有鑑於基因治療的潛力，發展效率高又具安全性的基因輸送系統是首要工作。蛋白質轉導區(Protein transduction domain, PTD)是種具有穿透細胞膜能力且對細胞傷害性低的胜肽，所以本實驗室設計了PTD-TP重組融合蛋白來發展DNA輸送系統。PTD-TP以富含疏水性胺基酸的蛋白質轉導區做為穿透細胞膜的工具；在C端融合人類拓樸異構酶第一型(human topoisomerase I)胺基端第3至第200胺基酸片段，可與DNA結合且具有nuclear localization signals (NLS)能將DNA送至細胞核內。本論文利用EMSA實驗證實重組融合蛋白PTD-TP與DNA的結合並不受DNA序列和構形影響。In vitro實驗方面，藉由報導基因luciferase活性分析得知，重組融合蛋白PTD-TP的大小及His-Tag均會影響轉染效果；此外也證實富含疏水性胺基酸的蛋白質轉導區能直接轉運天然活性態的蛋白，不受重組融合蛋白構形的限制。也利用低溫及ATP抑制劑等實驗證實重組融合蛋白PTD-TP/DNA複合體是經由內吞路徑(endocytosis)的機制進入細胞中。由於PTD-TP C端融合了人類拓樸異構酶第一型的胺基酸片段，所以也嘗試以真核生物系統作表現。證實重組融合蛋白PTD-TP可在真核生物利什曼原蟲Leishmania tarentolae系統中表現。

The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. Here, We described a protein-based gene delivery system, PTD-TP, derived from the fusion peptide domain of tryptophan–rich peptide (KETWWETWWTEW) and DNA binding
sequences of 198 amino acids residues of N-terninal of human topoisomerase I.
Delivery of cargoes mediated by protein transduction domain (PTD) attracts a lot of interest due to its transfection efficiency and safety. In the present work, according to the gel retardation assays, PTD-TP can bind DNAs regardless of nucleotide sequences and conformation. By in vitro luciferase activity analysis, we have showed that His-Tag and particle size influences PTD-TP transfection activity. And the hydrophobic peptide can transduce efficiently without pre-treated into denature form. In addition, cellular uptake of PTD-TP/DNA complexes is energy- and temperature- dependent, indicating PTD-TP- mediated gene delivery is probably through the endosomal pathway. Finally, we have proved that PTD-TP can be expressed in Leishmania tarentolae.

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