跳到主要內容

臺灣博碩士論文加值系統

(44.221.70.232) 您好!臺灣時間:2024/05/30 21:14
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

我願授權國圖
: 
twitterline
研究生:高偉適
研究生(外文):Wei-Shih Kao
論文名稱:人類干擾素α2b在中國倉鼠卵巢細胞之表現效率
論文名稱(外文):Expression Efficiency of Recombinant Human Interferon Alpha 2b in Chinese Hamster Ovary cells
指導教授:徐祖安
指導教授(外文):Tsu-An Hsu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:69
中文關鍵詞:干擾素中國倉鼠卵巢細胞蛋白質表現
外文關鍵詞:IFN-α2bCHO cellprotein expression
相關次數:
  • 被引用被引用:0
  • 點閱點閱:152
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
干擾素α2b為近年來重要的抗病毒與抗癌用藥,然而,利用中國倉鼠卵巢細胞表現人類干擾素α2b,卻產生表現量低的問題。以產量1毫克來說,就需要用到1.1 x 1011 個細胞生產,規模相當於367個T75培養瓶所培養的細胞數目。對於干擾素α2b處理中國倉鼠卵巢細胞,進而探討干擾素所引起的細胞毒性,我們發現無明顯的生長抑制現象產生。因此,我們推測干擾素α2b在中國倉鼠卵巢細胞表現量低的原因,可能導因於轉錄效率或轉譯效率的不良。為了深入了解人類干擾素α2b表現量低是否由低轉錄效率所導致,本文首先以人類干擾素α2b與綠螢光蛋白進行轉錄效率比較分析。結果顯示,在細胞整體與細胞核的RNA總數上,人類干擾素α2b高於綠螢光蛋白4倍;其次,在細胞質及細胞核mRNA的數量上,人類干擾素α2b與綠螢光蛋白並無明顯差異;最後,在蛋白質表現量方面,人類干擾素α2b遠比綠螢光蛋白低。另外,在過去文獻上曾提及RNA splicing能有效提高蛋白質產量,當我們利用RNA splicing改善人類干擾素α2b表現時,也發現到mRNA的增加無法明顯增加蛋白質的表現量。所以,人類干擾素α2b在中國倉鼠卵巢細胞表現量低的原因可能不在於轉錄效率,而是mRNA的轉譯起始效率或轉譯的中間過程。未來,深入的研究與探討可朝蛋白質轉譯起始效率與合成速度方面進行,並搭配動力學分析人類干擾素α2b在中國倉鼠卵巢細胞表現的反應瓶頸,進而解決表現效率不佳的問題。
Most recently, IFN-α2b has become an important anti-virus and anti-cancer drug. However, expressed human IFN-α2b (HuIFN-α2b) at low levels in Chinese hamster ovary (CHO) cells were observed. 1 mg of IFN-α2b production will require 1.1 x 1011 cells which are equivalent to cultured cells in 367 of T75 flasks. For the cytotoxicity of CHO cells induced by HuIFN-α2b, the growth inhibition still has not been observed notably after HuIFN-α2b treatment. Hence, the problem that expressed HuIFN-α2b at very low levels in CHO cells results from inefficient transcription or translation is considered. In results, firstly, copies of HuIFN-α2b is 4- fold higher than that of EGFP reporter in comparison of the cell total and nuclear RNA. Secondly, the copy number difference between HuIFN-α2b and enhanced green fluorescence protein (EGFP) was not distinguished markedly in comparison of cytoplasmic mRNA and nuclear mRNA. Finally, the amount of expressed HuIFN-α2b was significantly lower than that of EGFP in CHO cells. On the other hand, effects of splicing on enhancing protein expression have been demonstrated in the published paper. In spite of the increase in the amount of mRNA levels, HuIFN-α2b expression could not be raised markedly as splicing was employed. Therefore, the reason expressed HuIFN-α2b at low levels in CHO cells might be mRNA inefficient translational initiation or the middle process of translation rather than the transcriptional efficiency. In the future, the translational initiation efficiency and the synthesis rate of HuIFN-α2b in CHO cells will be further studied. The bottleneck in this overall HuIFN-α2b expression process in CHO cells will also be analyzed by kinetics to optimize the expression efficiency.
Barnes, L. M., Bentley, C. M., and Dickson, A. J. (2003). Stability of Protein Production From Recombinant Mammalian Cells. Biotechnol Bioeng. 81: 631-637.
Brinster, R. L., Allen, J. M., Behringer, R. R., Gelinas, R. E., and Palmiter, R. D. (1988). Introns increase transcriptional efficiency in transgenic mice. Proc. Natl. Acad. Sci. 85: 836-840.
Bruns, G. A. and Gerald, P. S. (1976). Human glyceraldehyde-3-phosphate dehydrogenase in man-rodent somatic cell hybrids. 192: 54-56.
Buchman, A. R. and Berg, P. (1988). Comparison of intron-independent and intron-independent gene expression. Mol. Cell. Biol. 8:4395-4405.
Casey, P. J. (1992). Biochemistry of protein prenylation. J Lipid Res. 33: 1371-1740.
Cho, E. J., Takagi, T., Moore, C. R., and Buratowski, S. (1997). mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev. 11: 3319-3326.
Deng, T., Li, Y., and Johnson, L. F. (1989). Thymidylate synthase gene expression is stimulated by some (but not all) introns. Nucleic Acids Res. 17: 645-658.
Devos, R., Opsomer, C., Scahill, S. J., Van, D. H. J., Fiers, W. (1984). Purification of recombinant glycosylated human gamma interferon expressed in transformed Chinese hamster ovary cells. J Interferon Res. 4: 461-468.
Gebauer, F. and Richter, J.D. (1996). Mouse cytoplasmic polyadenylylation element binding protein: An evolutionarily conserved protein that interacts with the cytoplasmic polyadenylylation elements of c-mos mRNA. Proc. Natl. Acad. Sci. 93: 14602-14607.
Geisse, S., Gram, H., Kleuser, B., and Kocher, H.P. (1996). Review Eukaryotic Expression Systems: A comparison. Protein Expr Purif. 8: 271-282.
Ghilardi, B. N., Wiestner, A., and Skoda, R. C. (1998). Thrombopoietin production is inhibited by a translational mechanism. Blood. 92: 4023-4030.
Gonzalez, R., Asenjo, J. A., and Andrews, B. A. (2001). Metabolic Control Analysis of Monoclonal Antibody Synthesis. Biotechnol. Prog. 17: 217-226.
Herbert, T.P. and Hecht, N.B. (1999). The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs. Nucleic Acids Res. 27: 1747-1753.
Heuvel, M. V., Siemerink, M. G., Bosveld, I. J., Zwarthoff, E. G., and Trapman, J. (2004). Interferon-α-(IFN) producing CHO cell lines are resistant to the antiproliferative activity of IFN: A correlation with gene expression. J. Cell. Biochem. 38: 269-278.
Huang, M. T. –F. and Gorman, C. M. (1990). Intervening sequences increase efficiency of 3` processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 18: 937-947.
Huang, Y. and Carmichael, G. G. (1996). Role of polyadenylation in nucleocytoplasmic transport of mRNA. Mol. Cell. Biol. 16: 1534-1542.
Jonasch, E., and Haluska, F. G. (2001). Interferon in oncological practice:Review of interferon biology, clinical applications, and toxicities. Oncologist. 6: 34-55.
Jonsson, J. J., Goresman, M. D., Wilson, N., and Mclvor, R. S. (1992). Intron requirement for expression of the human purine nucleoside phosphorylase gene. Nucleic Acids Res. 20: 3191-3198.
Keene, J. D. (1999). Why is Hu where? Shuttling of early-response-gene messenger RNA subsets. Proc Natl Acad Sci U S A. 96: 5-7.
Kaufman, R.J. (2000). Overview of Vector Design for Mammalian Gene Expression. Mol Biotechnol. 16: 151-160.
Kim, C. H., Oh, Y., Lee, T. H. (1997). Codon optimization for high-level expression of human erythropoietin (EPO) in mammalian cells. 199: 293-301.
Kruys, V., Wathelet, M., Poupart, P., Contreras, R., Fiers, W., Content, J., and Huez, G. (1987). The 3’ untranslated region of the human interferon-β mRNA has an inhibitory effect on translation. Proc. Natl. Acad. Sci. USA. 84: 6030-6034.
Lopata, M. A., Cleveland, D. W., and Sollner-Webb, B. (1984). High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucl. Acids Res. 12: 5707.
Lucas, B. K., Giere, L. M., DeMarco, R. A., Shen, A., Chisholm, V., and Crowley, C. W. (1996). High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector. Nucl. Acids Res. 24: 1774-1779.
Luo, M. J. and Reed, R. (1999). Splicing is required for rapid and efficient mRNA export in metazoans. Proc. Natl Acad. Sci. USA. 96: 14937-14942.
Luthman, H. and Magnusson, G. (1983). High efficiency polyoma DNA transfection of chloroquine treated cells. Nucl. Acids Res. 11: 1295.
Lu, S. and Cullen, B. R. (2003). Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells. RNA. 9: 618-630.
Maniatis, T. and Reed, R. (2002). An extensive network of coupling among gene expression machines. Nature. 416: 499-506.
Makoff, A. J., Oxer, M. D., Romanos, M. A., Fairweather, N. F., Ballantine, S. (1989). Expression of tetanus toxin fragment C in E.coli : High level expression by removing rare codons. Nucleic acids Res. 17: 10191-10202.
Martin, T. E. (1973). A simple general method to determine the proportion of active ribosomes in
eukaryotic cells. Exptl. Cell Res. 80: 496–498.
Mathews, M. B., Sonenberg, N., and Hershey, J. W. B. (1996). Origins and targets of translational control. In: Translational Control (Hershey, J. W. B., Mathews, M. B., and Sonenberg, N., ed). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
McCracken, S. et al. (1997). The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature. 385: 357-361.
Merrick, W. C. (1994). Eukaryotic protein synthesis: an in vitro analysis. Biochimie. 76: 822-830.
Minich, W.B. and Ovchinnikov, L.P. (1992). Role of cytoplasmic mRNP proteins in translation. Biochimie 74: 477-483.
Myers, S. R., Yakubu, M. F. E., Johnson, W. T., Baker, J. E., Cusick, T. S., Williams, E. K., Tinsley, F. C., Kriauciunas, A., Manetta, J., Chen, V. J. (1997). Acylation of human insulin with palmitic acid extends the time action of human insulin in diabetic dogs. Diabetes. 46: 637-642.
Niwa, M., Rose. S. D., and Berget, S. M. (1990). In vitro polyadenylation is stimulated by the presence of an upstream intron. Genes Dev. 4: 1552-1559.
Nott, A., Meislin, S. H., and Moore, M. J. (2003). A quantitative analysis of intron effects on mammalian gene expression. RNA. 9: 607-617.
Nott, A., Hir, H. L., and Moore, M. J. (2004). Splicing enhances translation in mammalian cells: an additional function of the exon junction complex. GENES & DEVELOPMENT 18: 210-222
Page, M. J. (1985). Expression of amplified human beta interferon genes using heavy metal induction in Chinese hamster ovary cells. Gene. 37: 139–144.
Peng, S. S. –Y., Chen, C. Y. –A., Xu, N., and Shyu, A. B. (1998). RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein. EMBO J. 17: 3461-3470.
Pestka, S. and Langer, J. (1987). Interferons and their actions. Ann. Rev. Biochem. 56: 727-777.
Pisarev, A.V., Skabkin, M.A., Thomas, A.A., Merrick, W.C., Ovchinnikov, L.P., and Shatsky, I.N. (2002). Positive and negative effects of the major mammalian messenger ribonucleoprotein p50 on binding of 40 S ribosomal subunits to the initiation codon of β-globin mRNA. J. Biol. Chem. 277: 15445-15451.
Proudfoot, N. J. (1989). How RNA polymerase II terminates transcription in higher eukaryotes. Trends Biochem. Sci. 14: 105-110.
Puck, T. T., Cieciura, S. J., and Robinson, A. (1958).Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. J Exp Med. 108:945–956.
Rai, M. and Padh, H. (2001). Expression systems for production of heterologous proteins. Curr Sci. 80: 1121-1128.
Reed, R. (2000). Mechanisms of fidelity in pre-mRNA splicing. Curr. Opin. Cell Biol. 12: 340-345.
Rolfe, F. G. and Sewell, W. A. (1997). Analysis of human interleukin-5 transcription by a novel nuclear run on method based on the polymerase chain reaction. J. Immunol. Methods. 202: 143-151.
Schwer, B. (2001). A new twist on RNA helicases: DExH/D box proteins as RNPases. Nat. Struct. Biol. 8: 113-116.
Sonenberg, N. (1996). 5’ mRNA cap-binding protein eIF4E and control of cell growth. In: transcriptional control. (Hershey, J. W. B., Mathews, M. B., and Sonenberg, N., ed). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Walsh, G. (2003). Biopharmaceuticals: Biochemistry and Biotechnology. Wiley, Chichester.
Will, C. L. and Luhrmann, R. (2001). Molecular biology. RNP remodeling with DExH/D boxes. Science 291: 1916-1917.
Wills, R. J. (1990). Clinical pharmacokinetics of interferons. Clin. Pharmacokinet. 19: 390-399.
Wurm, F. M. and Petropoulos, C. J. (1994). Plasmid Integration, Amplification and Cytogenetics in CHO Cells: Questions and Comments. Biologicals. 22: 95-102.
Yoshikawa, T., Nakanishi, F., Itami, S., Kameoka, D., Omasa, T., Katakura, Y., Kishimoto, M., and Suga, K. I. (2000). Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes. Cytotechnology. 33: 37-46.
Zhang, g., Gurtu, V., and Kain, S. R. (1996). An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells. Biochem. Biophys. Res. Commun. 227: 707-711.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top