中文｜English
ISSN 1001-5256 (Print)
ISSN 2097-3497 (Online)
CN 22-1108/R
Volume 40 Issue 2
Feb.  2024
Turn off MathJax
Article Contents
Article Navigation >  Journal of Clinical Hepatology  > 2024  >  40(2): 278-283

Preliminary identification of the cloning， expression， and function of Marmota himalayana type I interferon receptor β subunit

DOI: 10.12449/JCH240210
  • 1.

    Department of Infectious Diseases，General Hospital of The Central Theater Command，Wuhan 430030，China

  • 2.

    Department of Infectious Diseases，Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology，Wuhan 430030，China

Research funding:

National Natural Science Foundation of China (3021170);

National Major Basic Research Projects （973） (2005CB522900)

More Information
  • Corresponding author: FAN Hebin， 296592559@qq.com （ORCID： 0000-0002-9602-7755）
  • Received Date: 2023-04-11
  • Accepted Date: 2023-07-04
  • Published Date: 2024-02-19
    Abstract
  •   Objective  To clone the gene of Marmota himalayana type ‍Ⅰ interferon receptor β subunit （mhIFNAR2）， and to perform antibody preparation and functional identification.  Methods  RT-PCR was used for amplification in the spleen tissue of Marmota himalayana to obtain the sequence， which was cloned to the prokaryotic expression vector pRSET-B to express the recombinant protein. Electrophoresis and Western blot were used for identification. BALB/c mice were immunized with the recombinant protein to prepare the polyclonal antibody of its extracellular domain； immunohistochemistry， immunofluorescence assay， and Western Blot were used for identification， and the method of siRNA blockade was used to investigate its function. An analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for comparison between two groups.  Results  A fragment of mhIFNAR2 （149‍ ‍—‍ ‍1 ‍300 bp） was obtained from spleen tissue， which showed the highest homology of 98.05% in marmot. A prokaryotic expression plasmid was successfully constructed for expression of the extracellular domain of the mhIFNAR2（50-181aa） and was named pRSET-B.mhIFNAR2， and the recombinant protein expressed by this plasmid had a molecular weight of 27 kD， a purity of about 95% after purification， and a concentration of 160 μg/mL. After BALB/c mice were immunized with the purified recombinant protein， 1∶1 000 specific polyclonal antibodies were obtained， and immunohistochemistry and immunofluorescence assay showed the expression in cell membrane and cytoplasm. Among the three siRNAs synthesized， the siRNA starting from the 277 locus （siRNA277） could silence the expression of target genes and weaken the interferon signaling pathway compared with the blank control group and the negative control group （both P<0.05）.  Conclusion  The fragment of mhIFNAR2 is obtained， and the polyclonal antibody for the extracellular domain of mhIFNAR2 is successfully prepared， with relatively high titer and specificity， and can be used for immunohistochemistry， immunofluorescence assay， and Western blot.

     

    • FullText(HTML)
  • loading
    • References(22)
  • [1]
    ERSVAER E, SKAVLAND J, ULVESTAD E, et al. Effects of interferon gamma on native human acute myelogenous leukaemia cells[J]. Cancer Immunol Immunother, 2007, 56( 1): 13- 24. DOI: 10.1007/s00262-006-0159-1.
    [2]
    MERLI P, QUINTARELLI C, STROCCHIO L, et al. The role of interferon-gamma and its signaling pathway in pediatric hematological disorders[J]. Pediatr Blood Cancer, 2021, 68( 4): e28900. DOI: 10.1002/pbc.28900.
    [3]
    SCHRODER K, HERTZOG PJ, RAVASI T, et al. Interferon-gamma: An overview of signals, mechanisms and functions[J]. J Leukoc Biol, 2004, 75( 2): 163- 189. DOI: 10.1189/jlb.0603252.
    [4]
    BHAT MY, SOLANKI HS, ADVANI J, et al. Comprehensive network map of interferon gamma signaling[J]. J Cell Commun Signal, 2018, 12( 4): 745- 751. DOI: 10.1007/s12079-018-0486-y.
    [5]
    WANG BJ, TIAN YJ, MENG ZJ, et al. Establishing a new animal model for hepadnaviral infection: Susceptibility of Chinese Marmota-species to woodchuck hepatitis virus infection[J]. J Gen Virol, 2011, 92( Pt 3): 681- 691. DOI: 10.1099/vir.0.025023-0.
    [6]
    GUO WN, ZHU B, AI L, et al. Animal models for the study of hepatitis B virus infection[J]. Zool Res, 2018, 39( 1): 25- 31. DOI: 10.24272/j.issn.2095-8137.2018.013.
    [7]
    FAN HB, ZHU ZN, WANG Y, et al. Molecular characterization of the type I IFN receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus(WHV)[J]. Cytokine, 2012, 60( 1): 179- 185. DOI: 10.1016/j.cyto.2012.05.013.2012.
    [8]
    SCHREIBER G. The role of type I interferons in the pathogenesis and treatment of COVID-19[J]. Front Immunol, 2020, 11: 595739. DOI: 10.3389/fimmu.2020.595739.
    [9]
    MCNAB F, MAYER-BARBER K, SHER A, et al. Type I interferons in infectious disease[J]. Nat Rev Immunol, 2015, 15( 2): 87- 103. DOI: 10.1038/nri3787.
    [10]
    MURIRA A, LAMARRE A. Type-I interferon responses: From friend to foe in the battle against chronic viral infection[J]. Front Immunol, 2016, 7: 609. DOI: 10.3389/fimmu.2016.00609.
    [11]
    BASTARD P, MANRY J, CHEN J, et al. Herpes simplex encephalitis in a patient with a distinctive form of inherited IFNAR1 deficiency[J]. J Clin Invest, 2021, 131( 1): e139980. DOI: 10.1172/JCI139980.
    [12]
    KHANMOHAMMADI S, REZAEI N, KHAZAEI M, et al. A case of autosomal recessive interferon alpha/beta receptor alpha chain(IFNAR1) deficiency with severe COVID-19[J]. J Clin Immunol, 2022, 42( 1): 19- 24. DOI: 10.1007/s10875-021-01166-5.
    [13]
    PLATANIAS LC. Mechanisms of type-‍I‍- and type-‍II-interferon-mediated signalling[J]. Nat Rev Immunol, 2005, 5( 5): 375- 386. DOI: 10.1038/nri1604.
    [14]
    TOUGH DF, SUN S, ZHANG X, et al. Stimulation of naïve and memory T cells by cytokines[J]. Immunol Rev, 1999, 170: 39- 47. DOI: 10.1111/j.1600-065x.1999.tb01327.x.
    [15]
    World Health Organization. Global Hepatitis Report 2017[EB/OL].( 2017-04-19)[ 2023-04-11]. https://www.who.int/hepatitis/publications/global-hepatitis-report2017/en/. https://www.who.int/hepatitis/publications/global-hepatitis-report2017 /en/
    [16]
    LU YP, XU Y, YANG DL, et al. Molecular characterization of woodchuck type I interferons and their expression by woodchuck peripheral blood lymphocytes[J]. Cytokine, 2008, 41( 2): 127- 135. DOI: 10.1016/j.cyto.2007.11.002.
    [17]
    WANG BJ, LOHRENGEL B, LU YP, et al. Molecular characterization of woodchuck interleukin 15(wIL-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus(WHV)[J]. Cytokine, 2005, 32( 6): 296- 303. DOI: 10.1016/j.cyto.2005.11.007.
    [18]
    YANG Y, HUANG H, ZHANG ZH, et al. Cloning, expression and polyclonal antibody preparation of the asialoglycoprotein receptor of Marmota Himalayan[J]. J Huazhong Univ Sci Technol(Med Sci), 2007, 27( 4): 411- 414. DOI: 10.1007/s11596-007-0415-4.
    [19]
    SHEEHAN KC, LAI KS, DUNN GP, et al. Blocking monoclonal antibodies specific for mouse IFN-alpha/beta receptor subunit 1(IFNAR-1) from mice immunized by in vivo hydrodynamic transfection[J]. J Interferon Cytokine Res, 2006, 26( 11): 804- 819. DOI: 10.1089/jir.2006.26.804.
    [20]
    BENOIT P, MAGUIRE D, PLAVEC I, et al. A monoclonal antibody to recombinant human IFN-alpha receptor inhibits biologic activity of several species of human IFN-alpha, IFN-beta, and IFN-omega. Detection of heterogeneity of the cellular type I IFN receptor[J]. J Immunol, 1993, 150( 3): 707- 716.
    [21]
    COLAMONICI OR, D’ALESSANDRO F, DIAZ MO, et al. Characterization of three monoclonal antibodies that recognize the interferon alpha 2 receptor[J]. Proc Natl Acad Sci U S A, 1990, 87( 18): 7230- 7234. DOI: 10.1073/pnas.87.18.7230.
    [22]
    UZÉ G, LUTFALLA G, EID P, et al. Murine tumor cells expressing the gene for the human interferon alpha beta receptor elicit antibodies in syngeneic mice to the active form of the receptor[J]. Eur J Immunol, 1991, 21( 2): 447- 451. DOI: 10.1002/eji.1830210229.
    • Relative Articles
    • Supplements(0)
    • Cited By
  • 加载中

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Figures(7)  / Tables(4)

    Get Citation
    PDF
    XML

    Article Metrics

    Article views (249) PDF downloads(30) Cited by()
    Proportional views
    Related
        

    The first journal specializing in hepatobiliary and pancreatic diseases in China

    Supervisor：Ministry of Education of the People's Republic of China

    Sponsor：Jilin University

    Academic Support: Chinese Society of Hepatology，Chinese Medical Association

    Address: 461 Xinjiang Road, Changchun

    Submit：0431-88782044

    Peer review：0431-88783542

    Email：lcgdb@vip.163.com

    About Journal

    Submission Guideline

    Journal Online

    Hepatobiliary College

    Guidelines

    YesterdayIP[]YesterdayPV[]TodayIP[]TodayPV[]Online[]

    Website Design © 2020 Editorial Board of Journal of Clinical Hepatology 吉ICP备10000617号-1 Supported by: Beijing Renhe Information Technology Co. Ltd  

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return