大鼠Prestin-3'UTR双荧光素酶报告基因载体的构建与鉴定

Constructing and identifying of rat Prestin-3'UTR dual-luciferase reporter gene vector

目的构建大鼠Prestin基因(SLC26A5)3'端UTR区双荧光素酶报告系统,为研究Prestin基因的microRNA调控提供基础平台。方法以C6细胞基因组DNA为模板PCR扩增SLC26A5基因的3'UTR序列,并连接至pmri-RBREPORTTM双荧光素酶报告载体多克隆位点,测序验证插入序列并转染入293T细胞检测其荧光素酶活性。结果测序结果表明PMIR-3'UTR的插入序列正确,在转染入293T细胞后其荧光素酶能正常表达。结论大鼠Prestin-3'UTR双荧光素酶报告基因载体构建成功。

Objective To study the regulation of microRNA on Prestin gene, a rat Prestin-3＇UTR dual-luciferase reporter gene vector was constructed. Methods The 3＇untranslated region （3＇UTR） fragment of SLC26A5 gene was amplified by PCR from genomic DNA of C6 cells. PCR products were cloned into pmir-RB-REPORTTM dual-luciferase reporter gene vector. DNA sequencing identified 3＇UTR of SLC26A5 and the dual-luciferase reporter gene vector was transferred into 293T cells to quantitate the reporter activity by the Dual-Luciferase Reporter Assay System. Results The results showed that a 3＇ UTR fragment of SLC26A5 gene was successfully cloned into the pmir-RB-REPORTTM dual-lueiferase reporter gene vector, which authenticated by DNA sequencing, and the luciferase activity of 293T cells which was transfected vector could expressed availably. Conclusion Rat Prestin-3＇UTR dual-luciferase reporter gene vector was successfully constructed.