以甘藷生產 老鼠顆粒細胞-巨噬細胞集落刺激因子

Production of Mouse Granulocyte Macrophage -Colony Stimulating Factor (mGM-CSF) in Sweet Potato

黃昱欽(Yu-Chin Huang)

指導教授 ： 黃麗芬

元智大學/工程學院/生物科技與工程研究所/碩士(2011年)

摘要

甘藷 (Ipomoea batatas (L.) Lam)是世界七大重要作物之一，不同品系甘藷的塊根或葉子可供給人類的良好的營養來源。甘藷對生長環境的要求低，所以甘藷是未來分子農場的重要作物。我們成功建立甘藷的基因轉殖系統，因此可利用高單價外源蛋白的表現，增加甘藷的經濟價值。已知老鼠顆粒細胞-巨噬細胞集落刺激因子(mouse granulocyte -macrophage colony stimulating factor, mGM-CSF)影響小鼠白血球的增殖和分化，目前發現mGM-CSF在動物組織中的影響層面很廣，在利用小鼠為研究模式動物的基礎醫學上，包括癌症治療和惡性腫瘤的免疫療法等研究，需求量大也昂貴，目前5 ug mGM-CSF價格為8000元台幣。我們將 mGM-CSF基因構築於能廣泛表現的玉米泛素(Ubiquitin)啟動子或是在根部能大量表現的啟動子(SPOA1或PGI1)下游，並轉殖到甘藷中，藉此大量表達mGM-CSF。
本論文建立了五個帶有玉米泛素啟動子調控mGM-CSF外源基因的甘藷轉殖細胞株，藉由genomic DNA PCR、RT-PCR，GUS報導基因酵素活性分析和西方墨點法，確定外源基因的存在與表現。本論文成功將其中的兩株轉殖細胞株 (CSF-4和CSF-25)再生成植株，同時也分析了民國98年由林惠文碩士成功再生的兩株甘藷轉殖株，確認此二植株的葉片依然有mGM-CSF基因的存在與表現。另一方面，我們也成功將mGM-CSF構築在甘藷塊根中表現的SPOA1啟動子下游，並嘗試利用甘藷的基因轉殖系統建立轉殖株，同時也利用在阿拉伯芥根部能夠表現的PGI1啟動子，希望能藉由甘藷的基因轉殖系統，分析PGI1啟動子所調控GUS報導基因在轉殖甘藷不同部位的表現情況，期望mGM-CSF能夠表現在甘藷塊根中，利用塊根的優勢增加以甘藷表現外源蛋白的競爭力。

關鍵字

甘藷 ； 老鼠顆粒細胞-巨噬細胞集落刺激因子 ； 外源蛋白

並列摘要

Sweet potato, Ipomoea batatas (L.) Lam., is the 7th most important crop in the world. The tuberous roots and leaves for certain varieties of sweet potato are good nutrient source for human beings. Because of low requirement for growth environments, sweet potato becomes one of prospective host for plant molecular farming. We successfully developed gene transformation system in sweet potato. Therefore, we offer a new approach to increase the economic value of sweet potato in Taiwan through molecular farming proposed here. We apply this gene transformation system to produce the recombinant mouse Granulocyte-macrophage colony stimulating factor (mGM-CSF)proteins in transgenic sweet potato, the mouse GM-CSF can affect the mouse white blood
increase and growth, In rent year, the mGM-CSF use in mouse modern rescute, including therapy cancer and tumor, It is so expensive and necessary much more to research and rescute. It，s 5 ug sold NT 8000 dollars now. by either constitutive strong ubiquitin promoter or root overexpression promoters. (SPOA1 or PGI1) transformation to sweet potato, hope to overexpression recombination protein mGM-CSF. In our research, we establish five sweet potato transgenic cell lines pUBI::mGMCSF, use the genomic DNA PCR、RT-PCR、GUS reporter gene check and western blot analysis to check the recombination protein overexpression in sweet potato also succeed regeneration two lines to a sweet potato transgenic lines (CSF-4 and CSF-25),analysis had two regeneration transgenic sweet potato lines, the two lines still have the mGMCSF overxpression.
In the other hand, we succeed construction the SPOA1::mGMCSF，and try to use the sweet potato transformation system, and try to obtain the transgenic line.In other hand,we use the PGI promoter from the Arabidopsis, we hope use the transformation of sweet potato system, try to understand where the PGI1 driven GUS can overexpression in sweet potato. In future, we hope to overexpression in sweet potato tuberous root. To use the advantage of tuberous root production of mGM-CSF protein.

並列關鍵字

Sweet potato ； mGM-CSF ； recombination protein

參考文獻

Ahmad, M., Rees, R.C., McArdle, S.E., Li, G., Mian, S., Entwisle, C., Loudon, P., and Ali, S.A. (2005). Regulation of CTL responses to MHC-restricted class I peptide of the gp70 tumour antigen by splenic parenchymal CD4(+) T cells in mice failing immunotherapy with DISC-mGM-CSF. Int J Cancer 115, 951-959.

Arteaga, H.J., Mohamed, A.J., Christensson, B., Gahrton, G., Smith, C.I.E., and Dilber, M.S. (2001). Expression and release of stable and active forms of murine granulocyte-macrphage colony-stimulating factor (mGM-CSF) targeted to different subcellular compartments. Cytokine 14, 136-142.

Baguma, Y., Sun, C., Boren, M., Olsson, H., Rosenqvist, S., Mutisya, J., Rubaihayo, P.R., and Jansson, C. (2008). Sugar-mediated semidian oscillation of gene expression in the cassava storage root regulates starch synthesis. Plant Signal Behav 3, 439-445.

Barreda, D.R., Hanington, P.C., and Belosevic, M. (2004). Regulation of myeloid development and function by colony stimulating factors. Dev Comp Immunol 28, 509-554.

Blackburn, G.L., and Waltman, B.A. (2005). Pharmacotherapy to reduce visceral fat. Clin Cornerstone 7, 52-60.