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研究生(外文):Chia -Chen Pai
論文名稱(外文):Studies on the Fungal Immunomodulatory Protein and Superoxide Dismutase from Antrodia camphorata
指導教授(外文):Chi-Tsai Lin
外文關鍵詞:Antrodia camphorataFungal immunomodulatory proteinSuperoxide dismutasePolymerase chain reaction
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牛樟芝 ( Antrodia camphorata )為一台灣特有菌種,生長於牛樟樹 ( Cinnamomun kanehirai )中空樹洞中,會造成牛樟樹產生褐腐病(brown rot disease)。據民俗療法的記載,牛樟芝具有保肝解毒的能力,其保肝的機轉目前已知與免疫調節蛋白與抗氧化酵素的功能有極大的關係。
藉由聚合酶連鎖反應( PCR ),進行樟芝Fips與SODs的cDNA與基因選殖,並進行蛋白質表現。依據松杉靈芝、草菇、金針菇的Fips胺基酸序列,在其保守區域設計7組primers,經由多次不同條件的PCR及次選殖之後,目前尚未發現與Fips類似的序列。
在SOD選殖方面,在樟芝的子實體中選殖出3條SOD基因的部分片段,分別命名為AcaMn SOD I、AcaMn SOD II 及AcaFe SOD 。AcaMn SOD I 為genomic DNA,長度為420 bp,可轉譯出140 個胺基酸;AcaMn SOD II 為樟芝cDNA序列,長度為489 bp,可轉譯為105個胺基酸;AcaFe 為樟芝cDNA序列,長度為423 bp,可轉譯出129個胺基酸。
此外,自中研院蕭介夫老師實驗室所建構的樟芝菌絲體EST中取得一條全長Mn SOD的cDNA序列,其全長為954 b.p.,coding region 為693 bp,可轉譯出230 個胺基酸,命名為AcaMn SOD III 。以樟芝子實體的cDNA為模板,依據AcaMn SOD III表現區序列合成2段primers ( AcaMn-a, AcaMn-b )進行PCR,可得到與AcaMn SOD III中相同的Mn SOD 表現區。經由胺基酸比對,得知其與靈芝屬菌類及白枯真菌皆有很高的相似性,並且具有與文獻中所述Mn 離子結合有關的4個胺基酸( His59, His104, Asp189 , His193 ) 與活性區8個胺基酸中提供鍵結形成dimer型式的2個胺基酸 ( Glu192, Tyr197 ) 。將之接在pET-20b(+)中,以BL21(DE3 )pLysS為表現宿主,在C-端未接His-tag表現的plasmid中,可表現具有活性的SOD。另以樟芝子實體或菌絲體genomic DNA為模板,分別與AcaMn-a及AcaMn-b primer進行PCR,兩者均可得到具有4個 intron的SOD基因,且分析其基因序列完全相同。這些結果有助於未來進一步探討Fips與SODs。
Antrodia camphorata one kind of the medicinal mushrooms, can naturally parasite in the punk of Cinnamomun kanehirai Hay. In ethnologic medicine, A. camphorata has the activation of detoxification, anticancer and curing liver complaint. Evidences have showed that Fip and SOD are known on the physiologically active component in mushrooms. This motivates we to clone DNA of Fip and SOD from A. camphorata.
In accordance with the conserve amino acid of Fip or SOD from other species, we try to clone DNA of Fip and SOD from A. camphorata,3 clones of SOD (AcaMn SOD I, AcaMn SOD II, AcaFe SOD) were screened. AcaMn SOD I is a genomic DNA contains 420 bp, and its open reading frame ecoding 140 amino acid residues; AcaMn SOD II is a cDNA contains 489 bp, and its open reading frame ecoding 105 amino acid residues. AcaFe SOD is a cDNA contains 423 bp, and open reading frame ecoding 129 amino acid residues.
Meanwhile, another full-length Mn SOD sequence was obtained from A. camphorata mycelia (kindly given from Dr. Shaw’s Lab) named AcaMn SOD III containing 954 bp, and its open reading frame ecoding 230 amino acid residues. According to AcaMn SOD III sequence, 2 primers were synthesized, when using A. camphorata fruit bodies cDNA as a template, an 0.6 Kb coding region was amplified by PCR. Computer analysis of the residues required for coordinating the single trivalent manganese iron and 11 residues putatively involve in the active center were well conserve among all reported Mn SOD. Whereas, using A. camphorata genomic DNA as a template, an 0.8 Kb was amplified .Sequence analysis revealed that 4 introns were intervened in the AcaMn SOD III.
To furthermore characterize the A. camphorata Mn SOD, the coding region was subcloned into BL21(DE3)pLysS. The active SOD was expressed of the recombinat protein which no His-tag at C-terminal.
中文摘要……………………………………………………………. II
壹、 前言………………………………………………………… … 1
貳、 實驗材料與方法……………………………………………….33
參、 結果與討論…………………………………………………….58
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