猪细小病毒2型VP蛋白主要抗原表位区的原核表达及间接ELISA检测方法的建立

Prokaryotic expression of the structural protein VP of porcine parvovirus type 2 and establishment of an indirect ELISA for the detection of antibody against porcine parvovirus type 2

为了有效监测猪细小病毒2型(PPV2)的抗体水平,选择PPV2VP蛋白主要亲水区及高抗原指数区进行原核表达。以表达的重组tVP蛋白为包被抗原建立了检测PPV2抗体的间接ELISA。优化后的反应条件为:抗原包被浓度为0.8μg/mL,血清的最佳稀释度为1∶100,酶标二抗的工作浓度为1∶20 000,检测的临界值为0.400 5(D450nm≥0.400 5判定为阳性)。特异性试验证明,与猪细小病毒1型、猪瘟病毒、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪圆环病毒2型、口蹄疫病毒O型血清抗体无交叉反应,批内、批间重复性试验变异系数均小于10%。应用此方法对采自江苏省的480份临床血清样本进行了检测,PPV2抗体阳性率为31%。试验结果表明,tVP蛋白能很好地获得表达,以此蛋白作为包被抗原建立的间接ELISA具有较高的特异性和敏感性,可以用于临床血清PPV2抗体的检测。

In order to effectively monitor porcine parvovirus type 2(PPV2)antibody levels,the mainly hydrophilic regions of PPV2 VP protein with high antigenic index were prokaryotically expressed,and an indirect ELISA was established using the recombinant truncated VP(tVP)protein as coating antigen.The reaction conditions were optimized by using 0.8μg/mL of the purified tVP protein as coating antigen and by means of 1∶100dilution of the tested serum and 1∶20 000 dilution of SPA-HRP with a cut off-value of0.400 5(D450nm).ELISA analysis of the specificity of PPV2 serum showed that it had no cross-reactions with the positive sera to porcine parvovirus type 1,classical swine fever virus,pseudorabies virus,porcine circovirus type 2and foot-and-mouth disease virus type O,respectively.Its intra-assay and inter-assay coefficients of variation were within 10%.In application of the indirect ELISA to the detection of 480 clinical serum samples collected in Jiangsu Province,the positive rate of PPV2 antibody was 31%.Above results revealed that the tVP protein can be effectively expressed in the prokaryotic vector,and the indirect ELISA was sensitive and specific,which can be used in the detection of PPV2 serum antibodies in the clinic.