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主。部分的致病菌，如流行性感冒嗜血桿菌(Haemophilus influenzae)可分泌IgA1蛋白酶水解IgA1，使IgA1失去作用，導致後續免疫反應失效產生嚴重後果。前驅IgA1蛋白酶主要由信號肽、IgA1蛋白酶區域、連接區域、自體運輸蛋白質區域組成。過去對於IgA1蛋白酶的研究，主要集中在蛋白酶如何水解IgA1等作用以及羧基端的自體運輸功能等，對於連接區域蛋白並無太多著墨。因此本研究以不分型-流行性感冒嗜血桿菌的iga (Gene Bank DQ683357)為模板，使用PCR擴增連接區域蛋白的DNA片段，再以大腸桿菌UT5600品系表達重組連接區域蛋白，利用人類淋巴癌細胞進行細胞增生及聚集等試驗進而評估其影響。實驗結果顯示，相較於medium control和GST control，重組連接區域蛋白在1 μg/ml的濃度下對人類淋巴癌細胞生長沒有影響，然而在5 μg/ml和10 μg/ml的蛋白濃度下可以促進細胞生長。本研究結果指出，重組的連接區段蛋白可以使人類淋巴癌細胞增生。本實驗會對後續細胞模型提供研究之價值。

Immunoglobulin A（IgA）, the principal antibody class in secretions that bathe mucosal surfaces, acts as an important first line of defense. However, some pathogenic bacteria such as Haemophilus influenzae can produce IgA1 proteases to impair IgA1, especially in human mucosal immune system. IgA1 proteases are characterized by a polypeptide precursor containing four domains, the signal peptide, protease, linking region and the β-domain. The function of the protease and the β-domain (β core) had been studied extensively, but the linking region is less defined, let alone its function. To complete the project, the DNA fragment for linking region was amplified by PCR from iga gene (Gene Bank DQ683353) spanning from NT3130 to NT4686, and then transferred to pGEX-2T for expression. Recombinant linking region-protein was purified using glutathione-Sepharose column. The proliferation assay showed that purified recombinant protein did not enhance cell growth significantly at the concentration of 1 μg/ml compared to either the negative control or GST control; but when the concentration of the recombinant protein was increased to 5 μg/ml or 10 μg/ml, the cell proliferation was significantly stimulated. These results suggest that recombinant linking region-protein contains special element that stimulates the jurkat cell.

英文摘要
中文摘要
第一章
概論
1.1免疫球蛋白A1蛋白酶
1.2免疫球蛋白A1蛋白酶的結構、功能及自動轉運
1.3連接區域
1.4免疫球蛋白A在人類免疫系統的角
1.5不分型流行性嗜血桿菌
研究目的
第二章
材料及方法
實驗材料來源
實驗方法
2.1 分子生物學方法
2.1.1. 引子的設計
2.1.2. 聚合酶連鎖反應
2.1.3. 瓊脂凝膠電泳分析
2.1.4. 接合反應
2.1.5. 勝任細胞的製備
2.1.6 轉化作用
2.1.7. 質體的純化
2.1.8.限制酶切割及質體片段從凝膠中的回收
2.2 蛋白質分析方法
2.2.1 細菌的培養及基因表達的誘導
2.2.2 重組蛋白的親和層析純化
2.2.3 測量蛋白質液的消光值
2.2.4 蛋白質透析
2.2.5 蛋白質電泳
2.2.6 西方墨點法
2.2.7液相層析/質譜儀
2.3細胞培養及分析方法
2.3.1. 活化冷凍細胞
2.3.2. Jurkat細胞株培養
2.3.4. 細胞增生分析
2.4 統計分析
第三章 重組連接區域蛋白質的設計、基因改造、表達及純化
3.1不分型流行性感冒嗜血桿菌IgA1蛋白酶連接區域的基因片段聚合酶鏈反
3.2 選殖不分型流行性感冒嗜血桿菌IgA1蛋白酶連接區域基因片
3.3 基因定序
3.4 蛋白質表達
第四章 細胞測試
第五章 討論與結論
参考文獻

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