Construction and identification of recombinant adenovirus expressing S protein receptor binding domain and N protein of SARS-CoV-2 Delta variant
10.13200/j.cnki.cjb.003881
- VernacularTitle:表达SARS-CoV-2 Delta突变株S蛋白受体结合域和N蛋白的重组腺病毒的构建及鉴定
- Author:
Xue fu
- Publication Type:Journal Article
- Keywords:
Severe acute respiratory symptom coronavirus 2(SARS-CoV-2);
S protein;
Receptor binding domain(RBD);
N protein;
Adenovirus
- From:
Chinese Journal of Biologicals
2023;36(9):1025-1031
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo construct and identify a recombinant adenovirus expressing S protein receptor binding domain(RBD)and N protein of severe acute respiratory symptom coronavirus 2(SARS-CoV-2)Delta variant.MethodsThe RBD and N gene fragments of SARS-CoV-2 were cloned into pcDNA3.0BA vector respectively to construct recombinant plasmid pcDNA3.0BA-RBD-N. The RBD-CMV-N fragment was amplified by PCR and inserted into shuttle vector pShuttle-CMV. The shuttle plasmid pShuttle-RBD-N was then homologously recombined with pAdeasy-1 to obtain recombinant plasmid pAdeasy-1-RBD-N,which was transfected into HEK293 cells for recombinant adenovirus Ad-RBD-N packaging. The transcription of RBD and N genes of recombinant adenovirus in HEK293 cells was detected by RT-PCR,while the expre-ssion of RBD and N proteins by Western blot and immunofluorescence assay. 12 female BALB/c mice were immunized with Ad-RBD-N by intramuscular injection at a dose of 5 × 109copies per mouse. Blood samples were collected 14 d after immunization,and the serum antibody titers were measured by ELISA.ResultsThe RBD and N genes of recombinant adenovirus were transcribed normally in HEK293 cells,and the RBD and N proteins were expressed normally in MA104 cells. Mice immunized with the recombinant adenovirus produced specific IgG antibodies against RBD and N proteins.ConclusionThe recombinant adenovirus expressing S protein RBD and N protein of SARS-CoV-2 Delta variant was succe-ssfully constructed,which laid a foundation of the follow-up research on Delta variant vaccines.
