臺灣博碩士論文加值系統

根據文獻報告指出，當寄生蟲感染宿主時，蟲體所分泌的天門冬胺酸蛋白酶具消化分解宿主血紅蛋白的功用。本實驗由廣東住血線蟲（Angiostrongylus cantonensis）的表現序列標幟（Expressed Sequence Tag, EST）資料庫比對發現，編號AcL5-C160的序列與其他線蟲所分泌的天冬胺酸蛋白酶（aspartic protease）序列極為相似。藉由5’RACE找出5’端缺失的部份得到AcL5-C160的序列全長，得到長度為1,279 bp的開放讀架，轉譯出的蛋白質長度為425個胺基酸，分子量約為46 kDa、pI值約為5.8。分別與Caenorhabditis elegans、 Necator americanus與 Haemonchus contortus的天門冬胺酸蛋白酶作胺基酸序列的比較，其相似性介於42–52%之間，且立體結構極為相似。經過胺基酸序列分析後，刪除蛋白質訊號序列，找出抗原決性較強的區域，以人工方式合成15個胺基酸的胜肽鏈，並將之以皮下注射免疫兔子，抽取兔子血清得到多株抗體，分別與第五期幼蟲體抗原和蟲體排泄分泌產物（ESP）進行西方墨點法分析，發現在蟲體的本身與蟲體排泄分泌產物中可能都存在著天門冬胺酸蛋白酶。

Aspartic proteases play a pivotal role when the helminthes invade the host. Helminth aspartic proteases facilitate the invasion of host tissue and digest host hemolglobin as a food source. An aspartic protease homologues EST, AcL5-C160 has been isolated form our Angiostrongylus cantonensis EST library. 5’RACE was used to obtain the full length mRNA of this gene. The complete open reading frame（ORF）of the AcL5-C160 is 1,278 bp. The putative ORF encode protein of 425 amino acid residues with predicted molecular weight of 46 kDa, isoelectric point 5.8. The amino acid sequence of this aspartic protease from A. cantonensis showed similarity between 42–52% with Caenorhabditis elegans、Necator americanus and Haemonchus contortus. 3D model also showed similarity by ExPASy Molecular Biology Sever. We forecast the antigenic sequence and synthesize a 15 amino acids polypeptide. Rabbits were immunized with this synthetic polypeptide and obtained polyclonal antibodies. Western blotting was used to recognize the AcL5-C160 aspartic protease. We find aspartic proteases may exist in the somatic antigen and excretory/secretory products (ESP) of the fifth-stage larvae of A.cantonensis.

目 錄 …………………………………………… I
中文摘要 …………………………………………… II
英文摘要 …………………………………………… III
序 論 …………………………………………… 1
材料與方法 …………………………………………… 9
結 果 …………………………………………… 18
討 論 …………………………………………… 24
參考文獻 …………………………………………… 29
附圖與表格 …………………………………………… 35
附 錄 …………………………………………… 55

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