美洲型与欧洲型猪繁殖与呼吸综合征病毒Nsp7蛋白的截短表达和鉴定

Expression and identification of truncated Nsp7 protein of North American and Europe genotype porcine reproductive and respiratory syndrome virus

摘要:

猪繁殖与呼吸综合征病毒 (PRRSV) Nsp7蛋白具有较强的免疫原性，且在美洲型 (NA) 和欧洲型 (EU) PRRSV之间免疫原性差异显著，是抗体分型检测的理想靶抗原。本研究采用原核表达系统分别表达和纯化了NA和EU PRRSV Nsp7蛋白，Western blotting分析表明重组蛋白与相应血清型抗体有较强的免疫反应，但特异性较差，与另一血清型抗体仍存在一定的免疫反应，预示两种血清型PRRSV Nsp7蛋白存在免疫交叉反应抗原表位，全长表达不能用于分型诊断。利用生物学软件分析NA和EU PRRSV Nsp7的相似抗原表位，采用融合PCR缺失其编码序列后，经表达纯化获得NA-?Nsp7和EU-?Nsp7两种截短蛋白，蛋白大小约为43 kDa，Western blotting分析表明NA-?Nsp7和EU-?Nsp7可分别与相应血清型抗体发生特异性反应，且无免疫交叉反应，为NA和EU PRRSV分型抗体检测试剂的研制奠定了基础。

Abstract:

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 7 (Nsp7) plays an important role in the induction of host humoral immune response and could serve as an ideal antigen for serological genotyping assay for PRRSV based on the significant difference in immunoreactivities of North American (NA) and European (EU) PRRSV Nsp7. In this study, Nsp7 of NA and EU PRRSVwas separately expressed and purified using prokaryotic expression system. The purified recombinant Nsp7 proteins reacted with serum antibodies against corresponding genotype PRRSV in Western blotting. However, nonspecific reaction of whole recombinant Nsp7 with antibodies against another genotype PRRSV was observed, indicating that whole NA PRRSV Nsp7 and EU PRRSV Nsp7 have similar antigenic epitopes and recombinant proteins could not be used for genotyping of antibodies against PRRSV. Based on the analysis of similar antigenic epitopes at the hydrophilic B region of NA PRRSV Nsp7 and EU PRRSV Nsp7 by bioinformatics assessment, partial Nsp7 gene region deleted sequences encoding similar antigenic epitopes was constructed by fusion PCR. The recombinant truncated Nsp7 (NA-?Nsp7 and EU-?Nsp7, about 43 kDa) was expressed and the molecular weight was about 43 kDa. The results of Western blotting showed that NA-?NSP7 and EU-?NSP7 could be specifically recognized by positive serum to NA or EU PRRSV individually and nonspecific reaction was eliminated. This study provided a basis for further development of serological genotyping assay for North American and European genotype PRRSV infection.