Construction and expression of multivalent bispecific single chain Fv antibody gene specific for human ?-seminoprotein and CD_3 molecule / 中华泌尿外科杂志

ABSTRACT

Objective To construct multivalent bispecific single chain Fv antibody(Bi scFv) gene specific for human ? seminoprotein (? Sm) and CD 3 molecule and to detect its expression in HeLa cells. Methods Human IgG3 upper hinge/human p53 tetramerization domain fusion gene was obtained by recursive polymerase chain reaction (PCR),and was inserted into pUC19 to construct cloning plasmid pUC19/IgG3/p53.The anti CD 3 scFv and anti human? Sm scFv was cloned into pUC19/IgG3/p53 subsequently to construct multivalent bispecific anti human ? Sm /anti CD 3 scFv fusion gene,scFv 2(? Sm/CD 3),which was then subcloned into the pSecTag2 B expression plasmid.The pSecTag2 B plasmids including the fusion gene were then transfected into HeLa cells.The expression products were analyzed by both SDS PAGE and Western blot and were purified with Ni 2+ NTA superflow affinity chromatography.The binding affinity for PC 3 cells and peripheral blood mononuclear cells (PBMCs) was measured by flow cytometry. Results The multivalent scFv 2(? Sm/CD 3) gene consisted of 1638 bp encoding 546 amino acid residues,and was the same as that reported previously.The expression products of the multivalent scFv 2(? Sm/CD 3) with a relative molecular mass ( Mr ) of about 67 000,as confirmed by SDS PAGE and Western blot. After purified with Ni 2+ NTA superflow affinity chromatography,the multivalent scFv 2(? Sm/CD 3) showed significantly stronger binding to PC 3 cells and PBMCs than Bi scFv. Conclusions The multivalent scFv 2(? Sm/CD 3) which could bind to PC 3 cells and PBMCs has been successfully gained providing a laboratory foundation for clinical use.