SARS-CoV-2重组痘苗病毒疫苗rVTT△TK-RBD的构建、筛选及免疫原性研究

Construction,screening and immunogenicity of the recombinant poxvirus vaccine rVTTΔTK-RBD against SARS-CoV-2

目的 构建重组痘苗病毒载体疫苗rVTT△TK-RBD,并评价其安全性和免疫原性。方法 参考严重急性呼吸综合征冠状病毒2(SARS-CoV-2)基因序列合成受体结合域(RBD)基因,并将其插入自主构建的重组质粒pSTKE的多克隆位点上,构建重组痘苗病毒穿梭载体pSTKE-RBD,并转染到预先感染天坛株痘苗病毒(VTT)的BHK-21细胞内,经多轮的荧光噬斑筛选成功获得重组痘苗病毒rVTT△TK-RBD;通过滴鼻方式免疫小鼠后,检测rVTT△TK-RBD对BALB/c小鼠体质量的影响;通过肌肉免疫小鼠后,分析rVTT△TK-RBD对BALB/c小鼠产生的特异性抗体和中和抗体的水平;通过流式细胞术检测rVTT△TK-RBD对BALB/c小鼠T细胞亚群的影响。结果 利用同源重组、增强型绿色荧光蛋白(EGFP)筛选标记和多次荧光噬斑筛选,成功筛选获得了表达RBD的胸腺激酶(TK)基因缺失型重组痘苗病毒rVTT△TK-RBD,且PCR验证成功。BALB/c小鼠体内实验表明rVTT△TK-RBD具有较好的抗SARS-CoV-2的免疫原性且相比于亲本株VTT明显降低了对机体的毒性作用。结论 成功构建并获得SARS-CoV-2重组痘苗病毒疫苗rVTT△TK-RBD并通过各项试验证明其安全性和免疫原性。

Objective To construct a recombinant poxvirus vector vaccine,rVTT△TK-RBD,and to evaluate its safety and immunogenicity.Methods The receptor-binding domain(RBD)gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE,to construct the recombinant poxvirus shuttle vector pSTKE-RBD.This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain(VTT).The recombinant poxvirus rVTT△TK-RBD was successfully obtained after several rounds of fluorescence phage screening.The effect of rVTT△TK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination.The levels of specific and neutralizing antibodies produced by rVTT△TK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly.The effect of rVTT△TK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry.Results Through homologous recombination,enhanced green fluorescent protein(EGFP)screening marker,and multiple rounds of fluorescent phosphorescence phage screening,a recombinant poxvirus rVTT△TK-RBD,expressing RBD with deletions in the thymidine kinase(TK)gene,was successfully obtained,which was validated by PCR.The in vivo experiments on BALB/c mice showed that rVTT△TK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT.Conclusion The recombinant poxvirus vaccine rVTT△TK-RBD against SARS-CoV-2 is successfully constructed and obtained,with its safety and immunogenicity confirmed through various experiments.