臺灣博碩士論文加值系統

Streptococcus gordonii是草綠色鏈球菌種( viridans group strepto- cocci )之一，主要會在牙齒表面上形成牙菌斑引發齲齒。 超氧化歧化酵素( superoxide dismutases，SODs )為一種抗氧化酵素，會去除O2-以避免細胞遭受侵害。 本研究抽取S. gordonii細菌溶解液偵測SOD酵素活性，並用H2O2及DDC ( diethyldithiocarbamate )進行SOD活性抑制實驗，結果證實此菌的SOD為MnSOD。 SOD酵素活性在S. gordonii生長至靜止期時，達到最高，從指數期開始至靜止期止，增加了3到4倍。 為了探討金屬離子對S. gordonii SOD酵素活性之影響，將細菌培養在含有不同濃度Fe離子及Mn離子之培養基中，偵測培養24小時之菌體中SOD的活性。 結果證實S. gordonii的SOD蛋白質可以用Mn離子或Fe離子作為輔助因子而使SOD酵素活性恢復，其中，Mn離子恢復的酵素活性比Fe離子高，此SOD蛋白質為變異酵素( cambialistic enzyme )。 此結合Mn離子或Fe離子的酵素，其活性都不會被H2O2抑制。 本研究依據其他細菌SOD胺基酸序列的保守序列設計的一對引子，經PCR得到sod基因片段，為480 bp的部份序列，再利用genome Walker系統的方法選殖S. gordonii的sod基因的完整序列。 此基因含有603 bp的open reading frame，可轉譯出25.3 kD的SOD蛋白質。 此S. gordonii sodA基因的核酸序列與S. pneumoniae比對有高達81.51﹪的相同性，與S. agalactiae則有76.4﹪的相同性，SOD胺基酸序列與S. pneumoniae及S. mutans比對有89.05﹪及78.6﹪的相同性，相似性則高達90.54﹪及80.59﹪。將嵌入有S. gordonii sodA基因的pDESTTM15表現載體，轉形至E. coli BL21-SITM competent cells。 經誘導表現，生成約23 kD的蛋白質，且仍具有SOD酵素活性。

Streptococcus gordonii is a species of the viridans group streptococci, and is the major pathogen of dental caries. Superoxide dismutase, SOD, is an antioxidant enzyme, which scavenges O2-to protect cells from damage. In this study, we characterized the SOD of S. gordonii as an MnSOD by H2O2 and diethyldithiocarbamate inhibition test. Furthermore, we also found that the SOD has activity as a cambialistic enzyme, which can accept either iron or manganese as the cofactor to present enzyme activity. The activity of manganese —containing SOD was higher than that iron-containing SOD. The SOD activity of S. gordonii was variable in the growth phase of the organism, and was found three to four folds increasing from the exponential phase to the stationary phase.
The sodA gene of S. gordonii was cloned in this study. The gene contains 603 bp nucleotides. The nucleotide sequence of sodA has 81.51﹪identity to that of S. pneumoniae, and 76.4﹪to that of S. agalactiae. The amino acid sequence of this gene has 89.05﹪identity to that of S. pneumoniae, and 78.6﹪to that of S. mutans. It also has 90.54﹪similarity to that of S. pneumoniae, and 80.59﹪to that of S. mutans. The coding region of gene was recombined with native expression vector pDESTTM15 and was transformed into E. coli BL21-SI strain. After induction, the transformant expressed about 23 kD protein, which showed SOD activity by native PAGE gel analysis.

中文摘要………………………………………………I
英文摘要……………………………………………..III
緒論……………………………………………………1
實驗方法……………………………………………..10
實驗材料……………………………………………..25
結果…………………………………………………..31
討論…………………………………………………..37
圖表…………………………………………………..46
參考文獻……………………………………………..70

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