[1]韩凯凯,赵冬敏,毕可然,等.小鼠转录因子STAT1真核表达质粒的构建及生物学功能分析[J].江苏农业学报,2019,(01):136-141.[doi:doi:10.3969/j.issn.1000-4440.2019.01.020]
　HAN Kai-kai,ZHAO Dong-min,BI Ke-ran,et al.Eukaryotic expression plasmid construction and biological function analysis of mouse transcription factor STAT1[J].,2019,(01):136-141.[doi:doi:10.3969/j.issn.1000-4440.2019.01.020]
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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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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2019年01期
页码:
136-141
栏目:
畜牧兽医·水产养殖
出版日期:
2019-02-26

文章信息/Info

Title:
Eukaryotic expression plasmid construction and biological function analysis of mouse transcription factor STAT1
作者:
韩凯凯; 赵冬敏; 毕可然; 章丽娇; 刘青涛; 刘宇卓; 黄欣梅; 杨婧; 李银
（江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心，江苏南京210014）
Author(s):
HAN Kai-kai; ZHAO Dong-min; BI Ke-ran; ZHANG Li-jiao; LIU Qing-tao; LIU Yu-zhuo; HUANG Xin-mei; YANG Jing; LI Yin
(Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biologicals Engineering and Technology, Ministry of Agriculture/National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China)
关键词:
小鼠; 信号转导子和转录激活子1; α干扰素
Keywords:
mouse; signal transduction and activators of transcription 1(STAT1); IFN-α
分类号:
S852.3
DOI:
doi:10.3969/j.issn.1000-4440.2019.01.020
文献标志码:
A
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pDsRed-N1中，构建pDsRed-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pDsRed-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pDsRed-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。
Abstract:
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 pDsRed-N1 to construct recombinant plasmid pDsRed-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 pDsRed-N1-STAT1 was successfully expressed in BHK-21 cells. The DsRed-STAT1 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 pDsRed-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.

参考文献/References:

[1]CHEN S, CHENG A, WANG M. Innate sensing of viruses by pattern recognition receptors in birds[J]. Veterinary Research, 2013, 44(1): 82.
[2]GORAYA M U, WANG S, MUNIR M, et al. Induction of innate immunity and its perturbation by influenza viruses [J]. Protein Cell, 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 of the Stat1 transactivating domain is required for the response to type I interferons[J]. Embo Reports, 2003, 4(4):368-373.
[5]GUO J, HAYASHI J, SEEGER C. West Nile virus inhibits the signal transduction pathway of alpha interferon [J]. Journal of Virology, 2005, 79(3):1343-1350.
[6]HO L J, HUNG L F, WENG C Y, et al. Dengue virus type 2 antagonizes IFN-α but not IFN-γ antiviral effect via down-regulating Tyk2-STAT signaling in the human dendritic cell[J]. Journal of Immunology, 2005, 174(12):8163-8172.
[7]LIN R J, LIAO C L, LIN E, et al. Blocking of the α interferon-induced Jak-Stat signaling pathway by Japanese encephalitis virus infection [J]. Journal of Virology, 2004, 78: 9285-9294.
[8]LEE C K, RAO D T, GERTNER R, et al. Distinct requirements for IFNs and STAT1 in NK cell function[J]. Journal of Immunology, 2000, 165(7):3571-3577.
[9]QIU X, FU Q, MENG C, et al. Newcastle disease virus V protein targets phosphorylated STAT1 to block IFN-I signaling.[J]. PLoS ONE, 2016, 11(2):e0148560.
[10]RODRIGUEZ J J, WANG L F, HORVATH C M. Hendra virus V protein inhibits interferon signaling by preventing STAT1 and STAT2 nuclear accumulation [J]. Journal of Virology, 2003, 77(21): 11842-11845.
[11]ROTHLISBERGER A, WIENER D, SCHWEIZER M, et al. Two domains of the V protein of virulent canine distemper virus selectively inhibit STAT1 and STAT2 nuclear import[J]. Journal of Virology, 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.

备注/Memo

备注/Memo:
收稿日期：2018-05-08 基金项目：国家自然科学基金项目（31502101）；国家重点研发计划项目（2017YFD0500804） 作者简介：韩凯凯(1983-),男，河南新乡人，博士，副研究员，主要从事家禽病毒分子生物学研究。（E-mail）hankk0917@126.com 通讯作者：李银，（E-mail）muziyin08@163.com
更新日期/Last Update: 2019-02-27