抗PSA单链抗体/p53四聚功能域融合基因的构建及表达

Construction of the Tetramerizing Single Chain Fv Antibody Gene Specific for Human Prostate Specific Antigen and Its Expression in HeLa Cells

目的 :构建抗人前列腺特异抗原 (PSA)单链抗体 (scFv) /人 p5 3四聚功能域融合基因 ,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 pUC19载体中构建pUC19/IgG3/p5 3克隆载体。将抗PSAscFv克隆入pUC19/IgG3/ p5 3载体中 ,构建抗PSAscFv/人p5 3四聚功能域融合基因。经酶切鉴定及序列测定证实后 ,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 ,表达产物纯化后利用流式细胞仪进行活性测定。结果 :获得了抗PSAscFv/人p5 3四聚功能域融合基因 ,基因全长 891bp ,可编码 2 97个氨基酸 ,与已发表的抗PSAscFv、人IgG3上游铰链区和人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 35kD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细胞特异结合的抗PSAscFv四聚体 。

Objective: To construct anti human prostate specific antigen (PSA) single chain Fv antibody (scFv)/human p53 tetramerization domain fusion gene and express fusion protein in HeLa cells. Methods: The 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 PSA scFv was then cloned into pUC19/IgG3/p53 to construct anti PSA scFv /human p53 tetramerization domain fusion gene which was then subcloned 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 PC 3 cells was measured by flow cytometry. Results: The anti PSA scFv/human p53 tetramerization domain fusion gene consisted of 891bp encoding 297 amino acid residues, and was the same as that reported before. The expression products of the tetrameric anti PSA scFv, which relative molecular mass (Mr) was about 35 000, were confirmed by SDS PAGE and Western blot. After purified with Ni 2+ NTA superflow affinity chromatography, the tetrameric anti PSA scFv showed significantly stronger binding to PC 3 cells than scFv.Conclusion: The tetrameric anti PSA scFv which could bind to PC 3 cells has been successfully gained for the potential use in clinical studies.