口疮病毒抗体间接ELISA检测方法的建立及初步应用

为建立检测口疮病毒(orf virus,ORFV)抗体的间接ELISA方法,原核表达口疮病毒B2L蛋白与6×组氨酸标签的重组蛋白;对纯化复性后的重组B2L蛋白进行Western-blot鉴定;用复性后的重组B2L蛋白作为包被抗原,优化反应条件,建立检测ORFV抗体的间接ELISA,并进行特异性、灵敏度和重复性评价,最后应用所建立的间接ELISA对来自四川部分地区的384份山羊血清样品进行检测。结果显示,成功原核表达出42 ku的重组ORFV B2L蛋白。Western-blot结果表明,重组蛋白具有良好的抗原反应性。间接ELISA最佳反应条件为:包被抗原质量浓度为5μg/m L,1∶100稀释血清作用2 h,酶标二抗以1∶2000稀释作用30 min,TMB显色时间为15 min。在该优化条件下,D450≥0.31为阳性。该方法具有良好的特异性、灵敏度和重复性。临床样品的间接ELISA检测结果显示,总阳性率为66.67%(256/384),其中未接种过疫苗的羊只抗体阳性率为34.81%(55/158)。上述结果表明,建立的间接ELISA可用于ORFV抗体的检测,四川部分地区山羊场羊只感染野毒的概率较大,疫苗免疫效果不佳。

Establishment and preliminary application of an indirect ELISA for detection of orf virus antibody

To establish an indirect ELISA method for detection of orf virus(ORFV)antibodies,recombinant ORFV B2L and 6×His·tag protein was prokaryotically expressed.The expressed and purified recombinant B2L protein was identified by Western-blot.An indirect ELISA for detecting goat serum ORFV antibody level was established by using the recombinant B2L protein as coating antigen and the reaction conditions were optimized.Specificity,sensitivity and repeatability tests were carried out.Finally,total 384 goat serum samples from some areas of Sichuan Province were detected by the established indirect ELISA.In results,42 ku recombinant ORFV B2L protein was successfully expressed in prokaryotic cells.Western-blot results showed that the recombinant protein had good antigenic reactivity.The optimum reaction conditions were as follows:coating antigen mass concentration was 5μg/m L,serum diluted by 1∶100 reacting to coated antigen for 2 hours,enzyme-labeled antibody diluted by 1∶2000 combining for 30 min,and TMB colour developing time was 15 min.Under the optimum conditions,D450(≥0.31)was positive.The method had good specificity,sensitivity and repeatability.The total positive rate of the 384 samples was 66.67%(256/384),of which 34.81%(55/158)were not vaccinated.In conclusion,the indirect ELISA established in this study can be used for the detection of ORFV antibody,and the goat farms in some areas of Sichuan had a higher probability of wild ORFV infection.The immune effect of the vaccine is not good.