猪细小病毒VP2蛋白主要抗原表位区的原核表达及检测PPV抗体的间接ELISA的建立

Prokaryotic expression of a major antigenic epitope region of porcine parvovirus (PPV) VP2 protein and development of an ELISA for detection of PPV antibodies

利用大肠杆菌表达51ku的猪细小病毒(PPV)VP2蛋白主要抗原表位区,通过Western-blot检测,表达蛋白具有良好的反应原性。利用此表达蛋白建立了检测PPV抗体的间接ELISA。通过检测32份阴性血清最终确定本ELISA的判定标准为:血清抗体效价>0.18,判为PPV血清抗体阳性;血清抗体效价≤0.18,判为血清抗体阴性。此ELISA的批内重复性试验的变异系数小于5%,批间重复性试验的变异系数小于10%。用此ELISA与商品化PPV IgG检测试剂盒做符合性比较试验;结果,感染猪血清抗体检测符合率为100%,免疫猪血清抗体检测符合率为82%,非免疫健康猪血清抗体检测符合率为78%,样本总体检测符合率为80%。用建立的ELISA检测田间猪血清的PPV抗体,阳性率介于28.8%~37.5%之间,提示上述地区可能存在不同程度的PPV感染情况。本研究为PPV免疫或感染状况的检测提供了一种有用的定性诊断方法。

A major antigenic epitope region of porcine parvovirus(PPV)VP2protein was expressed in Escherichia coli.The expressed product was a 51 ku recombinant protein which had good immune reactivity with positive serum anti PPV by Western-blot analysis.Using the purified protein,an indirect ELISA was developed for detection of PPV antibodies.The cut-off value of ELISA was determined by detection of 32 sera from the non-immunized healthy pigs.For qualitative judgments,the serum sample is considered to be positive for PPV antibody if the calculated antibody titer is more that 0.18,otherwise serum sample is negative for PPV antibody if the antibody titer is lower than or equal to 0.18.The variation coefficient of intra-batch and inter-batch are lower than 5%and 10%,respectively.According to the criterion,the performance of this ELISA method was compared with a commercial PPV IgG ELISA kit.The coincident rate were 100%,82%and 78%for detection of sera samples from PPV infected pigs,vaccinated pigs and nonimmunized and healthy pigs,respectively.The overall coincident rate was 80%for all serum samples tested by two methods.The result for the detection of field serum samples showed that PPV antibody positive rate were among 28.8%to 37.5%,indicating that different levels of PPV infection might be present in the sampling region.The result here provided a useful qualitative diagnosis method for the detection of PPV immunization and infection status.