| 唐焱，周宏，钟警，姜浩.基因的miRNA载体构建及其活性鉴定.[J].中南医学科学杂志.,2012,40(4):360-363. |
| 基因的miRNA载体构建及其活性鉴定 |
| Construction and Activity Identification of miRNA Expressing Eukaryotic Vector of PRMT2 |
| 投稿时间：2012-05-09 |
| DOI： |
| 中文关键词: miRNA 蛋白精氨酸N-甲基转移酶 雌激素受体α 转染 |
| 英文关键词:miRNA PRMT2 ERα transfection |
| 基金项目: |
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| 中文摘要: |
| 目的构建蛋白质精氨酸甲基转移酶2（PRMT2）基因的miRNA真核表达载体，鉴定其转染乳腺癌MCF7细胞株后的生物活性。方法根据PRMT2基因序列设计合成4对pre-miRNA片段，定向克隆到pcDNATM6.2-GW/EmGFP-miR真核表达载体上，采用菌落的PCR和测序分析鉴定插入序列的完整性；并将重组载体转染至MCF7细胞株中，采用Western blot方法鉴定重组载体转染MCF7细胞后对PRMT2蛋白表达的干扰效果以确定其生物活性。结果构建的重组体插入片段的碱基序列完全正确，并且重组体瞬时定转染MCF7细胞后成功干扰PRMT2的表达。结论成功构建了靶向PRMT2基因的pcDNATM6.2-GW/EmGFP-miR真核表达载体，且在瞬时转染MCF7细胞后能下调PRMT2的表达。 |
| 英文摘要: |
| ObjectiveTo construct PRMT2 gene expressing eukaryotic miRNA recombiants，and to identify biological activity of recombinat in breast cancer cell line MCF7 cells after transfection. MethodsAccording to sequence of PRMT2gene,the pre-miRNA was designed and synthesized, then cloned into the GFP reporter pcDNATM6.2-GW/EmGFP-miR, and transfected into MCF7 cell line. The integrity of inserted fragment sequence was tested through colony PCR and sequence analysis , and the biological activity of recombinants was identified by detecting the interference efficiency of PMRT2 employing Western blotting.ResultsSequences of insert fragment in four miRNA expressing recombinants proved to be correct, and the expression of PRMT2 was inhibited after the recombinants transient transfection in MCF7 cells. ConclusionThe expressing eukaryotic miRNA recombinants of reporter pcDNATM6.2-GW/EmGFP-miR of PRNT2 were constructed successfully, and the biological activity is stable after transfecting MCF7 cells. |
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