四聚功能域提高前列腺特异抗体亲和力的作用

p53 tetramerization domain in improving functional affinity of bispecific single chain Fv antibody gene specific for human prostate specific antigen and CD3 molecule

目的探讨人p53四聚功能域在提高抗前列腺特异抗原(PSA)／抗人CD3双特异性单链抗体(scFv)亲和力方面的作用。方法利用递归聚合酶链反应(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 PSA／CD3双特异性scFv克隆入pUC19／IgG3／p53载体中,构建多价抗PSA/CD3双特异性scFv融合基因。将融合基因克隆入真核表达载体pSeeTag2-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。结果获得了多价抗PSA/CD3双特异性scFv融合基因,基因全长1638 bp,可编码546个氨基酸,与已发表的抗PSA／CD3双特异性scFv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67×103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细胞和人外周血单个核细胞(PBMC),亲和力高于双特异性scFv。结论人IgG3上游铰链区／p53四聚功能域基因与抗PSA／CD3双特异性 scFv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 bispecific single chain Fv antibody gene specific for human prostate specific antigen （PSA） and CD3 molecule [ scFv2 （PSA/ CD3） ], 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 scFv2 （PSA/CD3） was cloned into pUC19/IgG3/p53 to construct multivalent scFv2 （PSA/CD3） which was then subeloned 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 PC-3 cells and peripheral blood mononuclear cells （PBMCs） was measured by flow cytometry. Results The multivalent scFv2 （PSA/CD3） gene consisted of 1 638 bp encoding 546 amino acid residues, and was the same as that reported before. The expression products of the multivalent scFv2 （PSA/CD3） were confirmed by SDS-PAGE and Western blot. After purified with Ni^2＋ -NTA superflow affinity chromatography, the multivalent scFv2 （PSA/CD3） showed significantly stronger binding to PC- 3 cells and PBMCs than scFv2 （PSA/CD3）. Conclusion The multivalent scFv2 （PSA/CD3） exhibits much higher functional affinity than scFv2 （PSA/CD3）, which may break a new path to the improvement of functional affinity of antibody.