Construction and immunogenicity of a recombinant adenovirus expressing a fusion protein of SARS-CoV-2 RBD and N protein
10.3969/j.issn.1002-2694.2025.00.113
- VernacularTitle:融合表达新型冠状病毒RBD与N蛋白的重组腺病毒构建及免疫原性研究
- Author:
Peng ZHANG
1
;
Tongyao MAO
;
Surui JIANG
;
Dandi LI
;
Zhaojun DUAN
Author Information
1. 传染病溯源预警与智能决策全国重点实验室,国家卫生健康委医学病毒和病毒病重点实验室,中国疾病预防控制中心病毒病预防控制所,北京 102206
- Publication Type:Journal Article
- Keywords:
severe acute respiratory syndrome coronavirus 2 vaccine;
adenovirus-based vector;
cellular immunity;
humoral immunity
- From:
Chinese Journal of Zoonoses
2025;41(8):845-851
- CountryChina
- Language:Chinese
-
Abstract:
This study investigated the immunogenicity of type 5 replication deficient recombinant adenovirus comprising the fused receptor binding domain protein(RBD)and capsid protein(N)of the SARS-CoV-2 Omicron strain.The overlap PCR method was used to obtain the RBDgs3N fragment.The plasmid pKAd5ES-RBDgs3N was obtained through homologous recombination,and re-combinant virus was amplifiedand harvested after transfection of HEK293 cells.Cesium chloride density gradient centrifugation was used to purify the recombinant adenovirus,and the viral titer was determined through limited dilution analysis.The fusion protein ex-pression was identified with the western blot(WB)method.Recombinant adenovirus was injected intramuscularly into BALB/c mice,and the specific immune responses of the mice were detected with indirect ELISA and enzyme-linked immunosorbent spot technology.The titer of the recombinant adenovirus pKAd5ES-RBDgs3N virus seed was 1.168×1010 IU/mL.The RBDgs3N fusion protein was suc-cessfully expressed in HEK293 cells.Single dose intramuscular immunization significantly induced production of IgG antibodies against wild type(WT)SARS-CoV-2 virus and variant(Delta and Omicron)RBDs in mice,with antibody titers of 103.677 8,103.878 5,and 104.454 9,respectively.The specific antibody titer against N protein was 104.942 2.The level of IFN-γ secretion for RBD specific cellu-lar immunity was 1 452 SFC/106 cells.The type 5 replication deficient recombinant adenovirus with RBD and N gene fusion constructed in this study elicited high levels of specific antibodies against RBD and N protein and RBD specific cellular immunity in BALB/c mice,thus providing a basis for further evaluation of the recombinant adenovirus as a potential SARS-CoV-2 vaccine.
