摘要
应用两对人工合成的寡核苷酸引物，分别通过ＰＣＲ扩增，得到了ＣＮＴＦＲα－Ｉ５ＮＢＲＥ序列两侧的两个扩增片段，将其和在ＥｃｏＲⅤ位点切开的ｐＴ７ｂｌｕｅ一起定向连接，得到了插入在ｐＴ７ｂｌｕｅ的ＥｃｏＲⅤ位点的缺失了ＮＢＲＥ序列的ＣＮＴＦＲα－Ｉ５，然后再将其切下，插入到具有ＳＶ４０起动子的ＣＡＴ基因表达载体的ＢｇｌⅡ位点，构建了ＣＡＴ报道基因．细胞转染和ＣＡＴ实验表明，缺失ＮＢＲＥ后，ＣＮＴＦＲα－Ｉ５仍具有增强子功能，ＴＲ３通过该增强子对ＣＮＴＦＲα的表达具有诱导作用，说明这种诱导作用并不是单一通过ＮＢＲＥ序列进行的．
Ciliary neurotrophic factor (CNTF) is a member of the cytokine superfamily.It plays a very important role in the development and regeneration of the nervous system.CNTF utilizes a three component receptor system mainly consisting of a CNTF specific binding protein,known as CNTFRα.Orphan receptors is a category of receptors which cognate ligand is still unknown and belongs to nuclear receptor superfamily.TR3(NGFI B,Nur 77) is one of most important orphan receptors found so far.It has been known that TR3 has important functions in regulating some physiological processes in vivo .In order to investigate the interaction between TR3 and cis acting elements in CNTFRα gene,the NBRE sequence of CNTFRα I5 was deleted by PCR using 2 pairs of synthesized oligonucleotide primers.The resulting two PCR fragments in the flank of the NBRE sequence were ligated and cloned in EcoRⅤ site of pT7 blue vector and then in BglⅡ site of pCAT promoter vector,so reporter genes with NBRE deleted CNTFRα I5[CNTFRα I5 NBRE(-)] inserting into pCAT promotor vector at upstream sites relative to the CAT gene with the orientation the same or opposite to CAT expression were constructed.The cell transfection and reporter gene assay using chloramphenicol acetyltransferase demonstrated that CNTFRα I5 NBRE(-) still had an enhancer activity which could be induced by TR3 in a dose dependent manner.It shows that the induction of CNTFRα gene expression by orphan receptor TR3 is not completely through NBRE site,and other NBRE like sequences in CNTFRα I5 may also play some roles.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1998年第5期485-491,共7页
Chinese Journal of Biochemistry and Molecular Biology
