beta-Bungarotoxin純化自台灣雨傘節(學名Bungarus multicinctus，俗名Taiwan banded krait)蛇毒，由A chain與B chain兩個Subunits以雙硫鍵鍵結組成。 本實驗經由離子交換樹脂及高壓液相層析儀自雨傘節蛇毒中純化出BM12與BM13兩個新的beta-Bgt isotoxins，並利用質譜分析及N-termianl sequencing和peptide mapping得知BM12與BM13可能由相同A chian但不同B chain所組成。 將BM12及BM13進行抗原性、Phospholipase A2活性、二級結構及螢光分析，顯示可能由於B chain影響使得BM13抗原性大於BM12，同時也使得BM13 PLA2活性較BM12高，在與二價離子反應上BM12與BM13之酵素活性會受Sr2+、Ba2+離子所抑制，而且BM12與BM13對Mg2+、Ca2+、Sr2+、Ba2+之結合能力並不相同，可知不同A chain及B chain subunits的組合方式，會造成b-Bgt活性及結構上的差異。
Yeast-Two-Hybrid系統中，beta-Bgt B chain會和一potassium channel binding protein作用，將之轉形於E. coli系統中表現。 二級結構及螢光分析顯示，自E. coli系統表現之potassium channel binding protein具有Ca2+結合能力，證實其EF hand為具有功能之單元結構，但在In vitro cross-linking及Co-immunoprecipitation實驗中並無法顯現B chain可能和此一蛋白產生結合反應。
以PCR方式自雨傘節染色體放大出四段不同A chain genes，分別為A1 chain、A1a chain、A2 chain及A8 chain genes，四段基因之核苷酸序列同源性高達97.5%，與cDNA進行分析比對發現A1 chain之Signal peptide region有一段Intron的Insertion，使得A1 chain gene具有4個Exon及3個Intron，而A2 chain gene則使用相對於A1 chain gene鄰近的Intron 1序列與exon 2 region形成其exon 1，造成A2 chain只有3個Exon與2個Intron，也因此導致A2 chain signal peptide序列只有25個胺基酸，不同於A1 chain有27個胺基酸的Signal peptide。 然而，A1及A2 chain genes之 Promoter region序列及Transcription factor binding-sites具有極高的保留性，認為A chain genes可能由相同的機制所調控。 5’-RACE的分析指出，Bungarus multicinctus毒腺內之mRNAs中，27個胺基酸的Signal peptide 是A chain mRNAs中Pre-dominant species。 將目前已發表之Phospholipase A2 genes和cDNAs進行分析比較，證實此為首次提出蛇毒蛋白藉由Exon skipping方式產生不同的Signal peptide sequence。

beta-Bungarotoxin (beta-Bgt), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of the A chain and the B chain, cross-linked by an interchain disulfide bond. In this study, two novel beta-Bgt isotoxins were purified from Bungarus multicinctus venom by the combinations of ion-exchange chromatography and reverse phase HPLC. Amino acid sequencing, peptide mapping and mass analyses revealed that they probably contained the same A chain, but their B chain differed. Consequently, the discrepancies in their biological activity and fine structure reflected the role of B chain in intact of beta-Bgt. In Yeast-Two-Hybrid system, a potassium channel binding protein was identified to interact with the B chain of beta-Bgt. Although the recombinant potassium channel binding protein functionally bound with Ca2+, but it could not prove to bind with BM12 and BM13 as revealed by in vitro cross-linking assay.
The A chain genes including A1 chain, A2 chain and A8 chain genes were amplified by PCR reaction. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with A chain cDNAs revealed that the A1 chain gene was organized with four exon and three intron, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponds to the first exon of A1 chain gene is skipped instead of inclusion of intronic sequence adjacent to the second intron. The resulting A2 chain mRNA encoded a 25 residues signal peptide, which different from A1 chain mRNA with a 27 residues signal peptide. Comparative analyses on phospholipase A2 genes and cDNAs suggest that this is the first report on skipping of exon changes the signal peptide sequence of snake venom proteins.

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英文摘要------------------------------5
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實驗材料------------------------------18
實驗方法------------------------------20
實驗結果------------------------------34
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參考文獻------------------------------91

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