抗人CD3单链抗体四聚体基因在HeLa细胞中的表达和活性鉴定

目的　将抗人CD3单链抗体 (scFv) /人 p5 3四聚功能域融合基因转染到HeLa细胞 ,进行真核表达和活性测定。方法　将抗人CD3scFv /人 p5 3四聚功能域融合基因克隆入真核分泌表达载体 pSecTag2 B中 ,转染HeLa细胞进行表达 ,表达产物纯化后利用流式细胞仪进行活性测定。结果　表达产物经SDS PAGE和Westernblot证实 ,为Mr约35 0 0 0的特异蛋白条带。纯化后经流式细胞仪检测 ,可特异性地结合人外周血单个核细胞 (PBMCs) ,亲和力高于scFv。结论　获得了可与PBMCs特异性结合的抗人CD3scFv四聚体 ,为进一步临床应用奠定了基础

Expression of tetramerized single chain Fv antibody gene specific for human CD3 molecule in HeLa cells and identification of binding affinity

Objective To express anti-human CD3 single chain Fv antibody (scFv)/human p53 tetramerization domain fusion gene in HeLa cells and identify its binding affinity for peripheral blood mononuclear cells (PBMCs). Methods The anti-human CD3 scFv /human p53 tetramerization domain fusion gene was cloned into the pSecTag2-B expression plasmid. Then the pSecTag2-B plasmids concluding the fusion gene were transfected HeLa cells. The expression products were analyzed by both SDS-PAGE and Western blot, then were purified with Ni 2+-NTA superflow affinity chromatography. The binding affinity for PBMCs was measured by flow cytometry. Results The expressed products of the fusion gene with relative molecular mass (Mr) of about 35 000 were confirmed by SDS-PAGE and Western blot. The purified tetrameric anti-human CD3 scFv showed significantly stronger binding to PBMCs than scFv. Conclusion The tetrameric anti-human CD3 scFv which can bind with PBMCs has been successfully obtained for the potential use in clinical studies.