基于重组核衣壳蛋白的猪德尔塔冠状病毒间接ELISA检测方法的建立

Establishment of indirect ELISA detection method for porcine deltacoronavirus(PDCoV)based on recombinant nucleocapsid protein

目的:建立一种关于猪德尔塔冠状病毒(Porcine Deltacoronavirus, PDCoV)抗体的检测方法。方法:对猪德尔塔冠状病毒的核衣壳蛋白(N)进行原核表达,通过Western Blot、SDS-PAGE等方法,对重组蛋白进行鉴定;在获得正确表达的重组蛋白后,以此为抗原,对ELISA反应条件进行优化,并对其特异性、敏感性、重复性进行测试,最终建立ELISA检测方法,并对临床样品进行检测。结果:优化后的抗原包被浓度为2μg/孔,37℃孵育1 h;1%BSA 37℃封闭1 h;一抗稀释最佳浓度为1∶1 600,孵育时间为1 h;酶标二抗最佳孵育时间为60 min。重组蛋白能与猪德尔塔冠状病毒血清发生特异性反应,所建立的检测方法具有优良的敏感性、特异性、重复性。结论:本研究建立了一种针对猪德尔塔冠状病毒的间接ELISA检测方法,为猪德尔塔冠状病毒的抗体水平检测及疾病的防控提供了一种有效的方法。

Objective:To establish a method for detecting antibodies against Porcine Deltacoronavirus(PDCoV).Methods:The nucleocapsid(N)protein of Porcine Deltacoronavirus(PDCoV)was expressed in a prokaryotic system,and the recombinant protein was subsequently identified through techniques such as Western Blot and SDS-PAGE.After obtaining the correctly expressed recombinant protein,it was used as an antigen to optimize the ELISA reaction conditions.The specificity,sensitivity,and reproducibility of the assay were tested,leading to the establishment of an ELISA detection method.Finally,this method was applied to detect clinical samples.Results:The optimized conditions for the ELISA assay included an antigen coating concentration of 2μg/well,followed by incubation at 37℃for 1 h.Blocking was achieved using 1%BSA at 37℃for 1 h.The optimal dilution concentration for the primary antibody was 1∶1600,with an incubation time of 1 h.The incubation time for the enzyme-labeled secondary antibody was 60 minutes.Notably,the recombinant protein demonstrated specific reactivity with sera from pigs infected with PDCoV.The established ELISA detection method exhibited excellent sensitivity,specificity,and reproducibility.Conclusion:This study has established an indirect ELISA detection method specifically designed for Porcine Deltacoronavirus(PDCoV),providing an effective approach for detecting antibody levels against PDCoV and supporting the prevention and control of the disease.