摘要
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 the transcription factor(s) that is essential for activation of mfgl2 prothrombinase/fibroleukin gene in response to nucleocapsid protein of murine hepatitis virus type 3 (MHV-3). Methods Western blotting was performed to investigate whether HNF4 is expressed in macrophages of Ba1b/c mice where mfgl2 is expressed. Confocus microscope immunofluoresence was performed to show whether N protein of MHV enters into the nucleus of infected cells, which is a critical step for the N protein to facilitate its transactivation property. To facilitate the identification of three candidate factor(s) including hepatocyte nuclear factor 4 (HNF4)/liver factor A1 (LF-A1), cytomegalovirus immediate early gene 1.2 (IE1.2) regulatory element and granulocyte- macrophage colony stimulating factor (GM-CSF) in response to mfgl2 activation upon the stimulation of MHV-A59 N protein, gel mobility shift assay (GMSA), competition experiments and site directed mutagenesis were performed. Results Western blotting displayed that HNF4 was constitutively expressed in macrophages and did not show significant change under the stimulation of different MHV. Confocus microscope immunofluoresence clearly showed that N protein of MHV entered into the nucleus of infected cells. GMSA and competition experiments demonstrated binding to both HNF4 and IE1.2 fragments could be competed with the cold specific oligonucleotides but not with the same amount of non-specific oligos nucleotides. A super shift band was observed when HNF4 antibody was pre-incubated with the nuclear extracts indicating the interaction between the HNF4 element and mfgl2 promoter. Site directed mutagenesis of cis-elements HNF4 (pfgl2HNF4mut) and HNF4/IE1.2 (pfgl2HNF4/IE1.2mut) mutations abolished over 75% of transcription from wild-type mfgl2 promoter. However the pfgl2IE1.2mut displayed almost wild-type promoter activity (75%～80%). Conclusions The factor HNF4 binds to mfgl2 promoter and serves as an essential transcription factor for mfgl2/fibroleukin expression in respons to MHV-3 N protein.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2003年第8期678-683,共6页
National Medical Journal of China
基金
国家自然科学基金资助项目 (NSFC30 170 846 )
国家杰出青年科学基金项目 (NSFC30 2 2 5 0 40
NSFC30 12 5 0 19)
