H5亚型AIV HA抗原表位重组蛋白McAbs的制备及应用

目的以H5亚型禽流感病毒(avian influenza virus,AIV)的血凝素(HA)抗原表位重组表达蛋白为抗原,探索H5亚型AIV单克隆抗体制备的简便有效途径。方法利用H5亚型AIV的HA抗原表位原核表达重组蛋白为抗原,4次免疫8～10周龄雌性BALB/c小鼠后,取小鼠脾细胞与SP2/0骨髓瘤细胞融合,用间接ELISA方法筛选能分泌单克隆抗体的杂交瘤细胞株,并对单克隆抗体的特性及初步应用价值进行测试。结果得到一株能稳定分泌针对H5亚型AIVHA抗原表位的特异性单克隆抗体杂交瘤细胞株,单克隆抗体ELISA效价达1:5×104,为具有IgK轻链的IgM亚类。该单克隆抗体能特异性地与H5亚型AIV产生肉眼可见的WesternBlot免疫印迹反应,与其它供试的禽病抗原无反应。H5N1亚型AIV阳性血清能有效阻断辣根过氧化酶标记的单克隆抗体与HA抗原表位重组蛋白的结合。结论本研究探索出一条利用H5亚型AIV的HA抗原表位重组表达蛋白为抗原的单克隆抗体制备的简便、高效途径,所制备的单克隆抗体稳定性好、效价高、特异性强,在H5亚型禽流感病毒及其血清学检测中有较高的应用价值。

Production and Application of Monoclonal Antibodies Against Recombinant Protein of H5 subtype AIV HA Epitopes

Objective To develop an easier and more efficient way for monoclonal antibodies （McAbs） production by using the recombinant protein of H5N1 subtype avian influenza virus （AIV） hemagglutinin （HA） epitopes. Method 8 to 10-week-old female BALB/c mice were immunized four times with prokaryotic expression recombinant protein of H5N1 subtype AIV HA epitopes. Spleen cells from the immunized mice were fused with SP2/0 myeloma cells line. McAbs hybridoma cell lines were screened by indirect enzyme linked immunosorbent assay （ELISA）, and the McAbs profiles as well as their application value were evaluated. Result A hybridoma cell line was abtained stably secreting anti-HA epitopes McAbs specific for H5N1 subtype AIV. The McAbs were of IgM subtype with IgK light chain and the titres were up to 5×10^4 by indirect ELISA. In visible Western blot, they could react specifically with H5 subtype AIV and not with other avian pathogens. H5N1 subtype AIV positive serum could block conjugations of McAbs horseradish peroxidase labeled with HA epitopes recombinant protein. Conclusion A convenient and efficient way was found to develop McAbs by HA epitopes recombinant protein for H5 subtype AIV. The prepared McAbs were stable, and of high titre and specificity. They could be used as detective reagent either in pathogenic or serological diagnostic techniques for H5 subtype AIV.