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Expression profile of human immune-responsive gene 1 and generation and characterization of polyclonal antiserum

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Abstract

Murine immune-responsive gene 1 (IRG1) plays significant roles in embryonic implantation and neurodegeneration. The expression pattern of the human IRG1 gene, however, has not yet been established, and the predicted gene sequence has been revised several times according to computed expressed sequence tags (ESTs). To determine the human IRG1 gene expression profile, human fetal tissue samples, peripheral blood mononuclear cells (PBMCs) from normal healthy subjects, and the human leukemia cell lines THP-1 and K-562 challenged with lipopolysaccharide (LPS) were subjected to RT-PCR using degenerate primers. The results indicated that the IRG1 gene is differentially expressed in human fetal PBMCs and LPS-stimulated adult PBMCs. The amplified gene fragment was cloned into the pET32a(+) vector and fusion-expressed with a His-tag in a prokaryotic system. After affinity chromatography, human IRG1h fusion proteins were isolated by SDS-PAGE and identified by mass spectrometric analysis for use as an immunogen to immunize rabbits. The titer and specificity of the purified rabbit antiserum were sufficient to measure human IRG1 gene expression in various tissues and cultures. This purified polyclonal antiserum will allow us to initiate studies to elucidate the biological roles of the human IRG1 gene.

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Acknowledgments

This study was supported by the grants from the National Natural Science Foundation of China (30730054, 30572119, 30670937, 30971279, 30901363), the Hi-tech Research and Development Program of China from Ministry of Science and Technology (2007AA02Z120), the Ministry of Education (20060486008, 20090141120011), National Innovation Experiment Program for College Students (WU2007061), Provincial Department of Science and Technology of Hubei (2007ABC010), China, and Chang Jiang Scholars Program from Ministry of Education, China and Li Ka Shing Foundation, Hong Kong, China (Chang Jiang Scholar T.J.).

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Authors and Affiliations

  1. Department of Immunology, Wuhan University School of Medicine, Wuhan University, Dong Hu Road 115, Wuchang, 430071, Wuhan, China

    Wei Xiao, Lan Wang, Mengjun Wu, Jinquan Tan & Yuling He

  2. Laboratory of Allergy and Clinical Immunology, Institute of Allergy and Immune-Related Diseases, Centre for Medical Research, Wuhan University School of Medicine, Wuhan, People’s Republic of China

    Ruijing Xiao, Jinquan Tan & Yuling He

Authors
  1. Wei Xiao
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  2. Lan Wang
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  3. Ruijing Xiao
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  4. Mengjun Wu
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  5. Jinquan Tan
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  6. Yuling He
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Corresponding authors

Correspondence to Jinquan Tan or Yuling He.

Additional information

Wei Xiao, Lan Wang, and Ruijing Xiao are contributed equally to this work.

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Xiao, W., Wang, L., Xiao, R. et al. Expression profile of human immune-responsive gene 1 and generation and characterization of polyclonal antiserum. Mol Cell Biochem 353, 177–187 (2011). https://doi.org/10.1007/s11010-011-0784-7

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  • DOI: https://doi.org/10.1007/s11010-011-0784-7

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