抗人CD3单链抗体四聚体基因的构建及表达

Construction and Expression of Tetramerized Single Chain Fv Antibody Gene Specific for Human CD3 Molecule

为构建和表达抗人CD3单链抗体 (scFv) 人p5 3四聚功能域融合基因 ,选用人IgG3上游铰链区作为抗人CD3scFv和人p5 3四聚功能域之间连接的linker .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pUC18载体中构建pUC18 IgG3 p5 3克隆载体 .将抗人CD3scFv克隆入pUC18 IgG3 p5 3载体中 ,构建抗人CD3scFv 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亲和活性测定 .获得了抗人CD3scFv 人p5 3四聚功能域融合基因 ,基因全长 882bp ,可编码 2 94个氨基酸 ,与已发表的抗人CD3scFv、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人外周血单个核细胞 (PBMC)细胞 ,亲和力高于scFv 。

To construct and express anti human CD3 single chain Fv antibody (scFv)/human p53 tetramerization domain fusion gene, human IgG3 upper hinge was selected to be the linker between anti human CD3 scFv and human p53 tetramerization domain gene. The human IgG3 upper hinge/human p53 tetramerization domain fusion gene was obtained by recursive polymerase chain reaction (PCR), and was inserted into pUC18 to construct cloning plasmid pUC18/IgG3/p53. The anti human CD3 scFv was then cloned into pUC18/IgG3/p53 to construct anti human CD3 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 blotting, then were purified with Ni 2+ NTA superflow affinity chromatography. The binding affinity for peripheral blood mononuclear cells (PBMCs) was measured by flow cytometry. The anti human CD3 scFv/human p53 tetramerization domain fusion gene consisted of 882 bp encoding 294 amino acid residues, as that reported before. The expression products of the tetrameric anti human CD3 scFv with relative molecular mass ( Mr ) of about 35 000 were confirmed by SDS PAGE and Western blotting. After purified with Ni 2+ NTA superflow affinity chromatography, the tetrameric anti human CD3 scFv showed significantly stronger binding to PBMCs than scFv. The tetrameric anti human CD3 scFv capable of binding to PBMCs was successfully obtained for its potential use in clinical applications.