Activity Analysis Of Bovine IRF3 And IRF7 Promoters

Interferon regulatory factor 3(IRF3)and interferon regulatory factor 7(IRF7)are members of the IRF transcription factor family and are key molecules in the natural immune response to antiviral infections.They are mainly involved in inflammatory reactions and induce the production of type I interferons by immune responses.In order to study the role of IRF3 and IRF7 in the regulation of expression,the tissue distribution of IRF3 and IRF7 in yaks was examined.IRF3 was found to be the highest in spleen and least in liver,and IRF7 was induced to express.The content in each tissue is extremely low.In this study,IRF3 and IRF7 proteins were cloned and expressed,and high titer polyclonal antibodies were prepared.Provide materials for subsequent experiments.In order to explore the transcriptional regulation mechanism of IRF3 and IRF7,the IRF3 and IRF7 promoters were cloned using the bovine liver genome as a template,and dual-luciferase reporter vectors for the IRF3 and IRF7 promoters were constructed,respectively,using dual-luciferase gene reports.The system detected the activity of the IRF3 and IRF7 promoters and constructed a dual-luciferase reporter vector containing truncated fragments of different lengths in the 5' flanking region of the IRF3 and IRF7 promoters.The results showed that-1511 was upstream of the IRF3 gene.There is a positive regulatory region for transcription factor binding to-1012 bp and a negative regulatory region for transcription factor binding at-12 to +40 bp.There is a positive regulatory region of transcription factor binding at-550 to-311 bp upstream of the IRF7 gene.Bioinformatics software predicts that there is a transcription factor SP1 and STAT1 binding site between-550 and-311 bp of IRF7.In order to explore the effect of the transcription factor SP1 and STAT1 in IRF7 promoter,they were cloned to connect into the eukaryotic expression vector pCMV-HA and co-transfected with the IRF7 promoter to detect double fluorescence.The enzyme activity was found to enhance the activity of the IRF7 promoter after transfection with pCMV-HA-SP1 and pCMV-HA-STAT1.What's more,overexpression SP1 and STAT1,the expression level of IRF7 protein were increased.To further verify the effect of SP1 and STAT1 on the IRF7 promoter,small interfering RNA-mediated blockage of SP1 and STAT1 were constructed in this study,We found that transfecting interfering RNA pSH-SP1 and pSH-STAT1 can reduce the promoter activity of IRF7 and also inhibit the protein expression level of IRF7.This study demonstrate that there is a positive regulatory region for transcription factor binding from-1511 to-1012 bp upstream of the IRF3 gene,a negative regulatory region with binding of a transcription factor from-12 to +40bp,and the transcription factor SP1 and STAT1 were vital to regulate the IRF7 promoter.The above results lay a foundation for the in-depth study of IRF3 and IRF7 in the regulation of natural immune transcription.