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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 miR-33b eukaryotic expression vector and test its expression activity. Methods: The miR-33b mature cDNA sequence was synthesized and the miR-33b eukaryotic expression vector was constructed with GV251 as a vector. The recombinant clone was sequenced to ensure successful construction of the vector. The miR-33b expression vector was introduced into In human gastric cancer cell line MKN-28, the transfection efficiency was observed by inverted fluorescence microscopy, and the expression of miR-33b was detected by real-time quantitative RT-PCR to detect the expression activity of the foreign gene. Results: The eukaryotic expression vector of miR-33b was constructed successfully. The expression of GFP in transfected cells was observed by inverted fluorescence microscope. The results of real-time quantitative RT-PCR showed that compared with the control group, miR-33b The expression of miR-33b in the transfection group was significantly increased, with significant difference (P <0.05). Conclusion: The miR-33b eukaryotic expression vector was successfully constructed and laid the foundation for further study on the mechanism of miR-33b involved in gastric carcinogenesis.