抗犬瘟热病毒犬源化嵌合单链抗体scFv-Fc的制备与活性分析  

Preparation and Activity Analysis of Canine Derived Chimeric Single Chain Antibody scFv-Fc against Canine Distemper Virus

作　　者：刘雅坤 毕振威[2,3] 夏兴霞 莫菲[2,3] 徐司雨 钱晶 谭业平 诸玉梅[2,3] 赵建军 LIU Yakun;BI Zhenwei;XIA Xingxia;MO Fei;XU Siyu;QIAN Jing;TAN Yeping;ZHU Yumei;ZHAO Jianjun(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319 [2]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室,南京210014 [3]兽用生物制品(泰州)国泰技术创新中心,泰州225300

出　　处：《畜牧兽医学报》2024年第12期5762-5773,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划项目(2022YFD1800603)。

摘　　要：为减少和改善鼠源单克隆抗体在犬临床治疗中的异源性和功效,用RT-PCR方法从分泌抗犬瘟热病毒(CDV)中和单克隆抗体的鼠源杂交瘤细胞中扩增抗体重链可变区(V H)和轻链可变区(V L),通过Linker将V H和V L连接获得单链抗体(scFv)基因,选择犬源抗体IgG B恒定区(Fc)连接到scFv的C末端获得犬源化嵌合单链抗体(scFv-Fc)基因;分别构建鼠源scFv与犬源化scFv-Fc的原核表达载体,通过大肠杆菌表达、纯化,用ELISA、IPMA、细胞融合抑制和中和试验检测表达抗体的特异性和中和活性。结果显示:CDV鼠源scFv、犬源化scFv-Fc的ELISA最低检出浓度分别为14.38和102.75μg·mL^(-1);与表达的CDV H蛋白和CDV均发生特异性反应;能够抑制CDV H和F蛋白介导的细胞膜融合,完全保护细胞不出现病变的最低浓度分别为0.72和0.64μg·mL^(-1)。本研究成功制备了鼠源scFv与犬源化scFv-Fc嵌合抗体,为犬瘟热的临床治疗提供了候选抗体药物。To reduce and improve the heterogeneity and efficacy of mouse-derived monoclonal antibodies in clinical treatment of dogs,the variable region of heavy chain(V H)and variable region of light chain(V L)were amplified from mouse-derived hybridomas secreting monoclonal antibody against canine distemper virus(CDV)using RT-PCR,the single chain antibody(scFv)gene was obtained by linking V H and V L through Linker,and the canine-derived chimeric single chain antibody(scFv-Fc)gene was obtained by selecting the constant region(Fc)of canine IgG B to connect to the C-terminus of scFv;The prokaryotic expression vectors for expressing mouse-derived scFv and canine-derived scFv-Fc were respectively constructed,which were expressed in Escherichia coli for purification,and the specificity and neutralizing activity of the expressed antibodies were detected by ELISA,IPMA,cell fusion inhibition,and neutralization assay.The detection limit of ELISA for CDV mouse-derived scFv and canine-derived scFv-Fc are 14.38 and 102.75μg·mL^(-1),respectively;Both of scFv and scFv-Fc specifically react with the expressed CDV H protein and CDV;The cell membrane fusion mediated by CDV H and F proteins can be inhibited by scFv and scFv-Fc,and the minimum concentrations of them for complete protection of cells from CPE formation are 0.72 and 0.64μg·mL^(-1),respectively.The mouse-derived scFv and canine-derived chimeric antibody scFv-Fc are successfully developed,providing candidate antibody drugs for the clinical treatment of canine distemper.

关 键 词：犬瘟热病毒 中和活性 单链抗体 嵌合抗体 

分 类 号：S852.43[农业科学—基础兽医学]