title: Expression profile of human immune-responsive gene 1 and generation and characterization of polyclonal antiserum.
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.
authors.affiliation: Department of Immunology, Wuhan University School of Medicine, Wuhan University, Dong Hu Road 115, Wuchang, 430071 Wuhan, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xiao
authors.forename: Lan
authors.initials: L
authors.lastname: Wang
authors.forename: Ruijing
authors.initials: R
authors.lastname: Xiao
authors.forename: Mengjun
authors.initials: M
authors.lastname: Wu
authors.forename: Jinquan
authors.initials: J
authors.lastname: Tan
authors.forename: Yuling
authors.initials: Y
authors.lastname: He
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002262:Carboxy-Lyases; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007106:Immune Sera / Q000276:immunology*; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D010802:Phylogeny; D011506:Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D007106:Immune Sera; D008070:Lipopolysaccharides; D011506:Proteins; D011994:Recombinant Proteins; C561544:ACOD1 protein, human; D002262:Carboxy-Lyases