臺灣博碩士論文加值系統

在生物的訊息傳導路徑中,已經證明蛋白質激脢可以影響胚胎的發育、免疫反應、細胞的生長分化、癌細胞的增生、細胞的自裁作用，然而在昆蟲體內與免疫相關的蛋白質激脢，所知卻仍十分有限。目前在埃及斑蚊體內發現了一個可能與免疫反應有關的蛋白質激脢Aedes aegypti sterile20 (AaSte20)，這個AaSte 20激脢的cDNA是3,361 bps並包含了963個胺基酸，推測其分子量為107 kDa，在此peptide的N端具有完整的11個激脢的催化區段(domain)，根據它的胺基酸序列分析推斷， AaSte20是一個絲胺酸/酥胺酸的蛋白質激脢，而且含有一典型的ATP-binding區段，在北方墨點分析中也顯示AaSte20的mRNA為3.3 kb，而AaSte20的部分基因序列也在本研究中被確定。利用融合蛋白質生產了一個抗AaSte20的多株抗體，它能在埃及斑蚊和其細胞株(Aag2)中雜合到一個107 kDa的蛋白質，雖然AaSte20的mRNA和蛋白質不會因為細菌的刺激而增加，但也許他針對細菌刺激的反應和一般免疫相關激脢一樣，是在改變激脢磷酸化的活性，而非激脢的總量。

Protein kinases are known to signal messages involved in embryonic development, cell proliferation, differentiation, tumorgenesis, and apoptosis. Studies in immune related protein kinases in insects are still limited. A novel protein kinase, Aedes aegypti sterile 20 kinase (AaSte20), was isolated and characterized preliminary in this study. The cDNA of AaSte20 is 3,361 bps encoding 963 amino acids with a predicted Mr of 107 kDa. The N-terminal of the deduced peptide contains 11 catalytic domains of protein kinases. The sequence of AaSte20 reveals characteristics of a serine/threonine kinase with a putative ATP-binding motif. The Northern blot analysis indicates that the AaSte20 mRNA is 3.3 kb as well. A partial sequence of the AaSte20 gene was resolved in this study. Polyclonal antibodies against the AaSte20 were generated using a fusing protein, and they recognize a 107 kDa protein in mosquitoes and an Aedes aegypti cell line (Aag2). The expression of AaSte20 at levels of mRNA and proteins is not corresponding to bacterial challenge, but as the most of immune regulation kinase, its phosphorylation activity may be affected post bacterial infection instead.

目錄
頁次
中文摘要------------------------------------------------------1
英文摘要------------------------------------------------------2
壹、前言
（一）蛋白質激脢(protein kinase)---------------------------3
（二）訊息傳導路徑(The signal transduction pathway)--------4
（三）Ste20蛋白質激脢(sterile 20 protein kinase)-----------8
（四）研究動機---------------------------------------------9
（五）實驗設計--------------------------------------------10
貳、材料與方法
（一）實驗材料--------------------------------------------12
（二）藥品及器材------------------------------------------12
（三）實驗方法--------------------------------------------14
（四） 本研究所需使用到的試劑成分與配方----------------------30
參、結果
（一）埃及斑蚊AaSte20 cDNA的選殖--------------------------38
（二）埃及斑蚊AaSte20 genomic DNA的選殖-------------------40
（三）埃及斑蚊的AaSte20 mRNA在細菌刺激後表現量的分析------41
（四）抗AaSte20 mRNA多株抗體的製備------------------------42
（五）埃及斑蚊AaSte20的蛋白質表現-------------------------43
（六）AaSte20的激脢活性測定-------------------------------43
肆、討論------------------------------------------------45
伍、誌謝------------------------------------------------49
陸、參考文獻--------------------------------------------50
柒、圖--------------------------------------------------60
捌、附表------------------------------------------------93
玖、附錄------------------------------------------------97

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