開發增強醣類免疫抗原性之載體

1. 開發增強醣類免疫抗原性之載體 • Notch 是個演化上具高度保留，穿過細胞膜一次的受體蛋白，在哺乳類的Notch 受體蛋白有Notch1∼4四種，被認為可調控基因的轉錄、細胞的增生、分化及早期細胞命運的決定，而Notch訊息傳遞的異常或突變會造成某些疾病的發生。當相鄰細胞表現的ligand 與Notch 受體蛋白的細胞外區域結合時，Notch 訊號傳遞系統就被活化，整個細胞內區域在靠近內膜處被蛋白分解脢切斷，而形成活化型Notch (Notch IC, 細胞內區域)。此活化型Notch會轉移至細胞核中，並且和一些可與DNA結合的轉錄因子，如：RBP-Jκ/CBF1，以及其他細胞因子結合，進一步調控標的基因的表現。所以這些參與的因子，對於Notch 訊息傳遞路徑影響甚巨。因此，為了進一步探討Notch 訊息傳遞路徑，本論文首要目標著重於探索參與Notch1 下游訊息傳遞路徑仍未知的結合因子，及初步分析這種結合所產生的生物功能。本實驗參考葉添順老師與本實驗室先前利用GST-N1IC-ANK repeats之融合蛋白，與K562細胞萃取液混合，進行GST pull down Assay 來分析該細胞中有何蛋白質可與GST-N1IC-ANK融合蛋白做結合，並收集與融合蛋白結合的複合體，用2-D電泳及質譜儀分析此複合體的成員為何。在分析蛋白質體的數據中發現有數個候選蛋白質包括鋅手指蛋白的存在。分析比對之結果發現ZNF74的吻合度最高。ZNF74屬於C2H2 type的鋅手指蛋白。在其N端部份包含了一段稱為Krueppel-associated box (KRAB)的區域，在C端部份包含12個zinc finger motifs。具有KRAB 區域的蛋白主要功能到目前為止所知均具有抑制基因表現之功能，而zinc finger區域則是與特定的DNA序列做結合。因此本實驗有興趣繼續探討ZNF74與Notch 1之訊號傳遞是否有關。在實驗第一部份我們利用專一性引子以反轉錄聚合酶連鎖反應(RT-PCR)證實在K562細胞之中ZNF74基因是以isoform I存在。實驗第二部份，我們利用將HA-ZNF74基因植入表現N1IC的人類細胞HEK293，獲得細胞萃取液後再利用anti-HA或anti-N1IC抗體來進行共同免疫沉澱法(co-immunoprecipitation)反應，結果顯示ZNF74確實可以跟N1IC結合。為了更進一步找出ZNF74蛋白上是利用那個區域與N1IC做結合，我們利用可表現MBP融合的不同短縮型ZNF74之蛋白與表現N1IC或Ankyrin(Ank)的細胞萃取液混合。結果顯示，ZNF74的zinc finger區域與Ank的結合是必需的。實驗最後部份，我們利用SELEX(systematic evolution of ligands by exponential enrichment )來找出ZNF74結合的特定核苷酸序列為何。經過軟體序列比對結果相當複雜並無一致性。因此，根據上述的結果顯示，本實驗仍需要進一步的研究才能夠釐清在K562細胞中ZNF74蛋白參與Notch訊息傳遞下所扮演的生物角色為何。

2. Development of the carrierfor the preparation of potent carbohydrate immunogen • 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 antibody.