Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFNβ) gene regulation. Besides that IRF-2 is involved in immunomodulation,...Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFNβ) gene regulation. Besides that IRF-2 is involved in immunomodulation, hematopoietic differentiation, cell cycle regulation and oncogenesis. We have done molecular sub-cloning and expression of recombinant murine IRF-2 as GST (Glutathione-S-Transferase)- IRF-2 fusion protein in E. coli/XL-1blue cells. Recombinant IRF-2 with GST moiety at N-terminus expressed as GST-IRF-2 (~66 kd) in E. coli along with different low molecular mass degradation products revealed approximately 30, 42, 60 and 62 kd by SDS-PAGE and Western blot, respectively. We further confirm that degradation takes place at C-terminus of the fusion protein not at N-terminus as anti-GST antibody was detecting all bands in the immunoblot. The recombinant IRF-2 was biologically active along with their degradation products in terms of their DNA binding activity as assessed by Electrophoretically Mobility Shift Assay (EMSA). We observed three different molecular mass DNA/protein complexes (1 - 3) with Virus Response Element (VRE) derived from human Interferon IFNβ gene and five different molecular mass complexes (1 - 5) with IRF-E motif (GAAAGT)4 in EMSA gel. GST only expressed from empty vector did not bind to these DNA elements. To confirm that the binding is specific, all complexes were competed out completely when challenged with 100-X fold molar excess of IRF-E oligo under cold competition. It means degradation products along with full-length protein are able to interact with VREβ as well as IRF-E motif. This means degradation products may regulate the target gene (s) activation/repression via interacting with VRE/IRF-E.展开更多
Objective:To investigate the expression levels of interferon regulatory factor 3(IRF-3)and miRNA146a in peripheral blood of children with infectious mononucleosis and the effect of miRNA146a on IRF-3 expression.Method...Objective:To investigate the expression levels of interferon regulatory factor 3(IRF-3)and miRNA146a in peripheral blood of children with infectious mononucleosis and the effect of miRNA146a on IRF-3 expression.Methods:In an in vivo experiment,the expression levels of IRF-3 and miRNA146a in peripheral blood of 45 children with infectious mononucleosis and 34 healthy controls were detected by real-time PCR.The in vitro experiment was performed to examine the effect of miRNA146a on IRF-3 expression levels by transfecting miRNA146a mimics and their inhibitors.Results:There was significant difference in IRF-3 gene expression in peripheral blood between healthy controls and children with infectious mononucleosis(t=30.340,P<0.001)while miRNA146a expression was significantly increased compared with healthy controls(t=34.659,P<0.001),and there was a negative correlation between the two groups(r=-0.960,P<0.05).In HeLa cells,transfection with miRNA146a mimics significantly decreased the expression of IRF-3 mRNA(t=8.270,P<0.001)and protein(t=46.170,P<0.001),while miRNA146a inhibitor significantly up-regulated the expression of IRF-3 mRNA(t=8.582,P<0.001)and protein(t=25.891,P<0.001).Conclusion:miRNA146a may be involved in the down-regulation of IRF-3 gene expression by Epstein-Barr virus in children with infectious mononucleosis and then involved in the pathogenesis of infectious mononucleosis.展开更多
文摘Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFNβ) gene regulation. Besides that IRF-2 is involved in immunomodulation, hematopoietic differentiation, cell cycle regulation and oncogenesis. We have done molecular sub-cloning and expression of recombinant murine IRF-2 as GST (Glutathione-S-Transferase)- IRF-2 fusion protein in E. coli/XL-1blue cells. Recombinant IRF-2 with GST moiety at N-terminus expressed as GST-IRF-2 (~66 kd) in E. coli along with different low molecular mass degradation products revealed approximately 30, 42, 60 and 62 kd by SDS-PAGE and Western blot, respectively. We further confirm that degradation takes place at C-terminus of the fusion protein not at N-terminus as anti-GST antibody was detecting all bands in the immunoblot. The recombinant IRF-2 was biologically active along with their degradation products in terms of their DNA binding activity as assessed by Electrophoretically Mobility Shift Assay (EMSA). We observed three different molecular mass DNA/protein complexes (1 - 3) with Virus Response Element (VRE) derived from human Interferon IFNβ gene and five different molecular mass complexes (1 - 5) with IRF-E motif (GAAAGT)4 in EMSA gel. GST only expressed from empty vector did not bind to these DNA elements. To confirm that the binding is specific, all complexes were competed out completely when challenged with 100-X fold molar excess of IRF-E oligo under cold competition. It means degradation products along with full-length protein are able to interact with VREβ as well as IRF-E motif. This means degradation products may regulate the target gene (s) activation/repression via interacting with VRE/IRF-E.
基金Youth Medical Talents Project of Science,Education and Health of Jiangsu Province(No.QNRC2016615)Maternal and Child Health Key Talents Project of Jiangsu Province(No.FRC201736)。
文摘Objective:To investigate the expression levels of interferon regulatory factor 3(IRF-3)and miRNA146a in peripheral blood of children with infectious mononucleosis and the effect of miRNA146a on IRF-3 expression.Methods:In an in vivo experiment,the expression levels of IRF-3 and miRNA146a in peripheral blood of 45 children with infectious mononucleosis and 34 healthy controls were detected by real-time PCR.The in vitro experiment was performed to examine the effect of miRNA146a on IRF-3 expression levels by transfecting miRNA146a mimics and their inhibitors.Results:There was significant difference in IRF-3 gene expression in peripheral blood between healthy controls and children with infectious mononucleosis(t=30.340,P<0.001)while miRNA146a expression was significantly increased compared with healthy controls(t=34.659,P<0.001),and there was a negative correlation between the two groups(r=-0.960,P<0.05).In HeLa cells,transfection with miRNA146a mimics significantly decreased the expression of IRF-3 mRNA(t=8.270,P<0.001)and protein(t=46.170,P<0.001),while miRNA146a inhibitor significantly up-regulated the expression of IRF-3 mRNA(t=8.582,P<0.001)and protein(t=25.891,P<0.001).Conclusion:miRNA146a may be involved in the down-regulation of IRF-3 gene expression by Epstein-Barr virus in children with infectious mononucleosis and then involved in the pathogenesis of infectious mononucleosis.
