人類超氧岐化酵素Mn-SOD的基因選殖及其蛋白質的表現

活性氧分子以及其代謝產物（含氧自由基）具有高度氧化反應能力，可攻擊生物體內分子，如 : DNA、RNA、蛋白質、脂質、醣類與酵素等，造成細胞或組織的傷害。而研究中證實含氧自由基不僅與老化過程有關，並直接或間接的與許多疾病相關，包含: 缺血後灌流、癌症、類風濕性關節炎、呼吸困難症狀、動脈粥狀硬化。另外還有神經方面的疾病:阿滋海默症、帕金森氏症、脊索側柱硬化症 (ALS) 等。人類超氧歧化酵素Mn-SOD為一內生性的清除自由基之抗氧化酵素，對於保護細胞組織免受氧化傷害有明顯之作用，所以我們依此假設如果能定序出人類 Mn-SOD的基因，將其轉殖、純化而能取得具有活性之酵素蛋白，將可能對於氧化相關的疾病之治療有相當的價值。首先從人類肝臟組織中抽取出全部的RNA，藉由反轉錄酵素(Reverse transcriptase)的作用形成cDNA，由洋菜膠電泳分析得知Mn-SOD基因唯一具有667 bp大小的DNA。由此形成20 kDa的蛋白質。配合電泳純析出此片段，再轉形 (transformation)至大腸桿菌來複製基因片段。由西方點墨法我們可知在加入IPTG後，Mn-SOD的酵素可明顯被 IPTG大量表現，而由pyrogallo方法可以測定純化後之 Mn-SOD的酵素活性。我們的實驗結果顯示Mn-SOD的酵素活性和金屬錳離子濃度，是呈現dose-dependent的關係。我們同時以真核生物Baculovirus 蛋白質表現系統，用以表現人類Mn-SOD 基因，實驗結果相同。因此我們由此結論出要定序人類Mn-SO基因並合成具有活性之Mn-SOD酵素，並予純化是可行的，而且可能具有進一步運用在治療與氧化相關之疾病上。

關鍵字

人類超氧岐化酵素

並列摘要

Reactive oxygen species and other oxidants cause DNA damage, lipid peroxidation, protein denaturation, cell death and resultant organ injury. Substantial evidence suggests that oxygen free radicals are associated with not only aging process but also degenerative disorders, such as Parkinson’s disease, Amyotrophic Lateral Sclerosis, atherosclerosis and Rheumatoid Arthritis. Free radicals are also reported to be associated with malignancy, ischemia/reperfusion and inflammation. Manganese superoxide dismutase (Mn-SOD), an endogenous superoxide radical-scavenging mitochondrial enzyme, is crucial for protecting cells and tissues from oxidant injury and hyperoxia. Enhancement of endogenous cellular anti-oxidation capacity by introduction of Mn-SOD cDNA or full-length active Mn-SOD protein into cells may, therefore, offers attractive therapeutic strategy for diseases associated with oxidative stress. We therefore sought to sequence the human Mn-SOD gene and purify the active human Mn-SOD protein. Human Mn-SOD gene library was established by reverse transcription and polymerase chain reaction (RT-PCR) of the total mRNA extracted from human liver tissue. Agarose gel electrophoresis indicated that Mn-SOD gene is a 667 base pairs DNA which encodes a 20 kDa protein. SOD expression vector was thus constructed and transformed into Escherichia coli (E. Coli) for gene expression. Western blotting analysis indicated that Mn-SOD could be over-expressed in the presence of IPTG. After isolation and purification, the enzyme activity of recombinant human Mn-SOD was assayed by using pyrogallo method. Our data indicated that Mn-SOD enzyme activity was Mn2+ ion concentration dependent. Baculovirus expression system was also employed to test the expression of Mn-SOD gene. Similar results were observed. We, therefore, concluded that sequencing of human Mn-SOD gene and synthesis of active recombinant human Mn-SOD protein are feasible and can contribute to future therapeutic strategies against diseases associated with oxidative stress.

並列關鍵字

Mn-SOD

參考文獻

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人類超氧岐化酵素Mn-SOD的基因選殖及其蛋白質的表現

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