當要進行基因改造，或外來生物材料進入生物體時；例如：DNA疫苗或基因治療等，都需藉由外在物理、化學和生物性物質或精密儀器的協助。而其中DNA疫苗為1990年代新興的方法，不但可設計所要的特定蛋白抗原，且能有效的引起細胞性，以及體液性的免疫反應，形成長時間的保護作用。然而DNA疫苗要發揮功效，至少必須先突破細胞膜和核膜這兩道屏障，才能使DNA能經轉錄、轉譯，產生抗原蛋白，引起免疫反應。所以發展一個無細胞種類、無DNA序列、DNA構形等限制的運送DNA系統，是很重要的。近年來的研究中提到，帶正電的胜肽能有效穿透細胞膜，而這些胜肽都與protein transduction domain (PTD)有相關性，提供了外來物質輸送到活體細胞，或體外細胞的可能性。在此，我們分三個方面，來進行輸送蛋白的功能和特性分析。就保存而言：融合蛋白Pep-1-TP在pH值7±0.5左右的緩衝溶液中，置於4℃、-20℃、-80℃環境下，至少能穩定保存六個月，無明顯降解現象。而在轉染哺乳類細胞所需的37℃下，24小時後，仍有結合的能力。再者：從膠體延滯分析的結果可知道，Pep-1-TP和外來質體DNA結合是有特異性的，不受限於DNA大小(3kb~8kb)。最後，In vitro實驗方面，從螢光顯微鏡下觀察所表現的綠螢光蛋白，來判斷傳送情形。結果顯示，Pep-1-TP:質體DNA-EGF P-N1，在莫耳濃度(molar ratio)20:1下轉染細胞，有最佳的傳送效率。而且，傳送效率也會受到細胞株種類的影響。在融合蛋白對於細胞株的傷害方面，藉由比色法分析(MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide, based)得知，Pep-1-TP對細胞的毒性，相較於市售脂質體小。In vivo實驗方面，以母小鼠為實驗動物，依照莫耳濃度20:1混合的Pep-1-TP:pCMV-Mx-EGFP-N1，進行肌肉注射法(muscular injection, i.m.)。每隔兩週再注射一次，共重複兩次，收集鼠血清，進行酵素連結免疫吸收分析(enzyme-linked immunosorbent assay, ELISA) 。結果可知，Pep-1-TP在初次注射時，的確有協助外來質體DNA傳送到細胞內，然後引起動物自身的免疫反應。 因此，對於外來具生物活性材料的傳送，也許是快速且安全的方法。

　When you proceed to genetically modified organisms researches or foreign biological materials delivered studies; such as DNA vaccines or gene therapies, it’s essential that external physicals or chemicals means, biological vectors or precision instruments. A DNA vaccine being invented around 1990, it can induce cellular and humoral immunity and you can design the DNA sequence for your wanted by this way. However, a DNA vaccine must surmount two barriers, membranes and nuclear membranes, at least, thus DNA can be transcribed and translated. Therefore, it’s important to develop a DNA delivery system without cell types, DNA sequence and size. In recent researches, cationic peptides which can transfer to cells are related to protein transduction domain. It’s potential that foreign materials are delivered to cells in vitro or in vivo by this mean. Therefore, we experimented with functional and characteristic assays of the delivered protein in three aspects. Firstly, about storage, keeping in 4℃, -20℃, -80℃ refrigerators, fusion protein, Pep-1-TP, in pH7 buffer could be stable without significant degeneration at least in six months. And, we treated Pep-1-TP in a 37℃ environment where it transfer to mammalian cell lines and could still keep its binding ability with plasmids after a treated period of 24hrs. Besides, according to gel retardation assays, Pep-1-TP bound specifically with foreign plasmid DNA and without the size of it. Finally, by a fluorescent microscopy for observations, it was the best delivery efficiency as we proceed to the delivery experiments in vitro with the molar ratio of Pep-1-TP: plamid-EGFP-N1 of 20:1. In addition to, the cytotoxicity of Pep-1-TP to cell lines was lower than the commercial lipofectamine. In vitro, distinguish delivery results: the amounts of GFP were determined by a fluorescent microscopy. It’s the best delivery efficiency of transferring to cells by the mixture of molar ratio of Pep-1-TP: plasmid DNA-EGFP-N1 of 20:1. Furthermore, the delivery efficiency was related to cell lines. A cytotoxicity of the fusion protein, Pep-1-TP, was lower than commercial lipofectamine according to colorimetry assay (MTT based) results. In vivo, injecting a molar ratio of Pep-1-TP: pCMV-Mx-EGFP-N1 of 20:1 into female mice by using muscular injection, collecting serum from mice for the experiments, enzyme-linked immunosorbent assay (ELISA), the results showed that Pep-1-TP certainly promoted the delivery of foreign plasmid DNA into cells with the first vaccination and induced animals’ autoimmunity. Therefore, it may be a fast and safely method to delivery biologically active cargo molecules.

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