ELISpot技术检测猪抗口蹄疫γ干扰素方法的建立

发布时间：2018-03-31 02:16

本文选题：ELISpot　切入点：口蹄疫　出处：《中国畜牧兽医》2017年01期

【摘要】：猪口蹄疫免疫防御是控制猪口蹄疫疫情的重要手段,免疫水平指标通常用免疫抗体水平判定。对于其细胞免疫水平检测尚缺乏成熟可靠的技术,本试验利用市售ELISpot试剂盒建立猪口蹄疫特异γ干扰素检测方法,刺激物分别采用疫苗全病毒颗粒(O/Mya98/XJ/2010)、口蹄疫病毒T细胞表位多肽池,以植物血凝素(PHA)为阳性对照,对细胞浓度、刺激物浓度、孵育时间等进行优化。优化条件为:外周血PBMC新鲜提取或冻存细胞成活率90%以上,最佳细胞数为2×105个/孔,最佳反应时间是16h,病毒粒子146S含量为100ng/mL,多肽最佳浓度10μg/mL,PHA的最佳浓度是20μg/mL。ELISpot技术检测口蹄疫病毒感染猪γ干扰素方法的建立,为口蹄疫免疫力评价及口蹄疫疫苗免疫效果评估及进一步研究其与疫苗保护力(PD50)的相关性奠定基础。
[Abstract]:The immune defense against foot-and-mouth disease in pigs is an important means to control the epidemic situation of swine foot-and-mouth disease. The immune level index is usually determined by the level of immune antibody. There is a lack of mature and reliable technology for the detection of cellular immunity level of swine foot-and-mouth disease. In this study, a ELISpot kit was used to establish a specific interferon 纬 assay for porcine foot-and-mouth disease (FMD). The stimulator was treated with whole virus particle O / Mya98 / XJ / 2010, FMDV T cell epitope polypeptide pool and phytohemagglutinin (PHAA) as positive control. The optimum conditions were as follows: the survival rate of fresh or frozen peripheral blood PBMC was more than 90%, and the optimum number of cells was 2 脳 105 / well. The optimum reaction time was 16h, the content of virus particles 146S was 100ng / mL, the optimum concentration of polypeptide was 10 渭 g / mL PHA, and the best concentration was 20 渭 g/mL.ELISpot technique to detect interferon 纬 (IFN- 纬) in pigs infected with foot-and-mouth disease virus (FMDV). It lays a foundation for evaluating the immunity of foot-and-mouth disease (FMD), evaluating the immune effect of FMD vaccine and further studying its correlation with vaccine protective ability (PD50).
【作者单位】：大连市动物疫病预防控制中心;大连大学生命科学与技术学院;
【基金】：国家自然科学基金(81172777) 大连市科技计划基金(2014B11NC096)
【分类号】：S852.4

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