抗人γ-精浆蛋白单链抗体四聚体的构建及表达

Construction and expression of tetramerizing single chain Fv antibody gene specific for human γ-seminoprotein

目的 构建抗γ 精浆蛋白 (γ Sm )单链抗体 (scFv) /p5 3四聚功能域 ( p5 3TD)融合基因 ,并进行真核表达和活性测定。方法 利用递归聚合酶链反应 (PCR)法扩增IgG3上游铰链区与p5 3TD融合基因 ,克隆入 pUC19载体中构建 pUC19/IgG3 /p5 3克隆载体。将抗γ SmscFv克隆入该载体中 ,构建抗γ SmscFv/p5 3TD融合基因并克隆入真核表达载体 pSecTag2 B ,转染HeLa细胞表达纯化后 ,利用流式细胞仪 (FCM )进行活性测定。结果 获得了抗γ SmscFv/p5 3TD融合基因 ,基因全长 891bp ,可编码 2 97个氨基酸。表达产物经十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和Western印迹实验证实为约 3 5× 10 3 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 ,亲和力高于scFv。结论 获得了可与PC 3细胞特异结合的四价抗γ SmscFv ,为进一步临床应用奠定基础。

Objective To construct anti-human γ-seminoprotein (γ-Sm) single chain Fv antibody (scFv)/human p53 tetramerization domain (p53TD) fusion gene and express fusion protein in HeLa cells.Methods The human IgG3 upper hinge/p53TD fusion gene was obtained by recursive polymerase chain reaction (PCR),and was inserted into pUC19 to construct cloning plasmid pUC19/IgG3/p53.The antihuman γ-Sm scFv was then cloned into pUC19/IgG3/p53 to construct antihuman γ-Sm scFv/p53TD fusion gene which was then subcloned into the pSecTag2-B expression plasmid and were transfected HeLa cells.The expression products were analyzed by both SDS-PAGE and Western blot.The binding affinity for PC-3 cells was measured by flow cytometry.Results The anti-humanγ-Sm scFv/p53TD fusion gene consisted of 891bpencoding 297 amino acid residues.The expression products,which relative molecular mass (Mr) was about 35?000,were confirmed by SDS-PAGE and Western blot.The tetrameric antihumanγ-Sm scFv showed significantly stronger binding to PC-3 cells than scFv.Conclusion The tetrameric antihumanγ-Sm scFv which could bind to PC-3 cells has been successfully gained for the potential use in clinical practice.