cGAS稳定表达细胞系的构建及功能鉴定  

Construction and Functional Identification of Cell Lines Stably Expressing cGAS

作　　者：赵呈彦 任豪杰 万博[1,2] 魏战勇 何文瑞 ZHAO Chengyan;REN Haojie;WAN Bo;WEI Zhanyong;HE Wenrui(International Joint Research Center of National Animal Immunology,College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Ministry of Education Key Laboratory for Animal Pathogens and Biosafety,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物医学院国家动物免疫学国际联合研究中心,郑州450046 [2]动物病原与生物安全教育部重点实验室,郑州450046

出　　处：《畜牧兽医学报》2024年第11期5317-5324,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划青年科学家项目(2022YFD1801300);国家自然科学基金项目(32102655,32272987)。

摘　　要：天然免疫系统会在病毒入侵机体后的第一时间对其进行识别,并合成分泌I型干扰素和炎症因子等细胞因子参与宿主抗病毒免疫反应。天然免疫应答的启动主要通过机体自身表达的模式识别受体对病毒来源的病原相关分子模式的识别来实现。cGAS是目前最公认的胞质DNA受体,在各种类型的细胞和组织中广泛表达。猪肾细胞PK-15是猪病毒性疾病研究过程中应用十分广泛的工具细胞,然而本研究前期的转录组学数据显示,PK-15细胞中cGAS的转录水平极低,蛋白水平几乎检测不到PK-15细胞系中cGAS的表达,这极大程度的限制了PK-15细胞在猪病毒性疾病感染与免疫机制探究中的应用。本研究旨在构建一株稳定表达cGAS的PK-15细胞系。通过慢病毒包装系统,将cGAS基因整合到靶细胞基因组,经嘌呤霉素加压筛选,获得能够稳定表达cGAS的PK-15细胞系,并以猪伪狂病病毒(porcine pseudorabies virus,PRV)和猪细小病毒(porcine parvovirus,PPV)为模式病毒,对cGAS在抗DNA病毒感染过程中的作用进行了验证。结果表明,cGAS基因成功整合到宿主细胞PK-15基因组,重组细胞系PK-15-cGAS能够稳定表达cGAS蛋白;PRV和PPV感染PK-15-cGAS细胞能诱导产生更高水平的干扰素相关基因,在PK-15-cGAS细胞中PRV和PPV的复制受到明显抑制。这些结果表明,稳定表达cGAS的PK-15细胞系构建成功,它可用于猪病毒性疾病感染与免疫机制相关探究,为后续探究该类病毒免疫逃逸机制提供了良好的试验材料。The pathogenic microorganisms can be sensed and recognized by innate immune system once invaded the animals.And the sensing of conserved structural components called pathogen-associated molecular patterns(PAMPs)by pattern recognition receptors(PRRs)of the hosts activates a series of signaling events,leading to induction of downstream antiviral effector proteins including type I interferons(IFNs)and proinflammatory cytokines to clear the invaders.The nucleotidyltransferase GMP-AMP(cGAMP)synthase(cGAS)is ubiquitously expressed in various types of cells and tissues,and is still the most crucial cytosolic DNA sensor till now.However,the transcription and expression of cGAS are almost undetectable in porcine kidney cell lines PK-15,which play central roles in the study of swine viral disease.To construct the PK-15 cell lines stably expressing cGAS,the cGAS gene was inserted into the genome of the target cells using lentivirus packaging system,following by the identification of gene transcription,expression,and distribution.Finally,the DNA viruses pseudorabies virus(PRV)and porcine parvovirus(PPV)were used as model viruses to confirm the role of cGAS in innate antiviral immunity.The results showed that cGAS is successfully integrated into the genome of the PK-15 cells,and can be detected in the recombinant cell lines PK-15-cGAS as early as generation 3.The infection of PRV and PPV induced markedly higher levels of interferon-related genes,and consistently the replication of PRV and PPV was dramatically inhibited in PK-15-cGAS cells.In summary,the PK-15 cell lines stably expressing cGAS were prepared successfully,which will help to explore the mechanisms of viral infection as well as immune evasion.

关 键 词：cGAS PK-15 稳定细胞系 慢病毒包装系统 

分 类 号：S855.3[农业科学—临床兽医学]