抗前列腺特异抗原/抗人CD3双特异性单链抗体基因在HeLa细胞中的表达及亲和活性测定

Expression of bispecific single chain Fv antibody gene against human prostate specific antigen and CD3 molecule in HeLa cells and its identification of binding affinity

目的 在HeLa细胞中表达抗前列腺特异抗原 (PSA ) /抗人CD3双特异性单链抗体(scFv)融合基因 ,并进行亲和活性测定。方法 在已经构建抗PSA/CD3双特异性scFv融合基因基础上 ,将融合基因克隆入真核表达载体pSecTag2 B中 ,并转染HeLa细胞进行表达。表达产物纯化后 ,用流式细胞仪进行亲和活性测定。结果 经SDS PAGE和Western印迹实验证实 ,表达产物的Mr约为 65 0 0 0。纯化后经流式细胞仪检测 ,可特异性地结合PC 3细胞和人外周血单个核细胞(PBMC)。结论 获得了可与PC 3细胞和PBMC特异结合的抗PSA/CD3双特异性scFv ,为进一步临床应用奠定了基础。

Objective To express the bispecific single chain Fv antibody (scFv) gene specific for human prostate specific antigen (PSA) and CD3 molecule [scFv 2(PSA/CD3)] in HeLa cells and identify its binding affinity for PC-3 cells and peripheral blood mononuclear cells (PBMCs).Methods The scFv 2(PSA/CD3) was cloned into the expression plasmid pSecTag2-B.Then the plasmids pSecTag2-B containing the fusion gene were transfected HeLa cells.The expressed products were analyzed by both SDS-PAGE and Western blot.After purification the binding affinity of the purified fusion protein for PC-3 cells and PBMCs was detected by flow cytometry.Results The expressed products of the fusion gene with relative molecular mass (Mr) being about 65 000 were confirmed by SDS-PAGE and Western blot.The purified scFv 2(PSA/CD3) showed significantly strong binding to PC-3 cells and PBMCs.Conclusion The bispecific scFv specific for PSA and CD3 which can bind to PC-3 cells and PBMCs has been successfully gained.