猪ASC及Ub基因的克隆及真核表达重组质粒的构建
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摘要
炎症小体不仅参与机体的天然免疫反应,同时还参与机体抗病毒等多种宿主防御反应。在多种疾病相关信号通路反应中都伴随着蛋白质的泛素化修饰。NOD样受体(NLRs)介导的炎症小体信号通路中,凋亡相关点状样蛋白(ASC)是关键的接头蛋白。为了解猪瘟病毒(CSFV)感染对ASC泛素化水平的影响,通过RT-PCR从猪血管内皮细胞(ECs)中扩增获得ASC和泛素(Ub)的全长cDNA双链片段,连接至克隆载体pMD-18T,获得重组克隆质粒pMD-ASC和pMD-Ub。双酶切pMD-ASC获得ASC目的序列,连接至pcDNA3.1-Flag,双酶切pMD-Ub获得Ub目的序列,连接至pcDNA3.0-HA,获得重组表达质粒pcDNA3.1-Flag-ASC和pcDNA3.0-HA-Ub。将pcDNA3.1-Flag-ASC和pcDNA3.0-HA-Ub转染ECs,Western blot在蛋白水平检测ASC和Ub的表达。结果表明,成功构建了带有Flag标签的ASC真核表达质粒和带有HA标签的Ub真核表达质粒,且在ECs中获得高效表达,为进一步体外研究CSFV调控猪血管内皮细胞ASC泛素化的机制提供了实验基础。
Inflammasome plays a key role in the body's anti-innate immunity,and also participates in many host defense responses such as antivirus.Ubiquitination of proteins is accompanied by a variety of diseaserelated signaling pathways.ASC is a linker protein in NLRPs mediated inflammasome signaling pathway.To find the effect of CSFV infection on ubiquitination of ASC,the full-length double-stranded cDNA fragments of ASC and Ub were amplified from porcine vascular endothelial cells(ECs)by RT-PCR and ligated with the cloning vector pMD-18 Tto obtain recombinant plasmids pMD-ASC and pMD-Ub.Plasmid pMDASC was identified by enzyme digestion to obtain ASC target sequence,which was connected to pcDNA3.1-Flag.Plasmid pMD-Ub was identified by enzyme digestion to obtain Ub target sequence,which was connected to pcDNA3.0-HA.The recombinant expression plasmids pcDNA3.1-Flag-ASC and pcDNA3.0-HAUb were obtained.The pcDNA3.1-Flag-ASC and pcDNA3.0-HA-Ub were transfected into ECs to detect the expressions of ASC and Ub at the protein level by Western blot.The Flag tagged ASC eukaryotic expression plasmid and the HA tagged Ub eukaryotic expression plasmid were successfully constructed,and they were highly expressed in ECs.This study provided experimental basis for the further study on the mechanism of CSFV regulating the ubiquitination of ASC in porcine vascular endothelial cells.
Inflammasome plays a key role in the body's anti-innate immunity,and also participates in many host defense responses such as antivirus.Ubiquitination of proteins is accompanied by a variety of diseaserelated signaling pathways.ASC is a linker protein in NLRPs mediated inflammasome signaling pathway.To find the effect of CSFV infection on ubiquitination of ASC,the full-length double-stranded cDNA fragments of ASC and Ub were amplified from porcine vascular endothelial cells(ECs)by RT-PCR and ligated with the cloning vector pMD-18 Tto obtain recombinant plasmids pMD-ASC and pMD-Ub.Plasmid pMDASC was identified by enzyme digestion to obtain ASC target sequence,which was connected to pcDNA3.1-Flag.Plasmid pMD-Ub was identified by enzyme digestion to obtain Ub target sequence,which was connected to pcDNA3.0-HA.The recombinant expression plasmids pcDNA3.1-Flag-ASC and pcDNA3.0-HAUb were obtained.The pcDNA3.1-Flag-ASC and pcDNA3.0-HA-Ub were transfected into ECs to detect the expressions of ASC and Ub at the protein level by Western blot.The Flag tagged ASC eukaryotic expression plasmid and the HA tagged Ub eukaryotic expression plasmid were successfully constructed,and they were highly expressed in ECs.This study provided experimental basis for the further study on the mechanism of CSFV regulating the ubiquitination of ASC in porcine vascular endothelial cells.
引文
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[2]吕其壮,刘娟,林谦,等.猪免疫抑制性疾病病因学研究进展[J].动物医学进展,2017,38(9):96-100.
[3] Gou H,Zhao M,Xu H,et al.CSFV induced mitochondrial fission and mitophagy to inhibit apoptosis[J].Oncotarget,2017,8(24):39382-39400.
[4] Ning P,Hu C,Li X,et al.Classical swine fever virus Shimen infection increases p53signaling to promote cell cycle arrest in porcine alveolar macrophages[J].Oncotarget,2017,8(34):55938-55949.
[5]Velazquez-Salinas L,Risatti G R,Holinka L G,et al.Recoding structural glycoprotein E2in classical swine fever virus(CSFV)produces complete virus attenuation in swine and protects infected animals against disease[J].Virology,2016,494:178-189.
[6] Lamkanfi M,Dixit V M.Mechanisms and functions of inflammasomes[J].Cell,2014,157(5):1013-1022.
[7] Shi C S,Shenderov K,Huang N N,et al.Activation of autophagy by inflammatory signals limits IL-1βproduction by targeting ubiquitinated inflammasomes for destruction[J].Nat Immunol,2012,13(3):255-263.
[8] Guan K,Wei C W,Zheng Z R,et al.MAVS promotes inflammasome activation by targeting ASC for K63-Linked ubiquitination via the E3ligase TRAF3[J].J Immunol,2015,194(10):4880-4890.
[9]吴丹丹,彭忠,陈焕春,等.猪瘟病毒基因型概述[J].动物医学进展,2017,38(11):107-111.
[10] Jamin A,Gorin S,Cariolet R,et al.Classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil,blood and slpeen of infected pigs[J].Vet Res,2008,39(1):7.
[11] Summerfield A,Alves M,Ruggli N,et al.High IFN-alpha responses associated with depletion of lymphocytes and natural IFN-producing cells during classical swine fever[J].J Interferon Cytokine Res,2006,26(4):248-255.
[12]徐磊,卢培成,牛更智,等.猪瘟兔化弱毒免疫猪淋巴细胞亚类及转化水平、IFN-γ的变化与分析[J].中国预防兽医学报,2015,37(3):185-189.
[13] Kim J K,Jin H S,Suh H W,et al.Negative regulators and their mechanisms in NLRP3inflammasome activation and signaling[J].Immunol Cell Biol,2017,95(7):584-592.
[14] Elliott E l,Sutterwala F S.Initiation and perpetuation of NLRP3inflammasome activation and assembly[J].Immunol Rev,2015,265(1):35-52.
[15] Park S C,Juliana S,Hong P,et al.The mitochondrial antiviral protein MAVS associates with NLRP3and regulates its inflammasome activity[J].J Immunol,2013,191:4358-4366.
[16] Stutz A,Horvath G L,Monks B G,et al.ASC speck formation as a readout for inflammasome activation[J].Methods Mol Biol,2013,1040:91-101.
[17] Jiang X,Chen Z J.The role of ubiquitylation in immune defence and pathogen rvasion[J].Nat Rev Immunol,2011,12(1):35-48.