Construction and Functional Analysis of Luciferase Reporter Plasmid Containing CD25 Gene Promoter

Abstract

CD25 is the alpha chain of IL-2R and IL-2R is distributed on the surface of activated T cells, B cells, and NK cell. The transcriptional factor CD25 plays an important role in the proliferation and differentiation of Treg cells. MKL-1 is also called MAL (megakaryocytic acute leukemia) and belongs to the Myocardin family which also includes MKL-2 (megakaryoblastic leukemia 2) and Myocardin. Studies have shown that in the process of mouse megakaryocyte differentiation, the expression of MKL-1 is up-regulated, and MKL-1 promotes the physiological maturation of human and mouse megakaryocytes. However, the exact mechanism of the CD25 on megakaryocytes is unclear. In this study, human CD25 promoter luciferase reporter construct was generated by PCR amplification. Then the PCR fragment was digested and cloned into pGL3 vector, and this promoter sequence was verified by sequencing. The result showed that luciferase reporter with CD25 promoter was successfully constructed. The expression vectors of human MKL-1 were bought from addgene. Then the activation of the CD25 promoter was detected in 293T cells by luciferase reporter assay after transfected expression vectors of human MKL-1. The result showed that transfected the expression vectors can enhance the transcriptional activity of CD25. Our research will reveal the effect of CD25 on megakaryocytes and may provide a theoretical basis and therapeutic method for diseases caused by megakaryocytes.