抗人肿瘤坏死因子-α单链抗体与其配体的相互作用  

Interaction Between an Anti-human Tumor Necrosis Factor-α Single Chain Antibody and Its Ligand

作　　者：陈萍[1] 邓健蓓[1] 韩骅[1] 药立波[1] 苏成芝[1] 

机构地区：[1]第四军医大学生物化学与分子生物学教研室,西安710032

出　　处：《中国生物化学与分子生物学报》2000年第4期430-433,共4页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金!项目 ( 3 9670 695 )

摘　　要：为了在大肠杆菌中表达纯化抗人 TNF- α单链抗体并检测其结合活性与中和活性 .利用GST融合蛋白系统在大肠杆菌中表达抗人 TNF- α单链抗体 E6Sc Fv;分离包含体后进行变性和复性 ,再用亲和层析法进行纯化 ;用 ELISA法和酵母双杂交系统检测 E6Sc Fv与配体的结合 ;用 L92 9细胞检测 E6Sc Fv对人 TNF- α细胞毒作用的中和活性 .经变性 ,复性与亲和层析 ,E6Sc Fv被纯化 ,在 SDS- PAGE上为单一蛋白带 ;体外结合与中和实验表明 ,表达纯化的 E6Sc Fv可与人 TNF-α结合并中和其细胞毒活性 ;进一步用酵母双杂交系统证明当表达于细胞内时 ,E6Sc Fv仍保持了与TNF-α相结合的能力 .An anti human tumor necrosis factor (hTNF) α single chain antibody(E6ScFv) was expressed and purified in E.coli to identify the characterization of its binding and neutralizing activity.E6ScFv was expressed using the GST fusion protein system.The expressed protein,which was in the inclusion body fraction,was re natured and purified by affinity chromatography.The binding activity of the purified E6ScFv peptide was tested by ELISA as well as yeast two hybrid system and its neutralizing activity tested using the TNF α sensitive cell line L929.The E.coli expressed E6ScFv was purified to homogeneity and the purified E6ScFv could bind and neutralize the activity of hTNF α,which further showed that the E6ScFv could bind with hTNF α within cytoplasm.These data are essential for E6ScFv to be used in clinical therapy.

关 键 词：人TNF-Α 单链抗体 酵母双杂交 E6ScFV 胞内抗体 

分 类 号：R392.1[医药卫生—免疫学] Q813.2[医药卫生—基础医学]