PRL-1基因3’UTR荧光素酶报告基因载体及突变体的构建

Construction of PRL-1 gene 3'UTR luciferase reporter vector and mutational vector

目的针对促肝细胞再生磷酸酶1(phosphatase of regenerating liver-1,PRL-1)3’端非翻译区(3-untranslatedregion,3’UTR)构建PRL-1荧光素酶报告基因载体及突变体,为研究miR-339-5p在结肠癌中调控其靶基因PRL-1提供有效的工具。方法 PCR扩增包含PRL-1的3’UTR的DNA片段,克隆至荧光素酶载体psiCHECK-2,构建psiCHECK-2/PRL-13’UTR载体;经双酶切及测序鉴定后,对psiCHECK-2/PRL-1 3’UTR重组质粒"种子区"的7个碱基进行定点突变,构建psiCHECK-2/PRL-1 3'UTR突变载体。结果克隆获得的psiCHECK-2/PRL-1 3'UTR载体中DNA片段大小及序列与GenBank报道的一致,且插入方向正确。"种子区"的7个碱基定点突变成功。结论成功构建了含PRL-1基因3'UTR区的荧光素酶报告基因载体及突变体,可用于后续功能研究。

Objective In the light of phosphatase of regenerating liver 1 （phosphatase of regenerating Liver-l, PRL-1） 3＇- untranslated region （3-untranslatedregion, 3＇UTR）, we constructs PRL-1 luciferase reporter gene vector and mutant vector, which provides an effective tool for the study of miR-339-Sp in colon cancer in regulation of its target gene PRL-1. Methods PRL-1 3＇UTR DNA fragment amplified by PCR was cloned into the luciferase vector psiCHECK-2, and psiCHECK-2/PRL-1 3＇UTR vector was constructed. After double enzyme digestion and DNA sequencing verification, 7 ＂seeds＂ of base point the recombinant plasmid psiCHECK-2/PRL-1 3＇UTR were mutated to construct psiCHECK-2/PRL-1 3＇UTR mutational vector. Results psiCHECK-2/PRL-1 3＇UTR vector obtaining the DNA fragment sequence was identified with that of GenBank reported, and the correct direction was inserted. 7 ＂seeds＂ showed the success of site directed mutation. Conclusion The luciferase reporter gene vectors containing PRL-1 3＇UTR or mutational 3＇UTR area were successfully constructed, which were used for further functional study.