关键词: miR-30a, 慢病毒表达载体, 载体构建

Abstract: Objective: To construct lentiviral miR-30a expression vector for the study of the mechanism of miR-30a in chronic myeloid leukemia and the impact to imatinib resistance. Methods: Human bone marrow genomic DNA was extracted, miR-30a precursor sequence was amplificated and cloned into the lentivirus expression vector plVTHM, then sended sequencing. The recombinant vector was packaged in 293T cells and tittered in K562 cells. GFP-positive K562 cells with green fluorescence were sorted by flow cytometry, miR-30a expression levels in sorted cells were further detected using real-time quantitative PCR. The proliferation of different cells was detected by CCK-8 assay. Results: Sequencing of the recombinant lentiviral expression vector was completely correct; viral titer was 6 × 106TU/mL; 160μL virus solution / 1mL medium was selected to infect K562 cells; the expression level of miR-30a in infected K562 cells was significantly enhanced(P<0.05); the proliferation levels of cells overexpressed miR-30a decreased significantly(P<0.05). Conclusion: MiR-30a lentivirus vector was successfully constructed and miR-30a could be stably expressed in K562 cells, and miR-30a could inhibit the proliferation level of K562 cells.

Key words: miR-30a, lentiviral vector, vector construction