Application of PCR-based tRNAval Pol III-shRNA expression cassettes in screening of efficient siRNA for inhibiting hepatitis B virus / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs).</p><p><b>METHODS</b>Based on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR.</p><p><b>RESULT</b>Co-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration.</p><p><b>CONCLUSION</b>The expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.</p>

Inhibition of HBs-GFP fusion gene expression by RNA interference / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop an effective report gene system to test the effect of small interfering RNA (siRNA).</p><p><b>METHODS</b>HBV S gene was fused with enhanced green fluorescent protein (EGFP) gene to form HBs-GFP and the plasmid containing HBs-GFP was constructed. A vector expressing small hairpin RNA (shRNA) pAVU6 + 4sh357 was also constructed. Two plasmids were co-transfected into HepG2 cells transiently. The fluorescence of HBs-GFP was detected by fluorescence-activated cell sorting (FACS). The mRNA expression in HepG2 cells was detected by conventional RT-PCR and real-time PCR.</p><p><b>RESULTS</b>siRNA inhibited the expression of HBs-GFP 72 hours post transfection. The fluorescence of HBs-GFP in HepG2 cells treated with pAVU6+4sh357 was reduced by 55.4% compared with that of controls. The HBs-GFP expression in HepG2 cells treated with pAVU6+4sh357 was reduced by 76.3% and 90% as measured with conventional RT-PCR and real-time PCR, respectively.</p><p><b>CONCLUSION</b>This investigation demonstrated siRNA derived from shRNA expression vectors can inhibit the expression of HBs-GFP in HepG2 cells.</p>

Establish a screening system for selection of mRNA target sites for HBsAg to construct siRNA with shRNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To find some effective short interfering RNA's sites targeting HBV surface gene sequence using shRNA expression vectors.</p><p><b>METHODS</b>Four shRNA expression vectors targeting HBV surface gene sequence were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with HBs-EGFP fusion gene plasmid. The changes of HBs-EGFP image were detected by FACS and microscopy. The HBs-EGFP mRNA expression was evaluated by RT-PCR.</p><p><b>RESULTS</b>Four shRNA expression vectors and HBs-EGFP fusion gene plasmid were successfully constructed. pAVU6 + 4sh579 vector inhibited the HBs-EGFP expression by 69.8% in AD293 and suppressed the HBs-EGFP mRNA expression by 74.6%.</p><p><b>CONCLUSIONS</b>The results showed that the 579 site of HBV surface gene sequence was an effective target and pAVU6 + 4sh579 vector could suppress the HBs-EGFP expression in AD293 cells</p>