中国兽医科学2024，Vol.54Issue(10)：1318-1326,9.DOI:10.16656/j.issn.1673-4696.2024.0167
非洲马瘟病毒VP7蛋白的可溶性表达及抗体阻断ELISA方法的初步建立
Soluble expression of African horse sickness virus VP7 protein and preliminary establishment of blocking ELISA for its antibody detection
摘要
Abstract
In order to achieve soluble expression of African horse sickness virus(AHSV)VP7 protein,prepare specific monoclonal antibodies(McAbs)against VP7 protein,and establish an AHSV VP7 blocking ELISA antibody detection method,mutation design and codon optimization were performed on the genesequence encoding AHSV VP7 protein,and the soluble VP7 protein was expressed using Escherichia coli expression system.BALB/c mice were immunized with purified VP7 protein to obtain McAbs against the VP7 protein using hybridoma technology.The specificity of McAbs was confirmed by immunofluorescence assay and Western-blotting.The McAbs were used as a blocking antibody to establish AHSV VP7 blocking ELISA.The results showed that the soluble recombinant AHSV VP7 protein was successfully expressed in E.coli,and two cell lines,3F7 and 7H8,which stably secreted McAbs against VP7 protein,were successfully prepared.These two VP7 McAbs can not only react with the recombinant VP7 protein ex-pressed in E.coli,but also with the recombinant VP7 protein in BHK-21 cells,showing good specificity and reactivity.A blocking ELISA for AHSV antibody detection was established using recombinant VP7 protein and monoclonal antibody 7H8 as the coating antigen and blocking antibody,respectively,and 277 horse serum samples were tested.The results were consistent with those of the commercial imported AHSV antibody detection kit.This study achieved soluble expression of AHSV VP7 protein in E.coli expression system,prepared specific McAbs against VP7 protein,and established AHSV VP7 blocking ELISA method,which will provide technical reserves for effective prevention and control of African horse sickness in China.关键词
非洲马瘟病毒/VP7蛋白/可溶性表达/单克隆抗体/阻断EL1SAKey words
African horse sickness virus/VP7 protein/soluble express ion/monoclonal antibody/blocking ELISA分类
农业科技引用本文复制引用
徐婧,独军政,户鑫兵,宋昱庆,张鸿歌,田占成,关贵全,罗建勋,殷宏,魏衍全..非洲马瘟病毒VP7蛋白的可溶性表达及抗体阻断ELISA方法的初步建立[J].中国兽医科学,2024,54(10):1318-1326,9.基金项目
国家重点研发计划项目(2021YFD1800500) （2021YFD1800500）
甘肃省技术创新引导计划项目(23CXNA0016) （23CXNA0016）
甘肃省基础研究创新群体项目(22JR5RA024) （22JR5RA024）
甘肃省科技厅项目(22CX8NA011) （22CX8NA011）
农业科技创新工程项目(CAAS-ASTIP-2016-LVRI) （CAAS-ASTIP-2016-LVRI）
