摘要
为获得能表达严重急性呼吸综合征冠状病毒2(SARS-CoV-2)刺突蛋白受体结合结构域(RBD)的体外转录mRNA,将设计、优化并合成的RBD基因片段插入真核表达载体pcDNA3.1(+),构建重组表达质粒pcDNA3.1(+)-RBD;重组质粒转染哺乳动物细胞293T后,Western blot检测重组蛋白RBD的表达;以表达RBD的重组质粒为模板,PCR扩增包含T7启动子的RBD基因片段作为体外转录模板,之后利用T7转录试剂盒体外转录获得修饰的RBD mRNA,添加多聚腺苷酸尾后使用RNA纯化试剂盒进行纯化,纯化后的RBD mRNA转染至293T细胞中,利用Western blot进行目的蛋白表达鉴定。结果显示,编码RBD的重组质粒构建成功并能在293T细胞中表达外源蛋白,体外转录获得修饰的RBD mRNA能在哺乳动物细胞中翻译,表达的RBD蛋白能与抗RBD的单克隆抗体反应,具有免疫反应原性,为SARS-CoV-2 RBD mRNA疫苗的研制奠定了基础。
To obtain the in vitro transcribed mRNA expressing the receptor-binding domain(RBD)of Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the RBD gene fragment was designed,optimized and synthesized into the eukaryotic expression vector pcDNA3.1(+)and then the resulting recombinant expression plasmid pcDNA3.1(+)-RBD was transfected into mammalian cells 293T.Western blot was used to detect the expression of the recombinant protein RBD.The recombinant plasmid expressing RBD was used as a PCR template for amplification of RBD gene fragment containing T7 promoter and the PCR product was acted as an in vitro transcribed template to obtain the modified RBD mRNA by in vitro transcription,tailing and purification.The purified RBD mRNA was transfected into 293T cells and the target protein was identified by Western blot.The results showed that the recombinant plasmid encoding RBD was constructed and expressed in 293T cells and the modified RBD mRNA transcribed in vitro were translated into the target protein in mammalian cells.The expressed RBD protein reacted with the anti-SARS-CoV-2 spike RBD monoclonal antibody.The study laid the foundation for the development of SARS-CoV-2 RBD mRNA vaccine.
作者
周雪
高磊
张瑾宬
袁思凌
操蓉
刘丽娜
ZHOU Xue;GAO Lei;ZHANG Jincheng;YUAN Siling;CAO Rong;LIU Lina(Key Laboratory of Infectious Immune and Antibody Engineering of Guizhou Province,Engineering Research Center of Cellular Immunotherapy of Guizhou Province,School of Basic Medical Science/School of Biology and Engineering,Guizhou Medical University,Guiyang 550025,China;Key Laboratory of Biological and Medical Engineering,Guizhou Medical University,Guiyang 550025,China;Key Laboratory of Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guiyang 550025,China)
出处
《中国动物传染病学报》
北大核心
2025年第3期123-129,共7页
Chinese Journal of Animal Infectious Diseases
基金
贵州省科技计划项目(黔科合支撑[2020]4Y217)
国家级大学生创新创业训练计划项目(202110660008)
贵州省大学生创新创业训练计划项目(S202010660060)。
