Construction, Concentration And Determination Of ShRNA Lent Virus Vector Expression In Bmi-1 Gene

Objective: To construct Bmi-1 gene lent viral vector. By transfecting 293 T cells, collect virus liquid. Concentrate liquid and determine virus titer of it. Methods: Based on short hairpin RNA(sh RNA) of Bmi-1 gene as a target, 3 sh RNA fragments of Bmi-1 gene are inserted into a lent viral vector, and screen and identify it. Transfected 293 T cells with high metastatic, collect virus liquid and concentrate. Assay titer viral titers by fluorescence. Results: Construct lent viral vector containing the Bmi-1-sh RNA. Plasmids were extracted after transformation of E. coli DH5α. After the Eco RI and Bam HI double digestion, assay agars gel electrophoresis. RNA sequencing Results confirme that recombinant lent viral vectors have some interference with the experimental design and synthesis of target chain match. Sequence analysis shows that success of the recombinant construct. The screened recombinant lent viral vector PLVX-sh RNA2-Bmi-1 with the packaging plasmid PLP1, PLP2 and PLP / VSVG transfect 293 T cells together, while the establish empty vector as control group. Virus concentration which collected after dilution infects 293 T cells, and successfully detect titer viral titers by fluorescence. Conclusion: Construct sh RNA lent viral vector of Bmi-1 gene successfully. By transfecting 293 T cells, obtain the virus liquid and concentrate, then packaged lent viral particles. Measure viral titer successfully by fluorescence after infected target cells in order to discuss function of Bmi-1 gene in nasopharyngeal carcinoma cells.