基于VP1重组蛋白的口蹄疫病毒O型间接ELISA检测技术

Indirect ELISA based on recombinant VP1 for the testing of antibodies against Foot and mouth Disease virus serotype O

采用RT-PCR方法扩增O型口蹄疫病毒VP1基因片段,将其克隆于pET-32a。将重组质粒转化宿主菌Rosetta,经1.0mmol/L IPTG诱导,外源基因获高效表达。通过Western-blot以及ELISA试验证明,重组VP1蛋白与标准阳性血清发生反应。以纯化的VP1蛋白作为检测抗原包被酶标板建立了检测口蹄疫病毒O型抗体的间接ELISA方法。对355份临床血清样品的检测结果显示,建立的方法与口蹄疫病毒O型液相阻断试剂盒符合率为98.87%。表明建立的方法能够用于口蹄疫病毒O型抗体的快速分型检测。

VP1 gene of Food-and-mouth disease virus serotype O was amplified by RT-PCR, then cloned into pET-32a. The VP1 was expressed at a very high level after the recombinant pET-32a-VP1 plasmid was transformed into host bacteria Rosetta and was induced with 1.0 mmol/L IPTG. The reactivity of the expressed product to the positive serum was demonstrated by Western-blot and ELISA. An indirect ELISA for testing antibodies against FMDV serotype O was established using the purified recombinant protein as the coating antigen. By parallelly detecting a total of 355 serum samples with the established ELISA and FMDV serotype O liquid-phase blocking ELISA Kit, the calculated coincidence rate was 98.87%. It suggested the established ELISA could be used to detect antibodies against FMDV serotype O.