【摘要】 目的·分离多能干细胞关键因子Oct4的RNA结合蛋白。方法·培养小鼠胚胎干细胞(R1),从中提取细胞总RNA,反转录为cDNA,PCR扩增Oct4并转录为m RNA。采用链霉素亲和层析和质谱技术筛选候选RNA结合蛋白,采用RNA免疫沉淀技术鉴定候选RNA结合蛋白。结果·经高效液相色谱-质谱法鉴定和筛选出121个能与Oct4 3’非翻译区(untranslated region,UTR)和Oct45’UTR相结合的RNA结合蛋白,肽段图谱匹配数≥2的有11个。并对其中的7个候选RNA结合蛋白进行了进一步验证,结果显示Oct4可与DSP、SOD1、LMNA、NPM1、PSIP1、NCL和HDGF蛋白发生相互作用,其中HDGF与Oct4结合能力最强。结论·Oct4RNA结合蛋白的鉴定将为Oct4的调控机制提供一定理论依据,为后续其功能研究提供基础。更多还原

【Abstract】 Objective · To identify the RNA binding proteins of Oct4, a key factor of embryonic stem cells. Methods · Total RNA was extracted from mouse embryonic stem cells(R1), which was reverse-transcribed into cDNA. Then PCR product of Oct4 mRNA were transcribed into Oct4 mRNA.Finally the candidate RNA-binding proteins were eluted applying the streptomycin affinity chromatography, and submitted for high-performance liquid chromatography combined with the mass spectrometry. The identified RNA binding proteins were further confirmed by RNA immunoprecipitation(RIP). Results · 121 RNA binding proteins of Oct4 3’ untranslated region(UTR) and Oct4 5’ UTR were identified with liquid chromatography/mass spectrometry. There were 11 proteins with the number of peptide spectrum matches more or equal to 2, and 7 of them were selected for additional confirmation using RIP method. RIP results showed that Oct4 interacted with DSP, SOD1, LMNA, NPM1, PSIP1, NCL and HDGF. Among them, HDGF had the strongest binding ability to Oct4 mRNA. Conclusion · Identification of Oct4 RNA binding proteins will provide a theoretical basis for the regulation mechanism of Oct4, and will be a basis for the further study of its function.更多还原