Identification Of Transcription Regulatory Elements In Porcine IFIT2 Gene

IFITs family is an important member of the interferon stimulated genes(ISGs) family, most of the IFITs genes are composed of 2 exons. Normally, the IFITs gene was not expressed in cells, but under the stimulation of interferon, viral, microbial pathogen associated molecular patterns or the like, IFITs gene efficiently, rapidly express. Research on IFIT2 is mostly carried out on human and mice, researches on porcine haven’t seen on report yet. In the early study by our group, we cloned porcine IFIT2 gene and preliminary analysis its promoter activity area, find that in-1836 ~-1661 bp and-389 ~-310 bp exists negative regulatory elements, in-1661 ~-1397 bp and-310 ~-126 bp exists positive regulatory elements. In order to determine the transcriptional regulatory elements of porcine IFIT2 genes, we analysis the impact of corresponding sequence deletion to the activity of promoter in cultured PK15 cells in vitro, based on bioinformatics prediction, under the use of eukaryotic expression and dual luciferase reporter system. The main research results are as follows:(1) Bioinformatics analysis revealed that, Delta EF1 exists in-1838 ~-1661 bp. An ISRE(ISREIII) element presents in-1661 ~-1579 bp. Id3 binding site exists in-389 ~-310 bp. Two ISRE elements(ISREI and ISREII) and two NF-kappa B binding sites(NF-kappa BI and NF-kappa BII) exist in-310 ~-126 bp.(2) We did the ΔDelta EF1、ΔISREIII、ΔId3-ttg、ΔId3、ΔISREI、ΔISREII、ΔISREI-II、 ΔNF-Kappa BI 、 ΔNF-Kappa BII 、 ΔNF-Kappa BI-II 、 ΔNF-Kappa BI+ISREI-II 、ΔNF-Kappa BII+ISREI-II、ΔNF-Kappa BI-II+ISREI-II deletion mutants of the different elements in the promoter region of porcine IFIT2 by using the method of overlap extension PCR; based on p GL3-Basic vector backbone we successfully constructed fluorescent reporter plasmids: p GL3-ΔDelta EF1 、 p GL3-ΔISREIII 、 p GL3-ΔId3-ttg 、 p GL3-ΔId3 、 p GL3-ΔISREI 、 p GL3-ΔISREII 、 p GL3-ΔISREI-II 、 p GL3-ΔNF-Kappa BI 、 p GL3-ΔNF-Kappa BII 、p GL3-ΔNF-Kappa BI-II 、 p GL3-ΔNF-Kappa BI+ISREI-II 、 p GL3-ΔNF-Kappa BII+ISREI-II 、p GL3-ΔNF-Kappa BI-II+ISREI-II.(3) Dual-Luciferase reporter system analysis shows, in porcine IFIT2 gene promoter region, Delta EF1 inhibit gene transcription, NF-kappa Bs facilitate gene transcription, ISREs are cis-acting elements which have positive regulation of gene transcription. And exist synergies in-310 ~-126 bp area, between two ISIREs and NF-Kappa Bs, between NF-Kappa B and ISRE.