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OBF-1是淋巴细胞特异性转录共激活因子,其蛋白主要有2种异构体分别为p34和p35。为观察OBF-1对p300基因表达的影响,分离OBF-1-/-小鼠的脾脏单个核细胞及B细胞,采用RT-PCR及Western blot方法分别检测p300 mRNA和蛋白水平。构建小鼠OBF-1(p34)表达质粒和p300报告基因质粒。在293T细胞中过表达p34,检测p34对内源性p300蛋白表达的影响。将p34表达质粒和p300报告基因质粒共同转染293T细胞,通过荧光素酶报告基因方法检测p34对p300基因启动子转录活性的影响。结果:与来源于OBF-1+/+小鼠的脾脏单个核细胞相比,来源于OBF-1-/-小鼠的单个核细胞中p300表达无论是mRNA还是蛋白水平均明显降低。分析显示,OBF-1(p34)能够促进p300基因转录及蛋白表达。这些结果提示,OBF-1正调p300转录及表达。
OBF-1 is a lymphocyte-specific transcriptional coactivator, and its two isoforms are p34 and p35, respectively. To observe the effect of OBF-1 on the expression of p300 gene, spleen mononuclear cells and B cells of OBF-1 - / - mice were isolated and the mRNA and protein levels of p300 were detected by RT-PCR and Western blot respectively. Mouse OBF-1 (p34) expression plasmid and p300 reporter plasmids were constructed. P34 was overexpressed in 293T cells and the effect of p34 on endogenous p300 protein expression was examined. The 293T cells were co-transfected with the p34 expression plasmid and the p300 reporter plasmid, and the effect of p34 on the transcriptional activity of the p300 gene promoter was detected by luciferase reporter assay. Results: Compared with spleen mononuclear cells derived from OBF-1 + / + mice, p300 expression in OBF-1 - / - mice-derived mononuclear cells was significantly reduced both in mRNA and protein levels. Analysis showed that OBF-1 (p34) can promote p300 gene transcription and protein expression. These results suggest that OBF-1 is upregulated in p300 transcription and expression.