摘要
为了深入研究牛分枝杆菌PPE13在病原-宿主相互作用中的具体机制,本研究利用慢病毒表达系统构建了PPE13稳定表达的THP-1细胞系。首先,采用分子克隆技术,以牛分枝杆菌基因组DNA为模板扩增出ppe13目的片段,构建含有ppe13片段的重组慢病毒表达质粒,将该质粒和慢病毒包装质粒转染至239T细胞中,72 h后收集病毒。然后,将获得的病毒感染THP-1细胞中,经1μg/mL嘌呤霉素筛选获得稳定表达PPE13的细胞株THP-1/PPE13。最后,使用ELISA方法检测稳定表达细胞系中TNF-α、IL-1β和IL-6分泌的情况,以验证细胞系中PPE13的生物学活性。结果显示,相较于对照细胞THP-1/Con,THP-1/PPE13中TNF-α、IL-1β和IL-6的分泌水平均显著升高。该细胞系的建立为深入研究PPE13调控细胞信号通路的功能提供了实验基础。
To further study the specifi c mechanism of Mycobacterium bovis(M.bovis)PPE13 in pathogen-host interaction,a THP-1 cell line stably expressing PPE13 was established by Lentivirus expression system.Firstly,the target fragment of PPE13 was amplifi ed using M.bovis genomic DNA as template by molecular cloning technology and then the recombinant Lentivirus expression plasmid containing PPE13 fragment was obtained by enzyme digestion,enzyme chain and transformation.The recombinant plasmid and Lentivirus packaging plasmids were transfected into 239T cells,and the virus was collected after 72 h.THP-1 cells were infected with the Lentivirus and screened by 1μg/mL puromycin.Finally,TNF-α,IL-1βand IL-6 were tested by ELISA in THP-1/PPE13 cells to verify the function of PPE13.The results showed that the secretion levels of TNF-α,IL-1βand IL-6 were signifi cantly increased in THP-1/PPE13 compared with control cells THP-1/Con.The establishment of this cell line provided an experimental basis for further study on the function of PPE13 in regulating cell signaling pathway.
作者
刘卓彤
毕斯琪
孟祥苗
胡燕萍
宋厚辉
杨杨
LIU Zhuotong;BI Siqi;MENG Xiangmiao;HU Yanping;SONG Houhui;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,Zhejiang A&F University,Hangzhou 311300,China)
出处
《中国动物传染病学报》
CAS
北大核心
2024年第5期179-184,共6页
Chinese Journal of Animal Infectious Diseases
基金
浙江省自然科学基金资助项目(Y21C180001)
浙江省属高校基本科研业务费(2020YQ008)。
