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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 investigate the role of human p53 tetramerization domain in enhancing the affinity of anti-prostate specific antigen (PSA) / anti-human CD3 bispecific single chain antibody (scFv). Methods The fusion gene of human IgG3 hinge region and human p53 tetramerization domain was amplified by recursive polymerase chain reaction (PCR) and cloned into pUC19 vector to construct pUC19 / IgG3 / p53 cloning vector. The anti-PSA / CD3 bispecific scFv was cloned into the pUC19 / IgG3 / p53 vector to construct a multivalent anti-PSA / CD3 bispecific scFv fusion gene. The fusion gene was cloned into eukaryotic expression vector pSeeTag2-B and transfected into HeLa cells for expression. The expressed product was purified and assayed by flow cytometry. Results The multi-valent anti-PSA / CD3 bispecific scFv fusion gene was obtained. The full-length cDNA was 1638 bp in length and encoded a 546 amino acid sequence. This gene was associated with the published cDNA sequence of the anti-PSA / CD3 bispecific scFv and human p53 tetramerization domain Consistent. The expressed product was confirmed by SDS-PAGE and Western blot analysis of about 67 × 103 specific protein bands purified by flow cytometry can specifically bind PC-3 cells and human peripheral blood mononuclear cells (PBMC) Affinity is higher than bispecific scFv. Conclusion The functional affinity of the fusion protein between the upstream hinge region / p53 tetramerization domain of human IgG3 and the anti-PSA / CD3 bispecific scFv gene is greatly improved, which opens up new ideas for improving the functional affinity of the antibody.