高致病性猪繁殖与呼吸综合征病毒HuN4株GP3单克隆抗体的制备及其抗原表位的鉴定

Identification of a B-cell epitope in GP3 of highly pathogenic porcine reproductive and respiratory syndrome virus HuN4 strain

为制备抗高致病性猪繁殖与呼吸综合征病毒(H P-PRRSV)G P3株的单克隆抗体(M A b),本研究将H P-PRRSV H uN 4株免疫BA LB/c小鼠,以该病毒感染的细胞及真核表达的G P3蛋白为检测抗原,经间接免疫荧光(IFA)筛选获得了一株稳定分泌M A b的细胞株,命名为4G 5。抗体亚类鉴定重链类型为IgG 1,轻链类型为κ。该M A b细胞培养上清及腹水IFA效价分别为1∶256和1∶1 280。IFA结果显示,MAb4G5能够识别CH-1R、JXA1-R、HP-PRRSVHuN4株及其疫苗株HuN4-F112,而不识别RespPRRS M LV病毒株。Western blot结果显示,4G5与HP-PRRSVHuN4株及原核表达的GP3蛋白均可以反应,表明其针对的抗原表位为线性表位,中和试验结果显示该M A b无中和活性。通过截短表达GP3蛋白鉴定该M A b抗原表位识别序列为74W CRIGHD RCS83。本研究获得的M A b为进一步研究HP-PRRSV GP3蛋白的结构及功能奠定了基础。

To prepare monoclonal antibodies （MAb） against the GP3 of highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV）, SP2/0 cells was fused with the splenic cell from BALB/c mice immunized with HP-PRRSV HuN4 strain. A hybridoma cell line that stably secreted MAb against HP-PRRSV GP3 was scanned by indirect immunofluorescence assay （IFA）. The titers of the MAb in cell culture medium and ascites were 1：256 and 1：1 280, respectively. The MAb was able to react specifically with PRRSV CH-1R, JXA1-R, HuN4 and HuN4-F112, but not with RespPRRS MLV strain detected by IFA. Western blot analysis showed that the MAb was capable of reaction with GP3 expressed in E.coli, but had no virus neutralizing activity. Furthmore, the recognized epitope of the MAb was mapped at 74WCRIGHDRCSS3 in HP-PRRSV GP3 by scanning with series of short peptides expressed in E.coli. The MAb could be useful for further study of the structure and function of PRRSV GP3.