臺灣博碩士論文加值系統

α-sarcin 已知是由一種名為Aspergillus giganteus絲狀真菌所分泌的核醣毒素，當核醣體作為受質時，α-sarcin會水解大次單元體，作用在鄰近28S rRNA 胺基端的一個磷酸雙脂鍵，並產生一含493個核苷酸的RNA片段，進而造成蛋白質生合成受抑制。為了設計一個能進入細胞、毒殺細胞並自行分解的潛力藥物，我們利用蛋白質工程學的方式將α-sarcin與tat多肽鏈鍵結在一起。tat多肽鏈來自HIV病毒的tat蛋白質，是一種蛋白質傳送區域(protein transduction domain, PTD)具有將與之鍵結的蛋白質帶入細胞之能力。我們希望藉由tat多肽鏈之特性將α-sarcin帶入細胞並執行毒殺細胞之功用。首先，利用幾個分析實驗來檢定PTD-α-sarcin的特性。實驗結果顯示PTD-α-sarcin 帶有較低水解核醣核酸之活性，不會特異地切在一個磷酸雙脂鍵但會水解大次單元體，並且進入細胞效率低。由於以上結果，我們在PTD與α-sarcin間加入GGGS胺基酸序列，改良PTD-α-sarcin為PTD-gggs-α-sarcin。而PTD-gggs-α-sarcin能夠特異性地水解核醣體並較PTD-α-sarcin更有效率地抑制蛋白質的生合成。同時，在共軛焦顯微鏡下觀察發現PTD-gggs-α-sarcin能夠進入到細胞內。

Previously, our laboratory has investigated in the action mode of α-sarcin. α-sarcin is a ribotoxin secreted by filamentous mold, Aspergillus giganteus. When ribosomes are the substrate, α-sarcin cleaves a single phosphodiester bond at a universally conserved sarcin domain of 28S rRNA, then inactivates ribosome and inhibits protein synthesis. In general, α-sarcin is incapable of entering cell which has greatly limited the potential usage of α-sarcin for medical purpose. In this study, we attempted to design a self destructive potential drug which has the ability of penetrating cell membrane and killing cell, protein transduction domain (PTD), tat peptide, is employed in this study. Tat-conjugated proteins can enter cells via lipid-raft dependent endocytosis, so we engineered a PTD-α-sarcin and characterize with functional assays. First, we have constructed a PTD-α-sarcin that put the tat peptide at the N-terminal of α-sarcin, and characterized its activity. We have found that the engineered PTD-α-sarcin carries low RNase activity, hydrolyzes ribosome nonspecifically, and enters cells with low efficiency. To improve these deficiencies, linker composed of GGGS is added between PTD and α-sarcin to make PTD-gggs-α-sarcin. The new construct hydrolyzes ribosome specifically, and inhibits protein synthesis more efficiently in vitro. By confocol microscopy, it also found that PTD-gggs-α-sarcin is capable of cell entry. Our data suggests that activity of PTD-gggs-α-sarcin is better than PTD-α-sarcin.

Abstract………………………………………………………………I
Abstract (Chines)…………………………………………………II
Introduction ……… ………………………………………………1
Materials and methods ……………………………………………5
Polymerase chain reaction………………………………………5
RNA impregnated gel………………………………………………6
Expression and purification of protein ……………………8
Expression and purification of 35S-labeled protein……14
Rabbit reticulocyte lysate cutting assay…………………16
In vitro translation……………………………………………17
Cell culture………………………………………………………18
Cell entry assay…………………………………………………19
Caspase-3 activity assay………………………………………19
In vivo translation……………… ……………………………20
Result ……………………………… ……………………………22
Discussion ……………………………… ………………………29
Reference …………………………………………………………34
Figure ………………………………………………………………41
Appendix ……………………………………………………………55

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