白纹伊蚊唾液蛋白rAlb-34k2-1诱导的特异性细胞免疫应答对BMDM细胞中DENV-2复制的影响  

Effect of specific cellular immune response induced by anti-Aedes albopictus salivary protein rAlb-34k2-1 on DENV-2 replication in BMDM cells

作　　者：覃燕春 李莹 胡欢 周璐 黄贞植 吴家红 商正玲 QIN Yanchun;LI Ying;HU Huan;ZHOU Lu;HUANG Zhenzhi;WU Jiahong;SHANG Zhengling(Department of Immunology,School of Basic Medicine,Guizhou Medical University,Guiyang,Guizhou 561113,China;Department of Parasitology,Guizhou Medical University,Guiyang,Guizhou 561113,China)

机构地区：[1]贵州医科大学基础医学院免疫学教研室,贵州贵阳561113 [2]贵州医科大学基础医学院寄生虫学教研室,贵州贵阳561113

出　　处：《中国热带医学》2024年第8期907-913,共7页China Tropical Medicine

基　　金：国家自然科学基金项目(No.81960295)。

摘　　要：目的体外探究白纹伊蚊唾液蛋白rAlb-34k2-1诱导的小鼠脾淋巴细胞特异性免疫应答对2型登革病毒(dengue virus type 2,DENV-2)感染小鼠骨髓来源巨噬细胞(bone marrow-derived macrophages,BMDM)的影响。方法采用CCK-8法分析rAlb-34k2-1蛋白对免疫/未免疫小鼠脾细胞的特异性增殖反应;制备荷载蚊唾液蛋白rAlb-34k2-1、rAalb_CTL1蛋白(蚊唾液C型凝集素蛋白)的抗原提呈细胞(antigen presenting cell,APC),与免疫/未免疫小鼠脾淋巴细胞进行混合淋巴细胞培养,取细胞培养上清处理DENV-2感染的BMDM细胞,实时荧光定量逆转录PCR(real-time quantitative reverse transcription PCR,qRT-PCR)检测DENV-2的核酸变化,分析rAlb-34k2-1蛋白诱导的特异性T淋巴细胞应答对BMDM内DENV-2复制的影响。将免疫/未免疫小鼠脾淋巴细胞与rAlb-34k2-1蛋白预处理的感染BMDM进行共培养,通过qRT-PCR检测培养48、72 h后DENV-2 E基因的表达,分析免疫/未免疫小鼠淋巴细胞对预处理的BMDM中DENV-2复制的影响。结果小鼠脾细胞体外特异性增殖实验显示,与未免疫组比较,rAlb-34k2-1蛋白能导致免疫小鼠脾细胞特异性增殖,刺激指数(stimulation index,SI)>2,P<0.0001;荷载rAalb_CTL1蛋白的混合淋巴细胞培养上清未影响BMDM细胞中DENV-2的复制,荷载rAlb-34k2-1蛋白的混合淋巴细胞培养上清可抑制BMDM中DENV-2复制(2^(-ΔΔCT) <0.5,P<0.05);与免疫/未免疫小鼠脾淋巴细胞混合培养后,rAlb-34k2-1预处理的BMDM中DENV-2复制无差异。结论rAlb-34k2-1蛋白诱导的小鼠T淋巴细胞应答上清可抑制BMDM细胞中DENV-2的复制,为探究蚊唾液蛋白诱导免疫效应在蚊媒病毒致病中的作用提供前期实验基础。Objective To investigate the effect of Aedes albopictus salivary protein rAlb-34k2-1-induced mouse splenocytes immune response on dengue virus type 2(DENV-2)-infected bone marrow-derived macrophages(BMDM)in vitro.Methods The specific cell proliferation response of splenic cells from immunized/non-immunized mice to Alb34k2-1 protein was analyzed using the CCK-8 assay.The antigen-presenting cells(APCs)loaded with mosquito saliva proteins rAlb-34k2-1 protein/rAalb_CTL1 protein were prepared and co-cultured with lymphocytes from spleens of immunized/non-immunized mice.On the one hand,the levels of DENV-2 nucleic acids in BMDM treated with cell supernatants from mixed lymphocyte co-cultures were analyzed by qRT-PCR,and the effect of specific T cell response induced by rAlb-34k2-1 on DENV-2 replication in BMDM was analyzed.On the other hand,the expression of the DENV-2 E gene in BMDM pretreated with rAlb-34k2-1 protein was also evaluated to determine the direct effect of immunized mouse spleen lymphocytes co-cultured at 48 h and 72 h and the effect of spleen lymphocytes from immunized or non-immunized mice on DENV-2 replication in pretreated BMDMs was analyzed.Results Compared to the non-immunized group,the rAlb-34k2-1 protein in vitro caused a specific proliferation of immunized-splenocytes,with a proliferation index(SI)>2(P<0.0001).Mixed lymphocyte culture supernatants loaded with rAlb-34k2-1 protein inhibited DENV-2 replication in BMDM,while the mixed lymphocyte culture supernatants loaded with rAalb_CTL1 protein had no such effect(2^(-ΔΔCT) <0.5,P<0.05).There was no difference in DENV-2 intracellular replication in rAlb-34k2-1 pretreated BMDM after co-culture with either immunized-lymphocytes or non-immunized-lymphocytes.Conclusions The supernatant induced by the mouse T cell response to the rAlb-34k2-1 protein can inhibit DENV-2 replication in BMDM cells,providing preliminary experimental evidence to explore the role of mosquito salivary protein-induced immune responses in the pathogenesis of mosquito-borne viru

关 键 词：rAlb-34k2-1蛋白 2型登革病毒 细胞免疫应答 

分 类 号：R373.33[医药卫生—病原生物学]