一株针对猪繁殖与呼吸综合征病毒NADC30-like毒株GP5蛋白的单克隆抗体的制备及鉴定

Preparation and Identification of a Monoclonal Antibody against GP5 Protein of Porcine Reproductive and Respiratory Syndrome Virus NADC 30-like Strain

关键词:  猪繁殖与呼吸综合征病毒  NADC30-lke毒株  GP5蛋白  单克隆抗体

Key words:  PRRSV  NADC30-like strain  GP5 protein  Monoclonal antibody

摘要:

为获得针对猪繁殖与呼吸综合征病毒NADC30-like毒株GP5蛋白的单克隆抗体，采用表达纯化的PRRSV NADC30-like毒株的GP5蛋白按常规方法免疫小鼠，采用间接免疫荧光方法筛选出1株阳性杂交瘤细胞（命名为3E10），经3次亚克隆后制备腹水并进行纯化鉴定。结果显示：该杂交瘤细胞连续传代20代后细胞上清液的IFA效价仍不低于1:40。纯化后的单克隆抗体腹水浓度为0.204 mg/mL，亚类鉴定为IgM，且未发现有中和活性。特异性检测显示3E10单克隆抗体能与2株PRRSV NADC30-like毒株发生荧光反应，而与4株高致病性PRRSV疫苗毒株、2株经典株PRRSV疫苗毒株以及CSFV、PCV2、BVDV及Marc-145细胞均无荧光反应，显示出较好的应用前景。

Abstract:

In order to obtain monoclonal antibody against GP5 protein of porcine reproductive and respiratory syndrome virus (PRRSV) NADC 30-like strain, mice were immunized with GP5 protein of PRRSV NADC 30-like strain by conventional method. A positive hybridoma cell (named 3E10) was screened by indirect immunofluorescence method,which was prepared and purified after three subclones. The results showed that the IFA titer of the cell supernatant was not less than 1:40 after 20 passages. The concentration of purified monoclonal antibody in ascites was 0.204 mg/mL. the subclass was identified as IgM, and no neutralizing activity was found. Specific detection showed that 3E10 monoclonal antibody could react with 2 PRRSV NADC30-like strains, but not with 4 highly pathogenic PRRSV vaccine strains, 2 classical PRRSV vaccine strains, CSFV, PCV2, BVDV and Marc-145 cells, showing a good prospect of application.