为建立检测猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus,PHEV)血清抗体的间接ELISA方法,构建了高效表达PHEV核衣壳蛋白(N)的重组质粒(pET28a-N),Western blot检测该重组蛋白具有较好的免疫原性。以纯化的N重组蛋白作为包被抗原,通过方阵试验确定了抗原的最适包被浓度为2mg/L,酶标二抗的最佳稀释倍数为1∶10 000,建立了用于PHEV抗体检测间接ELISA方法。此外,用该法(N-ELISA)对天津市地区采集到的200份猪血清样品进行检测,阳性检出率为83.5%,与前期工作中建立的全病毒作为包被抗原的间接ELISA诊断方法 (HEV-ELISA)检测结果的符合率为90.91%。结果表明:以重组N蛋白为包被抗原所建立的用于PHEV血清抗体检测的方法能够用于检测PHEV感染及相关的流行病学调查。 In order to establish an indirect ELISA method for detecting serum antibodies to porcine hemagglutinating encephalomyelitis virus (PHEV), a recombinant plasmid (pET28a-N) was constructed to express PHEV nucleocapsid protein (N) efficiently and was detected by Western blot The recombinant protein has better immunogenicity. The purified recombinant N protein was used as the coating antigen, and the optimal coating concentration of the antigen was 2 mg / L and the optimal dilution of the enzyme-labeled secondary antibody was 1:10 000 by square matrix test, and the optimal dilution for the PHEV antibody Detection of indirect ELISA method. In addition, 200 pig serum samples collected from Tianjin area were detected by N-ELISA. The positive detection rate was 83.5%. Compared with the indirect ELISA method (HEV-ELISA) test results in line with the rate of 90.91%. The results showed that the method for detecting PHEV serum antibody with recombinant N protein as coating antigen could be used to detect PHEV infection and related epidemiological investigation.