可標靶一型類志賀毒素的多功能金奈米粒子

Abstract

可標靶一型類志賀毒素的多功能金奈米粒子 學生：李俊賢 指導教授：陳月枝博士 國立交通大學應用化學系分子科學碩士班 摘要

由食物引發的致命性疾病常有所聞，使得食品安全問題已經變成急迫待解的議題。食物中可能含有致命性的細菌，例如：大腸桿菌O157:H7。大腸桿菌O157:H7所造成的致命性疾病主要是由類志賀毒素所造成。類志賀毒素可分為兩種：一型類志賀毒素及二型類志賀毒素。類志賀毒素是由一個會抑制蛋白質的合成的A單元及負責與宿主細胞的細胞膜上醣基Gb3結合的五個Ｂ單元所組成。類志賀毒素可造成溶血性貧血，急性腎功能衰竭及血小板數目過低等疾病。因此發展適合的分析方法並且使用適合方法中和類志賀毒素的毒性是相當重要的課題。在本研究中發展了對一型類志賀毒素具標靶能力的新型功能性金奈米粒子，並據此開發快速偵測一型類志賀毒素的分析方法及中和一型類志賀毒素毒性的策略。此功能性金奈米粒子是利用鴿子蛋蛋白中之蛋白質為材料，藉由一鍋反應合成表面包覆鴿子蛋蛋白的蛋白質之金奈米粒子。由於在鴿子蛋的蛋白中的蛋白質，卵鴿蛋白占約百分之六十左右，而卵鴿蛋白中含有醣基Gb3的類似物。根據我們的結果顯示，所合成的鴿子蛋的蛋白蛋白質包覆金奈米粒子，外層的蛋白質大部分的來源為卵鴿蛋白，因此研究結果顯示所合成的金奈米粒子對一型類志賀毒素具有親和性，此金奈米粒子可從複雜的大腸桿菌O157:H7的分解液及含有類志賀毒素的食物樣品中選擇抓取一型類志賀毒素。結合此奈米探針及基質輔助雷射脫附游離質譜法為分析方法，對一型類志賀毒素的偵測極限可以低至約0.18 pM左右。除此之外，我們的所合成出來的金奈米粒子還可以中和被一型類志賀毒素感染的細胞毒性。我們使用老鼠纖維母細胞及肝癌細胞為模型細胞。測試結果顯示，如果被類志賀毒素感染的細胞可及時在一小時內餵食此功能金奈米粒子，被感染細胞的存活率可以被提升至大於95%左右。但是如果細胞被一型類志賀毒素感染超過四小時之後才餵食此功能性金奈米粒子，細胞的存活率將被降至大約30%左右。這些結果顯示此功能性金奈米粒可當作中和類志賀毒素毒性的試劑。此外，實驗結果還指出治療類志賀毒素感染細胞的時間點是有效反轉細胞存活率的關鍵。本論文所開發的鴿蛋白修飾金奈米粒子的合成方法很簡單，並且使用的材料成本也相當低的。因此，我們相信利用此功能金奈米粒子為探針所開發的方法有潛力可實際用於快速分析一型類賀志毒素，並且應當在未來也有潛力將此功能性金奈米粒子開發成可抑制一型類賀志毒素毒性的中和試劑。

Multifunctional Gold Nanoparticles for Shiga-like Toxin 1 Student: Chun-Hsien Li Advisor: Yu-Chie Chen M.S. Program in Molecular Science, Department of Applied Chemistry Abstract Food safety has become an urgent issue because lethal cases resulting from foodborne illness outbreaks occur occasionally. Food can be contaminated by pathogens such as foodborne Escherichia coli O157:H7. The pathogenicity of E. coli O157:H7 is caused by the generation of Shiga like toxin (SLT) type 1 and SLT type 2, which can cause hemolytic anemia, acute kidney failure, and a low platelet count. SLTs are consists of an A subunit that is responsible for blocking protein synthesis, and five B subunits that are the binding moieties toward globotriaosylceramide (Gal-α(1→4)-Gal-β(1→4)-Glc-β-O-ceramide) (Gb3) on the cell membrane of host cells. It is essential to develop suitable analytical methods that can be used to rapidly identify the presence of SLTs from suspicious samples. Furthermore, to develop effective medical treatment that can deal with SLT-intoxication cases is also important. In this study, an analytical platform that can be used to rapidly identify SLT-1 and an approach that can be used to neutralize the toxicity of SLT-1 based on the use of functional gold nanoparticles (Au NPs) were proposed. Functional Au NPs were generated by using pigeon egg white (PEW) proteins as the starting materials to generate PEW protein immobilized AuNPs (AuNPs@PEW) from one-pot reactions. PEW is dominated by pigeon ovalbumin (POA) (~60%), which contains abundant Gb3 analogues, i.e. Gal-α(1→4)-Gal-β(1→4)-GlcNAc termini. On the basis of our results, AuNPs@PEW were dominated by POA immobilized AuNPs (AuNPs@POA). Thus, we demonstrated that the generated AuNPs@PEW are suitable affinity probes for selectively trapping SLT-1B from complex cell lysates derived from E. coli O157:H7 and SLT-1 contaminated samples. By combining the nanoprobes with matrix-assisted laser desorption/ionization mass spectrometry (MALDI), the LOD for SLT-1B was as low as ~0.18 pM. In addition, the generated AuNPs@PEW were used as neutralization agents to rescue the cell viability of SLT-infected cells. L929 mouse fibroblast cells and Hep G2 liver cancer cells were selected as model cells. The results showed that the cell viability of SLT-infected cells can be rescued to > 95% if AuNP@PEW were added to the cell samples after infected by SLTs within 1 h. The cell viability was dropped to ~30% if the neutralization agents were not added until the cells were infected by SLTs for 4 h. The results demonstrated that AuNPs@PEW can be used as suitable agents for neutralizing SLT toxicity. Furthermore, to reverse the toxicity caused by SLTs, the timing for treating SLT-infected cells by the neutralization agents is essential. AuNPs@PEW can be easily generated from the proposed method and the cost for generation of the AuNPs@PEW is extremely low. Thus, we believe that the proposed analytical platform and medical treatment based on the use of AuNPs@PEW can be potentially used in real world applications.