猪圆环病毒2型间接ELISA检测方法的建立

根据GenBank中猪圆环病毒2型(PCV-2)ORF2基因序列设计特异性引物,进行PCR反应,回收PCR产物,将其克隆入pMD18-T载体,再定向克隆到表达载体pET-32a中并转化大肠杆菌Rosetta,进行原核表达.表达产物经His-Bind蛋白纯化试剂盒纯化,平均浓度为1.10 mg/ml.以1.8 μg/ml蛋白包被酶标板,建立ELISA检测方法.建立的ELISA方法与北京世纪元亨公司试剂盒比较,两者检测结果符合率达85%以上.用该方法对常州某猪场36份母猪和156份10～160日龄猪群血清样品进行了检测,检测结果显示母猪阳性率为81.4%,仔猪阳性率随着日龄的增加而逐渐降低,之后由于受PCV-2的感染,阳性率逐渐升高.本试验建立的ELISA方法,具有较好的特异性、重复性和敏感性,可用于猪群PCV-2抗体的大规模检测.

Development of Indirect ELISA for Detection of Porcine Circovirus Type 2

Referring to the published whole genome sequence of porcine circovirus type 2(PCV-2),a pair of primers were designed for the cloning of ORF2 gene in PCV-2 by PCR.The PCR product was linked with pMD18-T vector,cloned into expression vector pET32 and then transferred into E.coli Rosetta for the prokaryotic expression of the ORF2 gene.The expression product(protein) was purified by His-Bind Purification Kit.1.80 ug/ml expression product(protein) was used as antigen to establish an ELISA for the detection of antibody in the serum of swine infected with porcine circovirus type 2.The results detected by the ELISA were 85% accordance with those detected by a company-made kit.The detection of serum samples of 36 sows and 156 piglets aged from 10 d to 160 d by the ELISA revealed that the positive rate of sow serum reached 81.4%,while that of piglet serum first declined with the aging of the piglets and then climbed up due to the infection of PCV-2.The ELISA developed in the study could be used in massive detection of antibody to PCV-2 due to its specifictiy,good repeatability and sensitivity.