基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用
Acta Veterinaria Et Zootechnica Sinica（2020）
Abstract
旨在建立蓝舌病病毒(BTV)血清学ELISA抗体检测方法,本研究以原核表达并纯化的BTV NS4重组蛋白为包被抗原,通过反应条件优化,建立了一种BTV重组NS4蛋白的间接ELISA抗体检测方法.SDS-PAGE结果显示,获得大小约52 ku的NS4重组融合蛋白,主要在上清中存在,Western blot显示,纯化后的重组蛋白具有良好的抗原性.通过方阵试验进行了ELISA反应条件优化,确定了重组蛋白抗原最佳包被量为3.0μg·孔-1;血清最佳稀释倍数为1:200,酶标二抗最佳工作浓度为1:4000,临界值分别为0.29和0.35.上述以NS4蛋白作为包被抗原建立的BTV抗体间接ELISA方法检测敏感性可达1:1600;批内和批间重复性变异系数均小于10％;检测76份重庆地区牛群血清样品,阳性符合率为98％,阴性符合率为100％.本研究建立的间接ELISA方法为临床BTV血清抗体检测及BTV血清流行病学调查奠定了基础.
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