人miR-134真核表达载体的构建及其鉴定

Construction and identification of human eukaryotic expression vector of hsa-miR-134

摘要:

目的：构建包含外源性miR-134前体的真核表达载体，并使其在人类少突胶质瘤（oligodendroglia，OL）细胞中表达，为进一步研究miR-134的功能及作用靶标提供技术支持。方法：从人类OL细胞基因组DNA扩增 miR-134 前体，将所得目的片段克隆到pEGFP-N1载体上，重组质粒经测序鉴定后将其转染至人类OL细胞中，荧光倒置显微镜观察增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）的表达情况，并采用荧光定量PCR检测 miR-134基因水平的表达。结果：pEGFP-miR-134重组质粒经酶切、PCR鉴定可见目的条带与预期结果一致，经测序可见插入序列正确。将重组质粒转染OL细胞后，荧光定量PCR结果示pEGFP-N1空质粒转染组与空白对照组的miR-134表达水平无统计学差异（P=0.882）；pEGFP-miR-134质粒转染组miR-134表达水平是pEGFP-N1空质粒转染组的52倍，两者具有统计学差异（P=0.023）。结论：通过构建真核细胞表达质粒可以高效表达外源性microRNA分子，为后续的研究奠定实验基础。

Abstract:

Objective：To construct the eukaryotic expression vector containing exogenous miR-134 precursor，to study its expression in the human oligodendroglia（OL） cells and to provide the technical platform for further studying the function of miR-134 and its target gene. Methods：miR-134 precursor sequence was amplified from the human genomic DNA and cloned into pEGFP-N1. After being confirmed by PCR and sequencing，recombinant plasmid was transfected into human OL cells and transcription level of miR-134 was detected by real-time PCR. In addition，transcription efficiency was monitored by the inverted fluorescence microscopy. Results：The target bands were consistent with the expected results by enzyme digestion and PCR identification，and the inserted sequence was correct in gene sequencing detection. After the recombinant plasmid being transfected into OL cells，fluorescence quantitative PCR detection showed that there was no difference between pEGFP-N1 empty plasmid group and control group in the expression level of miR-134（P=0.882）. Moreover，the miR-134 expression level of pEGFP-miR-134 plasmid transfected group was 52 times over that of pEGFP-N1 empty plasmid group，with significant statistical differences（P=0.023）. Conclusions：The miR-134 expression vector is successfully constructed，which paves the way for the future experiment.