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目的探讨人p53四价功能域在提高抗体功能性亲和力和生物学活性方面的的作用。方法利用基因克隆技术,将人IgG3上游铰链区/p53四价功能域融合基因与前列腺癌/抗CD3双特异性单链抗体基因融合,构建多价抗体(mBsAb),测序正确后,将其亚克隆入真核表达载体进行表达,纯化表达产物,并利用流式细胞仪及51Cr释放试验进行生物学活性测定。结果经酶切、测序分析证实mBsAb构建成功,其真核表达产物分泌于细胞培养上清,相对分子量为67 000,mBsAb与PBMC和PC 3细胞的阳性结合率分别为70. 4%和81%,明显高于BsAb组。51Cr释放试验结果显示, mBsAb激活的T细胞可以裂解PC 3细胞,裂解百分率明显高于IL 2组和BsAb组。结论人IgG3上游铰链区/p53四价功能域基因与抗前列腺癌/抗人CD3双特异性单链抗体基因融合后表达产物的功能性亲和力和生物学活性大大提高,为提高抗体的功能性亲和力和生物学活性开辟了新的思路。
Objective To investigate the role of the human p53 tetravalent domain in enhancing functional affinity and biological activity of antibodies. Methods The fusion gene of human IgG3 upstream hinge / p53 tetravalent fusion protein and prostate cancer / anti-CD3 bispecific single chain antibody gene were fused by gene cloning technique to construct multivalent antibody (mBsAb). After sequencing correctly, Cloned into eukaryotic expression vector for expression, purified expression product, and the use of flow cytometry and 51 Cr release assay for biological activity determination. Results The constructed mBsAb was confirmed by enzyme digestion and sequencing analysis. The eukaryotic expression product was secreted in the cell culture supernatant with the relative molecular weight of 67 000. The positive binding rates of mBsAb to PBMC and PC 3 cells were 70.4% and 81% , Significantly higher than BsAb group. 51Cr release test results showed that mBsAb-activated T cells can lyse PC 3 cells, the percentage of lysis was significantly higher than the IL 2 and BsAb groups. Conclusion The functional affinity and biological activity of the fusion protein of the upstream hinge region / p53 gene and the anti-prostate cancer / anti-human CD3 bispecific single chain antibody gene fusion gene are greatly enhanced. In order to improve the functional affinity of the antibody And biological activity opened up new ideas.