The Study Of Constitution Corelated Relationship Between CBP And STAT4/STAT6 In Asthmatic Th1/Th2 Disfuction

STAT4 and STAT6 are two chief transcription factors in the Th1/Th2 differentiation ,they separat induct Th1 or Th2 type cytokins.CBP is one of the most important co-activation factors in cell nucleus, to promote gene transcription,the cytokines need to bind with CBP rivalry. There are reports show that CBP play a very important role both in STAT4 and STAT6 induced gene transcription. STAT4 maybe competed with STAT6 when binding with CBP in genetic transcription.This study is to construct two eukaryotic expression plasimids of STAT4 and STAT6(eukaryotic expression plasimid of CBP is a gift from prof.Ruan Xiong zhong,London University) .By overexpressing STAT4 or STAT6, to study is there any co-rivalry between STAT4 and STAT6 to influence Th1/Th2 classifacation.Objective To construct two eukaryotic expression plasimids of STAT4 and STAT6, then cotransfection into COS7 cell as well as pcDNA3-CBP at defferent propotion. Analysis the free STAT4 or STAT6 with Western blot.Methods The cDNA of mouse STAT4 and STAT6 gene amplified with RT-PCR from normal mouse spleen tissue were cloned into pMD19-T vector by T/A ligation. After double digested, STAT4/STAT6 cDNA fragment was subcloned into pIRES2-EGFP vector to construct a eukaryotic expression plasimid . After restriction endonucleases analysis and sequencing , then cotransfection into COS7 cell with pcDNA3-CBP.24 hours later,disrupt cell and analysis the free STAT4 or STAT6 with Western blot.Results When the quantity of the DNA of CBP and STAT6 is fixed,the expressing of free STAT6 in cell discruption is to increase gradually while the DNA of STAT4 is increased. When the quantity of the DNA of CBP and STAT4 is fixed,the expressing of free STAT4 in cell discruption is to increase gradually while the DNA of STAT6 is increased.Conclution STAT4 and STAT6 are binding with CBP competely.