中文摘要
狗是人類最忠實的朋友，根據統計在美國平均每四個人就擁有一隻寵物狗，而且每年在狗醫療上的花費就高達上百億美金。在先前實驗室的研究結果當中發現-介白素二十單株抗體在乳癌以及骨質疏鬆症當中都有治療的效果，然而這些疾病也都會發生在寵物狗身上。過去十年，癌症是造成老年狗死亡率的第一名，其中乳癌又是在母狗當中佔最大比例。因此，我的目標就是要去生產出一個具有抑制介白素二十生物活性的單株抗體。我們從哺乳動物細胞以及大腸桿菌細胞表現並純化出犬類介白素二十重組蛋白，將此蛋白與佐劑混合當成抗原打入老鼠體內進行免疫反應，再利用細胞融合技術成功建立了三株單株抗體(8H, 148B以及1412E)，經由酵素連結免疫分析法(ELISA)、西方墨點法(western blot) 以及犬類細胞染色(IHC)證明三株抗體確實會專一性的辨認犬類介白素二十蛋白，同時他們也可以跨物種的辨認小鼠及大鼠的介白素二十，其中8H是擁有和介白素二十最好的結合能力(binding affinity)。除外，我們鑑定了抗體的序列，得知他們屬於mIgG1 subtype，並且找出8H與抗原的重要結合區，也利用5’RACE將8H變異區的序列解岀。最後，我們利用細胞實驗(in vitro)以及小鼠動物實驗(in vivo)證明8H確實有抑制乳癌的效果。在未來，我們會做出8H小鼠及犬類的嵌合抗體，並且進行犬類的臨床試驗。

Dogs are the most faithful friends for people. The statistics shows that every four Americans raise a dog. Every year the medical expense for canine diseases was 10 billion USD. Our previous studies showed that the IL-20 monoclonal antibody have protective function in osteoporosis and breast cancer. These diseases also occur in the dog. Over the past decade, cancer is the leading cause of death in older dogs. Breast cancer is the first leading cause of death in old female dogs. Thus, there is significant demand for new drug to treat canine cancer. Therefore, I was aimed to generate a neutralizing anti-IL-20 monoclonal antibody against dog. We expressed and purified the recombinant canine IL-20 protein from mammalian cells and E. coli cells. IL-20 protein and the adjuvant mixture used as an antigen was injected into mice to generate the anti-canine IL-20 antibody. I successfully established three stable hybridoma clones. ELISA, western blotting and IHC staining showed that each monoclonal antibody specifically recognized canine IL-20. They also cross-reacted with mouse and rat IL-20. In these three clones, 8H has the highest binding affinity to canine IL-20. Here I select 8H antibody for further characterization and functional analysis. It is immunoglobulin gamma 1 (mIgG1) subtype. I also mapped the epitope of canine IL-20 recognized by the 8H antibody using western blotting. I have sequenced the variable region of 8H antibody by 5’RACE. The preliminary result showed that the 8H antibody inhibited canine and mouse breast cancer cell migration in vitro and in vivo. In the future, I will construct 8H canine chimeric antibody and investigate its therapeutic potential in clinics

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