【作者】 张明香；

【导师】 符州；

【作者基本信息】 重庆医科大学 ， 儿科学， 2010， 博士

【摘要】 第一部分STAT4、STAT6与CBP相互作用酵母双杂交系统的建立与评价目的:构建STAT4、STAT6的酵母表达质粒和CBP的诱饵表达质粒,并检测诱饵蛋白的毒性、渗漏和自激活作用,为酵母双杂交检测STAT4、STAT6与CBP的相互作用做准备。方法:PCR扩增小鼠的STAT4、STAT6和CBP基因,分别克隆入酵母双杂交系统的AD和BD载体,测序正确后,将上述质粒分别转化酵母AH109细胞,提取细胞蛋白,western blot检测蛋白表达情况,同时检测诱饵蛋白CBP的毒性、渗漏和自激活作用。结果:成功扩增了小鼠的STAT4、STAT6和CBP基因,并分别正确克隆入AD和BD载体,测序结果符合要求,western blot证实上述蛋白均能在AH109中正确表达,诱饵蛋白CBP对酵母AH109细胞无毒性和渗漏作用,但检测到自激活作用。结论:成功构建了用于检测STAT4、STAT6与CBP相互作用的酵母双杂交系统,但检测到诱饵蛋白CBP有自激活作用。第二部分诱饵蛋白CBP自激活结构域的检测目的:将CBP蛋白的各主要结构域分别构建到诱饵表达载体pGBKT7上,分别检测其毒性、渗漏和自激活作用来明确CBP蛋白分子中起自激活作用的关键结构域。方法:将CBP蛋白按主要结构域分成CBP1-697,CBP967-1574和CBP1678-2175三个部分进行PCR扩增并克隆入诱饵表达载体pGBKT7,酶切和测序鉴定后将诱饵质粒pGBKT7-CBP1-697、pGBKT7-CBP967-1574和pGBKT7-CBP1678-2175分别转化酵母AH109细胞,提取细胞蛋白,western blot检测诱饵蛋白表达情况,同时行毒性、渗漏和自激活作用检测。将有自激活作用的CBP1-697所包含的TAZ1和KIX结构域进一步分为CBP1-436(包含TAZ1)和CBP529-1200(包含KIX)两部分,同前进行PCR扩增、质粒构建、转化、蛋白表达及毒性、渗漏和自激活作用检测。结果:成功的将CBP蛋白分子的各主要结构域分成几个部分克隆入诱饵表达载体,使其在酵母AH109细胞中正确表达,并发现CBP蛋白分子的TAZ1结构域有自激活作用。结论:成功构建了可用于酵母双杂交的CBP诱饵表达质粒,明确了TAZ1结构域是CBP起自激活作用的关键结构域。第三部分酵母双杂交检测STAT4、STAT6与CBP各主要结构域间的相互作用目的:酵母双杂交检测STAT4、STAT6与CBP各主要结构域(TAZ1结构域除外)间的相互作用。方法:将酵母表达质粒pGADT7-STAT4/6分别与pGBKT7-CBP529-1200、pGBKT7-CBP967-1574和pGBKT7-CBP1678-2175共转化酵母AH109细胞,铺相应营养缺陷的培养板,通过报告基因表达检测筛选阳性克隆。结果:pGADT7-STAT4分别与pGBKT7-CBP529-1200、pGBKT7- CBP967-1574共转化酵母AH109细胞时不能激活报告基因表达,与pGBKT7-CBP1678-2175共转化酵母AH109细胞后可激活报告基因表达,pGADT7-STAT6与上述任何诱饵质粒共转化酵母AH109细胞均不能激活报告基因表达。结论:酵母双杂交检测到STAT4与CBP1678-2175存在直接的相互作用,没有检测到STAT6与CBP各结构域(TAZ1结构域除外)间的相互作用。第四部分STAT4与CBP相互作用关键结构域的检测目的:检测STAT4蛋白分子与CBP相互作用中起关键作用的结构域。方法:PCR扩增N-末端结构域缺失的STAT4,并克隆入酵母表达载体pGADT7,酶切和测序鉴定后将pGADT7-STAT4-N123转化酵母AH109细胞,提取细胞蛋白,western blot检测融合蛋白表达情况,酵母双杂交检测STAT4-N123与CBP1678-2175间的相互作用。结果:成功构建了不含有STAT4 N-末端结构域的酵母表达质粒pGADT7-STAT4-N123 ,但pGADT7-STAT4-N123与pGBKT7- CBP1678-2175共转化酵母AH109细胞后不能激活报告基因表达。结论:酵母双杂交没有检测到STAT4-N123和CBP1678-2175存在相互作用。第五部分免疫共沉淀对酵母双杂交的阳性结果进行验证目的:免疫共沉淀验证STAT4与CBP1678-2175间的相互作用,同时分别检测STAT4、STAT6与CBP1-436的相互作用。方法:PCR扩增CBP1678-2175和CBP1-436并与C-Myc标签融合,然后转克隆入真核表达载体pIRES2-EGFP,酶切、测序鉴定后western blot检测融合蛋白在COS7细胞中的表达情况。将pGADT7-STAT4与pIRES2-EGFP-CBP1678-2175共转染COS7细胞,提取细胞蛋白,用HA标签抗体沉淀细胞裂解液,C-Myc标签抗体检测CBP1678-2175-C-Myc融合蛋白,同法检测STAT4、STAT6与CBP1-436的相互作用。结果:成功的将CBP1678-2175和CBP1-436与C-Myc标签融合后克隆入pIRES2-EGFP,且融合蛋白在COS7细胞中能正确表达。HA标签抗体可将CBP1678-2175与STAT4同时沉淀,但CBP1-436与STAT4和STAT6均不能同时沉淀。结论:免疫共沉淀检测到CBP1678-2175与STAT4存在相互作用,但没有检测到CBP1-436与STAT4和STAT6存在相互作用。更多还原

【Abstract】 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.更多还原