4404 (pHSA-1)、LBA4404 (pHSA-2)及LBA4404 (pHSA-3)轉殖入阿拉伯芥中。我們共得pHSA-1轉殖種子1669顆、pHSA-2轉殖種子2268顆及pHSA-3轉殖種子3124顆。經種植於含hygromycin 15mg/l的1/2 MS培養基後,篩選三週後轉殖HSA-1植株僅70棵存活,轉殖HSA-2植株僅59棵存活,轉殖HSA-3植株僅126棵存活,我們將這些生長情況較佳的植株轉移到不含hygromycin的1/2 MS培養基中培養兩週後,再由培養基中移出種植於培植土中繼續長大。吾人選取生長情況良好的基因轉殖株共45棵;其中轉殖HSA-1有6棵,轉殖HSA-2有2棵,轉殖HSA-3有37棵。抽取其genomic DNA,並以PCR增殖與南方吸漬法進行偵測。結果顯示PCR增殖並未觀察到有HSA DNA片段被增殖出,而南方吸漬法也未找到具有轉殖HSA基因的植株。
Abstract
The goal of this study is to produce human serum albumin (HSA) in Arabidopsis. For the past two decades, many scientists used biotechnology to produce biopharmaceutical proteins in transgenic plants. Using plants as a factory for producing biopharmaceutical proteins may avoid pollution of unknown viruses concomitance with the extraction of proteins from human or animals by traditional extraction methods. In addition, the cost for producing biopharmaceutical proteins in crops on an agricultural scale may be less than that by traditional methods. To make an edible HSA, we constructed the following vector: pHSA-1, pHSA-2, pHSA-3. The pHSA-1 contains the preproHSA gene in pCAMBIA1302, a disarmed Ti vector. The pHSA-2 contains a signal peptide on the N-terminus of the mature HSA. The signal peptide comes from the gene encoding thaumatin-like proteins, a pathogenesis-related protein from oats. The pHSA-3 contains a cholera toxin B subunit (CTB) on the N-terminus of the mature HSA. These three clones were transformed into Agrobacterium strain LBA4404. We then introduced the different clones of HSA gene into Arabidopsis by Agrobacterium-mediated transformation. A total of 1669, 2268, and 3124 transgenic seeds were obtained from Arabidopsis transformed with LBA4404 (pHSA-1), LBA4404 (pHSA-2), and LBA4404 (pHSA-3), respectively. After grown in medium containing hygromycin 15 mg/l for 3 weeks, 70, 59, and 126 plants transformed with HSA-1, HSA-2, and HSA-3, respectively, were survived from selection. These plants were then cultured in MS medium without hygromycin for another 2 weeks. The plants were then transferred to pots and incubated in growth chambers till the plants grew up. We chose 45 putative transgenic plants for PCR and southern blot analyses. Of these 45 plants, 6, 2, and 37 plants come from plants transformed with HSA-1, HSA-2, and HSA-3, respectively. However, no signal was detected by PCR nor southern blot analyses.
目錄
中文摘要.......................I
Abstract......................II
目錄..........................IV
一、研究背景及目的.............1
二、研究材料與方法............12
三、結果......................24
四、討論......................28
五、參考文獻..................31
圖表..........................37
附錄封面......................53
附錄中文摘要..................54
附錄Abstract..................55
一、附錄研究背景及目的........56
二、附錄研究材料與方法........61
三、附錄結果與討論............69
四、附錄參考文獻..............72
附錄圖表......................74
朱光文. 1998. 生技藥品專題研究. 生物技術開發中心 P92.
Arakawa, T., Chong, D. K. X.,and Langridge, W. H. R. 1998a. Toxin B subunit vaccine. Nat. Biotech. 16:292-297.
Arakawa, T., Yu, J., Chong, D. K. X., Hough, J., Engen, P. C., and Langridge, W. H. R. l998b. A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nature Biotech. 16:934-938.
Arntzen, C. J. 1995. Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268:714-716.
Beardsley, T. 1986. Genetic engineering. Hepatitis vaccine wins approval. Nature 322:396.
Bechtold, N., and Pelletier, G. 1998. In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82:259-266.
Bent, A. F. 2000. Arabidopsis in planta transformation. Uses, mechanisms, and prospects for transformation of other species. Plant Physiol. 124:1540-1547.
Chargelegue, D., Vine, N. D., van Dilleweerd, C. J., Drake, P. M., and Ma, J. K. 2000. A murine monoclonal antibody produced in transgenic plants with plant-specific glycans is not immunogenic in mice. Trans. Res. 9:187-194.
Clough, S. J., and Bent, A. F. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16:735-743.
Conrad, U., Fiedler, U., Artsaenko, O., and Phillips, J. 1998. High-level and stable accumulation of single-chain Fv antibodies in plant storage organs. J. Plant Physiol. 152:708-711.
Cuatrecasas, P. 1973. Gangliosides and membrane receptors for cholera toxin. Biochemistry 12:3558-3566.
Curtiss, R. I., and Cardineau, C. A. 1990. Oral immunisation by transgenic plants. World Patent Application WO 90/02484.
Desfeux, C., Clough, S. J., and Bent, A. F. 2000. Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip. Method Plant Physiol. 123:895-904.
FDA 1982. Human insulin receives FDA approval. FDA Drug Bull. 12:18-19.
FDA 1985. Genetically engineered human growth hormone approved. FDA Drug Bull. 15:38-39.
FDA 1986. Products of new biotechnologies. FDA Drug Bull. 16:19.
FDA 1989. FDA approves Eprex for anemia related to AIDS. Narse Pract. 14:6.
FDA 1991. Marketing of recombinant granulocyte-macrophage colony-stimulating factor approved by FDA. Clin. Pharm. 10:411.
FDA 1992. FDA approves release of recombinant interlukin-2 product. Clin. Pharm. 11:669-670.
Fischer, R., and Emans, N. 2000. Molecular farming of pharmaceutical proteins. Trans. Res. 9:279-299.
Flaharty, K. K., Grimm, A. M., and Vlasses, P. H. 1989. Epoetin: human recombinant erythropoietin. Clin. Pharm. 8:769-782
Furman, W. L., and Crist, W. M. 1991. Potential uses of recombinant human granulocyte-macrophage colony-stimulating factor in children. Am. J. Pediatr Hematol. Oncol. 13:388-399.
Garlick, R. L., and Mazer, J. S. 1983. The principal site of nonenzymatic glycosylation of human serum albumin in vivo. J. Biol. Chem. 258:6142-6146.
Giddings, G. 2001. Transgenic plants as protein factories. Curr. Opin. in Biotechnol. 12:450-454.
Giddings, G., Allison, G., Brooks, D., and Carter, C. 2000. Transgenic plants as factories for biopharmaceuticals. Nat. Biotechnol. 18:1151-1155.
Giroux, R. W., Bushnell, W. R., Nelson, A. J., and Lin, K. C. 1996. A transient expression assay for evaluating the effectiveness of introduced host response genes for disease resistance in cereals. Plant Physiol. 111:41.
Goeddel, D. V., Kleid, D. G., Bolivar, F., Heyneker, H. L., Yansura, D. G., Crea, R., Hirose, T., Kraszewski, A., Itakura, K., and Riggs, A. D. 1979. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc. Natl. Acad. Sci. USA 76:106-110.
Gomperts, E., Lundblad, R., and Adamson, R. 1992. The manufacturing process of recombinant factor VIII, recombinate. Transfus. Med. Rev. 6:247-251.
Hiatt, A. 1990. Antibodies produced in plants. Nature 344:469-470.
Hiatt, A., Cafferkey, R., and Bowdish, K. 1989. Production of antibodies in transgenic plants. Nature 342:76-78.
Higo, K., Saito, Y., and Higo, H. 1993. Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35S promoter in transgenic tobacco. Biosci. Biotechnol. Biochem. 57:1477-1481.
Hodgson, J. 1993. Expression system: a user’s guide. Bio/Technology 11:887-893.
Judah, J. D., Gamble, M., and Steadman, J. H. 1973. Biosynthesis of serum albumin in rat liver, evidence for the existence of ‘proalbumin’. Biochem. J. 134:1083-1091.
Kapusta, H., Modelska, A., Figlerowicz, M., Pniewski, T., Letellier, M., Lisowa, O., Yusibov, V., Koprowski, H., Plucienniczak, A., and Legocki, A. B. 1999. A plant-derived edible vaccine against hepatitis B virus. FASEB J. 13:1796-1799.
Kusnadi, A. R. Nikolov, Z. L., and Howard, J. A. 1997. Production of recombinat proteins on transgenic plants: practical consideration. Biotechnoil. Bioeng. 56:473-484.
Langridge, W. H. R. 2000. Edible vaccines. Sci. Am. 283:48-53.
Larrick, J. W., and Thomas, D. W. 2001. Producing proteins in transgenic plants and animals. Curr. Opin. in Biotechnol. 12:411-418.
Lawn, R. M., Adelman, J., Bock, S. C., Franke, A. F., Houck, C. M., Najarian, C., Seeburg, P. H., and Wion, K. L. 1981. The sequence of human serum albumin cDNA and its expression in E. coli. Nucleic Acid Res. 9:6103-6114.
Leite, A., Kemper, E. L., da Silva, M. J. Luchessi, A. D., Siloto, R. M. P., Bonaccorsi, E. D., El-Dorry, H. F., and Arruda, P. 2000. Expression of correctly processed human growth hormone in seeds of transgenic tobacco plants. Mol. Breed. 6:47-53.
Lin, K. C., Bushnell, W. R., Szabo, L. J., and Smith, A. G. 1996. Isolation and expresssion of a host response gene family encoding thaumatin-like proteins in incompatible oat-stem rust fungus interactions. Mol. Plant-Mcrobe Interact. 9:511-522.
Ma, J. K. C. 2000. Genes, greens and vaccines. Nat. Biotechnol. 18:1141-1142.
Ma, J. K. C., Hikmat, B. Y., Wycoff, K., Vine, N. D., Chargelegue, D., Yu, L., Hein, M. B., and Lehner, T. 1998. Characterization of a recombinate plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat. Med. 4:601-606.
Ma, J. K. -C., Lehner, T., Stabila, P., Fux, C. I., and Hiatt, A. 1994. Assembly of monoclonal anibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur. J. Immunol. 236:275-292.
Magnuson, N. S., Linzmaier, P. M., Reeves, R., An, G., HayGlass, K., and Lee, J. M. 1998. Secretion of biologically active human interleukin-2 and interleukin-4 from genetically modified tobacco cells in suspension culture. Protein Expr. Purif. 13:45-52.
Mason, H. S., Lam, D. M-K., and Arntzen, C. J. 1992. Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA 89:11745-11749.
Matsumoto, S., Ikura, K., Ueda, M., and Sasaki, R. 1995. Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Mol. Biol. 1995 27:1163-1172.
Matsumoto, S., Ishii, A., Ikura, K., Ueda, M., and Sasaki, R. 1993. Expression of human erythropoietin in cultured tobacco cells. Biosci. Biotechnol. Biochem. 57:1249-1252.
Mittelsten, S. O., Afsar, K., and Paszkowski, J. 1998. Release of epigenetic gene silencing by trans-acting mutations in Arabidopsis. Proc. Natl. Acad. Sci. USA. 95:632-637.
Moshage, H. J., Janssen, J. A. M., Franssen, J. H., Hafkenscheid, J. C., and Yap, S. H. 1987. Study of the molecular mechanism of decreased liver synthesis of albumin in inflammation. J. Clin. lnvest. 79:1635-1641.
Owen, M., Gandecha, A., Cockburn, B., and Whitelam, G. 1992. Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco. Bio/Technology 10:790-794.
Parmenter, D. L., Boothe, J. G., van Rooijen, G. J., Yeung, E. C., and Moloney, M. M. 1995. Production of biologically active hirudin in plant seeds using oleosin partitioning. Plant Mol. Biol. 29:1167-1180.
Ramirez, N., Ayala, M., Lorenzo, D., Palenzuela, D., Herrera, L., Doreste, V., Perez, M., Gavilond, J. V., and Oramas, P. 2002. Expression of a single-chain Fv antibody fragment specific for the hepatitis B surface antigen in transgenic tobacco plants. Transgenic Res. 11:61-64.
Rothschild, M. A., Oratz, M., and Schreiber, S. S. 1988. Serum albumin. Hepatology 8:385-401.
Safai, B., Bason, M., Friedman-Birnbaum, R., and Nisce, L. 1990. Interferon in the treatment of AIDS-associated Kaposi's sarcoma: the American experience. J. Invest. Dermatol. 95:166S-169S.
Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular cloning. Alaboratory manual. Cold Spring Harbor Laboratory Press.
Sherry, S. 1987. The FDA Advisory Panel's decision on rt-PA. J. Clin. Pharmacol. 27:730-734.
Staub, J. M., Garcia, B., Graves, J., Hardukiewicz, P. T. J., Hunter, P., Nehra, N., Paradkar, V., Schlittler, M., Carrol, J. A., Spatola, L., Ward, D., Ye, G., and Russell, D. A. 2000. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18:333-338.
Tami, J. A. 1991. Recombinant interleukin-2 and the quest for FDA approval. Clin. Pharm. 10:141-142.
Thanavakam, Y., Yang, Y-F., Lyons, P., Mason, H. S., and Arntzen, C. J. 1995. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc. Natl. Acad. Sci. USA 92:3358-3361.
Vaquero, C., Sack, M., Changler, J., Drossard, J., Schuster, F., Monecke, M., Schillberg, S. and Fischer, R. 1999. Transient expression of a tumor-specific single-chain fragment and chimeric antibody in tobacco leaves. Proc. Natl. Acad. Sci. USA 96:11128-11133.
Villa-Komaroff, L., Efstratiadis, A., Broome, S., Lomedico, P., Tizard, R., Naber, S. P., Chick, W. L., and Gilbert, W. 1978. A bacterial clone synthesizing proinsulin. Proc. Natl. Acad. Sci. USA 75:3727-3731.
Walmsley, A. M., and Arntzen, C. J. 2000. plants of delivery of edible vaccines. Curr. Opin. Biotechnol. 11:126-129.
Zhu, Z., Hughes, K. W., Huang, L., Sun, B., Liu, C., Li, Y., Hour, Y., and Li, X. 1994. Expression of human a-interferon cDNA in transgenic rice plants. Plant Cell Tissue Organ Culture 36:197-204.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 俞何興 (1997) 台灣海域地質區之特徵。地質,第17卷,第1-2期,第47-68頁。
2. 全中平(民85)。以教材發展形成性評量的觀點探討國民小學二年級學童學習自然科有關簡單電路之通路及電流概念。國教學報,8,165-172。
3. 江明修(民81)。社會科學多重典範的爭辯:試論質與量的研究方法的整合。國立政治大學學報,64,315-344。
4. 何仕仁(民88)。重建災後之課程教學。教育資料與研究,31,79-81。
5. 何耀坤(民89)。探討大地震的預測對策。科學教育,227,24-33。
6. 邱美虹(民89)。概念改變研究的省思與啟示。科學教育學刊,8(1),1-34。
7. 席家玉(民85)。國小自然科教學現況調查分析。國教輔導,36(1),27-30。
8. 張敬宜(民89)。大台北地區國小學童對空氣相關概念認知之研究。科學教育學刊,8(2),141-156。
9. 梁慧芳(民87)。建構主義的觀點如何應用在自然科教學。屏師科學教育,3,11-21。
10. 郭金美(民88)。國小學童天文學的概念發展。嘉義師院國民教育研究學報,5,95-124。
11. 郭重吉(民81)。國中學生能量和波動概念另有架構之研究。彰化師範大學學報,3,505-527。
12. 陳世輝(民85)。原住民兒童對國小自然科教材「生物」概念理解之分析。花蓮師院學報,6,217-240。
13. 劉伍貞(民85)。國小學生月相概念學習之研究。未出版碩士論文。屏東:國立屏東師範學院國民教育研究所。
14. 蔡慧敏(民89)。永續減災的環境教育。環境教育季刊,41,63-70。
15. 鄭石岩(民88)。教師如何輔導震災後的兒童。學生輔導,64,120-127。