应用动物试验筛选适用於猪只之CpG ODN 佐剂.doc

應用動物試驗篩選適用於豬隻之CpG ODN 佐劑 In vivo Screening of CpG ODN as a DNA Adjuvant Suitable for Pig 研究生：吳炯毅 Wu, Jung-Yi 指導教授：鍾文彬 Chung, Wen-Bin 【摘要】 豬生殖與呼吸綜合症病毒 porcine reproductive and respiratory syndrome virus；PRRSV 屬於動脈病毒科，具有封套之單股RNA病毒。主要造成母豬繁殖障礙以及保育期豬隻呼吸症狀。細菌的DNA中含有多未甲基化雙核苷酸cytosine-phosphate-guanosine CpG ，稱為’ CpG motifs ’。動物的免疫系統會將 CpG motifs 辨識為外來物質，引發被動性免疫反應，Th1免疫路徑。人工合成包含CpG motifs 之寡去氧核糖核苷酸 oligodeoxynucleotides；ODNs ，。本目的將CpG佐劑免疫豬隻， peripheral blood mononuclear cells; PBMCs 以酵素免疫分析 enzyme-linked immunospot assay ; ELISPOT 檢測IFN-γ之活性。本試驗以磷硫化之CpG ODN 簡稱CpG 1 、包含套數之和酵素免疫分析 enzyme-linked immunospot assay ; ELISPOT 檢測分泌IFN-γ之細胞數量。結果顯示，豬隻經2次不活化疫苗施打後2週，無論疫苗中是否填加 CpG ODN其血清中和抗體皆無顯著差異，但施打疫苗中填加CpG 2、CpG 3及CpG 4者，其PRRSV特異性IFN-γ分泌細胞數量CpG motifs；佐劑；IFN-γ 【Abstract】 Porcine reproductive and respiratory syndrome virus PRRSV is an enveloped, single-stranded RNA virus and belongs to the Arteriviridae family. The disease caused by PRRSV is characterized by reproductive failure in sows and respiratory problem in pigs of all age. Bacterial DNA differs from vertebrate DNA by having a higher percentage of unmethylated cytosine-phosphate-guanosine CpG dinucleotides. These base contexts are termed ‘CpG motifs’. The recognition of CpG motif by vertebrate immune system can trigger protective immune responses. These responses can be induced by using the synthetic CpG oligodeoxyribonucleotides CpG ODNs , which have been demonstrated to be remarkably enhanced the vaccine efficacy and thus can be used as an adjuvants and immunotherapeutics. The objective of this study was to evaluate the immunomodulatory effects of phosphorothioate CpG ODN designated as CpG 1 , plasmids with 32 or 64 copies of CpG ODN designated as CpG 2, CpG 3 and CpG 4 as a potent adjuvant in the development of PRRSV vaccine in pigs. The two-week-old pigs were vaccinated intramuscularly with PRRSV vaccine with or without CpG ODNs and boosted 2 weeks post-primary inoculation. Animals were challenged wi