猪繁殖与呼吸综合征病毒BJ3毒株N蛋白的原核表达及其单克隆抗体的制备

Prokaryotic expression of N protein of porcine reproductive and respiratory syndrome virus BJ3 strain and preparation of its monoclonal antibody

为表达猪繁殖与呼吸综合征病毒(PRRSV)BJ3毒株N蛋白及制备抗N蛋白单克隆抗体,将N基因克隆至原核表达载体pColdⅠ,利用大肠杆菌BL21表达出了N蛋白。将IPTG诱导的细菌反复冻融3次后超声裂解,离心后分别收集上清和沉淀,用镍柱亲和层析分别纯化,SDS-PAGE结果显示,N蛋白主要以包涵体的形式存在。用纯化的N蛋白免疫小鼠,取免疫小鼠脾细胞和SP2/0骨髓瘤细胞融合,间接ELISA法筛选阳性杂交瘤细胞,3次亚克隆后获取1株PRRSV N单抗,命名为F4。F4可与大肠杆菌BL21、不同PRRSV分离株表达的N蛋白发生特异性反应。本试验为进一步研究N蛋白的结构、功能及建立快速诊断PRRSV方法奠定了基础。

In order to express the N protein of porcine reproductive and respiratory syndrome virus(PRRSV)BJ3 strain and prepare anti-N protein monoclonal antibody,the N gene was cloned into the prokaryotic expression vector pColdⅠ,and the N protein was expressed using E.coli BL21.Bacteria liquidinduced by IPTG were repeatedly frozenand thawed 3 times before being lysed by ultrasound.After centrifugation,the supernatant and precipitate were collected respectively.The supernatant and precipitate were purified by nickel column affinity chromatography.The results of SDS-PAGE showed that the N protein mainly existed as inclusion bodies.The purified N protein was used to immunize mice.The spleen cells of the immunized mice were fused with SP2/0 myeloma cells.The positive hybridoma cells were screened by indirect ELISA.After 3 subclones,a PRRSV N monoclonal antibody was obtained and namedas F4.F4 can react specifically with the N protein expressed by E.coli BL21 and different PRRSV isolates.The results of this experiment can lay a certain material research foundation for further research on the structure and function of N protein and the establishment of rapid diagnosis of PRRSV isolates.