赤羽病病毒Gc aa465~704的杆状病毒表达和单克隆抗体制备  

Baculovirus Expression and Monoclonal Antibody Preparation of Akabane Virus Gc aa465-704

作　　者：魏方 陈冬杰 邓俊花[1] 王晶晶 吴绍强[1] WEI Fang;CHEN Dong-jie;DENG Jun-hua;WANG Jing-jing;WU Shao-qiang(Institute of Animal Inspection and Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China)

机构地区：[1]中国检验检疫科学研究院动物检验与检疫研究所,北京亦庄100176

出　　处：《中国兽医杂志》2023年第12期49-53,共5页Chinese Journal of Veterinary Medicine

基　　金：基本科研业务费项目(2022JK11);“十四五”国家重点研发计划(2022YFD1800505)。

摘　　要：为制备针对赤羽病病毒(AKAV)糖蛋白Gc的单克隆抗体(mAb),本试验选取AKAV Gc蛋白第465~704位氨基酸的基因序列进行密码子优化并合成至pFastBac HTB载体;通过蓝白斑筛选和昆虫细胞杆状病毒表达系统制备重组AKAV Gc aa465~704蛋白,纯化后免疫小鼠;通过细胞融合和亚克隆筛选阳性杂交瘤细胞,利用Western blot和间接免疫荧光试验(IFA)对mAb特性进行鉴定。结果显示,本试验成功筛选出1株可稳定分泌抗AKAV Gc aa465~704蛋白mAb的杂交瘤细胞株4D1,Western blot结果显示,mAb 4D1可特异性识别AKAV Gc aa465~704蛋白;IFA结果显示,mAb 4D1能够与AKAV感染的BHK21细胞发生反应。结果表明,本试验成功制备抗AKAV Gc aa465~704蛋白mAb,为进一步研究AKAV Gc蛋白生物学功能和建立AKAV检测方法提供技术支持。To prepare monoclonal antibody(mAb)against the glycoprotein Gc of Akabane virus(AKAV),thegenesequence of AKAV Gc protein from amino acid 465 to 704 was codon-optimized and synthesized into the pFastBac HTB vector.After blue-white screening,recombinant AKAV Gam46s-04 was produced using the baculovirus expression system in insect cells.Then,Gaus-7o was purified and used forthe immunization of mice.Posive hybridoma cells secreting mAb were obtained through cell fusion and subcloning,and the mAb characteristics were identified by Western blot and indirect immunofluorescence assay(IFA).The results showed that the hybridoma cell line 4DI stably secreting mAb against AKAV Gcau4s-04 was successfully screened.Westerm blot results demonstrated that the mAb 4DI could specifically recognize AKAV Gca46s-704.And IFA results showed that mAb 4DI could react with AKAV-infected BHK21 cells.This study successflly prepared mAb against AKAV Gca46-704,which provides technical support for further research on the biological functions of AKAV Gc and the establishment of AKAV detection methods.

关 键 词：赤羽病病毒 Gc蛋白 杆状病毒表达 单克隆抗体 

分 类 号：S855.3[农业科学—临床兽医学]