摘要
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.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2011年第10期866-870,共5页
Chinese Journal of Zoonoses
基金
国家高技术研究发展计划(863计划)(No.2008AA10Z411)
北京市科委项目(Z07010501780701)
国家公益行业项目(No.200830-14
200903037-2)
中央级公益性科研院所基本科研业务费专项(0032007008)
关键词
狂犬病病毒
N蛋白
表达
单克隆抗体
rabies virus
N protein
expression
monoclonal antibody