基于单个B细胞抗O型口蹄疫病毒单克隆抗体的研制

Development of mouse monoclonal antibodies against type O FMDV based on single B cell technology

为了建立基于单个B细胞制备抗体技术,本研究以O型灭活FMDV为抗原,采集健康C57/BL小鼠脾细胞,经过磁珠富集、流式B220+/CD38+/Ig G1+/抗原+分选,获得单个抗原特异性的B细胞。经单细胞巢式PCR反应扩增获得抗体完整轻、重链可变区序列后,分别构建轻/重链表达质粒,然后共转染HEK-293细胞后,成功获得3株特异性单克隆抗体,且证明均属于Ig G1型抗体;ELISA结果显示,获得的3株抗体均能与O型FMDV抗原结合;免疫荧光试验证明,3株抗体能够特异性结合细胞内复制的O型FMDV。本研究成功建立了基于单个B细胞直接制备抗FMDV单抗的方法,为后期开发特异性单克隆抗体打下了坚实的基础。

In order to develop a new method for producing mouse monoclonal antibodies against FMDV serotype O based on single B cell sorting,B cells from spleen of healthy C57/BL mice were sorted at the gate of B220+/CD38+/IgG1+/antigen+after magnetic beads enrichment.The single B cell against FMDV type O was sorted into 96 well plate and the variable c DNA regions of the light and heavy chains of the antibodies were cloned by single-cell nested PCR and expressed in HEK-293 cells.Three anti-type O FMDV Ig G1 antibodies were successfully expressed.The ELISA and immunofluorescence assay showed that these antibodies could specifically bind to type O FMDV antigen as well as the infectious type O FMDV in PK cells.Our results indicated that the established method could be used to efficiently screening FMDV specific antibodies.