抗狂犬病病毒N蛋白单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against the nucleoprotein of rabies virus

目的制备抗狂犬病病毒N蛋白单克隆抗体,为建立快速准确的狂犬病病毒抗原检测方法奠定基础。方法将狂犬病病毒Flury LEP株N基因克隆至pGEX-6P-1表达载体中,将纯化后的pGEX-6P-1-RV-N蛋白免疫BALB/c小鼠3次。取小鼠脾脏细胞和SP2/0细胞融合后,用pET-32a-RV-N重组蛋白作为筛选抗原,经3次亚克隆筛选后得到1D9、2B6、3C5、6B5及5F2 5株单克隆抗体细胞株。亚类鉴定结果表明,其中1D9、2B6、6B5 3株亚类为IgG1,3C5、5F2的亚类为IgM。间接ELISA方法检测2B6单抗细胞腹水效价可达1∶106。经Protein G亲和层析柱纯化和脱盐后可得到浓度为2.5mg/mL的高纯度的单克隆抗体。其免疫荧光试验结果表明5株单克隆抗体均能特异地与狂犬病病毒结合。结论制备的单抗具有良好的特异性和敏感性,为后期诊断方法的建立奠定了基础。

In order to establish an immunological method of detecting rabies virus,we generated monoclonal antibodies against the nucleoprotein of rabies virus（RV）.In our study,the nucleoprotein（N） gene was cloned into prokaryotic expression vector pGEX-6P-1 and the protein was expressed in the Escherichia coli BL21（DE3） introduced by IPTG（isopropyl-β-D-thiogalactopyranoside）.The antigenic specificity of the RV N protein was confirmed by western blot.The purified pGEX-6P-1-RV-N was used as an antigen to immunize BALB/c mice.The spleen cells of the BALB/c mice were fused with the SP2/0 cells.Five hybridoma cell lines named 1D9,2B6,3C5,6B5,5F2,respectively,were screened and obtained by indirect ELISA with pET-32a-RV-N as an antigen.The subtype of 1D9,2B6,6B5 is IgG1 and the other two is IgM.The ascites from MAbs 2B6 reacted with purified pET-32a-RV-N protein in ELISA at a titer of 1∶106.The results of IFA indicated that these MAbs were specific to rabies virus.The results showed that this established method is rapidly and highly specific for the diagnosis of RV.