抗人AFP单链抗体的制备、鉴定及其免疫学活性的测定

目的 构建具有免疫学活性的抗人甲胎蛋白单链抗体ScFv (single chain fragment of variety region,ScFv),为进一步的研究奠定实验基础.方法 采用RT-PCR 技术,从分泌抗人AFP 单克隆抗体的杂交瘤细胞中扩增mAb 的VH、VL 基因,并进一步将其组装成VH-Linker-...

Construction, identification and determination of immunologic activity of murine anti-AFP ScFv

Objective To construct and identify the single-chain variable fragment(scFv) of monoclonal antibody against murine AFP and analyze the immunologic activity of recombinant ScFv protein.Methods The VL and VH genes were cloned by RT-PCR from anti-murine AFP mAb excreted by hybridoma cells.VH-linker-VL fragment(ScFv) was constructed and cloned into vector pUC19.The purified pUC19-ScFv plasmid was transformed into BL21 cells and induced by IPTG,the expression and activity of ScFv protein were analyzed with SDS-PAGE and ELISA.Results BL21 cells could express recombinant ScFv induced by IPTG.SDS-PAGE analysis revealed that the ScFv was expressed in inclusion body.Indirect ELISA results indicated that the expressed ScFv could bind specifically to recombinant protein of murine AFP,and the epitope recognized by the ScFv was same as the parental mAb of AFP.Conclusion Expression vector encoding murine anti-AFP ScFv is successfully constructed and it can efficaciously express ScFv with immune activity and specificity.