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博碩士論文 etd-0620103-142902 詳細資訊
Title page for etd-0620103-142902
論文名稱
Title
台灣眼鏡蛇蛋白酶抑制劑基因選殖及表現
Cloning and functional expression of Taiwan cobra chymotrypsin inhibitor
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
58
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2003-06-10
繳交日期
Date of Submission
2003-06-20
關鍵字
Keywords
抑制劑、台灣眼鏡蛇
taiwan cobra, chymotrypsin inhibitor
統計
Statistics
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The thesis/dissertation has been browsed 5674 times, has been downloaded 4111 times.
中文摘要
目前的研究顯示dendrotoxins和b-Bungarotoxin B chain的胺基酸序列相似於Kunitz-type protease inhibitors。實驗根據b-Bungarotoxin之B chain的cDNA設計引子以RT-PCR方式由台灣眼鏡蛇(Naja naja atra)毒腺total RNA中選殖出chymotrypsin inhibitor,命名為NNACI(Naja naja atra chymotrypsin inhibitor)。NNACI由57胺基酸組成內含六個Cys,前端有24個胺基酸的signal peptide。將NNACI裝載於pET29a(+)轉型入 BL21(DE3) E.coli,由於蛋白質表現於inclusion bodies,便以NTSB進行雙硫鍵重組。以chymotrypsin為作用酵素,進行NNACI抑制活性試驗,將fusion protein以thrombin切除後Ki值有顯著的增加(NNACI fusion protein Ki= 461.3 mM,NNACI Ki= 31.7 mM),進一步漸進移除N-terminal胺基酸,導致抑制活性下降最後完全喪失,因此初步認為N-terminal胺基酸與其抑制活性相關。依據b-Bungarotoxin之B chain基因序列設計引子,以PCR放大NNACI genomic DNA,將其genomic DNA與b-Bungarotoxin之B chain相較,兩者有高達83﹪的相似度,且一樣具有三個exons及三個introns的基因結構,顯示蛇毒protease inhibitor與B chain具有演化上的相關性。
Abstract
Previous studies showed that dendrotoxins and B chain of b-Bungarotoxin shared sequence and structural homology with Kunitz-type protease inhibitors. In the present study, the cDNA of Kunitz–type protease inhibitor was successfully amplified from Taiwan cobra venom gland total RNAs using the primers designed from the B chain of b-Bungarotoxin. The deduced amino acid sequence of the cDNA exhibited the structural character of chymotrypsin inhibitor, and the mature protein contained 57 amino acids with six Cys residues. The chymotrypsin inhibitor was subcloned into pET29a(+) and transformed into BL21(DE3) E.coli strain. The expressed protein was isolated from inclusion bodies of E.coli and subjected to refolding into its folded structure. The inhibitor potency of the recombinant protein on chymotrypsin activity had a Ki value of 461.3 mM. However, removal of its N-terminal fused peptide with thrombin further increased the Ki value to 31.7 mM. Removal of the N-terminal residues further reduced its inhibitory potency, and the inhibitory activity completely lost after deleting three residues at the N-terminus of mature protein. This reflects that the N-terminal region of protease inhibitor should be associated with its activity. The genomic DNA encoding the precursor of the inhibitor was also amplified using PCR. The genetic structure composed of three exons and three introns, which shared the same organization with the b-Bungarotoxin B chain gene. Moreover, the two genes showed a high degree of sequence identity up to 83%. This observation emphasizes the idea that the B chain of b-Bungarotoxin and protease inhibitor are evolutionarily related.
目次 Table of Contents
中文摘要……………………………………………………………… 1
英文摘要……………………………………………………………… 2
緒 論……………………………………………………………… 3
縮 寫……………………………………………………………… 9
實驗材料………………………………………………………………10
實驗方法………………………………………………………………12
實驗結果………………………………………………………………21
討 論………………………………………………………………28
圖 表………………………………………………………………34
參考文獻………………………………………………………………54
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