穿膜肽与抗猪流行性腹泻病毒膜蛋白单域抗体融合表达和入胞活性分析  

Expression and Cell-penetrating Activity Analysis of the Fusion Protein of Cell-penetrating Peptide and Anti-porcine Epidemic Diarrhea Virus Membran Protein Single Domain Antibody

作　　者：李丽 张辛铭 尹双辉[2] 张涛涛 杨顺利[2] 薛慧文[1] 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)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [3]石家庄市兽用生物制品供应站,河北石家庄050027

出　　处：《动物医学进展》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)。

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.

关 键 词：细胞穿膜肽 流行性腹泻病毒 膜蛋白 单域抗体 

分 类 号：S852.65[农业科学—基础兽医学] S858.28[农业科学—兽医学]