摘要
目的制备森林脑炎病毒mRNA疫苗,验证其能否在细胞内产生病毒样颗粒(virus-like particles,VLPs),并评价其在小鼠体内的免疫原性。方法设计含日本乙型脑炎病毒(Japanese encephalitis virus,JEV)信号肽、森林脑炎前体膜蛋白(premature membrane glycoprotein,prM)、森林脑炎E蛋白的基因序列,以此序列构建质粒,并通过体外转录方式合成森林脑炎病毒mRNA。将mRNA转染至HEK293T细胞,Western blot法检测mRNA在细胞内的表达,电镜检测mRNA在细胞内是否可自组装成VLPs。用纳米制备系统制备mRNA纳米脂质颗粒(lipid nanoparticles,LNPs),测量粒径及其分布。用2和10μg mRNA疫苗肌内注射免疫雌性BALB/c小鼠,每组6只,同时设阴性对照(PBS)组,加强免疫后1周,眼眶采血并分离血清,ELISA法检测结合抗体水平,噬斑减少试验检测中和抗体水平。结果mRNA转染HEK293T细胞后,Western blot分析显示,在相对分子质量约55000处可见单一条带,与E蛋白理论相对分子质量相符;电镜下可见直径约50 nm的球状颗粒,表面有明显刺突;制备的mRNA LNPs疫苗平均粒径为124.4 nm,聚合物分散性指数(polymer dispersity index,PDI)为0.049;与阴性对照组相比,2和10μg剂量mRNA诱导的结合抗体与中和抗体水平均显著提高(t分别为12.4和10.8、19.7和12.0,P均<0.001)。结论成功制备了能在细胞内表达VLPs的森林脑炎mRNA疫苗,该疫苗在小鼠体内具有较好的免疫原性,为VLPs技术与mRNA技术在新型森林脑炎疫苗中的研发提供了实验依据。
Objective To prepare an mRNA vaccine against tick-borne encephalitis virus(TBEV),verify its ability to produce virus-like particles(VLPs)in cells,and evaluate its immunogenicity in mice.Methods The gene sequence containing Japanese encephalitis virus(JEV)signal peptide,TBEV premature membrane glycoprotein(prM)and E protein was designed.The plasmids were constructed according to the sequence,and corresponding mRNA was synthesized by in-vitro transcription.The mRNA was transfected into HEK293T cells and then measured for the expression in cells by Western blot,and detected for the ability to self-assemble into VLPs in cells by electron microscopy.The mRNA lipid nanoparticles(LNPs)were prepared by Nano Assembler system,and the particle size and distribution of LNPs were measured.Female BALB/c mice were immunized by intramuscular injection at dosages of 2 and 10μg mRNA vaccine respectively,6 mice in each group,and the negative control group(PBS)was set.One week after booster immunization,orbital blood samples were collected and separated for serum.The binding antibody levels were detected by ELISA,and the neutralizing antibody levels were detected by plaque reduction assay.Results After mRNA transfection into HEK293T cells,a single band was found at the relative molecular mass of about 55000,which was consistent with the theoretical relative molecular mass of E protein.Under electron microscope,spherical particles with a diameter of about 50 nm were observed,and there were obvious spikes on the surface.The average particle size of mRNA LNPs vaccine was 124.4 nm and the polymer dispersity index(PDI)was 0.049.Compared with the negative control group,the levels of binding antibody and neutralizing antibody induced by 2 and 10μg mRNA significantly increased(t=12.4 and 10.8.19.7 and 12.0,respectively,each P<0.001).Conclusion A TBEV mRNA vaccine capable of producing VLPs in cells was successfully prepared,which demonstrated good immunogenicity in mice,providing experimental basis for the development of new generation TBEV vaccine in combination using of VLPs technology and mRNA technology.
作者
李状
郭一冰
赵琛
周航
吴业红
LI Zhuang;GUO Yibing;ZHAO Chen;ZHOU Hang;WU Yehong(Vaccine Research and Development Department,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)
出处
《中国生物制品学杂志》
2025年第8期897-901,907,共6页
Chinese Journal of Biologicals
基金
2023年度吉林省博士后择优资助。
关键词
森林脑炎
mRNA疫苗
病毒样颗粒
免疫原性
Tick-borne encephalitis
mRNA vaccine
Virus-like particles(VLPs)
Immunogenicity
作者简介
通信作者:吴业红,E-mail:wuyehong@sinopharm.com。
