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Regulatory Mechanisms of Chicken Il-12 in the Immune Responses to Infectious Bursal Disease Virus Vp2 and Newcastle Disease Virus Hn Proteins
|關鍵字:||畜牧獸醫類;基礎研究;Recombinant fowlpox virus;Il-12;Infections bursal disease virus;Newcastle disease virus;Dendritic cells||摘要:||
在我們先前的試驗中發現以重組禽痘毒，rFPV/chIL-12，表現的鷄細胞素chIL-12可促進由重組禽痘毒，rFPV/VP2，製造的VP2 引起雞隻體液及細胞性免疫反應，但卻會抑制由重組禽痘毒，rFPV/HN，製造的HN 蛋白質引起的抗體反應，雖然它也會促進雞隻對HN 蛋白質所引起的細胞性免疫。顯然相同細胞素chIL-12 對於不同抗原存在著不同的調控機制。因此，本計畫擬利用重組禽痘毒表現的VP2及HN 蛋白質分別和chIL-12 或油質佐劑做不同搭配組合，並於培養的細胞 (invitro) 及免疫雞隻 (in vivo)，分析和免疫反應相關基因mRNA 表現之消長，從而探討chIL-12 對VP2 及HN 蛋白質免疫反應調控機制。擬分三年進行。第一年擬從三種重組禽痘毒感染DF-1 細胞製備之溶解液中分別純化IBDV VP2、NDV HN及chIL-12 蛋白質並測試其生物活性；同時進行雞DC 細胞特異性標記蛋白質CD83 基因選殖，CD83 蛋白質表現純化。鑑定後，用以免疫小白鼠製備多價及單源抗體。第二年著重於脾臟DC 細胞、巨噬細胞及淋巴球的分離培養。接著利用不同組合的蛋白質/佐劑和細胞共同培養，並利用real-time RT-PCR 檢測受刺激後的細胞和免疫反應相關基因的mRNA 表現情形以及對Dextran-FITC 吞噬能力是否被激化。第三年將不同組合的蛋白質/佐劑和DC 細胞及淋巴球共同培養，再以ELISPOT 分析淋巴球分泌抗體數目是否增加。接著將不同搭組合之蛋白質/佐劑免疫雞隻，取脾臟分析DC 細胞、淋巴球，再依in vitro 方式進行DC 細胞和免疫反應相關基因mRNA的表現及ELISPOT 分析產生抗體淋巴球數目是否增加等。如此，比較分析細胞培養或體內試驗結果，預期可以找出chIL-12 於雞隻對VP2 及HN 蛋白質免疫反應的調控機制，對於更有效疫苗的研發或許可以進一步提供有用的資訊。
Our previous studies have demonstrated that recombinant rFPV/chIL-12 expressedchIL-12 enhanced both humoral and cell mediated immune responses against IBDVVP2 protein expressed by a recombinant rFPV/VP2; whereas, chIL-12 inhibitedhumoral immune response (HI antibody production) against NDV HN proteinexpressed by a recombinant rFPV/HN although chIL-12 also enhanced cell mediatedimmunity (CMI). Thus, different regulatory mechanisms exist among differentantigens by the same cytokine chIL-12. This research proposal is to prepare variousvaccine combinations consisting of proteins VP2 or HN with chIL-12 or oil adjuvant.The prepared vaccines are used to co-culture with splenic cell or to vaccinate chickens.The expression of several genes related to immune responses is assessed to insight theregulatory mechanisms of chicken immune responses against VP2 or HN protein bychIL-12. The time table for this proposal includes 3 years. In the first year, proteinsVP2, HN and chIL-12 will be purified from the recombinant virus-infected DF-1 cellsand their biological functions are analyzed. Meanwhile, the gene coded for a uniquemarker CD83, for chicken dendritic cells (DC) is cloned and CD83 is expressed. Thepurified CD83 will be further used to immunized BALB/c mice to prepare polyclonaland monoclonal antibody. In the second year, the DC, macrophage and lymphocyteare isolated from spleens and cultured. The cultured cells will be treated with variousvaccine combinations as described in the first year, followed by detecting several geneexpression related to immune responses against VP2 or HN protein by real timeRT-PCR. Also, phagocytosis capacity of the treated cells is assessed by theirendocytosis of Dextran-FITC. Finally, to assess if the numbers of chickenIgG-secreting cells increase, the cells treated with vaccine combinations are examinedby the ELISPOT techniques. The vaccine combinations are also used to vaccinatechickens. Spleens are removed and DC and splenic lymphocytes are prepared for theanalysis of gene expression related to immune responses against VP2 and HN and theincrease of numbers of chicken IgG-secreting cells by the ELISPOT, as described forin vitro study. The results obtained from those in cultured cells (in vitro) and inchickens (in vivo) are assessed and expected to figure out the mechanisms of immuneresponses against VP2 and HN proteins regulated by chIL-12. These findings mayprovide useful information for the development of more effective vaccines againstIBD and ND.
|Appears in Collections:||獸醫學系所|
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