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目的筛选HepG2细胞中转录因子RORγ(the retinoid-related orphan nuclear receptor gamma)相互作用蛋白,并对这些蛋白进行初步鉴定,为阐述RORγ介导调节HepG2细胞中各种生理学进程提供理论依据。方法从HepG2细胞中克隆RORγ基因并连接入载体pCeMM CTAP(SG)中形成RORγ-CTAP(SG)融合基因,再将其亚克隆入含嘌呤霉素抗性基因的载体质粒中,构建pMSCVpuro RORγ-CTAP(SG)质粒;稳定转染HepG2细胞并对RORγ-CTAP(SG)融合基因的表达和定位进行检测后,进行大规模扩增培养;用TAP(串联亲和纯化)方法捕获RORγ相互作用蛋白,通过银染找出差异性蛋白条带,质谱鉴定得到候选RORγ相互作用蛋白;用免疫共沉淀方法对候选蛋白RORγ相互作用蛋白进行验证鉴定。结果成功构建了质粒pMSCVpuro RORγ-CTAP(SG)并得到稳定转染HepG2细胞株,同时RORγ-CTAP(SG)融合基因能定位表达于细胞核内;通过TAP方法获得了RORγ蛋白复合物,然后通过串联质谱分析和数据库搜索从银染差异性显著蛋白条带中找到7个候选RORγ相互作用蛋白;用RORγ蛋白进行免疫共沉淀,对纯化出的7个RORγ相互作用蛋白分别检测,证实了RIP140,HSP90和RORγ在HepG2中有相互作用关系。结论筛选并鉴定了HepG2细胞中蛋白RORγ的相互作用蛋白RIP140和HSP90,这些研究发现支持了RORγ是共调解蛋白依赖的转录因子且以复合物形式行使功能的假说。其中,HSP90可能扮演分子伴侣角色,而RIP140在HepG2细胞中则可能充当RORγ蛋白的转录调控伙伴蛋白,具体机制有待进一步研究。
Objective To screen the proteins that interact with the retinoid-related orphan nuclear receptor gamma in HepG2 cells and to identify these proteins. METHODS: The RORγ gene was cloned from HepG2 cells and ligated into vector pCeMM CTAP (SG) to form RORγ-CTAP (SG) fusion gene. The recombinant plasmid was subcloned into vector plasmid containing puromycin resistance gene to construct pMSCVpuro RORγ- CTAP (SG) plasmid was constructed. HepG2 cells were stably transfected and the expression and localization of RORγ-CTAP (SG) fusion gene was detected. Then, large-scale expansion of HepG2 cells was performed. The RORγ interacting protein was captured by TAP (tandem affinity purification) , The differentially expressed protein bands were identified by silver staining and the candidate RORγ interacting proteins were identified by mass spectrometry. The protein RORγ interacting proteins were identified by immunoprecipitation. Results The plasmid pMSCVpuro RORγ-CTAP (SG) was successfully constructed and stably transfected into HepG2 cells. At the same time, the RORγ-CTAP (SG) fusion gene was localized in the nucleus. The RORγ protein complex was obtained by TAP method, Seven candidate RORγ-interacting proteins were found from the bands of significant differences in silver staining by mass spectrometry and database search. The co-immunoprecipitation with RORγ protein was used to detect the seven RORγ-interacting proteins respectively, which confirmed that RIP140, HSP90 And RORγ have an interaction in HepG2. Conclusion The proteins RIP140 and HSP90, a protein of RORγ in HepG2 cells, were screened and identified. These findings support the hypothesis that RORγ is a coslotone-dependent transcription factor and functions as a complex. Among them, HSP90 may play a role of molecular partner, and RIP140 may play a transcriptional regulatory partner of RORγ protein in HepG2 cells, and the specific mechanism remains to be further studied.