Construction and transcriptional activity of human CREG promoter reporter gene plasmid / 解放军医学杂志

ABSTRACT

Objective To construct the promoter enhanced green fluorescent protein (pEGFP1) reporter gene vector of different truncated fragments of human cellular repressor of E1A-stimulated genes (hCREG), and compare the transcriptional activity of each promoter to determine the hCREG core promoter region. Methods The promoter fragment with length of 2003 (–1925/+78) bp was obtained by querying the hCREG sequence from US National Center of Biotechnology Information (NCBI) database and combining with the characteristics of the promoter. Five promoter fragments were truncated by PCR and double enzyme digestion and cloned into pEGFP1 to construct pEGFP1_hCREG_2003, pEGFP1_hCREG_945, pEGF P1_hCREG_586, pEGFP1_ hCREG_478 and pEG FP1_hCREG_358 reporter gene vector plasmid. The 293T cells were transiently co-transfected with the internal reference plasmid pGL4.73 [hRluc/SV40] for 48 hours. The green fluorescence expression of pEGFP1_hCREG promoter reporter gene was observed under fluorescence microscope, and the mRNA expression of each promoter was detected by real-time quantitative PCR, and the core promoter region was determined. Bioinformatics was used to predict the transcription factors that might bind to the core promoter region. Results Five hCREG promoter reporter gene vectors were successfully constructed by double enzyme digestion and gene sequencing. The results showed that the transcription activity of pEGFP1_ hCREG_586 was the highest (P0.05), implying that –867/ –509 bp is a negative regulatory region, and there existed enhancer sequences in –400/–281 bp and –508/–401 bp, so the core promoter region of hCREG gene is located in the upstream sequence of –508/–281 bp. Bioinformatics predicted that the possibly bound transcription factors in key promoter region –508/–281 bp were Pax5/P53, C/EBPβ, GR-β, GATA-1, GR-α, c-Jun, PRB/ PRA, YY1, RXR-α, AP-2, FOXP3, GR, TFIID, STAT4 and c-Ets-1. Conclusion The recombinant plasmid of hCREG gene promoter has been successfully constructed, the core promoter of which is located in –508/–281 bp, where several transcription factors might be bound.