Page 92 - 南京医科大学自然版

南京医科大学学报（自然科学版） 第44卷第8期
·1120 · Journal of Nanjing Medical University（Natural Sciences） 2024年8月

·临床研究·

大别班达病毒mRNA疫苗的构建及初步评价

周婷婷 ，郁心怡 ，纪雪雯 ，王桂芹 ，吴梓菡 ，闻瑜婷 ，朱 进 ，刘新建 5*
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东部战区疾病预防控制中心，江苏 南京 210002；南京医科大学第一附属医院感染病科，江苏 南京 210029；南京中医
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药大学整合医学院 江苏 南京 210023；中科南京生命健康高等研究院，江苏 南京 211135；南京医科大学基础医学院
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病原生物学系，国家疫苗研发创新平台&卫健委抗体技术重点实验室 江苏 南京 211166
［摘 要］ 目的：基于mRNA疫苗策略构建大别班达病毒（Bandavirus davieense virus，DBV）疫苗，并免疫BALB/c小鼠，评价其
免疫学特征。 方法：优化并合成DBV糖蛋白Gn序列，克隆至pGEM⁃3Zf（+）载体，质粒通过BamHⅠ酶切线性化，进行体外转
录，酶法加帽和添加 A 尾制备 mRNA，瞬时转染真核 293FT 细胞，免疫印迹验证蛋白的体外表达。通过脂质纳米颗粒递送
mRNA，以低（2 μg/只）、中（5 μg/只）、高剂量（20 μg/只）每2周肌肉注射1次免疫BALB/c小鼠，ELISA测定小鼠血清抗体效价，
病毒中和实验评价小鼠血清阻断病毒感染细胞的能力。结果：制备的DBV mRNA疫苗能够进行体外转录和表达，低、中、高3
个免疫剂量均能诱导小鼠产生高水平的特异性抗体，其中高剂量免疫组可诱导稳定抗体表达至10周以上；病毒中和实验显
示，免疫血清能够与病毒结合，阻止病毒感染细胞。结论：获得了能够诱导小鼠产生特异性中和抗体的 DBV mRNA 疫苗，为
DBV感染的预防奠定了研究基础。
［关键词］ 大别班达病毒；mRNA疫苗；中和抗体；病毒感染
［中图分类号］ R392 ［文献标志码］ A ［文章编号］ 1007⁃4368（2024）08⁃1120⁃06
doi：10.7655/NYDXBNSN231187

Construction and evaluation of Bandavirus davieense virus mRNA vaccine
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ZHOU Tingting ，YU Xinyi ，JI Xuewen ，WANG Guiqin ，WU Zihan ，WEN Yuting ，ZHU Jin ，LIU Xinjian 5*
1 Huadong Medical Institute of Biotechniques，Nanjing 210002；Department of Infectious Disease，the first Affiliated
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Hospital of Nanjing Medical University，Nanjing 210029；School of Medicine & Holistic Integrative Medicine，
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Nanjing University of Chinese Medicine，Nanjing 210023；Nanjing Advanced Academy of Life and Health，Nanjing
211135；Department of Pathogen Biology，National Vaccine Innovation Platform & Key Laboratory of Antibody
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Techniques of National Health Commission，Nanjing Medical University，Nanjing 211166，China
［Abstract］ Objective：To construct a Bandavirus davieense virus（DBV）vaccine based on the strategy of mRNA vaccine，immunize
BALB/c mice，and evaluate its immunologic characteristics. Methods：The coding sequence of DBV glycoprotein Gn was optimized and
synthesized，then inserted into the pGEM⁃3Zf（+）plasmid. The linearization plasmids were enzyme digested by BamH Ⅰ，cap and
polyA tailing were added with polymerase to complete in vitro transcription. Western blotting verified the protein expression by
transient transfection of the mRNA into eukaryotic 293FT cells. The mRNA was delivered by lipid nanoparticles，and immunized
BALB/c mice by intramuscular injection of low（2 μg/mouse），medium（5 μg/mouse）and high（20 μg/mouse）dose once every two
weeks. The antibody titer in mice serum was detected by ELISA，and the ability of antibody to neutralize DBV in vitro was detected at
the cellular level by virus neutralization assay. Results：The prepared DBV mRNA vaccine could be transcribed and expressed in vitro.
All three immunization doses induced high levels of specific antibodies in mice，with the high⁃dose immunization group inducing stable
antibody expression for over 10 weeks. The virus neutralization assay showed that immune sera could bind to the virus and block virus
infection of cells. Conclusion：A DBV mRNA vaccine capable of inducing specific neutralizing antibodies in mice is obtained，laying
the foundation for research on the prevention of DBV infection.
［Key words］ Bandavirus davieense virus（DBV）；mRNA vaccine；neutralizing antibodies；virus infection
［J Nanjing Med Univ，2024，44（08）：1120⁃1125］

［基金项目］ 南京医科大学国家疫苗研发创新平台项目；东部战区疾病预防控制中心“逸启”自主科研基金（2024YQFH04）
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通信作者（Corresponding author），E⁃mail：liuxinjian@njmu.edu.cn；zhujin1968@njmu.edu.cn