牛副流感病毒3型核衣壳蛋白的原核表达及间接ELISA方法的建立
Chinese Journal of Preventive Veterinary Medicine（2019）
Abstract
为建立以牛副流感病毒3型(BPIV3)核衣壳蛋白(NP)为包被抗原的间接ELISA方法,本研究扩增BPIV3的NP基因并克隆于原核表达载体,获得重组表达质粒pET30-NP.将其转化至表达菌BL21(DE3),获得了可溶性表达的重组N蛋白,用镍柱在非变性条件下纯化后将其作为包被抗原,建立了检测BPIV3抗体的间接ELISA方法.特异性试验结果显示作为包被抗原的重组N蛋白仅与BPIV3阳性牛血清发生特异性反应,与牛传染性鼻气管炎病毒和牛病毒性腹泻病毒等牛的常见病原无血清学交叉反应,表明其特异性较强.牛BPIV3阳性血清在1:640倍稀释时按该方法检测仍为阳性,显示该方法具有较高的敏感性.重复性试验显示批内变异系数小于6％,批间变异系数小于12％,表明该方法具有较好的稳定性.对94份牛血清的比较试验显示,本研究建立的间接ELISA方法与病毒中和试验的符合率为98％.利用该方法检测了394份采自接种过BPIV3灭活苗牛场的血清样品和95份未接种疫苗牛场的血清样品,结果接种疫苗的牛血清均呈强阳性,而95份未接种疫苗牛血清的阳性率为80％.本研究建立的间接ELISA方法可以试用于国内BPIV3的流行病学调查和免疫监测,为国内BPIV3的防控提供技术支持.
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