Construction of a recombinant adenovirus for Mycobacterium tuberculosis c-di-AMP phosphodiesterase expression and induction of humoral immunity
10.3969/j.issn.1002-2694.2025.00.001
- VernacularTitle:结核分枝杆菌c-di-AMP磷酸二酯酶重组腺病毒的构建及其诱导的体液免疫应答
- Author:
Jia-hao HU
1
;
Huan-huan NING
;
Meng-juan DONG
;
Yan-zhi LU
;
Ting DAI
;
Cong-yue ZHANG
;
Zi-qing XU
;
Shu-yu WANG
;
Zheng-yan ZHOU
;
Yin-lan BAI
Author Information
1. 空军军医大学基础医学院微生物与病原生物学教研室,西安 710032;空军军医大学基础医学院学员队,西安 710032
- Publication Type:Journal Article
- Keywords:
Mycobacterium tuberculosis;
recombinant adenovirus;
c-di-AMP;
phosphodiesterase;
mucosal immunity
- From:
Chinese Journal of Zoonoses
2025;41(4):364-369
- CountryChina
- Language:Chinese
-
Abstract:
A recombinant adenovirus(rAd)for expression of Mycobacterium tuberculosis(M.tb)c-di-AMP phosphodiesterase CnpB was constructed,and its induced humoral immune response was detected.The codon-optimized gene of M.tb CnpB was cloned into the adenoviral plasmid pcADV.The recombinant plasmid pcADV-CnpB was transfected into HEK293T cells,and expression was detected with Western blot.The recombinant plasmid pcADV-CnpB and the backbone plasmid were co-transfected into HEK293T cells to obtain the recombinant adenovirus rAd-CnpB.rAd-CnpB was amplified in HEK293T cells,and the target protein expression of rAd-CnpB was detected with Western blot and immunofluorescence.Mice were immunized with rAd-CnpB intranasally,and their sera and bronchoalveolar lavage fluid(BALF)were collected.ELISA was used to detect levels of antigen-specific antibodies.Restriction enzyme digestion and sequencing indicated that the recombinant plasmid pcADV-CnpB was successfully constructed and led to protein expression in eukaryotic cells.rAd-CnpB was packaged and produced in HEK293T cells.After amplification and purification,rAd-CnpB with a titer of 5.53×1010 PFU/mL was obtained.rAd-CnpB led to CnpB expression in HEK293T cells.Intranasal immunization with rAd-CnpB increased levels of IgG and secretory IgA in BALF and led to high levels of IgG in sera.rAd-CnpB,the recombinant adenovirus for expression of c-di-AMP phosphodiesterase CnpB was successfully constructed,and was found to induce antigen-specific humoral and mucosal immune responses through mucosal immunization.Thus,rAd-CnpB may be used in further research on new TB vaccine strategies.
