小鼠转录因子STAT1真核表达质粒的构建及生物学功能分析
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摘要
本研究以小鼠组织的总RNA为模板,通过RT-PCR扩增得到小鼠信号转导子和转录激活子1(STAT1)基因完整开放阅读框的碱基序列,然后将其克隆到真核表达载体p DsRed-N1中,构建p DsRed-N1-STAT1重组质粒。测序成功的真核质粒转染至BHK-21细胞,通过α干扰素(IFN-α)分子刺激,检测细胞中STAT1分子的活化状态与定位,最终通过坦布苏病毒刺激,荧光显微镜检测STAT1分子的细胞内定位。结果表明,小鼠的STAT1基因开放阅读框为2 250 bp,编码749个氨基酸。同源性比对结果表明,小鼠STAT1与人、大鼠、猪、马等哺乳动物STAT1氨基酸序列的一致性分别为92%、97%、91%、91%。转染结果表明,构建的p DsRed-N1-STAT1真核表达质粒在BHK-21细胞中成功表达,无IFN-α刺激时,红色荧光只在BHK-21细胞的细胞质中出现,而加入IFN-α后,红色荧光则大多分布于细胞核内。用坦布苏病毒感染细胞后,红色荧光多分布于细胞质中。说明,构建的真核质粒p DsRed-N1-STAT1在哺乳动物细胞中能够正确表达融合蛋白质Red-STAT1,而且在IFN-α刺激下,Red-STAT1可由细胞质向细胞核转运,同时发现,坦布苏病毒感染能够有效抑制STAT1分子的核转运。
In this study,a full-length open reading frame( ORF) of signal transduction and activators of transcription 1( STAT1) gene was cloned from tissues and organs of mouse using RT-PCR. The mouse STAT1 gene was successfully cloned into the eukaryotic expression vector pD sRed-N1 to construct recombinant plasmid pD sRed-N1-STAT1. The successfully sequenced eukaryotic plasmid was transfected into BHK-21 cells,and the activation state and localization of STAT1 in the cells were detected by IFN-α molecule stimulation. The intracellular localization of mouse STAT1 was detected by fluorescence microscopy under the stimulation of duck tembusu virus( TMUV). The open reading frame of the STAT1 gene in mice was 2 250 bp and 749 amino acids were encoded. The amino acid sequence of STAT1 in mouse shared high identity with that in human( 92%),rat( 97%),wild pig( 91%),horse( 91%). Transfection results showed that eukaryotic expression vector pD sRed-N1-STAT1 was successfully expressed in BHK-21 cells. The DsRedSTAT1 fusion protein was predominantly located in the cytoplasmic compartment of the untreated BHK-21 cells. Under the stimulation of IFN-α,the DsRed-tagged STAT1 mainly localized in the nucleus. The constructed eukaryotic plasmid pD-sRed-N1-STAT1 can correctly express the fusion protein Red-STAT1 in mammalian cells,and under the stimulation of IFN-α,Red-STAT1 can be transported from the cytoplasm to the nucleus,and it is found that the infection of TMUV can effectively inhibit nuclear transport of STAT1 molecules.
In this study,a full-length open reading frame( ORF) of signal transduction and activators of transcription 1( STAT1) gene was cloned from tissues and organs of mouse using RT-PCR. The mouse STAT1 gene was successfully cloned into the eukaryotic expression vector pD sRed-N1 to construct recombinant plasmid pD sRed-N1-STAT1. The successfully sequenced eukaryotic plasmid was transfected into BHK-21 cells,and the activation state and localization of STAT1 in the cells were detected by IFN-α molecule stimulation. The intracellular localization of mouse STAT1 was detected by fluorescence microscopy under the stimulation of duck tembusu virus( TMUV). The open reading frame of the STAT1 gene in mice was 2 250 bp and 749 amino acids were encoded. The amino acid sequence of STAT1 in mouse shared high identity with that in human( 92%),rat( 97%),wild pig( 91%),horse( 91%). Transfection results showed that eukaryotic expression vector pD sRed-N1-STAT1 was successfully expressed in BHK-21 cells. The DsRedSTAT1 fusion protein was predominantly located in the cytoplasmic compartment of the untreated BHK-21 cells. Under the stimulation of IFN-α,the DsRed-tagged STAT1 mainly localized in the nucleus. The constructed eukaryotic plasmid pD-sRed-N1-STAT1 can correctly express the fusion protein Red-STAT1 in mammalian cells,and under the stimulation of IFN-α,Red-STAT1 can be transported from the cytoplasm to the nucleus,and it is found that the infection of TMUV can effectively inhibit nuclear transport of STAT1 molecules.
引文
[1] CHEN S,CHENG A,WANG M. Innate sensing of viruses by pat-tern recognition receptors in birds[J]. Veterinary Research,2013,44(1):82.
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[6] HO L J,HUNG L F,WENG C Y,et al. Dengue virus type 2 an-tagonizes IFN-αbut not IFN-γantiviral effect via down-regulatingTyk2-STAT signaling in the human dendritic cell[J]. Journal ofImmunology,2005,174(12):8163-8172.
[7] LIN R J,LIAO C L,LIN E,et al. Blocking of theαinterferon-in-duced Jak-Stat signaling pathway by Japanese encephalitis virus in-fection[J]. Journal of Virology,2004,78:9285-9294.
[8] LEE C K,RAO D T,GERTNER R,et al. Distinct requirementsfor IFNs and STAT1 in NK cell function[J]. Journal of Immunolo-gy,2000,165(7):3571-3577.
[9] QIU X,FU Q,MENG C,et al. Newcastle disease virus V proteintargets phosphorylated STAT1 to block IFN-I signaling.[J]. PLoSONE,2016,11(2):e0148560.
[10] RODRIGUEZ J J,WANG L F,HORVATH C M. Hendra virus Vprotein inhibits interferon signaling by preventing STAT1 andSTAT2 nuclear accumulation[J]. Journal of Virology,2003,77(21):11842-11845.
[11] ROTHLISBERGER A,WIENER D,SCHWEIZER M,et al. Twodomains of the V protein of virulent canine distemper virus selec-tively inhibit STAT1 and STAT2 nuclear import[J]. Journal of Vi-rology,2010,84(13):6328-6343.
[12]张守平. p-STAT1蛋白参与调节A型流感病毒的复制及炎性反应的研究[D].北京:中国农业大学,2015.
[2] GORAYA M U,WANG S,MUNIR M,et al. Induction of innateimmunity and its perturbation by influenza viruses[J]. ProteinCell,2015,6(10):712-721.
[3] KIM H S,LEE M S. STAT1 as a key modulator of cell death[J].Cellular Signalling,2007,19(3):454-465.
[4] PILZ A,RAMSAUER K,HEIDARI H,et al. Phosphorylation ofthe Stat1 transactivating domain is required for the response to typeI interferons[J]. Embo Reports,2003,4(4):368-373.
[5] GUO J,HAYASHI J,SEEGER C. West Nile virus inhibits thesignal transduction pathway of alpha interferon[J]. Journal of Vi-rology,2005,79(3):1343-1350.
[6] HO L J,HUNG L F,WENG C Y,et al. Dengue virus type 2 an-tagonizes IFN-αbut not IFN-γantiviral effect via down-regulatingTyk2-STAT signaling in the human dendritic cell[J]. Journal ofImmunology,2005,174(12):8163-8172.
[7] LIN R J,LIAO C L,LIN E,et al. Blocking of theαinterferon-in-duced Jak-Stat signaling pathway by Japanese encephalitis virus in-fection[J]. Journal of Virology,2004,78:9285-9294.
[8] LEE C K,RAO D T,GERTNER R,et al. Distinct requirementsfor IFNs and STAT1 in NK cell function[J]. Journal of Immunolo-gy,2000,165(7):3571-3577.
[9] QIU X,FU Q,MENG C,et al. Newcastle disease virus V proteintargets phosphorylated STAT1 to block IFN-I signaling.[J]. PLoSONE,2016,11(2):e0148560.
[10] RODRIGUEZ J J,WANG L F,HORVATH C M. Hendra virus Vprotein inhibits interferon signaling by preventing STAT1 andSTAT2 nuclear accumulation[J]. Journal of Virology,2003,77(21):11842-11845.
[11] ROTHLISBERGER A,WIENER D,SCHWEIZER M,et al. Twodomains of the V protein of virulent canine distemper virus selec-tively inhibit STAT1 and STAT2 nuclear import[J]. Journal of Vi-rology,2010,84(13):6328-6343.
[12]张守平. p-STAT1蛋白参与调节A型流感病毒的复制及炎性反应的研究[D].北京:中国农业大学,2015.