分析 Tat-SOD1 融合蛋白

1. 分析Tat-SOD1融合蛋白 Amyotrophic Lateral Sclerosis (ALS) 是一種進行性運動神經肌肉萎縮症，屬於運動神經元疾病( MND )的一種，目前病因不明，臨床上尚無有效治療藥物。ALS可分為兩型 : 85%~90%屬於偶發性( Sporadic, SALS ) ; 其他15%~10% 屬於遺傳性 ( Familial, FALS )。在FALS病人的sod1基因上，已發現約有50種點突變。推測ALS病因可能是 : 由於人體內sod1基因發生點突變，無法清除體內所產生過量的自由基hydroxyl radical (‧OH )，進而攻擊神經細胞造成細胞走向apoptosis死亡。Apoptosis被認為是造成ALS病人運動神經受傷萎縮的主因，因此我們探討突變型sod1基因與神經細胞死亡而誘發的運動神經元疾病ALS之致病機轉的關係。 HIV-1 病毒具有Tat蛋白，此Tat蛋白與HIV-1的複製有關。Tat蛋白主要是由4個domain所組成，其中以basic domain最重要，使病毒的Genome DNA具有轉位的能力。目前的研究已知Tat蛋白可攜帶其他的蛋白質，穿透細胞膜進入細胞內。 PC12細胞是一研究神經疾病良好的細胞實驗模式，可用來探討變異的sod1基因與 ALS 之間的相關性。因此我們以未分化的PC12 cell為細胞實驗模式進行protein ( Tat-SOD1 fusion protein ) transduction的實驗。目前已成功的建立了protein transduction delivery system，超越傳統transfection技術的缺點。 我的主要論文內容是以正常小鼠為動物實驗模式，研究Tat-SOD1 融合蛋白是否可以經由腹腔注射或藉由其他的方式進入老鼠體內，穿透細胞膜及血腦障壁，到達腦部及各個組織器官的細胞內，並觀察其分佈情形。

2. Analysis the distribution of Tat-SOD1 • The most frequent genetic causes of amylotrophic scelosis (ALS) determined so far are mutations occurring in the gene coding for copper/znic superoxide dismutase (Cu,Zn-SOD). The mechanism may involve the formation of hydroxyl radicals or malfunctioning of the SODprotein .The development of peptide drugs and therapeutic proteins is limitedby the pool permeability and the selectivity of the cell membrane. A series of small protein domains , termed protein transduction domains(PTDs) , have been shown to cross biological membranes efficiently and independently of specific transporters and receptors , and to promote the delivery of peptides and proteins into cells.The human immunodeficiency virus type 1 (HIV-1) Tat proteintransduction domain (PTD), which posses a characteristic positive chargeon the basis of their enrichment of arginine and lysine residues activates transcription by specifically binding a stem-loop element in the viral long terminal repeat and is responsible for highly efficient protein transductionthrough the plasma membrane.Denatured Tat-SOD1 fusion protein was observed to successfullytransduced into undifferentiated PC12 cells and retained its activity via protein refolding.It had been demonstracted that intraperitoneal injection of Tat-β-galactosidase fusion protein resulted in delivery of the biologicallyactive fusion protein to all tissues in mice, including the brain (Dowdy, 1999).According to it, we also purify the Tat-SOD1 fusion protein and want to observe that Tat-SOD1 fusion protein also can delivery efficiently to all tissue in mice by intraperitoneal injection.