hfgl2凝血酶原酶基因5′端调控序列的克隆及生物信息学分析

Coloning of 5′ regulatory sequence of hfgl2 prothrombinase gene and its bioinformatic analysis

目的扩增人纤维蛋白原样蛋白2(hfgl2)凝血酶原酶基因5′端调控序列,构建荧光素酶报告基因表达载体,对hfgl2基因5′侧翼启动子序列进行生物信息学分析,预测可能的调控区域及顺式作用元件。方法用聚合酶链反应(PCR)法从人外周血单个核细胞基因组DNA中扩增hfgl2凝血酶原酶基因5′端调控序列,PCR产物酶切后克隆至pGL3-Basic载体,重组质粒行酶切及测序鉴定,使用在线分析软件对5′端调控序列中的转录因子结合位点及启动子序列进行预测。结果扩增了长度为1 567bp的hfgl2凝血酶原酶基因5′端序列,酶切及测序鉴定证实构建的pGL3-hfgl2质粒插入片段正确无误,分析显示该基因序列共含有138个、31种顺式作用元件。结论构建hfgl2基因近端启动子转录调控序列荧光素酶报告基因的表达载体为研究hfgl2的转录调控奠定了基础。

Objective To amplify 5′ regulatory sequence of human fibrinogen-like protein 2（hfgl2） prothrombinase gene,construct expression vectors with luciferase reporter gene and predict the possible regulation regions and cis-acting elements through bioinformatic analysis of 5′ flanking promoter sequences of hfgl2 gene.Methods Polymerase chain reaction（PCR） was used to amplified the 5′ regulatory sequence of hfgl2 prothrombinase gene from genomic DNA of human peripheral blood mononuclear cells.PCR products was digested and cloned into pGL3-Basic vector.The recombination plasmid was subjected to enzyme digestion and sequencing,and the online analysis software was employed to predict the transcription factor binding sites and the promoter sequences in 5′ flanking regulatory sequences.Results A length of 1 567 bp 5′ flanking sequence of hfgl2 prothrombinase gene was amplified.Enzyme digestion and sequencing confirmed that the constructed plasmid pGL3-hfgl2 insert fragment was correct,and the analysis demonstrated that the gene sequence including a total of 138 cis-acting elements which were in 31 kinds.Conclusion Construction of luciferase reporter gene vector containing regulatory sequences of proximal promoter of hfgl2 gene lays a foundation for the study of transcription regulation of hfgl2 gene.