A型流感病毒PB1蛋白单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against PB1 protein of influenza A virus

以A型流感病毒A/Chicken/Zhejiang/YH/2/2013(H7N9)的PB1基因为模板,扩增其1519~2274bp间的片段,并构建原核表达载体pET-28a(+)-H7N9-sPB1。通过大肠杆菌原核表达获得重组蛋白His-sPB1后,以其作为抗原免疫BALB/c小鼠,通过细胞融合筛选获得3株能够稳定分泌单克隆抗体的细胞株5G5、2H3和5E8。Western-blot和IFA检测表明,3株单克隆抗体对于不同亚型的流感病毒均具有良好的反应性。免疫共沉淀结果表明,3株单克隆抗体能用于免疫共沉淀试验。A型流感病毒PB1蛋白单克隆抗体的制备为进一步研究PB1蛋白的结构与功能以及流感病毒的监测与诊断提供了必要的工具。

The polymerase basic protein 1(PB1)gene fragment(1519—2274 bp)of influenza A virus(IAV)A/Chicken/Zhejiang/YH/2/2013(H7 N9)was cloned into the prokaryotic expression vector p ET-28 a(+),named p ET-28 a(+)-H7 N9-s PB1,and transformed into E.coli BL21 which was used as the host for the expression of PB1 protein.The recombinant protein His-s PB1 was used as the immunogen for BALB/c mice to prepare monoclonal antibodies.Three cell lines that could steadily secrete monoclonal antibodies were obtained and named as 5 G5,2 H3 and 5 E8.Western-blot and indirect immunofluorescence assay(IFA)showed that all three monoclonal antibodies(m ABs)had reactivity to different subtypes of influenza viruses.Moreover,these m ABs could also be used to perform the immunoprecipitation(IP)experiment.In conclusion,the preparation of monoclonal antibody against IAV PB1 protein provides a necessary tool for the further study of the structures and functions of PB1 protein as well as the detection and diagnosis of influenza virus.