虹彩病毒DNA疫苗與結合細胞白介素-1β基因 對點帶石斑魚免疫之影響

Abstract

海水魚類虹彩病毒症近年來亞洲各國海水養殖的重要疾病之一。本實驗是採用DNA疫苗來做為防治虹彩病毒的方法。其中以虹彩病毒錐體膜蛋白基因（OSGIV-8L）為抗原基因之DNA疫苗，產生的免疫保護效果未達預期。因此本實驗在虹彩病毒DNA疫苗中，另外導入一段點帶石斑魚IL-1β基因，藉由檢測點帶石斑魚相關免疫指標，進而評估是否IL-1β基因能作為免疫佐劑而提升疫苗免疫效果。 本實驗首先利用免疫組織化學染色實驗證實建構出的DNA疫苗，經由肌肉注射後，能在石斑魚體中表現IL-1β蛋白與病毒錐體膜蛋白。觀察到搭配IL-1β基因之DNA疫苗，能提升石斑魚血清中IL-1β表現量，並能更為快速地誘發石斑魚產生特異性免疫反應，包含更早誘發石斑魚產生更好的專一性抗體，以及更高量的細胞型毒殺T淋巴球CD8α基因表現。而藉由觀察石斑魚淋巴細胞之超氧陰離子產生能力試驗、吞噬能力分析、與增生能力分析，發現給予含IL-1β基因之DNA疫苗確實能提升石斑魚免疫細胞活性，增加先天免疫能力。且IL-1β基因能調控TNF-α、IL-8之基因表現，並誘使Mx 基因表現。 本實驗報告中顯示，搭配IL-1β基因能使DNA疫苗能提升疫苗免疫效果。且IL-1β基因可能藉由提升石斑魚的先天免疫反應與刺激相關免疫基因表現，使DNA疫苗能更為迅速地誘發啟動特異性免疫反應，並使石斑魚免疫初期可能更具有抗病毒能力。

Orange-spotted grouper iridovirus（OSGIV） have been identified as one of the important pathogens of grouper culture. Vaccination is one of the useful methods against viral diseases. However, conventional vaccines usually induce poor immune protective effects. To increase DNA vaccine immunogenicity, a bicistronic vector separately encoding the myristilated membrane protein（MMP）and grouper interleukin-1β（IL-1β）was constructed. To investigate the expression of DNA vaccines, MMP and IL-1β protein expressed were demonstrated by immunohistochemistry. IL-1b protein expressed in serum was quantified by ELISA. Both antibody level and CD8α mRNA expression level were better in groupers immunized with bicistronic DNA vaccines. Furthermore, IL-1β gene injection increased proliferation of the lymphocytes and macrophage functions. IL-1β gene also significantly up-regulated of cytokines and Mx gene. These results clearly demonstrated that co-expression of an adjuvant cytokine from a bicistronic DNA vaccine may be an effective approach to increasing OSGIV DNA vaccine immunogenicity.