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
汪芋 1张守峰 2于婷婷 1米立娟 2么乃全 3缪发明 2扈荣良1
作者信息
- 1. 吉林农业大学动物医学院,吉林长春 130118;中国农业科学院长春兽医研究所,吉林 长春 130122
- 2. 中国农业科学院长春兽医研究所,吉林 长春 130122
- 3. 吉林农业大学动物医学院,吉林长春 130118
- 折叠
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.
Abstract
In order to obtain a cell line which high expressed of rabies virus glycoprotein the produc-tion 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 puro-mycin 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 poten-tial 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Key words
rabies/rabies virus glycoprotein/lentivirus/cell lines/immunological experiment/subunit vaccine引用本文复制引用
出版年
2024
NETL
NSTL
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