基于生物信息学分析的人ID4基因启动子表达载体的构建及鉴定

Construction and identification of expression vector of human ID4 gene promoter based on bioinformatics analysis

目的:基于生物信息学分析构建人ID4基因启动子表达载体,以其作为研究ID4基因启动子表达调控分析的工作基础。方法:在分析人ID4基因启动子结构和调控元件的基础上,据UCSC基因组生物信息学中人ID4基因转录起始点(TSS)上游启动子区-643 bp及5′非翻译区(5-′UTR)+164 bp序列设计并合成引物,进行PCR扩增;以PCR产物作为模板经巢式PCR扩增了TSS上游启动子区-621 bp片段,将PCR产物回收、纯化后再连接到pGEM-T Easy载体上并转化至DH5α感受态大肠杆菌中构建成pGEM-T Easy-人ID4基因启动子重组质粒。转化产物经半巢式PCR及酶切鉴定,对得到的阳性克隆进行测序。结果与结论:成功构建出含有糖皮质激素受体、cAMP结合蛋白及雌激素受体反应调控元件序列的人ID4基因启动子表达载体。该表达载体的构建为进一步研究人ID4基因的启动子活性及表达调控奠定了基础。

Objective ：To construct expression vector of human ID4 gene promoter on the basis of bioinformatics analysis. Methods： According to Internet-based bioinformatics analysis of human ID4 gene promoter structure and transcriptional regulatory dements, the upstream-621bp sequence of ID4 gene transcriptional start site which includes glucocorticoid receptor, cAMP binding protein and estrogen receptor response dements was amplified by PCR. The PCR product of the 621bp sequence was purified and linked to pGEM-T Easy vector by T4 DNA ligase. Recombinant of human ID4 gene promoter- pGEM-T Easy was transformed into DH5α competent E. coll. Then the positive recombinant was identified by nested PCR and DNA sequencing. Results and Conclusion：The human ID4 promoter-pGEM-T Easy recombinant plasmid was successfully constructed. The construction of expression vector of human ID4 promoter provides a platform for further researches on promoter activity and expression regulation of human ID4 gene.