抗bFGF人源性抗体的瞬时表达、纯化及鉴定  

Expression,purification and characterization of a humanized antibody to bFGF by HEK293T cell

作　　者：陶俊[1] 李丹[1] 邓宁[1] 王宏[1] 龚义平[1] 向军俭[1] 

机构地区：[1]暨南大学生命科学技术学院抗体工程中心,广州510632

出　　处：《中国免疫学杂志》2010年第10期876-880,共5页Chinese Journal of Immunology

基　　金：国家科技计划"863"专题课题(2009AA02Z112)

摘　　要：目的:将一株人源性中和性抗bFGF单链抗体基因转入真核表达载体,进行真核瞬时表达,通过细胞试验进一步验证其中和活性。方法:将1AscFv抗体基因克隆到真核表达载体pIgG中(编码全长人源性IgG1类抗体),T4连接酶连接后,转化大肠杆菌DH5α,挑取4个克隆,经测序其中有一株含有正确的scFv序列,命名为pIgG-1A2,将pIgG-1A2转染真核细胞HEK293T进行瞬时表达,表达产物经蛋白G纯化后,SDS-PAGE检测纯化抗体的纯度;ELISA和Western blot检测抗体的特异性;MTT法检测纯化抗体对人脐静脉血管内皮细胞(HUVEC)和人神经胶质瘤细胞株U87MG增殖的影响。结果:经酶切鉴定和测序发现pIgG-1A2含有正确的抗体轻重链基因,经293T细胞瞬时表达,表达产物经蛋白G亲和层析柱纯化后经SDS-PAGE鉴定,获得纯度为95%的电泳纯抗体,产量可以达到每升细胞培养上清10mg,交叉反应显示表达的抗体特异性针对bFGF,体外实验证实1A2能够抑制bFGF诱导的人脐静脉血管内皮细胞的增殖,同时还能抑制神经胶质瘤细胞株U87MG的增殖。结论:通过真核细胞成功地表达了抗bFGF人源性抗体,纯化后获得了高纯度的抗体,对HUVEC和神经胶质瘤细胞株U87MG具有抑制作用,为抗bFGF人源性抗体抗肿瘤研究奠定基础。Objective：To construct and express a naturalizing scFv （1 A） against bFGF and identify its neutralizing activity by in vitro cell experiment. Methods： 1A scFv was cloned into a eukaryotic expression vector plgG containing the genomic sequences of human IgG1 constant region, after ligated by T4 DNA Ligase, the plasmid was transformed into E. coil DH5a strains, and then we selected four clone for DNA sequencing. After confirmed by DNA sequencing, we found that one clone included the right scFv DNA sequence, the right clone was named pIGG-1A2 . Then the plasnrids were transfected in HEK293T cells by FuGene HD. The secreted antibody was identified by ELISA, and was purified by protein G affinity chromatography. The purity of the antibody was analyzed by SDS-PAGE, and the specificity of the antibody was assessed by ELISA and Western blot. The anti-tumor and anti-vascular endothelial cell effects of the antibody on glioma cells and human umbilical vein endothelial cells （HUVEC） were analyzed by MTT assay. Results：The eukaryotic expression vector for human anti-bFGF antibody was constructed correctly. After expressed by 293T cells, the antibody could bind with bFGF specifically. After purified by protein G affinity chro- matography,the purity of the antibody was assessed by SDS-PAGE and found to be 〉 95% pure,and we can obtain 10 mg purified antibody from 1 liter of cell culture medium. The in vitro experiment results showed that 1A2 was able to inhibit the proliferation of human umbilical vein endothelial cells induced by bFGF and to ihhibit the proliferation of glioma cells. Conclusion： The eukaryotic expression vector for hmnan antibFGF antibody gene is successfully constructed and expressed transiently in 293T cells. Expressed antibody could inhibit the proliferation of human umbilical vein endothelial cells induced by bFGF and inhibit the proliferation of glioma cells. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on bFGF signaling for growth

关 键 词：BFGF 人源性抗体 瞬时表达 中和活性 

分 类 号：R392.11[医药卫生—免疫学]