Construction and its expression of PRPS2 shRNA plasmid

Objective To construct a series of plasmids containing the shRNA for phosphoribosyl pyrophosphate synthetase 2 and provide a basis for the study of the function of PRPS2 gene. Methods A series of shRNA for PRPS2 were designed and synthesized,and then recombined with eukaryotic expression vector GV102 as shRNA recombinant plasmids( experiment groups) and identified by sequencing. The recombinant plasmids were named as GV102- PRPS2- 1,GV102- PRPS2- 2,GV102- PRPS2- 3,respectively. The plasmids were transfected to HCT116 cells. RT- PCR and Western blot were performed to detect the expression level of PRPS2 for selecting the best plasmid. Results The sequences of shRNA recombinant plasmids were confirmed correct. The HCT116 cells expressing green fluoresce increased time- dependently in 72 h after transfection and the transfection rate was 40%- 50%. The RT- PCR and Western blot results showed that the relative expression quantities of PRPS2 in experiment groups were decreased significantly compared with negative control group( P 0. 05). The mRNA expression of PRPS2 in GV102- PRPS2- 3 group decreased by 73% to 0. 27 ±0. 05 and PRPS2 protein level decreased by 70% to 0. 30 ± 0. 04,and its interference effect was better than GV102- PRPS2- 1( mRNA: 0. 61 ± 0. 03,protein: 0. 37 ± 0. 06) and GV102- PRPS2- 2( mRNA: 0. 89 ± 0. 02,protein: 0. 84 ± 0. 05). Conclusion The study makes it successful to down-regulate the expression of PRPS2 in HCT116 cells by RNAi and lay a foundation to study the effect of down-regulation of PRPS2 on cells.