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目的构建miR-145的真核表达载体,为研究miR-145在结肠癌中的生物学功能奠定基础。方法设计并应用PCR扩增miR-145基因片段,将其导入真核表达载体pCMV-myc中构建重组质粒pCMV-miR-145后,将重组质粒转染入结肠癌细胞系HCT116中,运用RT-PCR检测miR-145的表达情况。结果酶切及DNA测序证实miR-145被正确克隆入真核表达载体pCMV-myc中,该重组质粒能在HCT-116细胞中高效表达miR-145。结论成功构建了miR-145的真核表达载体pCMV-miR-145,该载体能有效高表达miR-145。
Objective To construct eukaryotic expression vector of miR-145 and lay a foundation for studying the biological function of miR-145 in colon cancer. Methods The gene fragment of miR-145 was amplified by PCR and inserted into eukaryotic expression vector pCMV-myc to construct recombinant plasmid pCMV-miR-145. The recombinant plasmid was transfected into human colon cancer cell line HCT116. RT- PCR detection of miR-145 expression. Results Enzyme digestion and DNA sequencing confirmed that miR-145 was correctly cloned into the eukaryotic expression vector pCMV-myc. The recombinant plasmid can efficiently express miR-145 in HCT-116 cells. Conclusion The eukaryotic expression vector pCMV-miR-145 of miR-145 was successfully constructed, which can efficiently express miR-145.