miR-33b表达载体构建及表达活性检测

Construction of miR-33b expression vector and expression activity detection

目的:构建miR-33b真核表达载体及其表达活性检测。方法:人工合成miR-33b成熟cDNA序列,以GV251为载体,构建miR-33b真核表达载体,并对重组克隆进行测序确保载体构建成功;采用脂质体转染法将miR-33b表达载体导入人胃癌细胞MKN-28中,通过倒置荧光显微镜观察转染效率,再利用实时定量RT-PCR检测miR-33b的表达,以检测外源基因的表达活性。结果:成功构建了miR-33b真核表达载体;转染细胞后采用倒置荧光显微镜观察表明载体中GFP的表达活性较好;实时定量RT-PCR结果表明,相较于对照组细胞,miR-33b转染组中miR-33b表达显著增加,且具有显著性差异(P<0.05)。结论:成功构建miR-33b真核表达载体,并为进一步研究miR-33b参与胃癌发生发展机制奠定基础。

Objective： To construct the miR-33b eukaryotic expression vector and detect its expression activity. Methods： The miR-33b mature c DNA sequences were composited manually and inserted into the GV251 vector,which was the construction of miR-33b eukaryotic expression vector,and the recombinant clone was sequenced to ensure the success of vector construction. miR-33b expression vector was transfected into human gastric cancer cells MKN-28 by liposome transfection method. Then,inverted fluorescence microscope was used to observe the transfection efficiency,and quantitative real-time PCR was used to detect miR-33 b expression and expression activity of exogenous gene. Results： miR-33b eukaryotic expression vector was constructed successfully. Inverted fluorescence microscope observation of transfection cell with GFP showed that expression activity was fine. Real-time quantitative PCR results showed that miR-33b expression in experimental cell groups were increased significantly compared with control groups（ P 〈 0. 05）.Conclusion： The results showed that miR-33b eukaryotic expression vector was constructed successfully,and established a foundation for the further study the mechanism of miR-33b in gastric cancer development.