人p53四聚功能域对提高抗人CD3单链抗体亲和力的作用

Study on p53 tetramerization domain in improving functional affinity of single chain Fv antibody gene specific for human CD3 molecule

目的探讨人p53四聚功能域在提高抗人CD3单链抗体(scFv)亲和力方面的作用。方法利用递归PCR法扩增人IgG3上游铰链区与人p53四聚功能域融合基因,克隆入pUC19载体中构建pUC19/IgG3/p53克隆载体。将抗人CD3scFv克隆入pUC19/IgG3/p53载体中,构建抗人CD3scFv/人p53四聚功能域融合基因。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。结果获得了抗人CD3scFv/人p53四聚功能域融合基因,基因全长882bp,可编码294个氨基酸,与已发表的抗人CD3scFv、人IgG3上游铰链区和人p53四聚功能域基因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。结论人IgG3上游铰链区/p53四聚功能域基因与抗人CD3scFv基因融合后表达产物的功能性亲和力大大提高,为提高抗体的功能性亲和力开辟了新的思路。

[Objective] To fuse the genes of p53 tetramerization domain and single chain Fv antibody gene specific for human CD3 molecule, and exploit a new way to improve the functional affinity of antibody. [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-CD3 scFv was then cloned into pUC19/IgG3/p53 to construct anti-CD3 scFv/human p53 tetramerization domain fusion gene which was then subcloned into the pSecTag2-B expression plasmid. Then the pSecTag2-B plasmids containing the fusion gene were transfected into 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 peripheral blood mononuelear cells （PBMCs） was measured by flow cytometry. [Results] The anti-CD3 scFv/human p53 tetramerization domain fusion gene consisted of 882 bp encoding 294 amino acid residues, and was the same as that reported before. The expression products of the tetrameric anti-CD3 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 chromatogra- phy, the tetrameric anti-CD3 scFv showed significantly stronger binding to PBMC cells than scFv. [Conclusion] The tetrameric anti-CD3 seFv exhibits much higher functional affinity than scFv,which may break a new path to the improvement of functional affinity of antibody.