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

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...

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

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.