SARS-CoV-2重组痘苗病毒载体疫苗的构建、筛选、安全性和免疫原性研究

Construction,screening,safety and immunogenicity of recombinant vaccinia virus vector vaccine against SARS-CoV-2

目的利用穿梭质粒通过同源重组构建表达SARS-CoV-2 RBDEPI蛋白重组痘苗病毒,并对其进行筛选、鉴定和免疫原性检测。方法参照SARS-CoV-2全基因组序列设计合成目的基因RBDEPI,通过PCR、测序、酶切连接将目的基因插入到痘苗病毒穿梭载体pSTKE中,构建pSTKE-RBDEP。将pSTKE-RBDEPI与天坛株痘苗病毒VTT共转染鼠肾细胞BHK-21中,通过同源重组,并以增强型绿色荧光蛋白EGFP为筛选标记,通过多次绿色荧光噬斑筛选获得重组病毒rVTT△TK-RBDEPI。利用RT-PCR和Western blot检测rVTT△TK-RBDEPI目的基因的转录和目的蛋白的表达情况;通过ELISA和ELISPOT检测rVTT△TK-RBDEPI免疫小鼠SARS-CoV-2与痘苗病毒载体特异性抗体水平和特异性淋巴细胞IL-4与IFN-γ的表达情况,确定rVTT△TK-RBDEPI的免疫原性。结果经过10次绿色荧光噬斑筛选、RT-PCR与Western blot鉴定获得1株能够表达SARS-CoV-2 RBDEPI的重组痘苗病毒rVTT△TK-RBDEPI,且rVTT△TK-RBDEPI具备良好的免疫原性。结论获得能够表达SARS-CoV-2 RBDEPI的重组痘苗病毒,该疫苗具有免疫原性,为以痘苗病毒为载体的SARS-CoV-2疫苗的进一步研究和临床治疗奠定了基础。

Obective To construct recom binant vaccinia virus expressing sars-cov-2 rbdepi protein,and to screen,identify,and detect its safety and immunogenicity.Methods The target gene RBDEPI was designed and synthesized according to SARS-CoV-2,the target gene was inserted into the vaccinia virus shuttle vector pSTKE by PCR、sequencing and enzyme digestion,to construct the recombinant shuttle vector pSTKE RBDEPI.Then,the vector pSTKE RBDEPI was transfected with Tiantan vaccinia virus VTT into BHK-21 cells for homologous recombination,EGFP was used as a screening marker,the recombinant virus was obtained by plaque screening.The recombinant virus was identified by RT-PCR and Western blot,the SARS CoV-2 and VTT specific antibody,IL-4,and IFN-γlevels were detected by ELISA and ELISPOT,to determine the immunogenicity of rVTT OTK-RBDEPI.Results The recombinant vaccinia virus rVTT△TK-RBDEPI which could express RBDEPI was obtained after 10 times of plaque screening,and rVTTOTK-RBDEPI has good immunogenicity.Conclusion The recombinant vaccinia virus expressing SARS CoV-2 RBDEPI was successfully obtained and also has good immunogenicity,this study laid a foundation for further research and clinical treatment of SARS-CoV-2 vaccine with vaccinia virus as vector.