摘要
目的:构建及表达抗人B7-1及B7-2双特异性抗体(anti-B7-1/B7-2-Bs Ab),并分析其与相应抗原结合的特性。方法:采用PCR的方法分别从重组质粒中克隆出抗人B7-1及B7-2单链抗体基因,将其克隆入p IRES2-EGFP表达载体,并用6×His为纯化的标签。用脂质体法将两个基因转染入中华仓鼠卵巢细胞(CHO),经G418加压筛选高表达株,扩大培养并收集上清纯化。分析抗体对膜型B7-1及B7-2的识别及与其相应抗原的结合能力。以天然高表达B7-1及B7-2分子的Raji细胞用丝裂霉素处理后作为APC,人PBMC为效应细胞,分析抗体通过阻断B7-CD28共刺激信号对PBMC增殖的影响。结果:获得了稳定表达抗人B7-1/B7-2-Bs Ab的CHO细胞株及相应的抗体。该抗体与B7-1及B7-2基因转染细胞株L929-B7-1及L929-B7-2的阳性结合率分别为99.5%及62.0%。Anti-B7-1/B7-2-Bs Ab对抗人B7-1及B7-2单链抗体的竞争结合率分别为0.4%及32.4%。Anti-B7-1/B7-2-Bs Ab通过阻断B7-CD28共刺激信号使PBMC的增殖效应仅为阴性对照组的59.7%。结论:成功构建了稳定分泌抗人B7-1/B7-2-Bs Ab的CHO细胞株(命名为SA-VI)。该抗体可以识别B7-1及B7-2分子并具有良好的生物学活性。
Objective：To construct and express the bispecific antibody against human B7-1 and B7-2,in order to study the biological functions of its binding with antigen. Methods： The anti-CD80-sc Fv gene and anti-CD86-sc Fv gene were cloned from recombinant plasmid by PCR,and then subcloned into the eukaryotic expression vector p IRES2-EGFP to construct recombinant p IRES2-EGFP /Bs Ab,for 6 × His as purification tag. Chinese hamster ovary（ CHO） cells were transfected by this recombinant plasmid through liposome-mediated methods and selected by G418. The anti-B7-1 / B7-2-Bs Ab were cultured and purified from culture supernatants. The identification of the anti-B7-1 / B7-2-Bs Ab to membrane B7-1 and B7-2 were performed. With Raji cells which expressed B7-1 and B7-2highly and were treated by mitomycin firstly as the APC,and human PBMC as the effect cells,analysing the influence of the antibody on the proliferation of PBMC by blocking the co-stimulatory signal. Results： The stable cells with highly secretion of the B7-1 / B7-2-Bs Ab harvested. The binding rate of the anti-B7-1 / B7-2-Bs Ab with L929-B7-1 and L929-B7-2 cells were 99. 5%,62. 0%,respectively. And the antibody could inhibit the combination of anti-B7-1 sc Fv and anti-B7-2 sc Fv with L929-B7-1 and L929-B7-2 cells,the inhibition rate were 0. 4%,32. 4%. Additionally,the antibody suppressed the proliferation of PBMC by blocking the co-stimulatory signal mediated by B7-CD28,the proliferation rate was 59. 7% as the contrast. Conclusion： Anti-B7-1 / B7-2-Bs Ab had been successfully expressed in CHO cells（ named as SA-VI） and the antibody could identify membrane B7-1 and B7-2 molecule in the cell surface and mediate the related biological functions.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2015年第7期927-931,共5页
Chinese Journal of Immunology
基金
国家自然科学基金(No.81373236)
