開發增強醣類免疫抗原性之載體
Development of the carrier
for the preparation of potent carbohydrate immunogen
中文摘要 醣類在疾病診斷上被認為是重要的標的，大部份細菌或病毒表面之醣類抗原被證 明是感染性疾病中最具臨床意義的抗原；而醣化程度的不同，也被視為正常細胞 是否轉型成腫瘤細胞的一個指標。因此，腫瘤相關之醣類抗原便相繼被選擇用來 作為開發抗癌免疫療法及癌症早期診斷的主要標的。由於醣類抗原的抗原性極 低，需要結合至蛋白質載體，以增加其抗原性，但就目前所使用的蛋白質載體中， 增強該醣類抗原所引發免疫反應的效益並不理想，使得以醣類抗原為主的抗癌疫 苗開發一直難以突破。故此篇論文探討，如何設計一可供醣類抗原有效結合之蛋 白質載體，並順利送至抗原呈現細胞產生免疫反應。為達此目的，我們利用線性 重覆抗體辨認區段 (Linear Array Epitope, LAE ) 的概念，合成一段可轉譯出富含 半胱氨酸 (Cysteine) 的 DNA 序列，希望能增加醣類在載體上結合的效率，並構 築成包含綠膿桿菌(Pseudomonas aeruginosa) 外毒素 A 之接受器結合區及富含半 胱氨酸片段的蛋白質載體。實驗的結果，我們設計的蛋白質載體，確實能讓醣類 抗原高效結合，且在西方點墨法及圓點點墨法的實驗中顯示，用此抗原所免疫的 老鼠血清，可辨認所結合的醣類抗原。因此，可預期的，我們所設計的抗原載體 應可提供一醣類相關疾病研究之良好平台，並解決因醣類低抗原性，而難以生產 醣類抗體的研究困難。 英文摘要Changes in glycosylation are often a hallmark of disease states. Recently,
carbohydrates have been proved to the most clinical relevant antigens of the many well-defined antigens for vaccines against infectious diseases. In fact, cancer cells also display glycans at different levels than normal cells. This also leads
tumor-associated carbohydrate antigens as one of the targets for immunotherapy and diagnosis of human cancer. Because the immunogenicity of carbohydrate is very weak, it is essential for elevating immune response by conjugating carbohydrate to a protein carrier. Unfortunately, the protein carriers used so far have low conjugating efficiency. It leads the development of carbohydrate-based cancer vaccine becomes a very
difficult job. In this study, we design and construct an antigen carrier for carbohydrate conjugating efficiently and delivering into antigen presenting cells. For this propose, we used the idea of Linear-Array-Epitope (LAE) to construct DNA fragments
encoding cysteine rich peptide for carbohydrate conjugation, followed by subcloning it into a protein expression vector. The final carrier protein is a fusion protein
containing the receptor binding domain of pseudomonas aeruginosa exotoxin A and cysteine rich peptide. Our preliminary study showed that the ratio of carbohydrate conjugated to the protein carrier is great increased when compared to commercial available carbohydrate carrier. We then apply it in animal study. After immunizing the BALB/c mice with this carbohydrate immunogen, the induced antibodies against carbohydrate were examined by western blotting and dot blotting. In this study, we demonstrate that our new designed carrier can offer high conjugating efficiency for various carbohydrates to solve the low immunogenicity of carbohydrate in the generation of anti-carbohydrate
