畸形表皮自调节因子1相关功能蛋白的筛选

Screening of deformed epidermal autoregulatory factor 1-related functional proteins

目的获得畸形表皮自调节因子1(DEAF1)稳定高表达的HEK293T细胞株,通过免疫共沉淀(Co-IP)和质谱分析技术筛选能与DEAF1相互作用的蛋白质因子。方法用Turbo Fect转染试剂将p EGFP-m DEAF1和p EGFP-N1质粒分别转染HEK293T细胞和2F-2B细胞、荧光显微镜观察DEAF1在2种细胞中的定位。将质粒p3×FLAG-m DEAF1分别转染HEK293T和2F-2B细胞株,同时设置相应未转染的空白对照组;通过反转录PCR法和实时定量PCR(qRT-PCR)检测DEAF1在2种细胞中mRNA表达水平,通过Western blot法检测DEAF1在2种细胞中蛋白表达水平。p3×FLAG-m DEAF1质粒转染HEK293T细胞,G418筛选阳性克隆,Western blot法、免疫荧光染色和流式细胞术鉴定DEAF1稳定表达的HEK293T-DEAF1细胞株。抽提HEK293T-DEAF1细胞和正常HEK293T细胞的核蛋白,用EZviewTMRed ANTI-FLAG M2亲和珠子进行Co-IP,SDS-PAGE分离,挑选明显富集和对照泳道没有的条带进行质谱分析,明确DEAF1的相关功能蛋白。结果荧光显微镜下可见DEAF1定位于细胞核;反转录PCR、qRT-PCR检测发现转染组DEAF1的mRNA表达水平明显高于未转染组;Western blot法检测发现转染组有融合蛋白m DEAF1-FLAG表达,成功获得稳定高表达DEAF1的HEK293T-DEAF1细胞。经Co-IP筛选和质谱分析,得到一些DEAF1的相关功能蛋白,如前mRNA处理所需的因子及酶,参与起始后RNA聚合酶,参与肽键断裂、蛋白空间结构形成的分子和其他转录因子等。结论成功获得稳定高表达DEAF1的HEK293T-DEAF1细胞,经Co-IP筛选和质谱分析获得了一些DEAF1相关的功能蛋白。

Objective To obtain HEK293 T cell line which expresses deformed epidermal autoregulatory factor-1（DEAF1） highly and stably,and to screen candidate proteins which can interact with DEAF1 by co-immunoprecipitation（Co-IP） and mass spectrometry（ MS）. Methods Plasmids p EGFP-m DEAF1 and p EGFP-N1 were transfected into HEK293 T and 2F-2B cell lines with Turbo Fect transfection reagent,respectively. The localization of DEAF1 was observed in the two kinds of cell lines by a fluorescence microscope. Plasmid p3 × FLAG-m DEAF1 was transfected into HEK293 T and2F-2B cell lines,respectively. Non-transfected cells were used as a control group. The expression levels of DEAF1 mRNA and protein were detected by reverse transcription PCR,real-time quantitative PCR（ qRT-PCR） and Western blotting,respectively. Plasmid p3 × FLAG-m DEAF1 was transfected into HEK293 T cells,and then HEK293 T cell line expressing stably DEAF1 was selected with G418 and identified by Western blotting,immunofluorescence and flow cytometry. Nuclear proteins of HEK293T-DEAF1 and HEK293 T cell lines were extracted and detected using Co-IP and SDS-PAGE with EZview^TM Red ANTI-FLAG M2 affinity gel. Bands,which were obviously enriched and did not exist in the control lanes,were analyzed with MS. Results DEAF1 was located in the nucleus under the fluorescence microscope. Reverse transcription PCR and qRT-PCR showed that the mRNA level of DEAF1 in DEAF1-expressed HEK293 T cells was higher than that in control cell line.Western blotting revealed the expression of m DEAF1-FLAG fusion protein in the transfected cells. HEK293 T-DEAF1 cells which expressed DEAF1 highly and stably were obtained successfully. Some candidate proteins which might interact with DEAF1 were screened by Co-IP and MS,such as cytokines and enzymes required for pre-mRNA processing,postinitiation RNA polymerase,and other transcription factors involved in peptide bond breaking and spatial structure formatting,and so on. Conclusion The study obtained HEK293T-DEAF1 cells which expressed DEAF1 highly and stably as well as a large set of candidate proteins which might interact with DEAF1 using Co-IP and MS.