Establishment Of A Dual-Glo Luciferase Assay For Detecting The Activity Of TPO

TPO plays an extremely important role in the growth,differentiation and mature process of hematopoietic stem cells and megakaryocyte.It has significant effects in clinical practice for the treatment of pancytopenia caused by chemotherapy or other diseases.Recent years,the synthesis of TPO analogs or TPO mimetic peptide has become research hotspot.Thus,a stable and efficient screening system is needed to investigate the large number of candidate compounds.In this study,we established a stable and specific screening method for TPO mimetic peptide throughthe activation of the JAK/STAT signaling pathway.4 plasmid:DNA,TPO receptor c-mpl,c-fos luciferase,control renilla and pAdvantage,simultaneously transfected to NIH 3T3 cells.After 48 h,add TPO or TPO mimetic peptide,TPO binding to mpl induces the formation of a productive mpl dimer,enabling the associated JAK kinase molecules to activate each other by transphosphorylation.Phosphorylation of STAT become a dimer,inter nucleus and binding to c-fos promoter,start the firefly luciferase expression.The fluorescence intensity of firefly luciferase and Renilla luciferase can be read and calculate the relative fluorescence ratio to show the activity.This experiment optimized the transfection parameters for NIH 3T3 cell by transfecting with GFP and detecting the amount of GFP expression.By exploring the cotransfected plasmids proportions and the positive control concentration,we optimized the dual luciferase assay method,and ultimately,the assay conditions is:the four plasmids cotransfected ratio of:c-mpl:c-fos:Renilla:pAdVAntage = 5:2:0.1:2.The dual luciferase assay system can bu used for the detection of positive control activity,successfully.Morevoer,we detected the TPO mimetic peptide activity using this system,and got a candidate with higher biological activity,Strain No.11#2-3.The Dual-GLO luciferase assay results showed that TPO mimetic peptide expressed by 11#2-3 strains showed a novel significant biological activity(P<0.001,compared with control)in a dose-dependent manner.