Detect The Interaction Of STAT4 And STAT6 With CBP And The Key Domain Contributed For Their Interaction

PART1 Construction and identification of the yeast two-hybrid system for detecting the interaction of STAT4 and STAT6 with CBPObjective:To construct the yeast expression plasmids of STAT4 and STAT6,the bait expression plasmids of CBP,detect the toxity,leakage and autoactivation of bait plasmid,which lay the foundations for detecting the interaction of STAT4 and STAT6 with CBP through the yeast two-hybrid system.Methods:The fragments of mouse STAT4,STAT6 and CBP were amplified by PCR, and then were cloned into vector AD and vector BD,respectively.Each plasmid was transformed into AH109 yeast cells,and the protein expression of STAT4,STAT6 and CBP was analyzed by western blot.Toxity,leakage and autoactivation of bait protein were detected simultaneously.Results:Mouse STAT4,STAT6 and CBP were amplified and cloned into vector AD and vector BD successfully,sequencing results met the requirements.The expression of above proteins in AH109 yeast cells were confirmed by western blot.No toxity and leakage but autoactivation was found with bait protein CBP.Conclusion:The yeast two-hybrid system for detecting the interaction of STAT4 and STAT6 with CBP was constructed successfully,but autoactivati- on of bait protein CBP was detected.PART2 Detect the domain which plays key role in autoactivation of bait protein CBPObjective:To clone all major domains of CBP into bait expression vector pGBKT7,and then toxity,leakage and autoactivation of each bait protein were detected to identify the domain which plays key role in autoactivation of CBP.Methods:CBP was divided into CBP1-697,CBP967-1574 and CBP1678-2175 three parts according to its principal domain,all fragements were amplified by PCR and then cloned into bait expression vector pGBKT7.Bait expression plasmids pGBKT7-CBP1-697,pGBKT7-CBP967-1574 and pGBKT7-CBP1678-2175 were transformed into AH109 yeast cells respectively after digestion and sequencing.Cell protein was extracted and the expression of bait protein was tested by western blot,at the same time,toxity,leakage and autoactivation of each bait protein were detected. Autoactivated CBP1-697 was further divided into CBP1-436(contain TAZ1 domain) and CBP529-1200(contain KIX domain) two different parts according to its two domain,PCR,plasmids construction,transformation,protein expression were carried out as before,as well as the detection of toxity, leakage and autoactivation.Results:All major domain of CBP protein were divided into several parts and then cloned into bait expression vector,respectively.Each bait protein can be expressed correctly in AH109 yeast cells.It was found that the TAZ1 domain of CBP protein is autoactivated.Conclusion:The TAZ1 domain was identified to play key role in autoactivation of CBP.Bait plasmids which can be used in yeast two-hybrid system to detect interaction of STAT4 and STAT6 with CBP were constructed successfully.PART3 Detect interaction of STAT4 and STAT6 with different major domain of CBP through yeast two-hybrid systemObjective:To detect interaction of STAT4 and STAT6 with different major domain of CBP (except TAZ1 domain) through yeast two-hybrid system.Methods:AH109 yeast cells were cotransformed with pGADT7-STAT4/6 and pGBKT7-CBP529-1200/CBP967-1574/CBP1678-2175 and then plated onto corresponding nutritional deficiency plates.Positive clones were screened through detecting the expression of reporter genes.Results:Expression of reporter genes can not be detected when pGADT7-STAT4 and pGBKT7-CBP529-1200/CBP967-1574 cotransformed into AH109 yeast cells,but can be detected when pGADT7-STAT4 cotransformed with pGBKT7-CBP1678-2175. Expression of reporter genes can not be detected when pGADT7-STAT6 cotransformed with anyone of the three.Conclusion:Direct interaction of STAT4 and CBP1678-2175 was detected by yeast two- hybrid system,no interaction was detected between STAT6 and different domain of CBP(except TAZ1 domain).PART4 Identify the domain of STAT4 which plays key role in the interaction between STAT4 and CBPObjective:To identify the domain of STAT4 which plays key role in the interaction between STAT4 and CBP.Methods:N-terminus deleted STAT4 was amplified by PCR and cloned into pGADT7, and then pGADT7-STAT4-N123 was transformed into AH109 yeast cells after digestion and sequencing.Yeast cell protein was extracted and the expression of fusion protein was tested by western blot.Interaction between STAT4-N123 and CBP1678-2175 was detected by yeast two-hybrid system.Results:STAT4 N-terminus domain deleted yeast expression plasmid pGADT7- STAT4-N123 was constructed successfully,but no reporter genes expression was detected after pGADT7-STAT4-N123 and pGBKT7-CBP1678-2175 cotransformed into AH109 yeast cells.Conclusion:No interaction was detected between STAT4-N123 and CBP1678-2175 by yeast two-hybrid system.PART5 Verify the positive results of yeast two-hybrid system by coimmunoprecipitationObjective:To verify the interaction of STAT4 and CBP1678-2175,and detect the interaction of STAT4 and STAT6 with CBP1-436 by coimmunoprecipitation. Methods:CBP1678-2175 and CBP1-436 were fusioned with C-Myc tag and cloned into eukaryotic expression vector pIRES2-EGFP,the expression of fusion protein in COS7 cell was analysed by western blot after digestion and sequenceing.The protein of COS7 cells which were cotransfected with pGADT7-STAT4 and pIRES2-EGFP- CBP1678-2175 was extracted,cell lysate was precipitated by HA tag antibody and detected by C-Myc tag antibody.Detect the interaction of STAT4 and STAT6 with CBP1-436 in the same way.Results:C-Myc tag was fused onto CBP1678-2175 and CBP1-436,then both were cloned into pIRES2-EGFP.Fusion proteins can be expressed correctly in COS7 cells. CBP1678-2175 and STAT4 can be precipitated by HA tag antibody at the same time,but CBP1-436 could not be precipitated either with STAT4 or with STAT6,simultaneously.Conclusion:Interaction of CBP1678-2175 and STAT4 was detected by coimmunoprecipitation,but no interaction of CBP1-436 with STAT4 or STAT6 was detected.