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

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。

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

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.