关键词: miR-196b, 慢病毒表达载体, 载体构建

Abstract: 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 plVTHM-miR-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 plVTHM-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. Conclusion The miR-196b overexpression vector had 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.

Key words: miR-196b, lentiviral vector, vector construction