人類白血球顆粒細胞增生因子（Human granulocyte colony stimulating factor；hG-CSF）是人體嗜中性白血球（neutrophilic granulocyte）生長、分化所必須的細胞激素，是由174個氨基酸所構成。主要是應用在化療所引起之嗜中性白血球缺乏症（neutropenia）。本實驗以基因工程的方法，將hG-CSF基因轉殖到酵母菌，如酵母菌可使hG-CSF分泌到細胞外，即可簡化之後之純化程序。實驗中，我們以聚合酶連鎖反應(PCR) 從pET25b-hG-CSF中擴增hG-CSF，並轉殖此因子之基因片段。此DNA片段有522 bp，並且在5’端設計有6個組織氨酸（histidine tag）和可被腸激酶所辨識（enterokinase cleavage site）的序列，需要時可將此酵素切割成hG-CSF蛋白質。PCR之後挑選clone帶有hG-CSF基因，並且將此基因再接到表現質體（pPIC9K）上，完成質體pPIC9K-hG-CSF的建構。最後將質體轉殖到酵母菌SMD1168內，挑選出生長於含有4 mg/ml G-418的YPDS固態培養基上的菌落，這可能是含有high copy number的轉型菌。 
    將誘導後的細胞及培養基，分別以SDS-PAGE及西方點墨法（Western blotting）來鑑定結果。可發現不管是在細胞內或培養基都有hG-CSF，而分泌到細胞外的hG-CSF約為每公升10毫克，表現的hG-CSF確實其分子量在20 kDa左右。
    將經由FPLC分子篩液相層析管柱（HiPerp Sephacryl S-200）初步純化後的Fraction I（12~19）和Fraction II（21~34），各分別添加到人類白血病細胞株（HL-60）中，發現Fraction II有助於HL-60細胞增生，而Fraction I卻不能使細胞增生，因此可判斷Fraction II具有生物活性。添加40 ng/ml的Fraction II，在第７天時，可以觀察到細胞數目比對照組增加了２倍；添加Fraction II由1 ng ~ 40 ng/ml有顯著增生，而在40 ng ~ 150 ng/ml達到最高的細胞數。

Human granulocyte colony stimulating factor (hG-CSF) is a 18.7 kDa glycoprotein , consisting 174 amino acids. It can stimulate granulocyte colony formation and affects proliferation, differentiation and activation of mature neutrophilic granulocytes. It was widely for treatment of neutropenia in cancer therapy. We cloned the hG-CSF gene into P. pastoris and hope to see the recombinant hG-CSF can secrete into medium. It will make the purification procedure much easiler. We amplified the hG-CSF gene from pET25b-hG-CSF by polymerase chain reaction (PCR). The size of the hG-CSF DNA contains 522 bp, and histidine tag (His6) and enterokinase cleavage site (Asp4Lys) were added at the N-terminus of the protein. The PCR product was first cloned to pOPtima™ cloning vector (TA- Cloning). The “insert” was further cloned into pPIC9K vector. pPIC9K-hG-CSF plasmid was integrated into the alcohol oxidase region of the SMD1168 genome. We selected the transformants that were selected from medium contains 4 mg/ml of G-418. Based on it can be selected from the plate contains 0.2 mg/ml of G-418 having 1 copy of insertion. We can predict this is a multicopy clone. 
  Under on culturing condition, the recombinant hG-CSF was able to be secreted into the medium, 10 mg/L of culture medium. After the medium was concentrated, we obtained the protein from FPLC-gel filtration column (Hiperp Sephacryl S-200). There were two fractions . 「Fraction I and Fraction II」 From SDS-PAGE, both were located at 20 kDa. But from the biological activity study for stimulation of granulocyte. We only found Fraction II has the activity. We concluded that Fraction I was obtain in the early portion of the gel filtration column. So it may exist as the aggregated form.

目 錄

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                                             1
第二節 人類白血球顆粒細胞增生因子                       3
第三節 嗜甲基酵母菌Pichia pastoris(P. pastoris)               5

第二章 實驗材料、設備
第一節 實驗材料                                        16
第二節 實驗設備                                        19
第三節 培養基配置                                      20
第四節 酵母菌表現質體及寄主選擇                        23

第三章 實驗方法
第一節  hG-CSF基因選殖                                26
第二節  pPIC9K表現質體和hG-CSF基因之重組             34
第三節  pPIC9K-hG-CSF質體轉殖到酵母菌                 39
第四節  hG-CSF基因表現                                42
第五節  hG-CSF重組蛋白質純化                          49
第六節  hG-CSF對細胞增生的測定                        52

第四章 結果與討論
第一節  製備人類白血球顆粒細胞增生因子基因             59
第二節  確認及純化P. pastoris所表現的hG-CSF蛋白質      61
第三節  檢測表現蛋白質hG-CSF的活性                    64

第五章 結論與未來展望                                  81

第六章 參考文獻                                        83


圖表目錄

表1.1  生物技術工業常用的蛋白質表現系統                2
表1.2  利用P. pastoris表現的異源蛋白質                  15
表3.1  PCR反應之各項成分                             27
表3.2  PCR反應條件                                   27
表3.3  TA ligation之各項成分                            31
表3.4  pOptima-hG-CSF DNA以限制酶(EcoRI、NotI)水解    34
之反應組成
表3.5  pPIC9K 質體DNA以限制酶(EcoRI、NotI)水解之反   35
應組成
表3.6  接合反應溶液                                    37
表3.7  pPIC9K-hG-CSF質體 DNA以限制酶(EcoRI、NotI)水  38
解之反應組成
表3.8  pPIC9K-hG-CSF質體 DNA以限制酶(SacI)水解之反應  40
組成

圖1.1  G-CSF蛋白質分子之三級結構                       11
圖1.2  P. pastoris代謝甲醇路徑圖                          12
圖1.3  異源蛋白質分泌至胞外示意圖                       13
圖1.4  表現載體整合進入P. pastoris染色體DNA的方式      14
圖2.1  pPIC9K質體圖                                    23
圖2.2  pPIC9K multiple cloning site                         24
圖2.3  pPIC9K-hG-CSF質體建構示意圖                    25
圖3.1  TA vector：pOptima™ cloning vector質體圖           30
圖3.2  轉漬槽示意圖                                48
圖3.3  血球計數盤                                       55
圖3.4  MTT還原成formazan示意圖                        56
圖4.1  洋菜膠電泳分析PCR反應擴增的重組基因            66
圖4.2  TA cloning 藍白篩選                               67
圖4.3  洋菜膠電泳分析重組基因限制酶片段                 68
圖4.4  洋菜膠電泳分析pPIC9K-hG-CSF的限制酶片段        69
圖4.5  Insert基因（上）與資料庫中人類白血球顆粒細胞增生因子基因（下）定序結果                                      70
圖4.6  重組人類白血球顆粒細胞增生因子基因DNA全長氨基酸轉譯圖                                                    71
圖4.7  以SDS-PAGE及西方點墨法來確認P. pastoris所表現之hG-CSF                                                 72
圖4.8  以SDS-PAGE及西方點墨法分析，通過親和性管柱之重組hG-CSF（native的管柱結果）                             73
圖4.9  以SDS-PAGE及西方點墨法分析，通過親和性管柱之重組hG-CSF（denatured的管柱結果）                          74
圖4.10  FPLC分子篩液相層析（HiPrep Sephacryl S-200）      75
圖4.11  以SDS-PAGE及西方點墨法分析，經分子篩（HiPrep Sephacryl S-200）液相層析管柱純化之hG-CSF               76
圖4.12  測試於培養基中有無添加1.25％ DMSO對HL-60細胞增生的影響                                               77
圖4.13  HL-60細胞經hG-CSF（denatured，實驗3.5.2）處理後，以細胞計數觀察細胞增生的變化                           78
圖4.14  HL-60細胞經hG-CSF（12~19）及hG-CSF（21~34）處理後，以細胞計數觀察細胞增生的變化                       79
圖4.15  利用MTT assay方法分析不同濃度的hG-CSF（21~34）對HL-60細胞增生的影響                                   80

Cereghino, J., Cregg, JM. (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 24, 45-66.

Cregg, JM., Madden, KR., Barringer, KJ., Thill, GP., Stillman, CA. (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9, 1316-1323.

Cregg, JM., Vedvick, TS., Raschke, WC. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnol. 11, 905–910.

Daly, R., Hearn, MTW. (2005) Expression of heterologous proteins in Pichia pastoris: A useful experimental tool in protein engineering and. production. J. Mol. Recognit. 18, 119–138.

Hill, C. P., Osslund, T. D., 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.

Koutz, P.J., Davis, G.R., Stillman, C., Barringer, K., Cregg, JM., Thill, G. (1989) Structural compsrison of the Pichia pastoris alcohol genes. Yeast. 5, 167-177.

Kubota, N., Orita, T., Hattori, K., Oh-eda, M., Ochi, N., Yamazaki, T. (1990) Structure characterization of natural and recombinant human granulocyte colony-stimulating factors. J. Biochem. 107, 486-492.

Lasnik, MA., Porekar, VG., Stalc, A. (2001) Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast Pichia pastoris. Pflugers Arch - Eur. J. Physiol. 442, 184-186.

Lu, H. S., Clogston, C. L., Narhi, L. O., Merewether, L. A., Pearl, W. R., 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. Science. 229, 16-22

Metcalf, D. (1990) The colony-stimulating factors: discovery, development and clinical application. Cancer. 65, 2185-2195.

Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 65, 55-63.

Nagata, S., Tsuchiya, M., Asano, S., Yamamoto, O., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., Yamazaki, T. (1986) The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor. EMBO J. 5, 575-581.

Narhi, LO., Arakawa, T., Aoki, KH., Elmore, R., Rohde, MF., Boone, T., Strickland, TW. (1991) The effect of carbohydrate on the structure and stability of erythropoietin. J. Biol. Chem. 266, 23022-23026.

Nomura, H., Imazeki, I., Oheda, M., Kubota, N., Tamura, M., Ono, M., Ueyama, Y., Asano, S. (1986) Purification and characterization of human granulocyte colony-stimulating factor (G-CSF). EMBO J. 5, 871–876.

Ostergaard, S., Olsson, L., Nielsen, J. (2000) Metabolic engineering of Saccharomyces cerevisiae. MMBR. 64, 34-50.

Romanos, M. (1995) Advances in the use of Pichia pastoris for high level gene expression. Curr. Opin. Biotechnol. 6, 527-533.

Scorer, CA., Buckholz, RG., Clare, JJ., Romanos, M. (1993) The intracellular. production and secretion of HIV-1 envelope protein in the methylotrophic. yeast Pichia pastoris. Gene. 136, 111-119. 

Shreekrishnna, K., Nelles, L., Potenz, R., Cruze, J., Mazzaferro, P., Fish, W., Fuke, M., Holden, K., Phelps, D., 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.

Towbin, H., Staehelin, T., Gordon, J. (1992) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnol. 24, 145-149.

Vedvick, T., Buckholz, RG., Engel, M., Urcan, A., Kinney, J., Provow, S., Siegel, RS., Thill, GP. (1991) High-level secretion of biologically active aprotinin from yeast Pichia pastoris. J. Ind. Microb. 7, 197-201.

Veenhuis, M., Van Dijken, JP., Harder, W. (1980) The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv. Microb. Physiol. 24, 1-82.

Wegner, G. (1990) Emerging applications of methylotrophic yeasts. FEMS Microbiol Rev. 87, 279-284.

Yamaguchi, T., Yamaguchi, T., Kogi, M., Yamamoto, Y., Hayakawa, T. (1997) Bioassay of human granulocyte colony-stimulating factor using human promyelocytic HL-60 cells. Biol. Harm. Bull. 20, 943-947.

Zsebo, KM., Lu, HS., Fieschko, JC., Goldstein, L., Davis, J., Duker, K., Suggs, SV., Lai, PH., Bitter, GA. (1986) Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha-factor leader region. J. Biol. Chem. 261, 5858-5865.