【摘要】 目的 制备呼吸道合胞病毒F蛋白的融合前构象(pre-F)mRNA疫苗，进行可行性验证。方法 对抗原基因进行密码子优化设计，分别连接至pGEM-3Zf-N3载体上，通过质粒线性化、体外转录、纯化和Cap1加帽处理，得到功能性mRNAs。在转染至293T细胞后，利用Western blot和间接免疫荧光试(IFA)来确定目标蛋白的表达情况。通过微流控装置制备LNP/mRNAs疫苗免疫C57BL/6小鼠，检测小鼠血清中抗原特异性IgG抗体效价，评估免疫效果。结果 成功构建重组质粒Pre-F-GCN4t和mDS-Cav1,通过双酶切验证条带符合预期大小。表达目的抗原大小为57.57 ku和64.35 ku。成功检测小鼠血清中抗原特异性IgG抗体，Pre-F-GCN4t免疫组的效果优于mDS-Cav1组，3次免疫较2次免疫提升了2倍，抗体效价最高可达1∶4×10~4。结论 两种抗原设计模式均具有可行性，Pre-F-GCN4t的设计方式对于激活机体的体液免疫应答更有优势，mRNA疫苗无需体内转录，为呼吸道合胞病毒mRNA疫苗的优化设计提供理论基础。更多还原

【Abstract】 Objective Preparation of pre conformational(pre F) mRNA vaccine for respiratory syncytial virus F protein fusion and feasibility validation. Methods Perform codon optimization design on antigenic genes, connect them to pGEM-3Zf-N3 vectors, and obtain functional mRNAs through plasmid linearization, in vitro transcription, purification, and Cap1 capping treatment. After transfection into 293T cells, Western blot and indirect immunofluorescence assay(IFA) were used to determine the expression of the target protein. Preparation of LNP/mRNAs vaccine for immunization of C57BL/6 mice using microfluidic devices, detection of antigen-specific IgG antibody titers in mouse serum, and evaluation of immune efficacy. Results Successfully constructed recombinant plasmids Pre-F-GCN4t and mDS Car1,and verified the expected size of the bands through double restriction enzyme digestion. The target antigen sizes for expression are 57.57 ku and 64.35 ku. Successfully detected antigen-specific IgG antibodies in mouse serum. The Pre-F-GCN4t immunization group showed better efficacy than the mDS Car1 group, with a 2-fold increase in antibody titer after 3 immunizations compared to 2 immunizations. The highest antibody titer could reach 1∶4×10~4. Conclusion Both antigen design modes are feasible, and the design approach of Pre-F-GCN4t has advantages in activating the body’s humoral immune response. mRNA vaccines do not require in vivo transcription, providing a theoretical basis for the optimization design of respiratory syncytial virus mRNA vaccines.更多还原