中国生物工程杂志

Objective:To construct NBS1 microRNA expressing eukaryotic recombinants, and identify biological activity of recombinants in Hela cell after transfection . Methods: According to sequence of NBS1mRNA, the NBS1 pre-microRNA was designed and synthesized, then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cell line. To detect integrity of inset fragment through colony PCR and sequencing analysis. To identify interference efficiency of NBS1 miRNA recombinants by way of Real-Time PCR 12h after thansfection and determine biological activity of recombinants . Results: Sequences of inset fragment in four microRNA expressing recombinants were correct . NBS1 mRNA expression of four microRNA recombinants were 0.24±0.17 (NBS1mi-1 recombinant), 0.12±0.12 (NBS1mi-2 recombinant), 0.41±0.97 (NBS1mi-3 recombinant), 0.48±0.93 (NBS1mi-4 recombinant),that is the lowest in the NBS1mi-2 group.Conclusion: Four NBS1-targeting microRNA expressing recombinants all have biological activity in Hela cell line, and NBS1mi-2 recombinant has the most interference efficiency. The microRNA expressing plasmidwhich were successfully constructed and lay foundation for the studies on the tumor gene therapy of microrna targeting NBS1.