臺灣博碩士論文加值系統

第一部份：
蟑螂，目前已知可引起許多由人類免疫球蛋白E（IgE）所媒介的過敏性反應，包含有過敏性鼻炎、氣喘…等，但至目前為止，並非所有的致敏原都己被研究透澈，許多來自蟑螂的蛋白，都顯現出IgE結合活性，但僅有少部分有被進一歩分析與研究。為了釐清這一類的蟑螂過敏原，本研究中利用了二維電泳技術並配合N端定序、質譜儀等蛋白質學的方式進行分析。再進一步結合了生物資訊學中過敏原資料庫的運算，來輔助並找尋具潛力的新德國蟑螂過敏原。利用上述的方式，本篇研究中，共鑑定出10種來自德國蟑螂新的IgE結合蛋白而其中aldolase、 arginine kinase、enolase、Hsp70、TIM及vitellogenin蛋白都已在其它可導致過敏的物種，例如：塵蟎、蝦、植物花粉或真菌中，曾被鑑定為該物種的過敏蛋白。但都未曾在德國蟑螂上有過相類似的報告。另一方面，更進一步利用FARRp過敏源資料庫分析的結果也指出：arginine kinase、enolase及TIM這三個來自德國蟑螂的IgE結合蛋白，個別在各個不同致敏物種間的同源致敏蛋白，均存在著相當高的可能性是具有免疫交互作用。在本篇研究中，同時以人體皮膚試驗方式，驗証了vitellogenin蛋白在人體上，確實能引起典型的過敏反應。進一步分析個人化過敏蛋白IgE結合圖譜時也發現：在不同過敏病人上存在著個人化獨特的結合圖譜：不只在人體IgE與測試蛋白間結合的頻度，另外在結合強度上也存在著個人化的差異。這些發現，除了提供更多的蟑螂過敏病研究材料也同時提供了一個可能性，為將來對此類過敏疾病進行個人化醫療或臨床診斷提供更正確的資訊。

第二部份：
德國蟑螂是一種常見的室內吸入性過敏原，並被認為與過敏性氣喘疾病有關。由於其重要性，故常可見於現行過敏血清醫療檢驗項目中。近幾年來，由於分子生物學技術的進步，有許多的過敏原蛋白都已被基因選殖，並提供良好的平台以進行過敏原特性的分析，例如過敏原決定點的分析。本篇的主角，Bla g 9過敏原蛋白，是由德國蟑螂上，藉由蛋白質體學配合免疫學方式，新發現的過敏蛋白。並已完成基因解序及重組表現。利用陰離子交換管柱，我們也成功由德國蟑螂蟲體中，順利純化到均質化的德國蟑螂Bla g 9蛋白。由於Bla g 9蛋白在蛋白序列分析時，被認為是屬於精胺酸激酶酵素家族的一員，經活性鑑定，純化得到的Bla g 9，也的確具有精胺酸激酶的酵素活性。在後續以蟑螂過敏病人血清所進行的ELISA篩檢試驗中，也再次證實了Bla g 9確實具有結合病人血清中IgE抗體的活性，並且具有約五成的盛行率。
利用純化的美洲蟑螂精胺酸激酶過敏原（Per a 9）對Bla g 9所做的免疫抑制實驗證實了，精胺酸激酶是美洲及德國蟑螂中共通的過敏蛋白，並很有可能具有共通的過敏原決定點。為了找尋在Bla g 9上可能的過敏原決定點，我們利用了分子生物學的方式，成功將Bla g 9蛋白利用大腸桿菌分段表現，並檢測了各片段對於人類IgE抗體的結合活性。在後續研究中也發現，當Bla g 9 胜肽片段不包含氨基酸序列280-300時，對IgE抗體結合的能力會受到明顯的影響。但在利用合成胜肽分別合成兩段，包含280-295及290-305的序列時，卻無法保有IgE的結合活性。這結果暗示了在Bla g 9過敏蛋白上重要的過敏原決定點可能是屬於構型性的過敏原決定點。

Part I:
Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach (B. germanica) show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins was separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase (TIM), and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the FARRp allergen database indicated that arginine kinase, enolase, and TIM showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.

Part II:
The German cockroach （B. germanica） is a well-known indoor aeroallergen, considered as one the causative agents of extrinsic bronchial asthma and frequently included in serum test panels for allergic diagnosis. Molecular characterization of allergens by recombinant DNA technology progressed rapidly during the last few years. The Bla g 9 protein, a novel allergen from B. germanica was identified by two-dimensional immunoblotting followed by the molecular cloning, and expression of this allergen as a 6 × His-tagged fusion protein in Escherichia coli. A purified Bla g 9 was obtained by anion exchange chromatography and had arginine kinase activity. It reacted with serum IgE from cockroach-allergic patients and shown high prevalence recognition. In ELISA inhibition assay, we observed that P. americana arginine kinase （Per a 9） inhibited the IgE-binding to B. germanica arginine kinase （Bla g 9）, which indicated the same IgE epitope between arginine kinase from different cockroach species. In order to identify the allergenic determinants of Bla g 9, recombinant （r）Bla g 9 and fragments were also generated, and IgE-binding epitopes were assessed by ELISA. Recombinant fragmented Bla g 9 proteins not containing 280-300 amino acid residues were significantly decrease the reactivity to sera from cockroach sensitized individuals, suggesting that this region contains the IgE-binding epitope. Despite strong IgE reactivity to rBla g 9, the pooled serum from 10 cockroach-sensitized patients did not show IgE reactivity to two synthetic peptides consisting of 15 residues covering 280-305 amino acids. These results suggest the possibility that Bla g 9 may have a conformational epitope in the C-terminal region.

目錄 I
表圖目錄 IX
縮 寫 i
1. 緒 論 1
1.1. 前言 1
1.2. 過敏的因素 3
1.3. 過敏反應機制 4
1.4. 過敏原的種類 9
1.5. 過敏的預防與治療 10
1.5.1. 預防與治療 10
1.6. 蟑螂過敏原 13
1.6.1. 蟑螂與過敏 13
1.6.2. 過敏原的分子特性 15
1.6.3. 蟑螂過敏原的未來研究方向 17
第一部份：利用蛋白質體學方法鑑定德國蟑螂過敏原，並分析過敏病人特異性過敏原反應圖譜 19
中文摘要 20
Abstract 21
2. 實驗材料與實驗方法 22
2.1. 血清樣本 22
2.2. 蟑螂蟲體來源 22
2.3. 儀器及裝置 22
2.4. 大腸桿菌及質體 23
2.5. 酵素 24
2.6. 藥品與試劑組 24
2.7. 德國蟑螂過敏原之鑑定 25
2.7.1. 二次元電泳分析 25
2.7.1.1. 德國蟑螂蛋白質粗萃取物之製備 25
2.7.1.2. 蛋白質濃度的測定 26
2.7.2. 偵測 IgE結合蛋白 27
2.7.2.1. SDS聚丙烯醯胺膠體電泳分析 27
2.7.2.1.1. 板膠的裝置： 27
2.7.2.1.2. 配製separating gel： 27
2.7.2.1.3. 配製3 % stacking gel 27
2.7.2.1.4. Running buffer的製備 28
2.7.2.1.5. 蛋白質樣品的處理 28
2.7.2.1.6. 電泳之操作方法 28
2.7.2.2. 免疫轉漬分析 29
2.7.2.2.1. 蛋白質轉印 29
2.7.2.2.2. 染色與退色 30
2.7.2.2.3. 西方墨點法 30
2.7.2.3. 二維電泳分析 31
2.7.2.3.1. IPG電泳膠片之重新水合化 31
2.7.2.3.2. 第一維的IEF分析 32
2.7.2.3.3. 第二維的SDS-PAGE分析 32
2.7.2.3.4. 免疫轉漬分析 33
2.7.3. 蛋白質定序分析 33
2.7.4. 質譜分析 34
2.7.4.1. In-gel digestion 34
2.7.4.1.1. 還原及 4-vinylpyridine基化反應 34
2.7.4.1.2. 膠體原位酵素切割 34
2.7.4.1.3. 萃取切割片段 34
2.7.5. 利用液相層析串聯式質譜儀進行蛋白質鑑定分析 35
2.7.5.1. 胜肽樣品的製備 35
2.7.5.2. 液相層析進樣 35
2.7.5.3. 電灑-四極棒-飛行式質譜分析 36
2.7.6. 德國蟑螂過敏原之選殖 36
2.7.6.1. RNA的製備及電泳 36
2.7.6.1.1.純化total RNA 36
2.7.6.1.2. RNA電泳 37
2.7.6.1.2.1. 配置10 × MOPS buffer 37
2.7.6.1.2.2. 配置loading dye 37
2.7.6.1.2.3. 純化mRNA 38
2.7.7. 建構cDNA library 38
2.7.7.1. 合成第一股cDNA 39
2.7.7.2. 合成第二股cDNA 39
2.7.7.3. Adaptor接合反應 40
2.7.7.4. 標的蛋白質基因的選殖及Rapid Amplification of cDNA Ends (RACE) 40
2.7.8. DNA電泳 40
2.7.8.1. 20 × TAE buffer的製備 40
2.7.8.2. 分析 agarose gel 41
2.7.8.3. 自瓊膠電泳片中純化PCR產物 41
2.7.8.4. pGEM-T Easy Vector的接合反應 41
2.7.8.5. 大腸桿菌轉型作用 42
2.7.8.5.1. Competent cells之製備 42
2.7.8.5.2. 轉形作用之步驟 42
2.7.8.5.3. 轉形基因庫之篩選 43
2.7.8.5.4. 微量抽取質體DNA 43
2.7.8.5.5. 同源性蛋白質之搜尋與排比 44
2.7.9. 利用過敏原資料庫進行致敏性評估 44
2.7.10. 蟑螂過敏原之純化 45
2.7.10.1. 萃取蟑螂蛋白質 45
2.7.11. 蟑螂過敏原之免疫特性分析 46
2.7.11.1. 個人化IgE抗體育過敏原蛋白結合圖譜分析 46
2.7.11.2. 皮膚測試反應 47
3. 結果 48
3.1. 以一維及二維電泳方式，偵測IgE結合蛋白 48
3.2. 蛋白身份鑑定結果 48
3.3. 線上過敏原資料庫分析結果 50
3.4. 過敏病人的個人化IgE抗體與過敏原結合圖譜 51
3.5. 過敏原皮膚測試 51
4. 討論 52
4.1. 蟑螂過敏原的重要性 52
4.2. 蟑螂過敏原的防治 52
4.3. 過敏病人特性分析 53
4.4. 蛋白質身份鑑定 54
4.5. 個別IgE結合蛋白特性分析 55
4.5.1. 卵黃前質蛋白（Vitellogenin） 55
4.5.2. 具交叉免疫反應過敏原潛力的蛋白 57
4.5.2.1. 精胺酸激酶（Arginine kinase） 57
4.5.2.2. 烯醇酶（Enolase） 57
4.5.2.3. 丙醣磷酸異構酶（triosephosphate isomerase, TIM） 58
4.5.2.4. 其它可能的過敏蛋白 58
4.5.2.5. 醛縮酶（aldolase） 58
4.5.2.6. 熱休克蛋白（heat shock protein） 59
4.6. 交叉免疫與過敏 59
4.7. 總結 60
第二部份：鑑定與分析德國蟑螂過敏原Bla g 9之過敏原決定點 62
中文摘要 63
Abstract 64
5. 動機與目的 65
6. 實驗材料與方法 67
6.1. 血清樣本 67
6.2. 蟑螂蟲體來源 67
6.3. 儀器及裝置 67
6.4. 大腸桿菌及質體 68
6.5. 酵素 68
6.6. 藥品與試劑組 69
6..7. 德國蟑螂之cDNA來源 70
6.8. 德國蟑螂與美洲蟑螂天然型精胺酸激酶（Arginine kinase）蛋白之純化 70
6.8.1. 蟑螂的處理 70
6.8.2. 緩衝液的製備 71
6.8.3. 粗萃與硫酸銨分劃 71
6.8.4. 陰離子交換樹酯管柱層析分離法 72
6.8.5. 西方墨點法 72
6.8.6. 利用液相層析串聯式質譜儀進行蛋白質確認分析 73
6.8.7. 德國蟑螂過敏蛋白Bla g 9之精胺酸激酶活性分析 73
6.8.8. 凝集素結合反應 74
6.8.8.1. 配置清洗緩衝液 74
6.8.8.2. 製備明膠填塞緩衝液 74
6.8.8.3. 製備凝集素反應液 75
6.8.8.4. 製備AEC呈色反應液 75
6.8.8.5. 凝集素結合反應 75
6.9. 分段選殖與表現德國蟑螂過敏原-Bla g 9 76
6.9.1. 德國蟑螂過敏原重組胜肽 rBla g 9 各重組胜肽片段之表現與純化 76
6.9.1.1. 重組胜肽基因建構與胜肽表現 76
6.9.1.1.1. 標的基因的 PCR 增幅 76
6.9.1.1.2. 選殖基因DNA片段與pQE30選殖質體的處理 77
6.9.1.1.3. 接合反應 77
6.9.1.2. 在大腸桿菌表現 6 × His-tagged peptide 78
6.9.1.3. 重組胜肽的純化 78
6.9.1.3.1. 配製鎳離子親合性管柱 78
6.9.1.3.2. 6 × His-tagged recombinant （r）Bla g 9 和各胜肽片段的純化 79
6.9.2. Tricine-SDS 聚丙烯醯胺膠體電泳分析 80
6.9.2.1. 配製 separating gel 80
6.9.2.2. 配製 spacer gel 80
6.9.2.3. 配製 stacking gel 80
6.9.2.4 電泳之操作 80
6.10. ELISA（Enzyme-linked immunosorbent assay）與ELISA抑制效應分析 81
6.10.1. ELISA篩檢試驗 81
6.10.2. 粗萃取蛋白的ELISA抑制反應試驗 82
6.10.3. ELISA 交叉反應抑制分析 82
6.10.4. 利用ELISA篩檢試驗各德國蟑螂Bla g 9重組或生合成胜肽片段與人類IgE結合能力 83
6.11. 德國蟑螂過敏原蛋白質Bla g 9 的分子模擬 83
6.11.1. 同源性蛋白之搜尋與排比 83
6.11.1.1 蛋白結構模板搜尋 83
6.11.1.2 相似序列的排比工作 84
6.11.1.3 三維立體結構之分子模擬 84
6.11.1.4 重要抗原決定點胜肽片段之呈現 84
7. 結果 86
7.1. 德國蟑螂過敏原Bla g 9之序列比對分析 86
7.2. 德國蟑螂與美洲蟑螂精胺酸激酶（Arginine kinase）之純化 86
7.3. 利用液相層析串聯式質譜儀進行蛋白質確認與N端蛋白質序列質量圖譜分析 87
7.4. 德國蟑螂Bla g 9之免疫特性分析 88
7.4.1. 病人間盛行率ELISA篩檢試驗 88
7.4.2. Bla g 9對德國蟑螂粗萃取蛋白的IgE結合抑制反應 88
7.4.3. ELISA交叉反應抑制分析 89
7.5. 利用二維電泳與凝集素結合法進行德國蟑螂過敏原Bla g 9的糖蛋白分析 89
7.6. 德國蟑螂過敏原Bla g 9人類IgEs抗體抗原決定基選殖與分析 90
7.6.1. 大片段過敏原決定點辨識圖譜 90
7.6.2. 小片段過敏原決定點辨識圖譜 90
7.7. 三維立體結構之分子模擬 91
7.8. 人類B 細胞過敏原決定點立體位置之呈現 91
8. 討論 93
8.1. 精胺酸激酶過敏原的重要性 93
8.2. 精胺酸激酶的免疫交叉反應 94
8.3. Bla g 9的三級結構預測與過敏原決定點分析 95
9. 參考文獻 100
10、附錄 118

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