PRRSV GP5和M抗原中和表位的融合表达与纯化以及间接ELISA检测方法的建立  

Fusion Expression and Purification of Neutralizing Epitopes of PRRSV GP5/M Proteins and Establishment of GP5/M-based Indirect ELISA

作　　者：李雷斌 李晓霞 王睿男[2] 周智[2] 刘玉良[2] 赵柏林[2] 孙航 冯冰 陈玲 王传彬[2] 李义平[1] 孙雨[2] Li Leibin;Li Xiaoxia;Wang Ruinan;Zhou Zhi;Liu Yuliang;Zhao Bolin;Sun Hang;Feng Bing;Chen Ling;Wang Chuanbin;Li Yiping;SunYu(College of Animal Science and Technology,Guangxi University,Nanning,Guangxi 530004,China;China Animal Disease Control Center,National/OIE Porcine Reproductive and Respiratory Syndrome Reference Laboratory,Beijing 102600,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]中国动物疫病预防控制中心,国家/OIE猪繁殖与呼吸综合征参考实验室,北京102600

出　　处：《中国动物检疫》2022年第3期71-79,共9页China Animal Health Inspection

基　　金：现代农业产业技术体系建设北京市创新团队项目(BAIC02-2021)。

摘　　要：为建立检测猪血清中猪繁殖与呼吸综合征病毒(PRRSV)中和抗体的间接ELISA方法,分析了PRRSV结构蛋白GP5和M的二级结构抗原指数、抗原性、亲水性、表面可及性以及潜在中和表位等参数,选取GP5的47~61、143~156和186~199位氨基酸,M蛋白的63~70、133~146和156~169位氨基酸作为免疫原,构建带接头序列的GP5/M中和表位融合表达质粒;通过优化大肠杆菌表达和蛋白纯化条件,免疫印迹鉴定GP5/M特异抗原,获得了可溶性的GP5/M中和表位融合蛋白。基于纯化的GP5/M中和表位融合抗原,建立了检测猪血清中PRRSV中和抗体的间接ELISA方法。采用建立的间接ELISA方法对6种其他猪源病原阳性血清进行检测,结果均无交叉反应;敏感性试验显示,将血清中和试验鉴定的PRRSV阳性对照血清稀释到1:12800时仍呈阳性;重复性试验显示,批内重复变异系数为2%~10%,批间重复变异系数小于15%;符合性试验显示,同时用建立的间接ELISA方法和血清中和试验对420份临床血清样品进行检测,结果符合率为95.24%。结果表明,该方法操作简单、耗时短,敏感性、特异性、重复性及符合性均良好,具有较好的应用推广价值。In order to establish an indirect ELISA method for detecting neutralizing antibodies against porcine reproductive and respiratory syndrome virus(PRRSV)in porcine serum,the secondary structure of GP5 and M proteins of PRRSV for antigenic index,antigenicity,hydrophilicity,surface accessibility,and potential neutralizing epitopes were analyzed.Amino acids 47~61,143~156 and 186~199 of GP5 protein and 63~70,133~146 and 156~169 of M protein were selected as immunogens to construct GP5/M fusion neutralizing epitope expression plasmid with linker sequence;following optimization of E.coli expression and protein purification conditions,the GP5/M specific antigens were identified by Western blot to obtain the soluble GP5/M fusion protein.Based on the purified GP5/M neutralizing epitope fusion antigen,an indirect ELISA method for detecting PRRSV neutralizing antibodies in pig serum was established.No cross reaction was observed when six positive serums of other porcine pathogens were detected by the established method.It was shown that,by the sensitivity test,the PRRSV positive control serum identified by serum neutralization test was still positive when diluted to 1:12800;by the repeatability test,the intrabatch repeated variable coefficient was 2%to 10%,and the inter-batch one was less than 15%;and by the coincidence test,the coincidence rate was 95.24%when 420 clinical serum samples were detected by the established method and serum neutralization test.In conclusion,the established method was characterized by the advantages of simple and rapid operation,good sensitivity,specificity,repeatability and consistency,which was valuable to be applied and popularized.

关 键 词：猪繁殖与呼吸综合征病毒(PRRSV) 中和表位抗原 融合GP5/M ELISA 

分 类 号：S851.3[农业科学—预防兽医学]