LGALS3BP 3’UTR荧光素酶报告基因载体构建及鉴定

Construction of a luciferase reporter gene vector for the LGALS3BP 3’UTR

目的构建LGALS3BP(lectin,galactoside-binding,soluble,3 binding protein,LGALS3BP)基因3’非编码区(3’-untranslated region,3’UTR)的野生型及突变型荧光素酶报告基因重组载体并进行鉴定,以备进一步研究miRNA对LGALS3BP基因的调控。方法Targetscan软件预测LGALS3BP 3’UTR上的miRNA结合位点;以人肝癌细胞系SMMC-7721基因组DNA为模板,进行PCR扩增,获得野生型LGALS3BP基因3’UTR片段,经双酶切将目的片段定向插入报告基因载体pGL3-Control中,将克隆好的重组载体转化大肠埃希菌DH5α感受态细胞进行扩增,采用菌落PCR及测序监定重组子。设计位点突变型LGALS3BP 3’UTR扩增引物,以构建的野生型LGALS3BP 3’UTR重组质粒为模板,采用重叠PCR及定点突变PCR技术进行扩增分别构建单独位点及双位点突变型重组荧光素酶报告基因载体。结果野生型LGALS3BP 3’UTR重组质粒经菌落PCR鉴定,可见871 bp的目的基因片段,大小与预期相符;测序结果表明,插入序列与LGALS3BP 3’UTR序列完全一致且插入方向正确。Targetscan软件预测到LGALS3BP 3’UTR区有两个感兴趣的miRNA结合位点。突变型LGALS3BP 3’UTR重组质粒测序鉴定结果表明,单独位点及双位点突变型重组质粒中均成功引入结合位点突变。结论成功构建LGALS3BP的3’UTR野生型(pGL3-LGAL-W-3’UTR)、两个单独位点突变型(pGL3-LGAL-M1-3’UTR和pGL3-LGAL-M2-3’UTR)及双位点突变型(pGL3-LGAL-M-3’UTR)重组荧光素酶报告基因载体,为后续研究miRNA对LGALS3BP基因的调控奠定基础。

Objective To construct a recombinant luciferase reporter gene vector for wildtype and mutant 3’⁃untranslated regions(UTRs)of the LGALS3BP gene to study regulation of the LGALS3BP gene and miRNAs.Methods TargetScan was used to predict the binding site of miRNAs in the 3’UTR of the LGALS3BP gene.The wildtype LGALS3BP 3’UTR was amplified by PCR using genomic DNA from SMMC⁃7721 hepatocellular carcinoma cells as a template.PCR products were digested by two restriction enzymes and inserted into the luciferase reporter vector pGL3⁃control.Recombinant plasmids were verified by colony PCR and sequencing.Primers for LGALS3BP 3’UTR with binding site mutations were designed.Single and double site mutant luciferase reporter gene vectors were prepared by overlapping PCR and site⁃directed mutagenesis PCR using the wildtype LGALS3BP 3’UTR recombinant plasmid as a template.Results Colony PCR showed a target gene fragment of 871 bp,which was consistent with the expected size.The inserted sequence was consistent with the LGALS3BP 3’UTR in sequencing.TargetScan indicated two miRNA⁃binding sites in the LGALS3BP 3’UTR.The single and double site mutant sequences were successfully inserted into the LGALS3BP 3’UTR.Conclusions Wildtype LGALS3BP 3’UTR(pGL3⁃LGAL⁃W⁃3’UTR),two single site mutants(pGL3⁃LGAL⁃M1⁃3’UTR and pGL3⁃LGAL⁃M2⁃3’UTR),and a double site mutant(pGL3⁃LGAL⁃M⁃3’UTR)luciferase reporter gene vectors were successfully constructed,which may facilitate studies of LGALS3BP gene regulation through miRNAs.