α-Bungarotoxin (α-Bgt) 屬於一種α-neurotoxin，由台灣雨傘節（Bungarus multicinctus）蛇腺中純化而得，長度為74個胺基酸，並由5對雙硫鍵構成以維持其結構。此次實驗為了探討雙硫鍵對此蛇毒蛋白結構之影響，我們利用定點突變（site-directed mutagenesis）的方法，分別去除5對雙硫鍵其中的一對。將未經突變與突變過後之α-Bgt cDNAs選殖到pET14b與pET32a(+)表現載體中，並轉型（transformation）到Escherichia coli, BL-21(DE3)菌株中。在pET14b表現系統中，重組蛋白是以包涵體（inclusion bodies）存在於中E. coli中，再將重組蛋白在in vitro進行重新折疊（refolding）。另外，在pET32a(+)表現系統中，重組蛋白為可溶性的fusion protein形式存在，並可透過His-Bind resin將其純化。此外，更進一步的以RP-HPLC將protein純化並以SDS-PAGE分析重組蛋白的性質。值得注意的是refolding實驗中，在去除第三對（C29S,C33S）後，對其結構的形成具有正面的幫助。雖然SDS-PAGE的分析中，各蛋白質的移動速率相同顯示其分子量並無差異。但經由native gel分析後，，判斷重組蛋白可能具有雙硫鍵交換的情形產生。根據此一結果研判：α-Bgt可能是透過多樣的路徑以達成雙硫鍵的形成以及結構的折疊。

α-Bungarotoxin (α-Bgt), a α-neurotoxin from Taiwan banded krait (Bungarus multicinctus), consists of a single polypeptide chain of 74 amino acid residues cross-linking with five disulfide bonds. In order to explore the structural dependence of the disulfide bonds in the toxin molecule, the mutants with deleting one out of the five disulfide bonds were prepared by site-directed mutagenesis. The mutated and wild-type cDNAs were subcloned into the expression vectors pET14b as well as pET32a(+), and then transformed into Escherichia coli, BL-21(DE3). The recombinant proteins derived from pET14b expression system were isolated from inclusion bodies of E. coli, and refolding into their folded structure in vitro. Alternatively, the proteins derived from pET32a(+) system were expressed as a fusion protein and purified on a His-Bind resin column. Moreover, the recombinant proteins were further purified using a reverse phase column, and the homogeneity of recombinant proteins was determined by SDS-PAGE analyses. Noticeably, the refolding reaction was beneficially achieved by deleting the disulfide bond Cys29-Cys33. Although SDS-PAGE analyses showed that the recombinant proteins were homogeneous in molecular weight, several disulfide isomers appeared in each protein preparation as revealed by native gel analyses. These observations suggest that the formation of disulfide bonds and folding ofα-Bgt may pass through multiple pathways.

中文摘要----1
英文摘要----2
縮寫--------3
緒論--------4
實驗材料----10
實驗方法----11
實驗結果----21
討論--------26
圖表--------28
參考文獻----49

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