Construction and Identification of NBS1-targeting microRNA Expressing Eukaryotic Vector / 中国生物工程杂志

ABSTRACT

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.The biological activity of recombinants through identify interference efficiency of NBS1 microRNA recombinants by way of Real-Time PCR and Western blot were determined.Results:Sequences of inset fragment in four microRNA expressing recombinants were correct.NBS1 mRNA and protein expression of four microRNA recombinants were decrease,which 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 plasmid which were successfully constructed and lay foundation for the studies on the tumor gene therapy of microrna targeting NBS1.