臺灣博碩士論文加值系統

以前的研究指出溶血蛋白（hemolysin）在各物種間的分佈範圍很廣，從細菌、蜜蜂、毒蛇、毒蛙到人類都有被發現。它們共同的特徵是會在紅血球培養基上造成b型溶血。其中，酵素型溶血蛋白的神經鞘磷脂水解脢算是比較少見的，但卻是近年來最受到重視的。
在1997年時Liu, B.和Pena, L.A.等人發現神經鞘磷脂水解脢在細胞凋亡（apoptosis）的訊息傳導上扮演了一個相當重要的角色，它能將細胞膜表面的sphingomyelin分解成ceramide + phosphocholine，而分解出來的ceramide被證實是一個secondary messenger，它具有調控細胞凋亡、細胞分化、細胞增生的作用。
本篇論文從具有溶血活性的細菌Bacillus subtilis的培養開始，利用FPLC（快速蛋白質液相層析儀）經過陰離子交換管柱、膠粒過濾管柱去純化分泌到培養基中的蛋白質，得到一個具有溶血活性的蛋白質。將此溶血蛋白做N-terminal定序並與蛋白質資料庫比對後發現可能是神經鞘磷脂水解脢。
藉由之前已知的Bacillus cereus的神經鞘磷脂水解脢的DNA序列設計一對引子（primer），從Bacillus subtilis的基因中釣出對應的基因，再送入pUC19做DNA的定序後，確定此一溶血蛋白是神經鞘磷脂水解脢。為了之後的研究方便，於是決定再將這個神經鞘磷脂水解脢的基因重組到一蛋白質表現質體，pET21a之中，並轉型至大腸桿菌BL21(DE3)之中以建立一個完整的表現及純化系統。

Previous reports point out that hemolysin has been found in a wide range of species from bacteria, bee, snack, frog to human. The common feature of hemolysin is one of the known hemolysin, b-hemolysis on blood-agar plate. Sphingomyelinase is rarer than other hemolysin, but it has attracted attention in recent years.
Liu, B. et al and Pena, L. A. et al in 1997 discovered that sphingomyelinase plays an important role in apoptosis.It can hydrolyse sphingomyelin on cellular membrane into ceramide and phosphocholine and the released ceramide acts as a secondary messenger to induce apoptosis, cell differentiation and cell proliferation.
This study starts from culturing of hemolytic Bacillus subtilis, purification of hemolysin from culture medium by anionic exchange column and gel-filtration column. The purified hemolysin was proved to be sphingomyelinase by protein N-terminal sequencing method and the alignment of the N-terminal sequence to a protein databank.
We designed a pair of primers based on the DNA sequence of Bacilus cereus sphingomyelinase that has been published. We then used PCR method to amplified the gene from Bacillus subtilis genomic DNA and cloned it into a pUC19 plasmid for DNA sequencing analysis. The DNA sequence data showed that the cloned gene is encoding sphingomyelinase. To express sphingomyelin, we inserted the gene into a expression vector, pET21a and transformed it into the E.Coli strain, BL21(DE3).
The recombinant sphingomyelinase with molecular weight of 36.7 kDa has hemolytic activity.

頁數
目錄 I
圖目錄 III
表目錄 V
中文摘要 IV
英文摘要 VII
重要名詞縮寫表 IX
第一章 緒論
第一節 溶血蛋白的簡介 1
第二節 神經鞘磷酯水解脢的研究現況 4
第二章 實驗材料及方法
第一節 溶血蛋白的純化、定量、氨基酸定序
及活性分析 7
第二節 神經鞘磷脂水解脢的基因選殖 11
第三章 結果 20
第四章 討論 23
圖表 27∼55
參考文獻 56∼59

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