基于gE蛋白的羊伪狂犬病病毒间接ELISA的建立与应用

Establishment and Application of Indirect ELISA for Detection of Goat PRV based on gE Protein

为建立羊源伪狂犬病病毒(Pseudorabies virus,PRV)抗体的检测方法,从PRV中对gE基因进行克隆,并构建重组载体对gE蛋白进行原核表达,以重组gE蛋白为包被抗原,建立PRV抗体间接ELISA检测方法,并进行临床样本检测。结果显示,重组gE蛋白大小为42 ku,以包涵体形式表达;Western blot检测表明重组蛋白具有良好的反应原性;重组蛋白作为包被抗原的最佳工作浓度为1μg/mL,待检血清100倍稀释,以HRP标记的兔抗山羊IgG 10000倍稀释作为二抗;检测临床样本时判定阴阳性的临界值为0.284;灵敏性试验结果显示,羊PRV阳性血清稀释2048倍时检测结果仍为阳性;批内变异系数(2.45%~5.00%)与批间变异系数(2.55%~7.41%)均小于10%;采用建立的方法检测其他常见羊源病毒阳性血清结果均为阴性;对收集的376份临床羊血清进行检测,PRV阳性率为10.6%。建立的间接ELISA检测方法可用于羊源样本中伪狂犬病病毒抗体的检测,该方法的建立为羊场PRV抗体检测提供了技术支持。

In order to establish the detection method of pseudorabies virus(PRV)antibody,the gE gene was cloned from PRV and its expression vector was constructed to perform prokaryotic expression of recombinant gE protein.With recombinant gE protein as coated antigen,the indirect ELISA detection method of PRV antibody was established and the clinical samples were tested.The results showed that,the recombinant gE protein size is 42 ku,expressed in the form of an inclusion body;Western blot indicated that the recombinant protein has good immunogenicity;The optimal antigen-coated concentration is 1μg/mL,a 100-fold dilution of the serum to be tested,HRP fold dilution of rabbit anti-goat IgG 10000 is used as secondary antibody;The critical value of negative and positive was determined as 0.284;The sensitivity test showed that,goat PRV positive serum was still judged as positive when diluted 2048 times;The intra-batch coefficient of variation(2.45%-5.00%)and the inter-batch coefficient of variation(2.55%-7.41%)were both less than 10%;Other virus positive antibody serum test results were negative;Of 376 goat sera collected in the clinic,the PRV positive rate was 10.6%.The indirect ELISA method established in this study can be used for the detection of PRV antibodies,and the establishment of this method provides technical support for PRV antibody detection in goat farms.