体外转录mRNA系统的建立及其在呼吸道合胞病毒疫苗中的应用  

Development of in vitro transcribed mRNA and its application in respiratory syncytial virus vaccine

作　　者：侯风萍 程亚慧 唐悦 吴爱莉 王云瑾 李晓萌 王剑 李雄雄 HOU Fengping;CHENG Yahui;TANG Yue;WU Aili;WANG Yunjin;LI Xiaomeng;WANG Jian;LI Xiongxiong(The Second Research Department,Lanzhou Insititute of Biological Products Co.,Ltd.,Gansu Provincial Vaccine Technology Innovation Center,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司第二研究室,甘肃省疫苗技术创新中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2024年第3期17-25,共9页Progress In Microbiology and Immunology

摘　　要：目的建立mRNA的体外转录系统,并用于评价呼吸道合胞病毒(respiratory syncytial virus,RSV)mRNA疫苗的免疫效果。方法以增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)为模式蛋白,将EGFP基因分别克隆入pcDNA3.1(+)和pmRV载体中,通过体外转录合成EGFP mRNA,以验证载体及mRNA框架结构的正确性;然后将RSV融合前F蛋白(RSV preF)序列构建于经验证的载体,体外转录合成RSV preF mRNA;转染细胞后,通过荧光显微镜、流式细胞术、免疫荧光和Western blot等方法验证蛋白表达;进一步通过微流控技术用脂质体纳米颗粒(lipid nanoparticles,LNP)包封体外转录合成的mRNA,经过粒径和多分散性指数检测后进行小鼠免疫和评价。结果经过验证发现,pcDNA3.1(+)的框架结构更适合mRNA;EGFP mRNA转染HEK 293T细胞24 h后,体外转染效率>90%;免疫荧光和Western blot等方法验证RSV mRNA能够在24 h内表达preF;包封后的RSV mRNA疫苗免疫小鼠,血清抗体滴度与RSV preF蛋白免疫抗体滴度相近(P>0.05),细胞因子IFN-γ和IL-4均有一定程度的上调。结论建立了正确表达目的蛋白的mRNA体外转录系统,以此为基础的RSV mRNA疫苗在小鼠体内可诱导强的免疫应答,为潜在的RSV mRNA候选疫苗奠定了基础。Objective To establish the in vitro transcription system for mRNA candidate vaccines and apply them to the respiratory syncytial virus mRNA vaccine to evaluate the immune responses of the mRNA vaccine.Methods EGFP was used for development of mRNA vaccine system,and cloned into pcDNA3.1(+)and pmRV.EGFP mRNA was transcribed in vitro.The better plasmid was selected for construction of RSV preF based on the ECFP protein expression.RSV preF mRNA was transfected into different cells,and the expression of target protein was verified by Western blot and immunofluorescence assay.The synthetic mRNA was encapsulated in lipid nanoparticles(LNP)by microfluidic method and characterized by dynamic light scattering(DLS).The RSV preF mRNA vaccine was administrated to mice to perform immunological evaluation.Results The transcribed ECFP mRNA in vitro was transfected into HEK 293T cells,and the transfection efficiency was more than 90%.RSV preF was cloned into pcDNA3.1(+),and transcribed mRNA was expressed and verified by Western blot and immunofluorescence assay.The results showed that RSV preF mRNA could accurately express RSV preF.The encapsulated mRNA vaccines were used to immunize mice,resulting in strong antibody levels in sera in mRNA groups that is similar to protein group,and up-regulated cytokines IFN-γ and IL-4 also to some extent.Conclusion In vitro transcribed mRNA was successfully expressed in cells and the RSV preF mRNA vaccine can induce strong cellular and humoral immune responses in mice,laying a foundation for potential RSV mRNA candidate vaccines.

关 键 词：mRNA疫苗 体外转录系统 呼吸道合胞病毒 脂质体纳米颗粒 免疫应答 

分 类 号：R183.3[医药卫生—流行病学]