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将人巨细胞病毒IE86cDNA克隆入pGEX-2T表达载体,在大肠杆菌中表达出GST-IE86融合蛋白.SDS-PAGE和Western blot分析表明,GST-IE86融合蛋白存在于细胞裂解上清中,为可溶性蛋白,分子量为92ku.通过亲和层析柱纯化,分别获得纯化的GST和GST-IE86融合蛋白.采用特异性亲和层析共分离技术研究HCMVIE86蛋白与转录调控蛋白及转录因子相互作用,表明IE86能与直接结合启动子DNA的转录激活因子SP1,AP1,AP2及转录因子TFⅡB相互作用而形成异源蛋白复合物.该转录激活因子及转录因子与IE86蛋白同时被吸留在亲和层析柱中,但IE86蛋白不能与NF-кB相互作用.提示IE86蛋白与转录激活因子及转录因子相互作用的活性与IE86蛋白的糖基化无关;IE86蛋白激活多种基因转录是由于IE86蛋白具有两个蛋白质结合位点,它们与激活转录的调控蛋白和转录因子结合,这种相互作用加速了转录起始复合物的装配.
The human cytomegalovirus IE86 cDNA was cloned into the pGEX-2T expression vector and expressed in E. coli GST-IE86 fusion protein.SDS-PAGE and Western blot analysis showed that the GST-IE86 fusion protein was present in the cell lysate supernatant and was soluble Protein with a molecular weight of 92ku.The purified GST and GST-IE86 fusion proteins were purified by affinity chromatography.The interaction of HCMVIE86 protein with transcriptional regulatory proteins and transcription factors was investigated by specific affinity chromatography co-separation technique IE86 can interact with transcriptional activators SP1, AP1, AP2 and TFⅡB that directly bind to promoter DNA to form a heterologous protein complex. The transcription activator and transcription factor are simultaneously occluded with IE86 protein in affinity chromatography IE86 protein could not interact with NF-кB, suggesting that the interaction between IE86 protein and transcriptional activator and transcription factor is not related to the glycosylation of IE86 protein. IE86 protein activates many kinds of gene transcription because IE86 protein has two Protein binding sites that bind to regulatory proteins and transcription factors that activate transcription, and this interaction accelerates the assembly of the transcriptional initiation complex.