人類顆粒細胞增生因子（Human Granulocyte Colony Stimulating Factor；hG-CSF）能刺激嗜中性白血球前驅細胞生長，並促進其增殖、分化，同時具有促進嗜中性白血球由骨髓釋出及增強成熟嗜中性白血球的機能，它被廣泛的應用在癌症化療和骨髓移植後，所引起的嗜中性白血球缺乏症（neutropenia）。本實驗將hG-CSF基因整合到pPIC9K質體，轉殖到酵母菌SMD1168，以4 mg/ml G-418抗生素篩選出multicopy inserted clones，並誘導使其表現hG-CSF。
使用搖瓶及發酵槽方式大量誘導表現hG-CSF，並以SDS-PAGE及西方點墨法（Western blottnig）分析，發現表現之hG-CSF分子量確實在20 kDa。比較以搖瓶和發酵槽方式表現，以發酵槽方式誘導之hG-CSF表現量為搖瓶方式的7倍。
將誘導後之濃縮培養基，以FPLC-DEAE Sepharose Fast Flow與Sephadex G-50管柱純化，回收率分別為76.5 %及65.7 %，純化倍率分別為3.6及10.4。之後以此hG-CSF樣品對HL-60細胞增生的測試，採用1.計算細胞總數及 2.定量細胞內酵素活性（succinate dehydrogenase），結果是1.hG-CSF確實有助於HL-60細胞增生，添加100 ng/ml hG-CSF於培養7天後，所增生之細胞數目為未添加hG-CSF的2倍；2.細胞內之succinate dehydrogenase酵素活性也增加，在培養7天後，加100 ng/ml hG-CSF約為加2 ng/ml濃度時的9~10倍；細胞所增生的數目，亦隨著hG-CSF添加量的增加而增加。

The recombinant human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that has been widely used for the treatment of neutropenia after chemotherapy and bone marrow transplantation. It can stimulate both proliferation, differentiation of neutrophils. In this study, we cloned the G-CSF encoding gene into pPIC9K vector and transformed it into SMD1168 yeast cells. The transformed cells were selected on the plate containing 4 mg/ml of G-418.
The recombinant hG-CSF was found to be secreted in the medium, and molecular weight was as predicted at 20 kDa on SDS-PAGE, corresponding to the commercial standard. Comparing with bioreactor and shake flask, the specific hG-CSF protein production increased 7 times.
After medium was concentrated, both DEAE Sepharose Fast Flow and Sephadex G-50 column chromatography were used to purify the hG-CSF protein. It resulted in the recovery of 76.5 % and 65.7 % respectively, and the purification of 3.6 and 10.4 fold respectively.
By using HL-60 cell (Human promyelocytic cell), the bioactivity of hG-CSF can be observed. For one: The effect of the purified hG-CSF to promote the total cell numbers to 2 fold when 100 ng of the purified hG-CSF was added to the cells. The proliferation rate was hG-CSF-dependent. The most efficient was at the inoculation size of 1 × 105 cells/ml for 100 ng of purified hG-CSF. The other was the MTT assay to estimate the stimulation of the cellular succinate dehydrogenase activity. When the addition of 2~100 ng purified hG-CSF to the cells, the enzyme activity increased 9~10 times.

封面內頁	
口試委員審議通過簽名表	
授權書	
致謝	
中文摘要........................................I
英文摘要........................................II
目錄............................................III
圖表目錄........................................V
	
第一章　緒論	
第一節　前言....................................7
第二節　人類顆粒細胞增生因子....................8
第三節　嗜甲基酵母菌 Pichia pastoris............12
	
第二章　實驗材料、設備	
第一節　實驗材料................................23
第二節　實驗設備................................26
第三節　培養基配置..............................28
	
第三章　實驗方法	
第一節　pPIC9K-hG-CSF質體之製備.................32
第二節　pPIC9K-hG-CSF質體轉殖到酵母菌...........36
第三節　hG-CSF基因表現..........................40
第四節　hG-CSF重組蛋白質之純化..................52
第五節　hG-CSF對細胞增生之測定..................54
	
第四章　結果與討論	
第一節　基因建構................................60
第二節　蛋白質表現..............................61
第三節　蛋白質純化..............................63
第四節　檢測純化後蛋白hG-CSF對HL-60的生物活性...64
	
第五章　結論與未來展望..........................82
	
第六章　參考資料................................86

圖表目錄 表 1.1 生物技術工業常用之蛋白質表現系統 21
表 1.2 利用P. pastoris系統所表現之異源蛋白質 22  
表 4.1 以搖瓶及發酵槽方式表現hG-CSF之表現量表 80
表 4.2 以DEAE Sepharose Fast Flow及Sephadex G-50純化hG-CSF之結果 81
圖 1.1 造血幹細胞分化圖 16
圖 1.2 hG-CSF 蛋白質分子的三級結構圖 17
圖 1.3 hG-CSF與其他相似結構之細胞激素比較圖 18
圖 1.4 嗜甲基酵母菌代謝甲醇路徑圖 19
圖 1.5 異源蛋白質分泌至胞外示意圖 20  
圖 2.1 pPIC9K質體圖 31  
圖 3.1 hG-CSF基因DNA序列及氨基酸轉譯圖 32
圖 3.2 發酵槽主體示意圖 44
圖 3.3 發酵槽裝置示意圖 45
圖 3.4 轉漬槽示意圖 51
圖 3.5 血球計數盤 56
圖 3.6 MTT還原成MTT formazan示意圖 57
圖 4.1 pPIC9K-hG-CSF經限制酶水解後之DNA片段。 67
圖 4.2 經由不同濃度的G-418抗生素篩選出的菌株，經PCR所合成之DNA片段。 68
圖 4.3 以SDS-PAGE和西方點墨法分析，以不同濃度G-418抗生素所篩選出的菌株，經誘導後所表現之hG-CSF。 69
圖 4.4 以SDS-PAGE分析，在1 ml培養基中經1~6天誘導所表現之hG-CSF。 70
圖 4.5 以SDS-PAGE和西方點墨法分析，經搖瓶方式誘導（400 ml）所表現之hG-CSF。 71
圖 4.6 以SDS-PAGE和西方點墨法分析，經發酵槽方式誘導（2 L）所表現之hG-CSF。 72
圖 4.7 以DEAE Sepharose Fast Flow管柱純化hG-CSF之流程圖。 
73
圖 4.8 以Sephadex G-50管柱純化hG-CSF之流程圖。 74
圖 4.9 以SDS-PAGE和西方點墨法分析，經DEAE Sepharose Fast Flow與Sephadex G-50管柱純化後之hG-CSF。 75
圖 4.10 以MTT assay分析不同濃度之純化hG-CSF對HL-60細胞增生的影響。 76
圖 4.11 測試培養基中有無添加1.25 % DMSO對HL-60細胞增生的影響。 77
圖 4.12 測試培養基中添加不同濃度之純化hG-CSF對HL-60細胞增生的影響。 78
圖 4.13 hG-CSF的添加量對起始細胞密度的影響。 79

Bhatacharya, P., Pandey, G., and Mukherjee, K.J. (2007) Production and purification of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) from high cell density cultures of Pichia pastoris. Bioprocess Biosyst. Eng. 30, 305-312.
Cregg, J.M., Madden, K.R., Barringer, K.J., Thill, G.P., and Stillman, C.A. (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9, 1316-1323.
Cregg, J.M., Vedvick, T.S., and Raschke, W.C. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnol. 11, 905-910.
Gemmill, T.R., and Trimble, R.B. (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochem. Biophys. Acta. 1426, 227-237.
Gellissen, G. (2000) Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 54, 741-750.
Hartner, F.S., and Glieder, A. (2006) Regulation of methanol utilisation pathway genes in yeasts. Microb. Cell Fact. 5, 39.
Hill, C.P., Osslund, T.D., and Eisenberg, D. (1993) The structure of granulocyte-colony-stimulating factor and its relationship to other growth factor. Proc. Natl. Acad. Sci. USA. 90, 5167-5171.
Houard, S., Heinderyckx, M., and Bollen, A. (2002) Engineering of non-conventional yeasts for efficient synthesis of macromolecules: the methylotrophic genera. Biochimie. 84, 1089-1093.
Koutz, P., Davis, G.R., Stillman, C., Barringer, K., Cregg, J., and Thill, G. (1989) Structural comparison of the Pichia pastoris alcohol oxidase genes. Yeast 5, 167-177.
Kubota, N., Orita, T., Hattori, K., Oh-eda, M., Ochi, N., and Yamazaki, T. (1990) Structure characterization of natural and recombinant human granulocyte colony-stimulating factors. J. Biochem. 107, 486-492.
Lasnik, M.A., Porekar, V.G., and Stalc, A. (2001) Human granulocyte colony stimulating factor (hG-CSF) expression by methylotrophic yeast Pichia pastoris. Pfluger Arch–Eur. J. Physiol. 442, 184-186.
Lu, H.S., Clogston, C.L., Narhi, L.O., Merewether, L.A., Pearl, W.R., and Boone, T.C. (1992) Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. J. Biol. Chem. 267, 8770-8777.
Metcalf, D. (1985) The granulocyte-macrophage colony stimulating factors. Cell 43, 5-6.
Metcalf, D. (1990) The colony stimulating factors. Discovery, development, and clinical applications. Cancer 65, 2185-2195.
Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assay. J. Immunol. Method 65, 55-63.
Nagata, S., Tsuchiya, M., Asano, S., Yamamoto, O., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., and Yamasaki, T. (1986) The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor. EMBO J. 5, 575-581.
Narhi, L.O., Arakawa, T., Aoki, K.H., Elmore, R., Rohde, M.F., Boone, T., and Strickland, T.W. (1991) The effect of carbohydrate on the structure and stability of erythropoietin. J. Biol. Chem. 266, 23022-23026.
Nicola, N.A., Metcalf, D., Matsumoto, M., and Johnson, G.R. (1983) Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor. J. Biol. Chem. 258, 9017-9023.
Nomura, H., Imazeki, I., Oheda, M., Kubota, N., Tamura, M., Ono, M., Ueyama, Y., and Asano, S. (1986) Purification and characterization of human granulocyte colony-stimulating factor (G-CSF). EMBO J. 5, 871-876.
Ostergaard, S., Olsson, L., and Nielsen, J. (2000) Metabolic engineering of Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 64, 34-50.
Sakai, Y., Tani, Y., and Kato, N. (1999) Biotechnological application of cellular functions of the methylotrophic yeast. J. Mol. Catal., B Enzym. 6, 161-173.
Shreekrishnna, K., Nelles, L., Potenz, R., Cruze, J., Mazzaferro, P., Fish, W., Fuke, M., Holden, K., Phelps, D., and Wood, P. (1989) High level expression, purification, and characterization of  recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Biochem. 28, 4117-4125.
Socolovsky, M., Lodish, H. F., and Daley, G.Q. (1998) Control of hematopoietic differentiation: lack of specificity in signaling by cytokine receptors. Proc. Natl. Acad. Sci. USA. 95, 6573-6575.
Souza, L.M., Boone, T.C., Gabrilove, J., Lai, P.H., Zsebo, K.M., Murdock, D.C., Chazin, V.R., Bruszewski, J., Lu, H., and Chen, K.K. (1986) Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 232, 61-65.
Shyu, W.C., Lin, S.Z., Lee, C.C., Liu, D.D., and Li, H. (2006) Granulocyte colony-stimulating factor for acute ischemic stroke: a randomized controlled trial. Can. Med. Assoc. J. 174, 927-933.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76, 4350-4354.
Vedvick, T., Buckholz, R.G., Engel, M., Urcan, M., Kinney, J., Provow, S., Siegel, R.S., and Thill, G.P. (1991) High-level secretion of biologically active aprotinin from the yeast Pichia pastoris. J. Ind. Microbiol. 7, 197-201.
Wang, C., Wang, L., and Geng, X. (2007) Renaturation with simultaneous purification of rhG-CSF from Escherichia coli by ion exchange chromatography. Biomed. Chromatogr. 21, 1291-1296.
Yamaguchi, T., Yamaguchi, T., Kogi, M., Yamamoto, Y., and Hayakawa, T. (1997) Bioassay of human granulocyte colony-stimulating factor using human promyelocytic HL-60 cells. Biol. Pharm. Bull. 20, 943-947.
Zsebo, K.M., Lu, H.S., Fieschko, J.C., Goldstein, L., Davis, J., Duker, K., Suggs, S.V., Lai, P.H., and Bitter, G.A. (1986) Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha-factor leader region. J. Biol. Chem. 261, 5858-5865.