Construction, Identification of Bcl-2 RNAi Expression Plasmid and Interference Effects on Raji Cells

Objective To construct short hairpin RNA (shRNA) interfering eukaryotic expression plasmid vector specific to bcl-2 gene and detect the interfering effects on B-lymphoma Raji cell. Methods Two pairs of complementary shDNA sequence were designed and synthesized according to bcl-2 cDNA from GenBank. After annealing, complementary double strands were formed and cloned into pGenesil-1 vector to construct recombinant plasmids, which were identified by restriction enzyme digestion and DNA sequence analysis. Recombinant plasmids were transfected into Raji cells mediated by 1ipofectamineTM 2000, then bcl-2 mRNA expression and cell apoptotic rate was detected by RT-PCR and flow cytometry respectively. Results The results of restriction enzyme digestion and sequence analysis showed that pGenesil-1-bcl-2-544 and pGenesil-1-bcl-2-1009 plasmid vectors specific to bcl-2 shRNA were successfully constructed. At the forty-eighth hour post-transfection, bcl-2 mRNAs expression of Raji cells were reduced to (31.95±3.02)% and (47.57±2.88)% in pGenesil-1-bcl-2-544 group and pGenesil-1 -bcl-2 -1009 group respectively. Compared with the control group, statistically significant difference was observed (P0.05). The ratio of apoptosis cells were (14.25±0.84) % and (11. 96±0.79)% in pGenesil-1-bcl-2-544 group and pGenesil-1-bcl-2-1009 group respectively, which was significantly higher than that in the control group(P0.05). Conclusion The eukaryotic shRNA plasmid expression vectors specific to bcl-2 gene were successfully constructed, which can silence bcl-2 gene and induce apoptosis effectively on B cell lymphoma.