Jiang Yun-Bo, Fang Liu-Rong, Xiao Shao-Bo, Xie Tian-Tian, Chen Huan-Chun
Laboratory of Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong Agriculture University, Wuhan 430070, China.
Sheng Wu Gong Cheng Xue Bao. 2005 Mar;21(2):259-64.
The cDNA fragment encoding the truncated GP5 and the full-length M protein of Porcine Reproductive and Respiratory Syndrone Virus (PRRSV) were orderly fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKG-56. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-GP5-M fusion protein was expressed in high level. Western-blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against PRRSV. The fusion protein was further purified and used as an antigen to establish a novel PRRSV ELISA diagnose assay (P56-ELISA). Comparison between P56-ELISA and the abroad kit IDEXX-ELISA showed the two methods had 94.1 percent agreement by detecting 205 serum samples, indicating that the indirect P56-ELISA was specific and sensitive. The correlation between virus neutralization antibody of the infected pigs (not convalescent pigs) and antibody response to the fusion protein GP5-M was further studied. The regression function analysis suggested that there was no significant correlation between ELISA antibody response (OD630 nm) to the fusion protein GP5-M in clinical serum and their specific neutralizing titers.
将编码猪繁殖与呼吸综合征病毒（PRRSV）截短型GP5和全长M蛋白的cDNA片段依次融合至pGEX-KG表达载体的谷胱甘肽S-转移酶（GST）下游，构建融合表达质粒pKG-56。将其转化至大肠杆菌BL21（DE3）中，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示GST-GP5-M融合蛋白获得高效表达。通过蛋白质免疫印迹法（Western-blot）证实表达的融合蛋白能与PRRSV抗血清发生特异性反应。进一步纯化该融合蛋白并将其作为抗原建立了一种新型PRRSV酶联免疫吸附测定法（P56-ELISA）。通过检测205份血清样本，比较P56-ELISA与国外试剂盒IDEXX-ELISA，结果显示两种方法的符合率为94.1%，表明间接P56-ELISA具有特异性和敏感性。进一步研究了感染猪（非康复猪）的病毒中和抗体与对融合蛋白GP5-M的抗体反应之间的相关性。回归函数分析表明，临床血清中对融合蛋白GP5-M的酶联免疫吸附测定抗体反应（OD630 nm）与其特异性中和效价之间无显著相关性。
