猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus，PRRSV）rHN4-Δ25+NP49株是新型基因标记弱毒疫苗候选株，为了配合对该基因标记疫苗免疫猪血清抗体的鉴别诊断，本研究以标记基因编码的NP49(新城疫病毒NP蛋白C末端49个氨基酸，即NP49)多肽作为包被抗原，建立标记疫苗免疫猪血清中NP49抗体的ELISA鉴别诊断方法。通过对NP49-ELISA工作条件的优化，结果显示NP49-ELISA的多肽抗原最适包被浓度为500 ng/孔，被检血清最佳稀释度为1：40。利用ROC曲线法确定S/P临界值为0.2，该方法批内与批间重复试验的变异系数均小于10%，与几种常见猪病阳性血清无交叉反应，具有较好重复性和特异性。本研究建立的NP49-ELISA鉴别诊断方法为猪繁殖与呼吸综合征新型基因标记疫苗的临床应用提供了有力保障。%Recombinant virus rHN4-Δ25+NP49 strain is a newly developed genetic marker vaccine against highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV). The marker gene NP49 represents 49 amino acids at C terminal of Newcastle disease virus NP protein. Reasonably, detection of the antibodies specific for marker gene NP49 would differentiate vaccinated pigs from field virus infected pigs. To develop a differental diagnosis, synthesized NP49 peptide was used as coating antigen in indirect ELISA. Then, the differential NP49-ELISA was optimized for coating antigen concentration, serum dilution, etc. The optimal coating concentration of NP49 antigen was 500 ng/well and optimal serum dilution was 1:40. The critical S/P value was determined at 0.2 according to the ROC curve. The intra-and inter-assay coefficients of variability between experiments were all less than 10%. There was no cross reaction with antibodies to other swine viruses, indicating good reproducibility and specificity. Results have demonstrated that NP49-ELISA is a reproducible and specific method for differential diagnosis of vaccinated pigs, paving a pathway for clinical application of PRRSV genetic marker vaccine rHN4-Δ25+NP49 strain.
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