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研究生:謝鎮安
研究生(外文):Hsieh, Chen-An
論文名稱:Expressionandpurificationofrecombinanthumaninterferon-γandinterleukin-12employingstarchbindingdomain
論文名稱(外文):利用澱粉吸附區域表現及純化重組人類干擾素-γ及介白素-12
指導教授:張大慈
指導教授(外文):Chang, Margaret Dah-Tsyr
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:147
中文關鍵詞:干擾素-γ介白素-12澱粉吸附區域
外文關鍵詞:interferon-γinterleukin-12starch binding domain
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Interleukin-12 (IL-12) has multiple biological effects on T cells and NK cells, including IFN-γ production leading to cellular-mediated immunity. Interferon-γ (IFN-γ) plays a role in inhibition of tumor growth and participates in immunoreactions. Among IFNs, IFN-γ is one of the most important therapeutic proteins since its immunodulation activity is better than the others. As is generally known that the expense for purification of recombinant agricultural, industrial, or therapeutical proteins is often quite high and the process is fairly complicated, hence a production scheme of high efficiency and low cost are practically needed. In this study, to develop an economic expression and purification process for two important cytokines, IL-12 and IFN-γ, fusion proteins AL*IL12 and AL*I consisted of an N-terminal starch binding domain (SBD) from Rhizopus oryzae (RoSBD) glucoamylase, a linker derived from the interdomain sequence of R. oryzae glucoamylase, and a C-terminal human IL-12 or IFN-γ were successfully produced in Pichia pastoris. The recombinant AL*IL12 and AL*I comprising the RoSBD can be secreted to culture medium and one-step purified using starch substance, a suitable material for affinity separation of proteins due to its low-cost, stability, and non-toxicity. The expression yield of AL*IL12 and AL*I was respectively 10 mg/L and 30 mg/L. The biochemical properties including molecular mass, glycosylation pattern, and oligomerization degree of AL*IL12 and AL*I have been characterized, and the regulatory effects of downstream genes by IL12 and IFN-γ have been verified by cellular treatment. Taken together, our recombinant IL-12 and IFN-γ have the potential to be further developed for diagnostic and therapeutic applications.
介白素-12 (interleukin-12) 能誘導T細胞和NK細胞產生干擾素-γ,干擾素-γ (interferon-γ)則具抑制腫瘤生長與調節免疫的重要功能,二者均能調控細胞性免疫作用。在數種干擾素中,干擾素-γ之免疫調節能力優於其他干擾素,為目前重要的醫療用蛋白質藥物。通常量產農工業及或藥用等級重組蛋白質產品時,純化回收的程序繁複且成本甚高,因此發展高效能、低單價的蛋白質產品的製程為全球生技產業的關鍵實務需求。本篇研究的目標為發展高效率介白素-12及干擾素-γ的重組蛋白質生產、純化、及應用,本研究利用嗜甲醇酵母菌(Pichia pastoris)成功表現胺基端含米根黴菌葡萄糖水解酵素之澱粉吸附區域(Rhizopus oryzae starch-binding domain, RoSBD)及其酵素功能區連結片段(Rhizopus oryzae glucoamylase linker)之人類介白素-12 (AL*IL12)和干擾素-γ (AL*I)。重組蛋白質AL*IL12和AL*I的表現產量分別約為10 mg/L和30 mg/L,二者之澱粉吸附區域使其能利用澱粉吸附的方式快速純化。分析重組蛋白質AL*IL12和AL*I顯示二者之分子量、醣基化程度、及聚合體比例等生化特性、及IL-12和IFN-γ誘導下游基因表現的生物活性。本論文研究之具體貢獻為開發重組IL-12和IFN-γ蛋白質成為生技製藥領域中分子檢測及疾病治療應用之潛力標的。
Acknowledgement........................................... I
中文摘要................................................. II
Abstract................................................ III
Table of Contents........................................ IV
List of Figures......................................... VII
List of Tables........................................... IX
List of Appendix.......................................... X
Abbreviations............................................ XI

Chapter 1 Introduction...............................................1
Chapter 2 Materials and Methods..........................11
2.1 Strains and expression plasmids......................11
2.2 Culture media composition............................11
2.3 Plasmid constructs...................................12
2.4 Preparation of competent cells.......................13
2.5 Transformation of E. coli............................13
2.6 Confirmation of colonies by in situ PCR..............14
2.7 Mini-preparation of plasmid..........................14
2.8 Recovery of DNA fragment.............................15
2.9 Restriction enzyme digestion.........................15
2.10 Ligation............................................16
2.11 Yeast transformation................................16
2.12 Expression of SBD, SBD-eGFP, eGFP-SBD and SBD-IFNγ (AI) in E. coli...........................................17
2.13 Expression of AI, ALI, AL*I and AL*IL12 in P. pastoris..................................................18
2.14 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)...............................18
2.15 Western blotting....................................19
2.16 Effects of starch on SBD adsorption.................20
2.17 Determination of N-linked glycosylation.............20
2.18 Purification of AI, ALI, AL*I and AL*IL12 by amylose resin chromatography......................................21
2.19 Purification of AL*I and AL*IL12 by corn starch.....22
2.20 Enterokinase digestion..............................22
2.21 Bicinchoninic acid (BCA) assay for concentration determination.............................................22
2.22 Cross-linking analysis..............................23
2.23 Analytical ultracentrifugation......................23
2.24 Cell culture and treatment..........................24
2.25 RNA isolation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)...................................25
2.26 Gene expression analysis by Real-Time PCR...........26
2.27 Mass spectrometry determination.....................26
2.28 In-gel digestion and LC-MS/MS.......................26

Chapter 3 Results...................................................28

Part I. SBD
3.1.1 Expression and purification of SBD, SBD-eGFP, and eGFP-SBD in E. coli.......................................28

Part II. Interleukin-12 (IL-12)
3.1.2 Construction of pPICZαA-AL*IL12 and pAO815-AL*IL12 genes.....................................................29
3.1.3 Expression of AL*IL12 in P. pastoris GS115 and protein identification ...................................30
3.1.4 Glycoprotein analysis..............................31
3.1.5 Purification of SBD-tagged IL-12 fusion protein by amylose resin or corn starch..............................32
3.1.6 IFN-γ gene was up-regulated by AL*IL12 in CCRF-CEM cells.....................................................33

Part III. Interferon gamma (IFN-γ)
3.2.1 Construction of pET23a-AI, pPICZαA-AI, pPICZαA-ALI, pPICZαA-AL*I, pAO815-AI, pAO815-ALI, and pAO815-AL*I.... 35
3.2.2 Expression of SBD-IFNγ in E. coli................ 36
3.2.3 Expression of AI, ALI and AL*I in P. pastoris GS115.................................................... 37
3.2.4 Identification of SBD-IFNγ variants by SDS-PAGE and Western blot............................................. 37
3.2.5 Glycoprotein analysis............................. 39
3.2.6 Purification of SBD-tagged IFNγ fusion proteins by amylose resin or corn starch........................... ..41
3.2.7 Enterokinase digestion............................ 42
3.2.8 Functional analysis of recombinant IFN-γ......... 43
3.2.9 Evaluation of genes up-regulated by IFN-γ in Beas-2B cells................................................. 44

Chapter 4 Discussion............................................... 46
Publication list..................................................... 53
References............................................... 54
Figures.................................................. 64
Tables...................................................109
Appendix.................................................131


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