免疫球蛋白IgA在免疫系統中扮演著重要的角色，尤以在黏膜表面對抗外來微生物病原體的侵入為著。但部分的致病菌，如流行性感冒嗜血桿菌(Haemophilus influenzae)及奈瑟氏腦膜炎雙球菌(Neisseria meningitides)等能夠分泌IgA1蛋白酶水解IgA1，使IgA1失去作用，甚至造成後續免疫反應失效的嚴重後果。前驅IgA1蛋白酶主要由四個不同的區域組成：(1)訊息肽，引導蛋白酶前驅物到達膜間質；(2)IgA1蛋白酶區域，約105kDa大小，可水解靈長類的IgA1；(3)α蛋白區域，其功能未知；(4)自體運輸蛋白質區域，可將蛋白酶及α蛋白質轉運到細胞外。過去對於IgA1蛋白酶的研究，主要集中在蛋白酶如何水解IgA1等的作用方面以及C端的自體運輸功能等。對於α蛋白區域並無太多的著墨。本研究以奈瑟氏肺炎雙球菌的iga (Gene Bank DQ683357)為模版，使用PCR擴增其α-protein的DNA片段，再以不具IgA1蛋白酶活性的大腸桿菌UT5600品系表達重組的α-protein，利用人類淋巴胞癌細胞進行細胞增生的試驗研究，借以評估IgA1蛋白酶的α-protein對淋巴癌細胞生長的影響。結果表明，包含medium control和GST control，在濃度1 μg/ml的α-protein處理下，對人類淋巴癌細胞生長沒有影響。但在第2、4、6天中，當α-protein濃度增加到5 μg/ml或10 μg/ml時，α-protein似乎可以促進細胞的生長，本研究結果指出，α-protein確實對人類淋巴癌細胞生長有所影響。

Immunoglobulin A (IgA), a major serum immunoglobulin and a predominant antibody in the external secretions that bathe mucosal surfaces, plays key roles in immune protection. Some pathogenic bacteria including Haemophilus influenzae and Neisseria meningitides, however, produce a protease called IgA1 protease to impair the function of IgA1. The iga mRNA is initially translated into a large precursor containing four distinct domains: a 31-amino acid signal peptide which leads the precursor to the periplasmic space, an 105-kDa protease domain which cleaves host IgA1 molecule, a β-domain responsible for autotransportation of the protease domain, and a linker α-protein between the protease and the β-domain. The hydrolytic function of the protease and the role of the β-core had been studied extensively, but the role of the α-protein has never been studied. Thus this study is designed to reveal the possible functions of α-protein in the proliferation of lymphocytes. To complete the project, PCR was used to amplify the DNA fragment for α-protein using iga gene (Gene Bank DQ683357) as template. The fragment spans nucleotide numbers of 1015-1405. The fragment was then cloned into pGEX-2T for expression. Recombinant α-protein was purified using glutathione -Sepharose column. The purified recombinant protein did not seem to affect the cell growth at the concentration of 1 μg/ml compared with the medium control or GST control. Interestingly, when the concentration was increased to 5 μg/ml or 10 μg/ml, α-protein seems to enhance the cell growth on the 2nd, 4th and 6th day assays, The results suggested that α-protein may enhance the cell growth in 4 days.

中文摘要
英文摘要
第一章 概論
一、IgA1蛋白酶的結構………………………………………………………4
二、免疫球蛋白A (IgA) …………………………………………………………6
三、IgA1蛋白酶的角色………………………………………………………8
四、IgA1蛋白酶的自動轉運………………………………………………………8
五、奈瑟氏腦膜炎雙球菌(Neisseria meningitides) ……………………………10
六、奈瑟氏腦膜炎雙球菌(Neisseria meningitides)的α蛋白區域………………11
七、研究目的………………………………………………………………………12
第二章 材料與方法
實驗材料來源……………………………………………………………………………14
實驗方法
一、分子生物學方法………………………………………………………………17
1. 聚合酶鏈反應(Polymerase Chain Reaction, PCR)
2. 瓊脂凝膠電泳(Agarose Gel Electrophoresis)檢測PCR產物
3. 接合反應(Ligation)
4. 勝任細胞(competent cell)的製備
5. 轉化作用(Transformation)
6. 質體的純化
7. 限制酶切割及質體片段從凝膠中的回收
二、蛋白質分析方法………………………………………………………………20
1. 細菌的培養及基因表達的誘導
2. 融合蛋白的親和層析純化
3. 測量蛋白質液的消光值
4. 蛋白質透析
5. SDS-聚丙烯醯胺膠體電泳 (SDS-PAGE)
三、細胞培養及分析方法…………………………………………………………22
1. 免疫細胞株之生長與培養
2. 細胞增生分析 (cell proliferation assay)
四、統計分析………………………………………………………………………22
第三章 重組α-蛋白質的設計、基因改造、表達及純化
一、α-蛋白區段的基因片段聚合酶鏈反應………………………………23
二、選殖α-蛋白區段……………………………………………………23
三、確認表達載體………………………………………………………24
四、表達載體在大腸桿菌株JM 109品系的接合反應…………………24
五、表達載體在大腸桿菌株UT5600品系的接合反應…………………24
六、基因定序……………………………………………………………24
七、重組蛋白的純化……………………………………………………25
八、蛋白質透析…………………………………………………………25
第四章 細胞測試…………………………………………………………………29
第五章 結論與討論………………………………………………………………33
参考文獻……………………………………………………………………………38

















圖表編號：
表一、IgA主要的理化特性和生物學特性…………………………………………6
圖1、瓊脂凝膠電泳分析PCR反應擴增的結果……………………………………39
圖2、瓊脂凝膠電泳分離α-蛋白片段與中間載體連接產物酶切結果圖…………40
圖3、瓊脂凝膠電泳分析重組基因的限制酶片段…………………………………41
圖4、在大腸桿菌JM109品系內，瓊脂凝膠電泳分析重組基因的限制酶片段……42
圖5、在大腸桿菌UT5600品系內，瓊脂凝膠電泳分析重組基因的限制酶片段……43
圖6、重組蛋白質部分純化物未透析前的SDS-PAGE電泳分析圖…………………44
圖7、重組蛋白質部分純化物透析後的SDS-PAGE電泳分析圖……………………45
圖8、各種濃度GST細胞增生分析結果………………………………………………46
圖9、以1 μg/ml的α-protein處理細胞後，細胞增生分析結果………………………47
圖10、以5 μg/ml的α-protein處理細胞後，細胞增生分析結果………………………48
圖11、以10 μg/ml的α-protein處理細胞後，細胞增生分析結果…………………49
圖12、以各種濃度(0、1、5、10 μg/ml)的α-protein處理細胞，細胞增生分析結果..50
圖13、細胞計數結果圖………………………………………………………………….51
附圖1A、絲胺酸免疫球蛋白A1蛋白酶結構示意圖………………………………52
附圖1B、含金屬離子的IgA1蛋白酶結構示意圖…………………………………52
附圖2、人類IgA1和IgA2鉸鏈區和各種細菌產生的IgA1蛋白酶的水解位點…..53
附圖3、前驅IgA1蛋白酶四大結構區的概要圖……………………………………54
附圖4、人類IgA………………………………………………………………………55
附圖5、Neisseria meningitides strain 430)IgA1蛋白酶的胺基酸全序…………………56
附圖6、第五型蛋白質分泌途徑(Type V protein secretion pathway) …………………57
附圖7、β區域形成管狀的二種型式……………………………………………………58
附圖8、WST-1分裂為formazan之機轉………………………………………………59
附表1、t-test多變異數測驗檢定組間差異之結果……………………………………66

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