甲型流感病毒核蛋白单克隆抗体制备及其初步应用

Preparation of monoclonal antibodies against the nucleoprotein of influenza A virus and its preliminary applications

目的 制备甲型流感病毒核蛋白（NP）单克隆抗体,为甲型流感病毒诊断试剂盒以及相关研究提供材料.方法 大肠埃希菌BL21 （DE3）表达NP作为免疫原,免疫Balb/c小鼠后取脾脏细胞与SP2/0细胞融合,筛选获得阳性细胞株,注射小鼠腹腔得到单克隆抗体,纯化后进行Western印迹法、ELISA法、细胞免疫染色和荧光法、双抗夹心ELISA法确定NP抗体的生物学特性,探索应用范围.结果 从Western印迹法、ELISA和细胞免疫染色法的结果可得知,抗NP单克隆抗体可以与免疫原特异性结合,也可以与H1N1、H3N2亚型流感病毒的NP特异性结合,具备检测流感病毒的能力.双抗夹心ELISA法进一步提升了抗体对抗原的检测灵敏度至128 pg.同时,通过细胞免疫荧光法观察到抗体可在细胞内显示NP从细胞核扩散到细胞质的过程.结论 制备的抗体成功检测H1N1、H3N2两种亚型的流感病毒,可作为用于流感防治的流感病毒诊断试剂盒的材料.

Objective To obtain monoclonal antibodies against nucleoprotein （NP） of influenza A virus,and provide materials for influenza A virus detection kits and related researches.Methods The Balb/c mice were immunized with the purified expression NP of Escherichia coli BL21 （DE3） and their spleen cells were fused with SP2/0 cells.The positive hybridoma cells were selected,and the monoclonal antibodies were obtained by intraperitoneal injection of these positive strains in mice.The purified antibodies were tested by Western blot,ELISA,cell-mediated immunity experiments,double-antibody sandwich ELISA method to confirm the biological characteristic and explore the preliminary applications.Results It was showed by Western blot,ELISA and cellmediated immunity experiments that the monodonal antibodies could specifically combine with both immunogeu and NP of H1N1,H3N2 subtypes of influenza virus,and had the influenza virus detection ability.By using doubleantibody sandwich ELISA method,it could improve the detection sensitivity up to 128 pg.Moreover,we could observe the location of NP from nuclear to cytoplasm in MDCK cell by using this antibody.Conclusions The prepared NP antibodies can successfully detect H1N1 and H3N2,which are predicted to be the materials of influenza diagnosis kits for flu prevention.