稳定表达猪BRD4-BD1/2蛋白的猪肺泡巨噬细胞传代细胞系的构建及其用于ASFV增殖的效果观察  

Construction of a Passaged Porcine Alveolar Macrophage Cell Line Stably Expressing the Porcine BRD4-BD1/2 Protein and Its Effects on ASFV Proliferation

作　　者：吴梦丽 孙华林 杨吉飞[1,2] 赵亚茹 关贵全 殷宏[1,2,3] 牛庆丽 WU Mengli;SUN Hualin;YANG Jifei;ZHAO Yaru;GUAN Guiquan;YIN Hong;NIU Qingli(State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;African Swine Fever Regional Laboratory of China(Lanzhou),Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonosis,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,兰州730000 [2]国家非洲猪瘟区域实验室(兰州)甘肃省病原生物学基础学科研究中心,兰州730046 [3]扬州大学,江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《畜牧兽医学报》2024年第10期4646-4659,共14页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然基金面上项目(32072830);国家重点研发计划(2021YFD1800101);甘肃省重大专项计划项目(22ZD6NA001);中央级公益性科研院所基本科研业务费专项(CAAS-ZDRW202409);甘肃省基础研究创新群体项目(22JR5RA024);特派团项目(22CX8NA011);中国农业科学院科技创新工程(CAAS-ASTIP-2021-LVRI)。

摘　　要：前期研究发现宿主表观遗传调控蛋白——含溴结构域蛋白质4(bromodomain-containing protein 4,BRD4)有助于非洲猪瘟病毒(African swine fever virus,ASFV)的复制,为了深入研究BRD4对ASFV复制的影响,通过筛选出显著促进ASFV复制的BRD4-BD1/2结构域,利用慢病毒表达系统成功构建稳定表达BRD4-BD1/2结构域的3D4/21细胞系,分析ASFV在3D4/21-BRD4-BD1/2细胞系与WT细胞系之间的复制差异。首先,以家猪基因BRD4-BD1/2为靶标,构建了含有3x Flag标签的重组质粒pLVX-IRES-puro-3x Flag-BRD4-BD1/2,并将其与质粒pMD2.G和pSPAX2共同转染至HEK-293T细胞进行慢病毒包装,获得具有感染能力的慢病毒。使用慢病毒感染3D4/21细胞后通过嘌呤霉素药物筛选,成功获得了稳定表达BRD4-BD1/2结构域的3D4/21细胞系。利用RT-qPCR和Western blot技术分别检测了ASFV感染3D4/21-BRD4-BD1/2细胞后CP204L和B602L基因的转录水平以及相应蛋白表达水平,并通过HAD50测定评价ASFV的复制能力。研究结果表明:成功构建了稳定表达BRD4-BD1/2结构域的3D4/21细胞系。与3D4/21-WT细胞系相比,BRD4-BD1/2稳定表达细胞系能够显著促进ASFV复制。本研究提供了深入研究BRD4蛋白在ASFV复制中功能的生物材料,并为ASFV疫苗候选株的开发提供了理论基础。Previous studies have identified that the host epigenetic regulator,bromodomain-containing protein 4(BRD4)facilitates the replication of the African swine fever virus(ASFV).To further investigate the impact of BRD4 on ASFV replication,the BRD4-BD1/2 domain,which significantly enhances ASFV replication,was identified.Using a lentiviral expression system,a stably expressing 3D4/21 cell line of the BRD4-BD1/2 domain was successfully constructed to analyze replication differences between the 3D4/21-BRD4-BD1/2 cell line and the wild-type(WT)cell line.Initially,a recombinant plasmid,pLVX-IRES-puro-3x Flag-BRD4-BD1/2,targeting the porcine gene BRD4-BD1/2 and containing a 3x Flag tag,was constructed.This plasmid,along with plasmids pMD2.G and pSPAX2,was transfected into HEK-293T cells for lentiviral packaging to obtain infectious lentiviruses.Following lentiviral infection of 3D4/21 cells and selection with puromycin,a cell line stably expressing the BRD4-BD1/2 domain was established.The transcription levels of ASFV genes CP204L and B602L,and the expression levels of their corresponding proteins in ASFV-infected 3D4/21-BRD4-/BD1/2 cells,were detected using RT-qPCR and Western blot techniques,respectively.ASFV replication capacity was assessed using the HAD50 assay.The results showed that the stably expressing BRD4-BD1/2 domain in the 3D4/21 cell line significantly enhanced ASFV replication compared to the 3D4/21-WT cell line.This study provides biological material for in-depth research on the function of the BRD4 protein in ASFV replication and lays a theoretical foundation for the development of ASFV vaccine candidates.

关 键 词：非洲猪瘟病毒 BRD4 BD1/2 3D4/21细胞 稳定表达细胞系 

分 类 号：S852.65[农业科学—基础兽医学]