猪繁殖与呼吸综合征病毒(PRRSV)GP5-M在大肠杆菌中的融合表达及其ELISA诊断方法的建立  

Expression of GP5-M Fusion Protein of Porcine Reproductive and Respiratory Synd rone Virus(PRRSV)and Establishment of ELISA Diagnose Based on the Recombinant Fusion Protein

作　　者：江云波[1] 方六荣[1] 肖少波[1] 谢甜甜[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物医学院动物病毒室,430070

出　　处：《生物工程学报》2005年第2期259-264,共6页Chinese Journal of Biotechnology

基　　金：家自然科学基金 (No .3 0 3 0 0 2 5 7);武汉市青年科技晨光计划 (No .2 0 0 2 5 0 0 10 41)资助项目~~

摘　　要：利用谷胱甘肽 S 转移酶 (GST)表达系统将猪繁殖与呼吸综合征病毒 (PRRSV)的ORF5和ORF6基因依序串联于GST下游 ,并在大肠杆菌中成功表达 ,获得了大小约为 6 0kD的融合蛋白GST GP5 M ,Westernblot检测证实表达的融合蛋白具有良好的生物学活性。以纯化的融合蛋白为抗原建立了猪繁殖与呼吸综合征P5 6 ELISA检测方法 ,与国外试剂盒IDEXX ELISA总符合率为 94 1% ,表明建立的P5 6 ELISA检测方法具有很好的特异性和敏感性。进一步分析了P5 6 ELISA检测结果与血清中和试验的相关性 ,经回归函数分析 ,发现在临床送检猪血清中的抗融合蛋白GP5 M抗体水平 (OD6 30nm)与中和抗体水平之间的相关性并不高。The cDNA fragment encoding the truncated GP5 and the full-length M pr o tein of Porcine Reproductive and Respiratory Syndrone Virus(PRRSV)were orderly fused to the downstream of glutathione S-transferase(GST) of pGEX-KG expressi on vector, resulting in the fusion expression plasmid pKG-56. After transformed in to E.coli BL21(DE3)and induced by IPTG, the results of SDS-PAGE showed t hat t he GST-GP5-M fusion protein was expressed in high level. Western-blot was per for med to confirm that the expressed fusion protein could specifically react with a ntiserum against PRRSV. The fusion protein was further purified and used as an a ntigen 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 a ntibody response to the fusion protein GP5-M was further studied.The regressio n function analysis suggested that there was no significant correlation between E LISA antibody response (OD 630nm) to the fusion protein GP5-M in cli nical serum and their specific neutralizing titers.

关 键 词：猪繁殖与呼吸综合征病毒 GP5-M融合蛋白 ELISA 中和抗体 相关性 

分 类 号：S852.65[农业科学—基础兽医学]