白纹伊蚊唾液蛋白促进巨噬细胞免疫应答及2型登革病毒的复制  

Salivary protein rAlb-34kDa-1 of Aedes albopictus modulates the immune response of Raw264.7 cells and promotes the replication of DENV-2

作　　者：旷晓元 肖秋秋 吴文诗 牟小会 吴家红 KUANG Xiaoyuan;XIAO Qiuqiu;WU Wenshi;MU Xiaohui;WU Jiahong(Department of Human Parasitology,Basic Medical College,Guizhou Medical University,Guiyang,Guizhou 550025,China;Guizhou Key Laboratory of Microbio and Infectious Disease Prevention&Control,Guiyang,Guizhou 550025,China;Department of Reproductive Medicine,the People's Hospital of Anshun City,Anshun,Guizhou 561000,China)

机构地区：[1]贵州医科大学基础医学院人体寄生虫学教研室,贵州贵阳550025 [2]贵州省微生物组与传染性疾病防控重点实验室,贵州贵阳550025 [3]安顺市人民医院生殖医学科,贵州安顺561000

出　　处：《中国热带医学》2025年第2期149-155,共7页China Tropical Medicine

基　　金：国家自然科学基金项目(No.81760374)。

摘　　要：目的 探讨白纹伊蚊唾液蛋白rAlb-34kDa-1对巨噬细胞(Raw264.7)的免疫调控作用及对Raw264.7细胞中2型登革病毒(Dengue virus type 2,DENV-2)复制的影响。方法 将原核表达质粒pET32a(+)-rAlb-34kDa-1转入大肠埃希菌(E.coli)BL21(DE3)后,按实验室前期建立的表达纯化条件获取唾液蛋白并通过SDS-PAGE及Western Blot(WB)方法鉴定;以CCK8法分析rAlb-34kDa-1蛋白对Raw264.7细胞的毒性。将0.02、0.20和2.0μg/mL浓度rAlb-34kDa-1作用Raw264.7细胞6、12和24 h,采用实时荧光定量逆转录PCR(real-time quantitative reverse transcription PCR, qRTPCR)检测细胞中白细胞介素-1β(interleukin-1β, IL-1β)、白细胞介素-6(interleukin-6, IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、趋化因子配体2(chemokine ligand 2, CCL2)、干扰素-β(interferon-β, IFN-β)、干扰素刺激基因15(interferon stimulated gene 15, ISG15)的表达,运用酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA)检测细胞培养上清IL-6和TNF-α的分泌;免疫荧光法观察rAlb-34kDa-1作用Raw264.7细胞后p65进入细胞核的情况,分析rAlb-34kDa-1蛋白是否通过NF-κB信号通路实现对Raw264.7细胞的免疫调控;按分组(DENV-2作用组、唾液蛋白+DENV-2共孵育组)加不同的处理因素作用6、12和24 h后收集细胞,利用qRT-PCR检测细胞内DENV-2的mRNA表达量及运用流式细胞术(FCM)检测不同处理因素作用24 h细胞内J2(dsRNA)的荧光强度。结果 SDS-PAGE及WB结果显示成功获得rAlb-34kDa-1蛋白且经CCK8法分析该蛋白对Raw264.7细胞无毒性;0.2μg/mL和2.0μg/mL的rAlb-34kDa-1均能诱导Raw264.7细胞中IL-1β、IL-6、TNF-α、CCL2、IFN-β、ISG15的表达;rAlb-34kDa-1作用Raw264.7细胞1 h后显著增强Raw264.7细胞核内的p65的荧光强度;rAlb-34kDa-1与DENV-2作用于Raw264.7细胞12 h时,2μg/mL的rAlb-34kDa-1显著增强细胞中DENV-2的mRNA表达量。作用24 h时,0.02、0.20和2μg/mL的rAlb-34kDa-1均能增强Raw264.7细胞中DENV-2的mRNA表�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 2μg/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-αsecretion 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 0.2μg/mL and 2μg/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

关 键 词：白纹伊蚊 rAlb-34kDa-1蛋白 2型登革病毒 免疫调控 

分 类 号：R384.1[医药卫生—医学寄生虫学]