人p53四价功能域对提高抗体功能性亲和力的作用  

Study on p53 tetramerization domain in improving functional affinity and biological activity of antibody

作　　者：王栋[1] 武国军[2] 谭建明[1] 王禾[2] 

机构地区：[1]南京军区福州总医院泌尿外科,350025 [2]第四军医大学西京医院泌尿外科

出　　处：《中华医学杂志》2005年第7期479-482,共4页National Medical Journal of China

基　　金：国家自然科学基金资助(39900180)

摘　　要：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 fuse the genes of p53 tetramerization domain and anti-CD3/anti-prostate-cancer bispecific single-chain antibody (BsAb), and exploit a new way to improve the functional affinity and biological activity of antibody. Methods Genes of p53 tetramerization domain and anti-CD3/anti-prostate-cancer BsAb was fused by technique of DNA sub-cloning. The fusion gene confirmed by sequencing was subcloned into the pSectag2-B plasmid. Then the recombinant plasmid was transfected into HeLa cells. The expression products, which were analyzed by both SDS-PAGE and western blotting, were purified with Ni^2+ -NTA superflow affinity chromatography. mBsAb-pSectag2-B plasmid was added into the suspensions of human peripheral blood mononuclear cells (PBMCs) and ~PC-3 cells respectively. Flow cytometry was used to examine the binding rate of multivalent anti-prostate-cander/anti-CD3 bispecific scFv with PBMCs and PC-3 cells. T cells were isolated from the PBMCs. PC-3 cells were labeled with Na_2[~51 Cr]O_4 used as target cells. Labeled PC-3 cells, T cells, and different concentrations of mBsAb were mixed. Natural release control well with labeled target cells only and maximum release control well with labeled target cells and 10% SDS were prepared. The supernatants were extracted. γ calculator was used to calculate the counts per minute (cpm) values to calculate the specific release rate of ~51 Cr. Results Sequencing showed a fragment from mBsAb-pSectag2-B with the size of 1.7 kb corresponding to the predicted value. SDS-PAGE and Western blotting showed expression of 67 000 D protein in the supernatant of culture fluid of HeLa cells transfected with MBsAb-pSectag2-B plasmid. The binding rates of multivalent anti-prostate-cancer/anti-CD3 bispecific scFv with PBMC and PC-3 cells were 70.4% and 81% respectively, significantly higher than those of anti-prostate-cancer/anti-CD3 bispecific scFv. In the presence of mBsAb the activated T cells lysed PC-3 cells in positive correlation with the antibody concentration and effective cell

关 键 词：双特异性单链抗体 生物学活性 PC-3细胞 人IGG 释放试验 前列腺癌 功能域 体功能 亚克隆 基因融合 

分 类 号：R73-3[医药卫生—肿瘤]