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为了探究兔多克隆抗体在猪流行性腹泻病毒检测和鉴定中的有效性。本研究通过Vero细胞大量培养了猪流行性腹泻病毒(PEDV)，并利用蔗糖密度梯度离心法对其进行了浓缩纯化。以纯化后的PEDV免疫新西兰白兔四次，采集收获了大量PEDV兔血清，用饱和硫酸铵沉淀法对其进行粗提，再用亲和层析法纯化得到PEDV兔多抗。ELISA检测结果显示，PEDV兔多抗的ELISA效价为1∶32000。间接免疫荧光试验结果表明，PEDV兔多抗能够识别PEDV-XD(G2a型)株、PEDV G1型CV777株，但不与猪其他易感病原猪瘟病毒C株(CSFV-C)、猪伪狂犬病毒K61株(PRVbartha-K61)发生反应，这提示，本研究制备的PEDV兔多抗具有检测的广谱性和特异性。Western blot鉴定结果显示，PEDV兔多抗能够与PEDV G2a和G1型的核衣壳蛋白N、刺突蛋白S、膜蛋白M发生特异性识别，其中，对N蛋白识别信号最强。此外，PEDV兔多抗具有体外中和活性，对PEDV-XD(G2a型)株的中和IC50为1.418μg/mL，对G1型CV777株的中和IC50为5.524 μg/mL，这表明PEDV兔多抗具有治疗潜力。综上，本研究为PEDV检测和鉴定提供了经济高效的多抗试剂，为PEDV治疗性抗体开发奠定了基础。
Abstract：To investigate the effectiveness of rabbit polyclonal antibodies in detecting and identifying porcine epidemic diarrhea virus (PEDV)， PEDV was propagated in Vero cells and purified by sucrose density gradient centrifugation. New Zealand White rabbits were immunized four times with purified PEDV to obtain high-titer antisera. Crude antibody extraction was performed using saturated ammonium sulfate precipitation， followed by affinity chromatography purification to obtain PEDV-specific rabbit polyclonal antibodies. The specificity and immunoreactivity of these antibodies were systematically evaluated through ELISA， Western blot， and immunofluorescence assays. ELISA results demonstrated an antibody titer of 1:32，000. Indirect immunofluorescence assay revealed broad-spectrum recognition of both PEDV-XD (G2a) and CV777 (G1) strains without cross-reactivity with other porcine pathogens， confirming detection specificity. Western blot analysis identified specific interactions with three structural proteins: nucleocapsid (N)， spike (S)， and membrane (M) proteins， with the strongest reactivity observed against the N protein. Notably， The PEDV rabbit-derived polyclonal antibodies demonstrate in vitro neutralizing activity against PEDV-XD (G2a) strain with an IC50 of 1.418 μg/mL and against CV777 strain with an IC50 of 5.524 μg/mL， indicating therapeutic potential of these antibodies. This study provides a cost-effective polyclonal antibody reagent for PEDV detection and establishes a foundation for developing therapeutic antibodies against PEDV. The findings demonstrate the dual utility of these polyclonal antibodies in both diagnostic applications and antiviral therapy development.
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基本信息:
中图分类号:S852.651
引用信息:
[1]方树人,蔡佳希,李子树,等.猪流行性腹泻病毒兔多克隆抗体的制备与鉴定[J].经济动物学报().
基金信息:
中国农业科学院科技创新工程（CAAS-ASTIP-ISAPS-2024）
2025-10-29
2025-10-29
2025-10-29