摘要
为获得能自主进入细胞的单域抗体(sdAb),将抗猪流行性腹泻病毒(PEDV)膜蛋白(M)的sdAb与一种细胞穿膜肽(TAT)融合表达,并纯化重组TAT-sdAb蛋白。用ELISA方法检测TAT-sdAb与M蛋白的结合活性,免疫荧光试验验证其穿Vero细胞膜活性、最适作用时间和剂量,并将TAT-sdAb与胞内PEDV进行共定位研究。结果证实,sdAb与TAT融合表达不影响其与M蛋白的结合活性,在使用剂量为50μg/mL、作用3 h时,能明显观察到其进入Vero细胞,并与胞内的PEDV呈现共定位分布。结果表明,融合TAT的sdAb具有抗原结合活性和穿膜活性,为基于单域抗体的药物研发提供了理论基础,为PEDV感染细胞的动态过程研究提供了可靠工具。
To obtain single-domain antibodies(sdAb)that can enter cells autonomously,an single domain antibody against the membrane protein(M)of porcine epidemic diarrhea virus(PEDV)was expressed as fusion with a cell-penetrating peptide(TAT).The recombinant TAT-sdAb was purified,and its M protein binding activity was detected by an enzyme-linked immuno sorbent assay.Meanwhile,the cell-penetrating activity,the best incubation times and dose for entering the cells,and the co-location with PEDV were identified in Vero cells by immunofluorescence assay.The results showed that the fusion of sdAb with TAT does not affect the binding activity to M protein,and TAT-sdAb can enter Vero cells observablely at the dose of 50μg/mL and 3 h incubation.In addition,the TAT-sdAb show co-localized distribution with PEDV in Vero cells.The results demonstrated that The TAT-sdAb fusion has both antigen-binding and cell-penetrating activity,which providing a theoretical basis for the development of the sdAb based drugs,and has the potential to be a powerful tool for the study on the dynamic process of PEDV interaction with cells.
作者
李丽
张辛铭
尹双辉
张涛涛
杨顺利
薛慧文
LI Li;ZHANG Xin-ming;YIN Shuang-hui;ZHANG Tao-tao;YANG Shun-li;XUE Hui-wen(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou,Gansu,730070,China;State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute Chinese Academy of Agricultural Sciences,Lanzhou,Gansu,730046,China;Shijiazhuang Veterinary Biological Products Supply Station,Shijiazhuang,Hebei,050027,China)
出处
《动物医学进展》
北大核心
2021年第6期8-13,共6页
Progress In Veterinary Medicine
基金
中国农业科学院农业科技创新工程项目(CAAS-ASTIP-2014-LVRI-09)。
关键词
细胞穿膜肽
流行性腹泻病毒
膜蛋白
单域抗体
cell-penetrating peptides
Porcine epidemic diarrhea virus
membran protein
single-domain antibody
