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National Chung Hsing University Institutional Repository - NCHUIR > 獸醫學院 > 微生物暨公共衛生學研究所 > 依資料類型分類 > 碩博士論文 >  評估小鼠IL-18同型異構物做為疫苗佐劑之能力及選殖小鼠CCL21

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/154123

標題: 評估小鼠IL-18同型異構物做為疫苗佐劑之能力及選殖小鼠CCL21
Evaluation of the ability of mouse IL-18 isoforms as the vaccine adjuvant and cloning of mouse CCL21
作者: 林佳琦
Lin, Jia-Chi
Contributors: 邱繡河
微生物暨公共衛生學研究所
關鍵字: 疫苗佐劑
vaccine adjuvant
日期: 2013
Issue Date: 2013-11-19 12:51:04 (UTC+8)
Publisher: 微生物暨公共衛生學研究所
摘要: 小鼠介白素-18(interleukin-18, IL-18)是一種促發炎的細胞素,具有誘導丙型干擾素(interferon-γ, IFN-γ)及其他促進免疫之活性,也可以與介白素-12(interleukin-12, IL-12)產生協同作用,調節體內的免疫反應。本研究室與另一個研究團隊於2005年相繼發現小鼠體內有較IL-18小片段的異構物,並且命名為小鼠介白素-18s(interleukin-18 small isoform, IL-18s)。本論文研究目的是評估小鼠IL-18及IL-18s做為疫苗佐劑的能力,以轉錄因子Nrf2(nuclear factor erythroid derived 2-related factor 2)當作抗原,以小鼠重組IL-18(recombinant mouse IL-18, rmIL-18)或IL-18s (rmIL-18s)蛋白做為佐劑,另亦比較合併使用傳統佐劑與否對於小鼠免疫反應之影響。本研究使用酵素聯結免疫吸附法(enzyme-linked immunosorbent assay, ELISA)檢測小鼠血清中辨識所施打抗原的IgG、IgG1及IgG2a專一性抗體,結果顯示rmIL-18比rmIL-18s更能提高辨識Nrf2抗原的IgG2a專一性抗體,這代表rmIL-18較能促進小鼠的第一型輔助T細胞(T helper 1, Th1)免疫反應。此外,我們以反轉錄聚合酶鏈反應(reverse transcription-polymerase chain reaction, RT-PCR)及定量聚合酶連鎖反應(quantitative PCR, qPCR)來檢測小鼠周邊白血球細胞之IFN-γ及CC趨化素-21(CC chemokine ligand 21, CCL21)的基因表現。結果顯示單獨使用rmIL-18做為佐劑,可以促使IFN-γ及CCL21基因表現量上升,而rmIL-18s在與佛氏佐劑(Freund’s adjuvant)合併使用時可以增加此二基因表現量。這些結果顯示rmIL-18及rmIL-18s單獨作用,或與佛氏佐劑一起作用時,各有不同的活性差異。此外,本研究亦選殖小鼠CCL21之cDNA基因,構築至原核表現載體pET32a。
Interleukin-18 (IL-18) is a proinflammatory cytokine with potent interferon-γ (IFN-γ)-inducing and other immunostimulatory activities. It plays important roles in mediating immune responses in synergism with IL-12. Previously, we and other group discovered a novel mouse IL-18 small (IL-18s) isoform. The purpose of this study was to evaluate the ability of mouse IL-18 and IL-18s as the vaccine adjuvant. Nuclear factor erythroid derived 2-related factor 2 (Nrf2), a transcription factor, was used as an immunogen and recombinant mouse IL-18 (rmIL-18) or IL-18s (rmIL-18s) protein was coadministered with or without Freund’s adjuvant into mice. We used enzyme-linked immunosorbent assay (ELISA) to detect antigen specific IgG, IgG1 and IgG2a antibodies in mice sera. The results showed that rmIL-18 had a higher activity than rmIL-18s to promote antigen-specific IgG2a antibodies, which may reflect Th1 immune response. Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) were performed to determine the gene expression of IFN-γ and CC chemokine ligand 21 (CCL21) in peripheral white blood cells. The results showed that rmIL-18 alone could induce IFN-γ and CCL21 gene expression whereas rmIL-18s, in combination with Freund’s adjuvant, could induce IFN-γ and CCL21. These results suggest that rmIL-18 and rmIL-18s have varied activities in the absence or presence of Freund’s adjuvant. On the other hand, mouse CCL21 was cloned and introduced into pET32a prokaryotic expression vector.
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