Cloning of ID4 gene promoter and construction of its expression regulatory vector

Objective To study the expression regulatory mechanism of ID4 gene.Methods The upstream 5' lateral wing region of 2 242bp from transcriptional start site(TSS) and downstream 5' non-coding region of 212bp on ID4 gene were searched out and downloaded from human genome databank of NCBI using the ID4 gene whole length cDNA as a probe.According to the targeted sequence,PCR amplification primers were designed and synthesized.Segmental amplification method was used to obtain two fragments of 1 829bp and 784bp in peripheral blood DNA samples from a healthy donor.The two fragments were inserted into pGL3 basic vector and ligation was successfully completed.After identification by sequencing,the ID4 promoter-pGL3bbasic recombinant was used as a template to subclone 5 fragments with identical 3' terminus and different 5' terminus.Then,the ID4 promoter subclone fragments were inserted into the pGL3 basic vector.Results The 6 identified ID4 promoter sequences with an interval of approximate 400bp were successfully cloned and 6 sub-ID4 promoter-pGL3 basic recombinants were constructed.Conclusion The human ID4 promoter we cloned and its expression regulatory vector we constructed lay a foundation for study on the promoter activity and expression regulation of ID4 gene.