摘要
目的 :构建抗人CD3单链抗体 (scFv) /p5 3四聚功能域融合基因 ,并进行真核表达及活性测定。方法 :在已经构建抗人CD3scFv和人IgG3上游铰链区 /p5 3四聚功能域融合基因基础上 ,将抗人CD3scFv克隆入载体pUC18/IgG3/p5 3中 ,构建抗人CD3scFv/p5 3四聚功能域融合基因。经酶切鉴定及序列测定证实后 ,将融合基因克隆入真核表达载体pSecTag2 B中 ,并转染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 :获得了抗人CD3scFv/p5 3四聚功能域融合基因 ,基因全长为 882bp ,可编码 2 94个氨基酸 ,与已发表的抗人CD3scFv、人IgG3上游铰链区和人p5 3四聚功能域基因cDNA序列相一致。表达产物经SDS PAGE和Westernblot证实 ,为Mr 约 35 0 0 0的特异蛋白条带。纯化后经流式细胞仪检测 ,可特异性地结合人外周血单个核细胞 (PBMC) ,亲和力高于scFv。结论 :获得了可与PBMC特异性结合的抗人CD3scFv四聚体 。
AIM: To construct anti-human CD3 single chain Fv antibody (scFv)/human p53 tetramerization domain fusion gene and express the fusion protein in Hela cells. METHODS: The anti-human CD3 scFv was cloned into the previously constructed plasmid pUC18/IgG3/p53 to construct anti-human CD3 scFv /human p53 tetramerization domain fusion gene. After enzyme digestion analysis and sequencing, the fusion gene was subcloned into the expression plasmid pSecTag2-B. Then the plasmid pSecTag2-B containing the fusion gene was transfected into Hela cells. The expressed products were analyzed by SDS-PAGE and Western blot. The binding of the purified fusion protein to human peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry. RESULTS: DNA sequencing showed the fusion gene was constructed successfully. The expressed product of the fusion gene with a relative molecular mass (M r) about 35 000 was confirmed by SDS-PAGE and Western blot. The purified tetrameric anti-human CD3 scFv showed significantly stronger binding to PBMCs than scFv. CONCLUSION: The tetrameric anti-human CD3 scFv which can bind to PBMCs has been successfully expressed and purified for potential use in clinical studies.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第5期560-562,567,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目 (No .3990 0 1 80 )
