AIM2炎症复合体活化体系的体外构建及初步应用  

Reconstruction and preliminary application of the AIM2 inflammasome activation system in vitro

作　　者：许锦霞 吴龙建 胡燕萍 孙敏捷 宋厚辉 杨杨 XU Jin-xia;WU Long-jian;HU Yan-ping;SUN Min-jie;SONG Hou-hui;YANG Yang(China-Australia Joint Laboratory for Animal Health Big Data Analytics,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology,Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,College of Animal Science and Technology&College of Veterinary Medicine,Hangzhou 311300,China)

机构地区：[1]浙江农林大学动物科技学院/动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程研究中心/浙江省动物医学与健康管理国际科技合作基地/中澳动物健康大数据分析联合实验室,浙江杭州311300

出　　处：《中国预防兽医学报》2024年第2期178-184,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(32272951);浙江省属高校基本科研业务费专项基金(2020YQ008)。

摘　　要：AIM2炎症复合体参与自身免疫性疾病和机体抵御病原体的过程,研究其激活机制具有重要的意义。为构建AIM2炎症复合体的体外活化体系,本研究通过PCR方法从人单核细胞(THP-1细胞)中扩增AIM2、ASC、pro-Caspase-1、pro-IL-1β基因片段后,构建重组质粒p3×Flag-AIM2、p3×Flag-ASC、p3×Flag-pro-Caspase-1和p3×Flag-pro-IL-1β,分别转染HEK293T细胞后采用western blot鉴定各蛋白的表达。结果显示,分别约在41.9 ku、34.1 ku、48.5 ku、24.6 ku处出现特异性条带,表明融合Flag标签的AIM2、ASC、Caspase-1和IL-1β蛋白在HEK293T细胞中获得了表达。除此之外,还可以在HEK293T细胞中检测到39 ku的内源性AIM2蛋白特异性条带,表明HEK293T细胞自身也表达内源性AIM2蛋白。将不同浓度比例的上述重组质粒转染或共转染HEK293T细胞,24 h后各组再转染poly(dA:dT),采用间接ELISA检测各组细胞中IL-1β的分泌水平。结果显示,空白对照组、p3×Flag-pro-IL-1β实验组和共转染p3×Flag-ASC、p3×Flag-pro-IL-1β实验组细胞上清中均未检测到IL-1β,共转染p3×Flag-ASC、p3×Flag-pro-Caspase-1、p3×Flag-pro-IL-1β和共转染p3×Flag-AIM2、p3×Flag-ASC、p3×Flag-pro-Caspase-1、p3×Flag-pro-IL-1β的实验组细胞上清中均能检测到高浓度的IL-1β,且共转染p3×Flag-AIM2的HEK293T细胞中IL-1β的分泌水平更高。此外,与对照组相比,poly(dA:dT)刺激后的各组HEK293T细胞中IL-1β的分泌水平极显著升高(P<0.001),表明AIM2炎症复合体的体外活化体系正确构建。将不同浓度的p3×Flag-AIM2与其它质粒共转染HEK293T细胞,并转染poly(dA:dT)作用,采用间接ELISA检测IL-1β的分泌水平,结果显示,无论是否转染poly(dA:dT),随着p3×Flag-AIM2的转染剂量的升高,IL-1β的分泌水平也显著或极显著升高(P<0.001、P<0.01、P<0.05、P<0.001),表明细胞中AIM2蛋白的表达量与AIM2炎症复合体的活性呈正相关。单核细胞增生性李斯特菌已被证实可以激活AIM2炎�The AIM2 inflammasome is involved in both autoimmune diseases and the host defense against pathogens,and the study of its activation mechanism will be very meaningful.To establish an activation system for the AIM2 inflammasome in vitro,the gene fragments of AIM2,ASC,pro-Caspase-1,and pro-IL-1βfrom human monocytic cells(THP-1 cells)were amplified by using PCR.Subsequently,the recombinant plasmids including p3×Flag-AIM2,p3×Flag-ASC,p3×Flag-pro-Caspase-1,and p3×Flag-pro-IL-1βwere constructed.These obtaining plasmids were transfected into HEK293T cells,and their expressions were confirmed by western blot analysis.The results were showed that the recombinant proteins of AIM2,ASC,Caspase-1,and IL-1βwere well-expressed tagged with Flag in the form of fusion expression in HEK293T cells,and the specific bands were shown at approximately 41.9ku,34.1ku,48.5ku,and 24.6ku,respectively.Notably,endogenous AIM2 protein was also detected at 39ku in HEK293T cells,indicating endogenous AIM2 was expressed in HEK293T cells.Co-transfection of plasmids with different concentrations into HEK293T cells,inluding or excluding poly(dA:dT)transfection,was performed,and IL-1βsecretion levels were assessed by indirect ELISA.The results demonstrated that IL-1βwas undetectable in the supernatants of the control group,the group transfected with p3×Flag-pro-IL-1βalone,or the group co-transfected with p3×Flag-ASC together with p3×Flag-pro-IL-1β.However,high concentrations of IL-1βwere detectable in the supernatants of the experimental groups co-transfected with p3×Flag-ASC,p3×Flag-pro-Caspase-1,p3×Flag-pro-IL-1β,and co-transfected with p3×Flag-AIM2,p3×FlagASC,p3×Flag-pro-Caspase-1,p3×Flag-pro-IL-1β,especially for p3×Flag-AIM2 co-transfection group exhibiting higher IL-1βsecretion levels in HEK293T cells.Additionally,after poly(dA:dT)stimulation,a significant increase in IL-1βsecretion levels was observed compared to the control group(P<0.001),confirming successful construction of the AIM2 inflammasome activation system in

关 键 词：质粒构建 AIM2炎症复合体 IL-1Β 

分 类 号：S852.6[农业科学—基础兽医学]