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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 the fusion gene of scFv / p5 3 tetramerization domain (p5 3TD) of anti-γ-seminiferous protein (γ Sm) and its eukaryotic expression and activity assay. Methods The fusion gene upstream of IgG3 and p5 3TD was amplified by recursive polymerase chain reaction (PCR) and cloned into pUC19 vector to construct pUC19 / IgG3 / p5 3 cloning vector. The anti-γ SmscFv was cloned into this vector, and the anti-γ SmscFv / p5 3TD fusion gene was constructed and cloned into the eukaryotic expression vector pSecTag2 B. After being transfected into HeLa cells and purified, the activity of the fusion protein was determined by flow cytometry (FCM). The results obtained anti-γ SmscFv / p5 3TD fusion gene, the full-length gene 891bp, encoding 2 97 amino acids. The expressed product was confirmed to be about 35 × 10 3 specific protein bands by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. The purified protein could be detected by flow cytometry Binding PC 3 cells with higher affinity than scFv. Conclusion Tetravalent anti-γ SmscFv, which can specifically bind to PC 3 cells, was obtained and laid the foundation for further clinical application.