College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Intervirology. 2012;55(1):12-20. doi: 10.1159/000322220. Epub 2011 Jan 14.
The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain. Recombinant protein was employed as the coating antigen to develop an indirect ELISA for PPRV antibody detection in the sera of infected small ruminants. Antibody detection was optimal at a 1:200 serum dilution and an antigen concentration of 3.2 μg/ml, and the positive threshold (cutoff) value of the assay was 2.18. Analysis of 697 serum samples revealed the sensitivity and specificity of the indirect ELISA to be 96.7 and 96.1%, respectively, compared with a commercially available ELISA test.
利用重叠 PCR 技术，无需病毒基因组 cDNA 作为模板，分两步合成了 2007 年西藏小反刍兽疫爆发中负责的病毒（PPRV）核衣壳（N）蛋白全长基因。全长 N 基因在大肠杆菌中成功表达，纯化的基因产物与针对 PPRV N 蛋白的单克隆抗体结合。此外，它能够替代重组 B-N 抗原作为包被抗原，用于使用另一种 PPRV 株制备的商业 ELISA 试剂盒。重组蛋白被用作包被抗原，开发用于检测感染小反刍动物血清中 PPRV 抗体的间接 ELISA。在血清稀释度为 1:200 和抗原浓度为 3.2μg/ml 时，抗体检测效果最佳，该检测方法的阳性阈值（截止值）为 2.18。对 697 份血清样本的分析表明，与商业 ELISA 检测相比，间接 ELISA 的敏感性和特异性分别为 96.7%和 96.1%。
