利用微陣列蛋白質晶片帥選GNRA tetraloop結合蛋白｜國立中央大學博碩士論文系統

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研究生：	陳寶興 CHAN BAO
論文名稱：	利用微陣列蛋白質晶片帥選GNRA tetraloop結合蛋白 Identification of GNRA tetraloop using proteome arrays
指導教授：	陳健生 Chien-Sheng Chen 高家誠 Chia-Cheng Kao
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 系統生物與生物資訊研究所 Graduate Institute of Systems Biology and Bioinformatics
畢業學年度：	98
語文別：	英文
論文頁數：	43
中文關鍵詞：	微陣列蛋白質晶片
外文關鍵詞：	microarray
相關次數：	點閱：10 下載：0
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微陣列晶片已經成為重要的研究工具，它已經被應用在系統性研究及高通量生物分析。由於越來越多生物體完成基因體的定序，現在已經可以做到把超過18000個人類蛋白質、5000個酵母菌蛋白質或4000 個大腸桿菌蛋白質利用精確的晶片點製手臂將這些個體的蛋白質體點印在一般大小及不同表面化學處理的載玻片上，利用這個高密度及涵蓋整體蛋白質的晶片來研究相關的生物議題，例如蛋白質與蛋白之間的交互作用、蛋白質與脫氧核醣核酸的交互作用、蛋白質與核醣核酸交互作用或蛋白質與化學分子交互作用的研究。我們利用化學的方法合成了常見的四種髮夾核醣核酸，這四個髮夾核醣核酸之間分別只有在第二個核甘酸不一樣，藉此探討出不一樣序列的髮夾核醣核酸有什麼不用的結合蛋白。在本論文的研究上我們以C型肝炎的髮夾核醣核酸來與大腸桿菌K-12的蛋白質體微陣列晶片來做反應，藉此找出大腸桿菌與C型肝炎的髮夾核醣核酸反應的蛋白質，後續再討論這些蛋白質在病毒入侵與宿主的免疫機制之間的關係。本研究對人類醫學上常見的病毒－C型肝炎病毒，利用高通量及系統性、及分子層次上的分析，可以發現生物標記與相關疾病的致病機轉。

Microarray has become an important technology for large-scale and high-throughput biology. More than 18000 human proteins, 5000 yeast proteins or 4000 E. coli proteins have been printed on different surface modified slides and implemented in research of protein-RNA interaction. RNA serves several essential functions for all life processes. Identifying proteins that preferentially bind to a hepatitis C virus (HCV) RNA hairpin may help explore functions involved in RNA viral activities or pathway of infection in the host. In our experiment, four synthesized RNA hairpin structures each with nucleotide(s) differences were probed with either yeast or E.coli proteome chip. By using anti-his antibody which was labeled with Cy3 or Cy5 to probe with the proteome chip, we estimated the amount of each protein on certain slides. This approach helps to identify and classify the cellular proteins that can recognize with the RNA hairpins exist in HCV, an important human pathogen. By using this miniature and high-throughput platform, we are able to discovery new biomarker that related to certain diseases.

TABLE OF CONTENT
Preface and acknowledgement ……………………………………………………… i
中文摘要......................................................................................................................ii
Abstract ..………………………………………………..…………………………..iii
CHAPTER 1   INTRODUCTION	1
1–1   Protein microarray technology	1
1–2   Types of protein microarrays	2
1–2–1   Analytical microarray	3
1–2–2   Functional protein microarray	4
1–3   Proteome libraries	5
1–3–1   Strategies of re-combinational cloning	5
1–3–2   Gateway cloning for Human ORFome collection	6
1–4   E. coli K-12 proteome and chip production	7
1–5   Microarray printing platforms	8
1–6   Different slide surfaces to print E. coli K-12 proteome	8
1–7   Quality of protein chip	9
1–8   Aim	9
CHAPTER   2     MATERIALS AND METHODS	11
2–1   Chemicals, reagents and materials	11
2–2   Cell culture and over-expression	11
2–3   Protein purification	12
2–3–1   Protein purification procedure.	13
2–4   Protein array assay	14
2–4–1   Probing protein chips with dual color anti-His antibodies.	14
2–4–2   Probing E. coli K-12 proteome chip with GNRA tetraloops	14
2–5   HS-4800 automatic hybridization chamber	15
2–6   Protein chip image analysis and data analysis	15
CHAPTER   3   RESULTS	16
3–1   GNRA tetraloop	17
3–2   Choosing the slide surfaces and probe concentration	18
3–3   RNA probing with the proteome chip	19
3–4   Optimization of TL2 concentration	20
3–4–1   Proteins that recognize TL2 tetraloop	21
3–4–2   TL2 binding protein on human proteome chip	24
3–5   Purification of candidate proteins	27
CHAPTER 4   CONCLUSION AND DISCUSSION	29
CHAPTER 5   REFERENCES	33

                                

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