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目的 研究鉴定mfg12凝血酶原酶 /纤维介素基因转录激活所必需的调控元件或转录因子。方法 应用免疫印迹法检测Ba1b/c小鼠的巨噬细胞内是否表达肝细胞核因子 4(HNF4) ,共聚焦显微镜免疫荧光技术检测鼠肝炎病毒 (MHV)的核心 (N)蛋白质是否进入被感染细胞核。应用DNA电泳迁移差异检测、竞争实验和定点诱变技术鉴定mfgl2基因转录激活所必需的调控元件或转录因子。结果 免疫印迹法表明巨噬细胞可持续表达HNF4。共聚焦显微镜免疫荧光技术证实MHV的N蛋白质可进入被感染细胞核内。凝胶移位分析和竞争实验显示HNF4和巨细胞病毒早期蛋白基因 1 2 (IE1 2 )均可与特异寡核苷酸竞争结合mfgl2启动子上特定位点而不与非特异寡核苷酸发生竞争。HNF4特异性多克隆抗体竞争试验可见超迁移带。定点突变mfgl2启动子上HNF4所结合的顺式调控元件可降低野生型mfgl2启动子的转录活动达 75 %,联合突变IE1 2结合位点未见协同效应。单纯突变IE1 2结合位点后 ,野生型mfgl2启动子的转录活动仍可保留 75 %～ 80 %。结论 HNF4具有与mfgl2 /fibroleukin启动子结合的特性 ,为MHV 3N蛋白质诱导mfgl2 /fibroleukin基因表达的必备转录因子。
Objective To identify regulatory elements or transcription factors necessary for the transcriptional activation of the mfg12 prothrombinase / fibronectin gene. Methods Western blotting was used to detect the expression of hepatocyte nuclear factor 4 (HNF4) in Ba1b / c mice and the nuclear protein (N) of murine hepatitis virus (MHV) by confocal microscopy. . DNA electrophoresis migration differential detection, competition experiments and site-directed mutagenesis were used to identify the regulatory elements or transcription factors necessary for the transcriptional activation of the mfgl2 gene. Results Immunoblotting showed that macrophages could sustainably express HNF4. Confocal microscopy immunofluorescence confirmed that N protein of MHV can enter the nucleus of infected cells. Gel shift assays and competition experiments showed that both HNF4 and cytomegalovirus early protein 1 2 (IE12) can compete with specific oligonucleotides for binding to specific sites on the mfgl2 promoter without interaction with non-specific oligonucleotides competition. HNF4-specific polyclonal antibody competition test showed hyper-migrating zone. Site-directed mutagenesis mfgl2 promoter HNF4 cis-regulatory elements can reduce the wild-type mfgl2 promoter transcriptional activity of 75%, the combined mutation IE1 2 binding sites no synergistic effect. After a simple mutation of the IE1 2 binding site, the transcriptional activity of the wild-type mfgl2 promoter can still be maintained at 75% to 80%. Conclusion HNF4 has the characteristics of binding to the mfgl2 / fibroleukin promoter and is an essential transcription factor for the MHV 3N protein to induce the expression of mfgl2 / fibroleukin gene.