制备新型冠状病毒(SARS-CoV-2)受体结合域(RBD)亚单位疫苗并评价其免疫原性.首先,通过PCR技术构建重组质粒pFastBacl-gp67-RBD,利用bac to bac技术,筛选获得重组杆状病毒P3-RBD.利用间接免疫荧光试验鉴定RBD蛋白的表达情况,经过Ni2+柱亲和层析纯化RBD蛋白,并鉴定其免疫反应性与血管紧张素转化酶2(ACE2)结合活性.最后,将RBD与鼠伤寒沙门菌鞭毛蛋白(FliC)混合免疫BALB/c小鼠,设置单独RBD组和混合铝佐剂(Alum)组作为对照,评价亚单位疫苗的免疫原性.结果显示,在P3-RBD重组杆状病毒感染的Sf9细胞中检测到特异性绿色荧光.RBD蛋白能与Anti-RBD多克隆抗体发生特异性反应,并与ACE2蛋白具有较高结合力.二免2周后ELISA结果显示,与单独RBD组相比,FliC和Alum佐剂组均可以显著提高血清中IgG抗体水平.此外,FliC佐剂组IgG亚型(IgG1和IgG2a)抗体水平均显著高于单独RBD组,表明FliC能促进RBD疫苗诱导Th1/Th2混合型免疫应答.综上,沙门菌FliC能增强新型冠状病毒RBD亚单位疫苗的免疫原性,具有良好的应用前景,为进一步研制安全有效的COVID-19亚单位疫苗奠定了基础.
Preparation and immunogenicity evaluation of SARS-CoV-2 receptor binding domain subunit vaccine
The aim of this study was to prepare a receptor binding domain(RBD)subunit vaccine of SARS-CoV-2 and evaluate its immunogenicity.Firstly,recombinant plasmid pFastBacl-gp67-RBD was con-structed by PCR.Recombinant baculovirus P3-RBD was obtained using bac to bac technique.The expression of RBD was identified by indirect immunofluorescence assay.After purification of RBD by Ni2+column affinity chromatography,its immunoreactivity and angiotensin-converting enzyme 2(ACE2)binding affinity were identified.Finally,the immunogenicity of the subunit vaccine was evaluated by immuniz-ing BALB/c mice with a mixture of RBD and Salmonella typhimurium flagellin(FliC),and setting up the RBD group and the aluminum adjuvant(Alum)group as controls,respectively.The results showed that green fluorescence was observed in Sf9 cells infected with P3-RBD,and the RBD protein could react with anti-RBD polyclonal antibody and ACE2 protein.After 2 weeks of second immunization,the results showed that both FliC and Alum adjuvant groups could significantly increase IgG titers in serum com-pared to the RBD group.In addition,the IgG subtypes(IgG1 and IgG2a)titers in the FliC adjuvant group were significantly higher than that in the RBD group,which demonstrated that FliC promotes the mixed Th1/Th2 immune response in the RBD vaccine.In conclusion,Salmonella FliC enhances the immunogenicity of the SARS-CoV-2 RBD subunit vaccine,which shows good application prospects and lays the foundation for the further development of a safe and effective COVID-19 subunit vaccine.
国家科技期刊平台
NSTL
万方数据
