猪繁殖与呼吸综合征病毒非结构蛋白NSP2的原核表达及其单克隆抗体的制备

Expression of Truncated NSP2 Protein of Porcine Reproduction and Respiratory Syndrome Virus in E.coli and Preparation of Monoclonal Antibodies Against NSP2 Protein

将猪繁殖与呼吸综合征病毒(PRRSV)S1株截短修饰的NSP2蛋白基因(tNSP2),克隆于pGEX-6P-1载体中,转化大肠杆菌后用IPTG诱导表达。Western blot结果表明,融合表达的GST-tNSP2蛋白能被PRRSV阳性血清特异性识别,大小约50kDa。经GST柱提取纯化蛋白GST-tNSP2,免疫Balb/c小鼠,取脾细胞与SP2/0骨髓瘤细胞进行融合,获得2株能稳定分泌抗NSP2蛋白抗体的杂交瘤细胞株,将其命名为2B5、3H3,亚型鉴定结果均为IgG1型,其轻链均为κ链。间接免疫荧光试验证明,2B5和3H3均能与PRRSV S1毒株产生特异性反应,而不能与SY0608毒株反应,从而为PRRSV分离株的鉴定及NSP2功能研究奠定了重要基础。

The NSP2 gene of porcine reproductive and respiratory syndrome virus （PRRSV） S1 strain was partly amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and a fusion protein GST-tNSP2 with molecular weight of 50 kDa was expressed in E. coli. The purified GST-tNSP2 protein showed a strong reaction with the PRRSV-positive sera in Western blot assay. Balb/c mice were immunized with the purified protein, and the splenocytes of the immunized mice were fused with murine myeloma cells SP2/0. After subcloning by 3 times, two hybridoma clones which produced McAbs steadily were screened by ELISA, named 3H3 and 2B5. They all reacted strongly with the PRRSV S1 infected Marc-145 cells in IFA, but not with the PRRSV SY0608 strain. Both of the McAbs belong to IgG1 isotype, and their light chains belong K type. The expressed GST- tNSP2 protein and McAbs could be used for identification of PRRSV isolates and functional analysis of NSP2.