猪繁殖与呼吸综合征病毒核衣壳蛋白的原核表达及间接ELISA抗体检测方法的建立

Prokaryotic expression of porcine reproductive and respiratory syndrome virus nucleocapsid protein and establishment of an indirect ELISA for detection of antibodies

以猪繁殖与呼吸综合征病毒(PRRSV)重组核衣壳蛋白(rN蛋白)为抗原,初步建立了检测PRRSV抗体的rN-ELISA方法。根据GenBank公布的PRRSV VR2332株编码核衣壳蛋白的ORF7基因序列设计合成了一对引物,成功扩增出ORF7基因。将其克隆到PET-30a构成原核表达载体,转化宿主菌BL21(DE3)并获得表达。通过SDS-PAGE和Western blot检测证实rN蛋白获得了高效表达,且具有良好的免疫学活性。以纯化的重组蛋白为抗原建立检测PRRSV抗体的间接ELISA法。试验证明,该方法与IDEXX公司生产的PRRSV抗体检测ELISA试剂盒对同批临床血清样本的检测结果完全相符,且对其他常见的6种猪疫病阳性血清检测均为阴性,无交叉反应。本研究建立的rN-ELISA方法敏感性高、特异性强,可用于猪繁殖与呼吸综合征常规诊断及流行病学的调查研究。

Established an indirect ELISA method for detecing PRRSV antibodies using the recombinant nucleocapsid protein（rN） of PRRSV expressed in E. coil （DE3） strain as antigen. Based on the ORF7 gene sequence encoding nucleocapsid protein of VR2332 strain in the GenBank, we designed a pair of primers and successfully amplified the ORF7 gene from PRRSV genome . The cloned gene was inserted into pET30a vector to obtain the recombinant prokaryotic expression plasmid. This recombinant plasmid was transformed into E. coil BL21 （DE3）,and expressed, rN protein was expressed in high level in BL21 as confirmed by SDS-PAGE, and showed strong immunological reaction with antiserum against PRRSV detected by western blot. An indirect rN-ELISA was developed for detecting PRRSV antibodies. We applied rN-ELISA and IDEXX kit to test for the same serous samples from clinic. Test results from the two kits were same. The rN-ELISA also used to detect the positive sera from six other common swine diseases was negative with no evident cross reaction. The results indicated that rN-ELISA was specific, sensitive and suitable for routine diagnosis of PRRS and also for epidemiological surveys.