牛口蹄疫病毒VP2结构蛋白抗体间接ELISA方法的建立

Development of VP2 indirect ELISA for detection of cattle antibodies against foot-and-mouth disease virus

为建立牛口蹄疫(FMD)抗体的检测方法,本研究将口蹄疫病毒(FMDV)的VP2基因,通过pPROEXTM HTb表达载体在大肠杆菌DH5α中表达,获得大小为35ku的重组VP2蛋白(rVP2),western blot证实rVP2可与FMDV5种血清型的牛阳性血清发生特异性反应。以纯化复性的rVP2为抗原建立了FMDVrVP2间接ELISA方法。重复性试验证实批内、批间变异系数均小于10%。特异性交叉试验表明,该抗原不与常见的其他7种牛病阳性血清发生交叉反应。检测非免疫无口蹄疫国家牛阴性血清的特异性为100%;检测感染血清敏感性为97.3%;检测O-AsiaⅠ的二价苗免疫牛血清,与4种商品化试剂盒比较,其符合率分别为69.0%、95.0%、90.4%和86.8%。实验结果表明建立的ELISA方法可以用于口蹄疫感染和免疫抗体检测。

The complete gene encoding the structural protein VP2 of FMDV was subcloned into expression vector pPROEXTM HTb and expressed in DH5α cells. Purified VP2 protein reacted positively with serotype-specific cattle sera of 5 serotypes of FMDV （O, A, C, SAT 2 and Asia I ） by western blot. An indirect ELISA （VP2-ELISA） was developed using purified protein as coating antigen to detect FMDV antibodies in cattle. The assay was highly specific and showed no cross-reaction with the positive sera of other bovine diseases. The sensitivity of the assay was 97.3 % against infected sera. Comparison with four commercial kit showed a coincidence rate of 69.0 %, 95.0 %, 90.4 % and 86.8 % against positive cattle serum, respectively.