Construction of Iuciferase reporter vectors harboring DC-SIGN promoters without AP-1 or ETS-1 transcription factor binding site and detection of their activity / 中华微生物学和免疫学杂志

ABSTRACT

Objective To detect the role of AP-1 or ETS-1 transcription factor binding sites (TFBS) in activity of DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) pro-moter. Methods Genomic DNA was extracted from peripheral blood. Complete DC-SIGN promoter and those without AP-1 or ETS-1 TFBS were amplified by PCR using designed primers. The PCR products were digested with MLu Ⅰ and Bgl Ⅱ and then ligated into MLu Ⅰ and Bgl Ⅱ digested pGL-3/Basic and pGL-3/En-hancer vectors. Transfection in Hacat and 293 cells was accomplished with Trans Fast liposome. Activity of luciferase was detected after 48 h. Results The DC-SIGN promoters without AP-1 or ETS-1 TFBS and the recombined pGL-3 vectors were correctly constructed. The activity of DC-SIGN promoters without AP-1 was reduced 20% (293) and 10% (Hacat), which was 40%-50% with enhancer. The activity of DC-SIGN pro-moters without ETS-1 was nearly vanished, no matter with or without enhancer. Conclusion ETS-1 TFBS, not AP-1 TFBS, plays an important role in activity of DC-SIGN promoter.