中和性流行性感冒病毒基因工程抗体在昆虫细胞中的全基因表达、纯化及鉴定  

Expression,Purification and Identification of Intact Recombinant IgG Antibody to Influenza Virus with Neutralizing Activity in Baculovirus/ Insect System

作　　者：杨贵宾[1] 吴淑华[1] 梁米芳[1] 赵小东[1] 王艳[1] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室,北京100052

出　　处：《病毒学报》2001年第4期313-317,共5页Chinese Journal of Virology

摘　　要：将中和性流行性感冒 (流感 )病毒基因工程抗体IV 2、IV 6的轻链和重链Fd段基因 ,分别克隆入全抗体表达载体 pAC L Fc ,构建成杆状病毒表达载体pAC L Fc Ⅳ 2和 pAC L Fc Ⅳ 6 ,转染昆虫Sf9细胞 ,利用杆状病毒 /昆虫细胞系统实现抗体的分泌型表达 ,表达产物进行亲和层析分离纯化。SDS PAGE电泳和Westernblot法证实有完整免疫球蛋白的表达 ,免疫印迹法证实它们能与流感病毒血凝素蛋白特异性结合。经间接竞争性抑制ELISA法测定 ,抗体与流感病毒抗原结合的解离常数KD 值分别为 2 5× 10 -9M和 3 0× 10 -9M。流感病毒基因工程全抗体经在昆虫细胞中的表达、纯化和抗体特性鉴定 ,获得了两株纯化的全抗体 ,可用于以后的动物模型呼吸道粘膜被动免疫抗感染的研究。The genes of light and heavy chain Fd fragments of recombinant MAb to influenza virus with neutralizing activity were separately inserted into baculovirus expression vector pAC-L-Fc to construct the expression vectors of intact IgG,pAC-L-Fc-Ⅳ-2 and pAC-L-Fc-Ⅳ-6.They were used to transfect insect Sf9 cell separately. Secreted products of this Baculovirus/Insect system were purified with affinity chromatography.SDS-PAGE and Western blot proved the intact IgG had been expressed and immunoblotting identified they could bind specifically with influenza virus HA.Indirect ELISA determined the K D of IgG-Ⅳ-2 and IgG-Ⅳ-6 with influenza virus antigen were 2.5×10 -9 mol/L and 3.0×10 -9 mol/L respectively.The purified IgG of these two MAbs to influenza virus with nuetralizing activity can be used for study of passive immunization of respiratory tract mucous membrane against viral infection in animal model.

关 键 词：杆状病毒 昆虫细胞 流感病毒 基因工程抗体 分泌性表达 纯化 鉴定 

分 类 号：R373.14[医药卫生—病原生物学] R392.2[医药卫生—基础医学]