首页|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

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

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  • 国家科技期刊平台
  • NETL
  • NSTL
  • 万方数据
为了实现非洲马瘟病毒(AHSV)VP7蛋白的可溶性表达,制备其特异性单克隆抗体,建立AHSV VP7阻断ELISA抗体检测方法,通过对编码AHSV VP7蛋白的基因序列进行突变设计与密码子优化,以大肠杆菌表达系统表达可溶性VP7蛋白并进行纯化,免疫BALB/c小鼠,利用杂交瘤技术获得VP7蛋白单克隆抗体.通过细胞免疫荧光和Western-blot鉴定单克隆抗体的特异性,并建立AHSV VP7阻断ELISA方法.结果显示,利用大肠杆菌表达系统成功表达了可溶性重组AHSV VP7蛋白,将纯化的VP7蛋白免疫小鼠筛选制备了2株稳定分泌VP7蛋白单克隆抗体的细胞株,分别为3F7和7H8.经鉴定证明这2株VP7单克隆抗体不仅能与大肠杆菌重组表达的VP7蛋白反应,而且也与BHK-21细胞表达的具有天然构象的重组VP7蛋白反应,具有良好的特异性和反应性.以重组VP7蛋白和单克隆抗体7H8分别为包被抗原和阻断抗体建立了AHSV VP7阻断ELISA方法,检测了277份马血清样本,结果与商品化进口 AHSV抗体检测试剂盒测定结果一致.本研究在大肠杆菌表达系统中实现了AHSV VP7蛋白的可溶性表达,制备获得了VP7特异性单克隆抗体,并建立了AHSVVP7阻断ELISA方法,为我国有效防控非洲马瘟的传入提供了技术储备.
Soluble expression of African horse sickness virus VP7 protein and preliminary establishment of blocking ELISA for its antibody detection
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.

African horse sickness virusVP7 proteinsoluble express ionmonoclonal antibodyblocking ELISA

徐婧、户鑫兵、宋昱庆、张鸿歌、田占成、关贵全、罗建勋、殷宏、魏衍全、独军政

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甘肃农业大学动物医学院,甘肃兰州 730070

中国农业科学院 兰州兽医研究所 兰州大学动物医学与生物安全学院 动物疫病防控全国重点实验室,甘肃兰州 730000

甘肃省病原生物学基础学科研究中心,甘肃 兰州 730046

非洲马瘟病毒 VP7蛋白 可溶性表达 单克隆抗体 阻断EL1SA

国家重点研发计划项目甘肃省技术创新引导计划项目甘肃省基础研究创新群体项目甘肃省科技厅项目农业科技创新工程项目

2021YFD180050023CXNA001622JR5RA02422CX8NA011CAAS-ASTIP-2016-LVRI

2024

10.16656/j.issn.1673-4696.2024.0167

中国兽医科学
中国农业科学院兰州兽医研究所

中国兽医科学

CSTPCD北大核心
影响因子：0.524
ISSN：1673-4696
年,卷(期)：2024.54(10)
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