人Tudor-SN UTR片段荧光素酶报告基因表达质粒的构建及活性检测

Construction of human Tudor-SN UTR luciferase reporter gene expression plasmid and its activity detection

目的为深入研究Tudor-SN蛋白本身在翻译水平的调控机制奠定基础。方法以HeLa细胞全基因组DNA为模板,PCR扩增出目的基因,利用双酶切的方法将目的片段连接到pGL3-Control载体上。再将构建成功的pGL3-5'UTR、pGL3-3'UTR重组质粒分别与内参海肾荧光素酶质粒瞬时共转染HeLa细胞,培养48h检测荧光素酶的活性。结果双酶切和基因测序法鉴定重组质粒构建成功,转染重组质粒后可检测到荧光素酶的活性;以pGL3-5'UTR质粒荧光素酶活性最高。结论成功构建了人Tudor-SN基因5'UTR和3'UTR序列的重组质粒,为研究Tudor-SN基因UTR区对翻译调控的影响奠定了基础。

Objective To lay a foundation for further study on the mechanism of Tudor-SN protein in translation . Methods The human Tudor-SN UTR fragments were amplified by PCR from genomic DNA extracted from HeLa cells , and then were inserted into pGL 3-Control expression vector .HeLa cells were co-transfected with the renilla luciferase plasmid and recombinant pGL3-5′UTR plasmid or pGL3-3′UTR plasmid.After 48 hours, luciferase activity was detected . Results Both double enzyme digestion and gene sequencing confirmed the construction of recombinant plasmid was suc -cessful.The luciferase activity was detected after transfection , and the luciferase activity of pGL3-5′UTR plasmid was the best.Conclusion The recombinant plasmids of pGL 3-5′UTR-luciferase and pGL3-3′UTR-luciferase are successfully con-structed, which lays a foundation for further study of regulatory mechanisms of Tudor -SN genetic UTR in translation .