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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 To construct PRL-1 luciferase reporter gene vectors and mutants targeting 3-untranslated region (3’UTR) of phosphatase of regenerating liver-1 (PRL-1) MiR-339-5p provides an effective tool for the regulation of its target gene PRL-1 in colon cancer. Methods The DNA fragment containing the 3’UTR of PRL-1 was amplified by PCR and cloned into vector psiCHECK-2. The psiCHECK-2 / PRL-13’UTR vector was constructed. After double enzyme digestion and sequencing, 2 / PRL-1 3’UTR recombinant plasmid “seed region ” of seven bases for site-directed mutagenesis to construct psiCHECK-2 / PRL-1 3’UTR mutation vector. Results The size and sequence of the DNA fragment cloned in psiCHECK-2 / PRL-1 3’UTR vector was consistent with that reported in GenBank and inserted in the correct orientation. The 7-bp site-directed mutagenesis of the “seed region” was successful. Conclusion The luciferase reporter gene vectors and mutants containing the 3’UTR region of PRL-1 gene were successfully constructed and could be used for further functional studies.