稳定表达狂犬病病毒糖蛋白293T细胞系的构建及免疫原性研究  

Construction and immunogenicity study of 293T cell line stably expressing rabies virus glycoprotein

作　　者：汪芋 张守峰 于婷婷[1,2] 米立娟 么乃全 缪发明[2] 扈荣良 WANG Yu;ZHANG Shoufeng;YU Tingting;MI Lijuan;YAO Naiquan;MIAO Faming;HU Rongliang(College of Veterinary Medicine,Jilin Agricultural University,Changchun 130118,China;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China)

机构地区：[1]吉林农业大学动物医学院,吉林长春130118 [2]中国农业科学院长春兽医研究所,吉林长春130122

出　　处：《中国兽医学报》2024年第11期2393-2399,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划基金资助项目(2022YFD1800101);国家自然科学基金资助项目(32172941)

摘　　要：为得到可用于狂犬病亚单位疫苗生产的高效表达狂犬病病毒(rabies virus, RABV)糖蛋白的细胞系,本研究以RT-PCR扩增获得RABV糖蛋白基因序列并克隆入慢病毒表达载体中。重组质粒pLV-RVG与包膜质粒pMD2.0G及包装质粒psPAX2共转染HEK-293T细胞,收获慢病毒并感染新的HEK-293T细胞,通过嘌呤霉素加压筛选获得293T-RVG细胞系。将此细胞系传代20代,间接免疫荧光法(IFA)及Western blot鉴定表明67 kDa的RABV-G蛋白在该细胞内能够高效表达;且细胞系冻融物293T-RVG辅以佐剂肌肉注射免疫小鼠,血清以FAVN法测定RABV中和抗体,在免疫14 d体内效价就可达到2.19 IU/mL,高于国际公认保护阈值(0.5 IU/mL)。本研究成功构建了稳定且高效表达RABV糖蛋白的细胞系293T-RVG,该细胞系能够对攻毒小鼠产生保护作用,可作为一种亚单位疫苗,具备用于大规模生产和应用潜能。In order to obtain a cell line which high expressed of rabies virus glycoprotein the production of rabies subunit vaccine,the RABV glycoprotein gene sequence was amplified by RT-PCR and cloned into the lentiviral expression vector.The recombinant plasmid pLV-RVG,envelope plasmid pMD2.0G and packaging plasmid psPAX2 were co-transfected into HEK-293T cells to harvest lentivirus and infect new HEK-293T cells.The 293T-RVG cell line was obtained by puromycin pressure screening.Indirect immunofluorescence assay(IFA)and Western blot showed that the 67 kD RABV-G protein was highly expressed in the cells.The neutralizing antibody titer reached 2.19 IU/mL within 14 days after immunization,which was higher than the internationally recognized protection threshold(0.5 IU/mL).This study showed that the cell line 293T-RVG with stable and high expression of rabies virus glycoprotein was successfully constructed.The cell line could protect mice against rabies virus challenge and could be used as a subunit vaccine with potential for large-scale production and application.

关 键 词：狂犬病 狂犬病病毒糖蛋白 慢病毒 细胞系 免疫试验 亚单位疫苗 

分 类 号：S851.3[农业科学—预防兽医学] S852.65[农业科学—兽医学]