可诱导表达结核分枝杆菌ESAT-6的THP-1细胞系的建立  

Establishment of THP-1 cell line that can be induced to express Mycobacterium tuberculosis ESAT-6

作　　者：唐亦然 戴鑫钧 胡燕萍 毕斯琪 孟祥苗 杨杨 宋厚辉 TANG Yiran;DAI Xinjun;HU Yanping;BI Siqi;MENG Xiangmiao;YANG Yang;SONG Hou-hui(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province/Zhejiang Provincial Engineering laboratory for Animal Health Inspection and Internet Technology,College of Animal Science and Technology/College of Veterinary Medicine Zhejiang A&F University,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实验室,浙江杭州311300

出　　处：《中国兽医学报》2021年第11期2148-2154,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31502034);浙江省自然科学基金资助项目(Y21C180001);浙江省万人计划(2017R52033);浙江农林大学学生科研训练项目(2020KX0162)。

摘　　要：利用Tet-On-3G系统构建可诱导表达ESAT-6的THP-1细胞系,研究结核分枝杆菌毒力蛋白ESAT-6在宿主细胞内的生物学功能。首先,利用聚合酶链反应(PCR)扩增ESAT-6片段和红色荧光蛋白mCherry片段,并与pLVX-TRE3G载体连接,构建pTRE3G-mCherry-ESAT-6。将其与调控质粒pLVX-Tet3G共同转染HEK293T细胞,Dox诱导后检测ESAT-6表达情况。然后,将质粒pTRE3G-mCherry-ESAT-6和pLVX-Tet3G分别与包装质粒psPAX2和pMD2.0G共转染HEK293T细胞,以获得慢病毒颗粒LV-TRE3G-ESAT-6和LVX-Tet3G。接着,将2种病毒感染THP-1细胞,通过Puromycin和G418进行筛选诱导表达ESAT-6的阳性细胞克隆,命名为THP-1/ESAT-6。利用Western blot检测THP-1细胞中ESAT-6的表达情况。结果表明,筛选获得了携带ESAT-6的THP-1诱导表达细胞系,并且ESAT-6表达量与Dox剂量和诱导时间呈正相关。最后,对细胞系中表达的ESAT-6进行生物活性检测,结果表明ESAT-6能够促进细胞的死亡和IL-1β的分泌。该细胞系的建立为深入研究ESAT-6调控细胞信号通路的功能提供了基础。In order to study the biological function of ESAT-6 in host cells,a THP-1 cell line that could induce the expression of ESAT-6 was constructed using Tet-On-3 G system.Firstly,the fragments of ESAT-6 and red fluorescent protein mCherry were amplified by PCR and then ligated with pLVX-TRE3 G vector to construct recombinant plasmid named pTRE3 G-mCherry-ESAT-6.The plasmids pTRE3 G-mCherry-ESAT-6 and pLVX-Tet3 G were co-transfected into HEK293 T,then ESAT-6 expression was detected after Dox induction.Secondly,the plasmids pTRE3 G-mCherry-ESAT-6 and pLVX-Tet3 G were co-transfected into HEK293 T cells with packaging plasmids psPAX2 and pMD2.0 G respectively to obtain lentiviral particles LV-TRE3 G-ESAT-6 and LVX-Tet3 G.THP-1 cells were infected with the two viruses and screened by puromycin and G418 to obtain the positive clones.The expression of ESAT-6 in THP-1/ESAT-6 cells was induced with different dosage of Dox or different time with certain Dox dosage.Western blot was used to detect the ESAT-6 expression.The results showed that ESAT-6 expressed by THP-1/ESAT-6 cell line could promote cell death and induce IL-1βsecretion.The establishment of this cell line provides an experimental basis for further study on the function of ESAT-6 in regulating cell signaling pathway.

关 键 词：结核分枝杆菌 ESAT-6 慢病毒包装 TET-ON 诱导表达 

分 类 号：S852.61[农业科学—基础兽医学]