多价抗人精浆蛋白/抗人CD_3双特异性单链抗体的构建及表达  

Construction and expression of multivalent bispecific single chain Fv antibody gene specific for human γ-seminoprotein and CD_3 molecule

作　　者：武国军[1] 王禾[1] 王栋 于磊[1] 郝晓柯[3] 白玉杰[4] 王智[4] 袁建林[1] 

机构地区：[1]第四军医大学西京医院泌尿外科,西安710033 [2]福州军区总医院 [3]第四军医大学西京医院检验科,西安710033 [4]第四军医大学基础部生物化学与分子生物学教研室

出　　处：《中华泌尿外科杂志》2004年第8期529-532,共4页Chinese Journal of Urology

基　　金：国家自然科学基金 ( 3 990 0 180 ) ;全军重点实验室研究基金 ( 1997 71 2 2 )

摘　　要：目的　构建多价抗人精浆蛋白 (γ Sm) /抗人CD3 双特异性单链抗体 (scFv)基因 ,并进行真核表达和活性测定。　方法　利用递归PCR法扩增人IgG3上游铰链区与人p5 3四聚功能域融合基因 ,克隆入pUC1 9载体中构建pUC1 9/IgG3/p5 3克隆载体。将抗人CD3 scFv(CD3 scFv)和抗人γ SmscFv(γ SmscFv)依次克隆入pUC1 9/IgG3/p5 3载体中 ,构建多价双特异性γ SmscFv/CD3 scFv[scFv2 (CD3 /γ Sm) ]融合基因。将融合基因克隆入真核表达载体pSecTag2 B中 ,转染HeLa细胞进行表达 ,表达产物纯化后流式细胞仪进行活性测定。　结果　获得了多价scFv2 (CD3 /γ Sm)融合基因 ,基因全长 1 6 38bp ,可编码 5 4 6个氨基酸 ,与已发表的γ SmscFv、CD3 scFv和人p5 3四聚功能域基因cDNA序列一致。表达产物经SDS PAGE和Western印迹实验证实为约 6 70 0 0的特异蛋白条带 ,纯化后经流式细胞仪检测可以特异性地结合PC 3细胞和人外周血单个核细胞 (PBMC) ,亲和力高于双特异性scFv。　结论　获得了可与PBMC和PC 3细胞特异结合的多价scFv2 (CD3 /γ Sm) 。Objective To construct multivalent bispecific single chain Fv antibody(Bi scFv) gene specific for human γ seminoprotein (γ 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 humanγ Sm scFv was cloned into pUC19/IgG3/p53 subsequently to construct multivalent bispecific anti human γ Sm /anti CD 3 scFv fusion gene,scFv 2(γ 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(γ 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(γ 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(γ Sm/CD 3) showed significantly stronger binding to PC 3 cells and PBMCs than Bi scFv. Conclusions The multivalent scFv 2(γ Sm/CD 3) which could bind to PC 3 cells and PBMCs has been successfully gained providing a laboratory foundation for clinical use.

关 键 词：多价抗人精浆蛋白 抗人CD3 双特异性单链抗体 基因表达 克隆 

分 类 号：R392[医药卫生—免疫学]