抗H5N1病毒嵌合IgA抗体基因的构建及其在CHO细胞中的表达

Construction of anti-H5N1 virus chimeric IgA antibody gene and its expression in CHO cells

摘要:

为了表达具有中和活性的抗禽流感H5N1病毒人-鼠嵌合IgA抗体, 采用RT-PCR法克隆具有中和活性的抗禽流感H5N1-HA鼠源单克隆抗体的轻重链可变区基因及相应的信号肽编码序列, 分别与人免疫球蛋白IgA2重链恒定区、Kappa恒定区基因拼接, 构建表达质粒pEF-IGHA9和pEF-IGK9, 共转染二氢叶酸还原酶缺陷型CHO(CHO-dhfr-)细胞,用ELISA检测培养上清中嵌合IgA抗体的表达, 对纯化的嵌合抗体进行SDS-PAGE、Western blotting 印迹分析。结果成功地在CHO细胞中表达了抗禽流感H5N1病毒人-鼠嵌合IgA抗体, 为制备抗H5N1重组分泌型IgA预防性抗体制剂奠定了良好的基础。

Abstract:

To express human-mouse chimeric IgA antibody directed against H5N1 virus, an anti-H5N1 chimeric IgA antibody gene was constructed by joining the light and heavy chain variable region genes and the corresponding signal peptide coding sequences of the anti-H5N1 mouse monoclonal antibody H5N1-HA with the coding sequences of the constant region of the human IgA2 heavy chain and Kappa chain respectively. Then the full-length chimeric light and heavy chain expressing plasmids pEF-IGHA9 and pEF-IGK9 were constructed and transfected into the CHO/dhfr- cells. The chimeric IgA antibody expression was confirmed by ELISA, SDS-PAGE and Western blotting. The successful expression of this anti-H5N1 chimeric IgA may help to provide a stand for developing passive immunological agents for H5N1 virus infection prophylaxis.