miR-196b慢病毒表达载体的构建和功能鉴定

Construction and functional identification of miR-196b lentiviral vector

目的构建携带miR-196b的慢病毒表达载体,为研究miR-196b在慢性粒细胞白血病中的功能及其对慢粒发生发展的作用奠定基础。方法以人骨髓细胞基因组DNA为模板,PCR法扩增miR-196b的前体序列,慢病毒表达载体plVTHM构建重组载体plVTHM-miR-196b,并包装病毒和进行滴度测定。用病毒液感染K562细胞,通过荧光观察转染效率和报告基因表达效率,用流式细胞仪分选感染后绿色荧光蛋白阳性的K562细胞,再用实时荧光定量PCR测定细胞内miR-196b的表达水平。最后用CCK-8法检测各组细胞的增殖情况。结果成功构建了plVTHM-miR-196b慢病毒表达载体;载体中绿色荧光蛋白有较好的表达活性;与对照组细胞相比,试验组miR-196b表达水平有显著提升(P<0.05);K562细胞稳定过表达miR-196b后增殖水平显著下降(P<0.05)。结论成功构建了miR-196b过表达载体,并在K562细胞内稳定高表达,miR-196b有抑制K562细胞增殖的作用。

Objective To construct miR-196b lentiviral expression vector to lay the foundation for the study of miR-196b function in chronic myeloid leukemia and its role in the development of CML.Methods The precursor sequence of miR-196b was amplified by PCR with human bone marrow genomic DNA as a template,vector plVTHMmiR-196b was recombinant with lentiviral vector plVTHM,packaged and titered.The transfection efficiency and reporter gene expression efficiency of K562 cells infected with the virus solution were observed by fluorescence,and the GFP-positive cells with a green fluorescence were sorted by flow cytometry,and intracellular miR-196b expression level was detected using real-time quantitative PCR.Finally,proliferation of cells in each group was detected by CCK-8 assay.Results lVTHM-miR-196b lentiviral vector was successfully constructed ; the carrier had a better expression of GFP activity; compared with control cells,miR-96b expression levels of the test group were significantly increased.The proliferation levels of K562 cells stably over-expressing miR-196b decreased significantly.Conclusions The miR-196b overexpression vector has been successfully constructed and highly and stably expressed in K562 cells,and miR-196b can inhibit the proliferation of K562 cells,these results conduct a foundation for the corresponding function of miR-196b in CML cells and in vivo.