基因免疫制备西尼罗病毒NS5蛋白特异性单抗

Preparation of Specific Monoclonal Antibodies against Protein NS5 of West Nile Virus by Genetic Immunization

研究了NS5蛋白在西尼罗病毒的特异性检测方面的应用及NS5在黄病毒复制中的作用机理。采用RT-PCR方法扩增了西尼罗病毒株的NS5基因片段,将其克隆至真核表达载体pVAX1,构建真核表达质粒。以重组质粒免疫BALB/c小鼠后取脾脏进行杂交瘤细胞融合,建立能稳定分泌西尼罗NS5单克隆抗体的杂交瘤细胞株。构建了真核表达质粒pVAX1-WNV-NS5,免疫动物后获得了2B2B9等4株稳定分泌特异性抗体的杂交瘤细胞株,均为IgM型。真核表达质粒免疫后成功地诱导了针对NS5蛋白的体液免疫应答,单抗特异性分析显示4株单抗与其他黄病毒存在一定交叉反应。

In order to study the application of anti-West Nile virus NS5 monoclonal antibodies （anti-WNV NS5 McAbs） to specific detection of West Nile virus, a fragment from NS5 gene of West Nile virus was amplified with RTPCR and cloned into an eukaryotic expression vector pVAX1 to construct an eukaryotic expression plasmid. BALB/c mice wee immunized with the recombinant plasmids by intramuscular injection. The mice with strong immune response were chosen and their spleen cells were taken and fused with hybridoma cells to establish hybridoma cells strain that could stably secrete anti-WNV NS5 McAbs. 2B2B9 etc. four strains that could stably secrete specific antibodies hybridolna cells lines were obtained after immunization of animal with the constructed eukaryotie expression plasmid pVAXI-WNV-NS5, and all of them belonged to IgM subclass/subtype/subgroup. Therefore, the immunization of eu- karyotic expression plasmid successfully induced and aimed at NS5 protein humoral immune response, and analysis on monoelonal antibodies specificity indicated that there were a certain cross responses of four monoelonal antibodies with other flaviviruses.