重组呼吸道合胞病毒G蛋白保守域的腺病毒载体疫苗的研究
Study on recombinant adenovirus vector vaccine in G protein conserved domain of respiratory syncytial virus
石艺 1柴鹏弟 2段招军 2章青 2孔翔羽 2王宏 2庞立丽 2李丹地2
作者信息
- 1. 甘肃中医药大学公共卫生学院,兰州 730000;中国疾病预防控制中心病毒病预防控制所传染病溯源预警与智能决策全国重点实验室 国家卫生健康委员会医学病毒和病毒病重点实验室,北京 102206
- 2. 中国疾病预防控制中心病毒病预防控制所传染病溯源预警与智能决策全国重点实验室 国家卫生健康委员会医学病毒和病毒病重点实验室,北京 102206
- 折叠
摘要
目的 以非复制型的5型人腺病毒(human adenovirus type 5,Ad5)为载体,构建重组呼吸道合胞病毒G蛋白(RSV-G)保守域的腺病毒载体疫苗,利用小鼠评价其免疫原性及免疫保护效果.方法 利用分子克隆方法构建插入能串联表达A亚型和B亚型RSV-G保守域(Gbcc和Gacc)的重组Ad5载体质粒(Ad5-Gbcc-Gacc)并转染HEK293A细胞,获得重组腺病毒Ad5-Gbcc-Gacc;通过多酶切鉴定病毒Ad5-Gbcc-Gacc的基因组,用Western blot验证Gbcc-Gacc基因的表达水平;重组腺病毒通过肌肉注射免疫BALB/c小鼠1次,在第6周用RSV Long毒株对小鼠以滴鼻的方式进行攻毒.用酶联免疫吸附试验(Elisa)检测和评价免疫小鼠血清IgG及抗体亚型,用微中和检测的方法测定中和抗体水平,同时每天记录小鼠攻毒前后的体重,并检测小鼠肺组织的病理变化、肺和鼻组织中RSV病毒拷贝数.结果 Western blot及多酶切鉴定结果与预期相符,表明重组腺病毒拯救成功.Ad5-Gbcc-Gacc在小鼠体内可以诱导较高滴度的特异性IgG、中和抗体水平及Th1/Th2平衡的免疫反应.与未免疫小鼠比较,经Ad5-Gbcc-Gacc免疫小鼠攻毒后肺部只有轻微病理损伤,且肺部及鼻甲的病毒拷贝数降低.结论 Ad5-Gbcc-Gacc具备良好的免疫原性,并能对RSV感染小鼠有一定的保护作用,为进一步研制基于G蛋白的RSV疫苗奠定了基础.
Abstract
Objective A recombinant adenoviral vector vaccine based on non-replicating human adenovirus type 5(Ad5),encoding the conserved domain of respiratory syncytial virus G protein(RSV-G)was constructed.The immunogenicity and protective efficacy of this vaccine were subsequently evaluated in mice.Methods The recombinant Ad5 vector plasmid(Ad5-Gbcc-Gacc)was constructed by inserted conserved domains of RSV A and RSV B.The recombinant adenovirus Ad5-Gbcc-Gacc was rescued in HEK293A cells.The genome of virus Ad5-Gbcc-Gacc was identified by multi-enzyme digestion,and the expression of Ad5-Gbcc-Gacc was verified by Western blot.Recombinant adenovirus was used to immunize BALB/c mice via intramuscular injection with signal dose,and then challenged with RSV Long strain at week 6.The levels of G specific IgG and antibody subtypes in serum were detected by enzyme-linked immunosorbent assay,the level of neutralizing antibodies was determined by micro-neutralization assay.After challenge,the mice's weight was recorded daily,the copies of RSV virus in the lung and nasal tissues were detected.Pathological changes in lung tissue were also examined.Results Western blot and multi-enzyme digestion identification confirmed the successful rescue of the recombinant adenovirus.Ad5-Gbcc-Gacc elicit high titers of specific IgG,robust neutralizing antibodies,and a balanced Th1/Th2 immune response in mice.In comparison to unimmunized controls,mice immunized with Ad5-Gbcc-Gacc reduced the viral copies in both lung and nasal tissue,and exhibited only minimal pathological damage of lung tissue following RSV challenge.In conclusion,Ad5-Gbcc-Gacc induced robust immunogenicity and offers protective effects against RSV infection in murine models.Conclusions Ad5-Gbcc-Gacc induce robust immunogenicity and can protect mice from RSV challenge,which lays a foundation for further development of RSV vaccine based on G protein.
关键词
腺病毒载体/呼吸道合胞病毒/G蛋白/中央保守域/疫苗Key words
Adenovirus vector/Respiratory syncytial virus/G-protien/Central conserved domain/Vaccine引用本文复制引用
基金项目
国家重点研发项目(2022YFC2304302)
出版年
2024
NETL
NSTL
万方数据