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目的构建小鼠miR-214的pcDNA3.1(+/-)真核表达载体并在COS-7细胞中转染检测其表达。方法小鼠基因组DNA扩增得到miR-214序列,将酶切后的miR-214克隆到真核表达载体pcDNA3.1(+/-),重组pcDNA3.1(+/-)-miR-214经过酶切和测序鉴定后转染COS-7细胞并应用萤光定量PCR法检测miR-214表达水平。结果小鼠miR-214真核表达载体序列分析完全正确;转染COS-7细胞后miR-214的表达水平可增加1.41倍。结论成功构建了pcDNA3.1(+/-)-miR-214真核表达载体并能在真核细胞中进行表达,为进一步进行miR-214的功能研究提供了实验基础。
Objective To construct the pcDNA3.1 (+/-) eukaryotic expression vector of mouse miR-214 and transfect it to detect its expression in COS-7 cells. Methods The miR-214 sequence was amplified from the mouse genomic DNA. The digested miR-214 was cloned into the eukaryotic expression vector pcDNA3.1 (+/-) and the recombinant pcDNA3.1 (+/-) - miR-214 The recombinant plasmid was transfected into COS-7 cells by restriction enzyme digestion and sequencing. Fluorescent quantitative PCR was used to detect the expression of miR-214. Results The sequence analysis of mouse miR-214 eukaryotic expression vector was completely correct. The expression of miR-214 in COS-7 cells increased by 1.41-fold. Conclusion The eukaryotic expression vector pcDNA3.1 (+/-) - miR-214 was successfully constructed and expressed in eukaryotic cells, which provided the experimental basis for the further study on the function of miR-214.