Salivary protein rAlb-34kDa-1 of Aedes albopictus modulates the immune response of Raw264.7 cells and promotes the replication of DENV-2
- Author:
KUANG Xiaoyuan
;
XIAO Qiuqiu
;
WU Wenshi
- Publication Type:Journal Article
- Keywords:
Aedes albopictus;
rAlb-34kDa-1 protein;
DENV-2;
immunoregulation
- From:
China Tropical Medicine
2025;25(2):149-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the immunomodulatory effects of Aedes albopictus salivary protein rAlb-34kDa-1 on macrophages (Raw264.7) and its impact on dengue virus type 2 (DENV-2) replication in Raw264.7 cells. Methods The prokaryotic expression plasmid pET32a(+)-rAlb-34kDa-1 was transfected into Escherichia coli (E.coli) BL21 (DE3), and salivary proteins were obtained according to the expression purification conditions established in the pre-laboratory stage and identified by SDS-PAGE and Western blot (WB). The toxic effects of rAlb-34kDa-1 protein in Raw264.7 cells were assessed by CCK8 assay. Raw264.7 cells were treated with rAlb-34kDa-1 at concentrations of 0.02, 0.2, and 2 μg/mL for 6, 12, and 24 h. The expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), chemokine ligand 2 (CCL2), interferon-β (IFN-β), and interferon-stimulated gene 15 (ISG15) within the cells was detected using real-time quantitative reverse transcription PCR (qRT-PCR). Enzyme-linked immunosorbent assay (ELISA) was used to measure IL-6 and TNF-α secretion in the cell culture supernatants. Immunofluorescence staining was used to observe the translocation of p65 into the nucleus of Raw264.7 cells following rAlb-34kDa-1 treatment, to analyze whether rAlb-34kDa-1 modulates the immune response in Raw264.7 cells through NF-κB signaling pathway. Cells were collected 6, 12, and 24 hours post-treatment under different experimental conditions (DENV-2 treatment group, salivary protein +DENV-2 co-incubation group) was measured using qRT-PCR, and the fluorescence intensity of J2 (dsRNA) in the cells was detected by flow cytometry (FCM) after 24 hours under different treatment factors. Results The results of SDS-PAGE and WB confirmed the successful acquisition of rAlb-34kDa-1 protein, and CCK8 analysis showed no cytotoxicity of this protein toward Raw264.7 cells. Both 0.2 μg/mL and 2 μg/mL rAlb-34kDa-1 induced the expression of IL-1β, IL-6, TNF-α, CCL2, IFN-β, and ISG15 in Raw264.7 cells. One hour after rAlb-34kDa-1 treatment, the fluorescence intensity of p65 in the nucleus of Raw264.7 cells was significantly enhanced. In Raw264.7 cells co-incubated with rAlb-34kDa-1 and DENV-2 for 12 hours, rAlb-34kDA-1 at a concentration of 2 μg/mL significantly increased the mRNA expression of DENV-2. After 24 hours of treatment, rAlb-34kDA-1 at concentrations of 0.02 μg/mL, 0.2 μg/mL, and 2 μg/mL all enhanced the mRNA expression of DENV-2 in Raw264.7 cells, and 2 μg/mL of rAlb-34kDa-1 co-incubated with DENV-2 significantly increased the J2 fluorescence signal in cells. Conclusion The rAlb-34kDa-1 protein can regulate the immune response in murine Raw264.7 cells and promote the replication of DENV-2 in these cells.
- Full text:20251117095122106674.Salivary protein rAlb-34kDa-1 of Aedes albopictus modulates the immune response of Raw264.7 cells and promotes the replication of DENV-2.pdf
