1株抗欧洲型和美洲型PRRSV核衣壳蛋白单克隆抗体的制备与鉴定

利用RT-PCR方法扩增出欧洲型猪繁殖与呼吸综合征病毒(PRRSV)SS-6毒株的核衣壳(N)蛋白基因,将其克隆到原核表达载体pGEX-6p-1上,构建了重组表达质粒pGEX-N,并将其转化到大肠杆菌BL21中,在诱导剂IPTG的诱导下获得高效表达,重组蛋白的分子量约为40 ku。用纯化的重组N蛋白免疫Balb/c小鼠,采用杂交瘤技术制备抗PRRSV N蛋白的单克隆抗体。经细胞融合获得一株可稳定分泌特异性单抗的杂交瘤细胞株,命名为1 B6,将其连续培养20代后仍能稳定分泌抗体。亚型鉴定结果为IgG1型,其轻链为κ链;间接免疫荧光试验和免疫印迹试验均证明,1B6单抗既能与欧洲型PRRSV反应,也能与美洲型PRRSV反应;ELISA检测杂交瘤细胞培养上清抗体效价为1:640,腹水的效价为1:256 000,为猪繁殖与呼吸综合征的诊断奠定了基础。

Preparation and identification of one monoclonal antibody against nucleocapsid protein of European and American-type porcine reproductive and respiratory syndrome virus

The nucleoeapsid (N) protein gene of European-type porcine reproductive and respiratory syndrome viIus (PRRSV) was amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and a fusion protein GST-N with molecular weight of 40 ku was expressed in E. coll. The Balb/c mice were immunized subcutaneously with the purified recombinant protein GST-N, and then hybridoma technique was utilized to develop monoclonal antibodies (McAb). A hybridoma cells steadily secreting specific McAb against N protein was screened and named as 1B6. It belonged to IgG1 isotype, and the light chain was κ chain. The analysis of indirect immunofluorescence assay (IFA) and Western blotting showed that 1B6 McAb could react with European-type PRRSV as well as North American PRRSV. The ELISA titer of cultural supernatant of hybridoma cells was 1:640 while the titer of aseites obtained fr6m mice was 1:256 000. This study will apply a basis for the detection of PRRSV.