Selection And Validation Of Optimal SIRNA Target Sites For Hepatitis B Virus X Gene Mrna By Use Of The PSOS Vector System

Objective: To construct the eukaryotic expression vector pSOS-X-siRNA and pSOS-siRNA targeting for Hepatitis B virus (HBV) X gene. After transfecting pSOS-x-siRNA and pSOS-siRNA into the hepatocellular carcinoma cell lines HepG2 and HepG2.2.15 respectively, We can screen and validate the HBx short interfering RNAs。Methods:To design and synthesis the specific four siRNAs for targeting HBx gene, the recombinant pSOS-x-siRNA was obtained by inserting the HBX gene and siRNA into pSOS. The pSOS-siRNA vector was produced through digesting by PacI and removing the HBx gene of pSOS-x-siRNA system. The plasmids pSOS-x-siRNA were transfected into HepG2 and the plasmids pSOS-siRNA were transfected into HepG2.2.15 respectively. The knockdown efficiency of siRNA can be assessed through observing the fluorescence intensity in HepG2 cells under the fluorescence microscope. ELISA detected the expression of HBsAg and HBeAg in supernatant,Western-blotting detected the expression of HBsAg and HBcAg in intracellular and Real Time PCR detected the expression of the HBs mRNA and HBx mRNA in HepG2.2.15 cells。Results:At 4 days after transfection , the fluorescence intensity of siRNA4 transfected HepG2 cells was lowest and reduced more than 90% . Similarly, The siRNA4 tranfected HepG2.2.15 cells, the expression of HBsAg and HBeAg in supernatant were decreased to 13.92±1.94%(p<0.05)and 21.69±4.92%(p<0.05). The levels of HBsAg and HBcAg in intracellular still were inhibited to 0.110±0.018 ( p<0.05 ) and 0.085±0.028(p<0.05), compared with the control. Furthermore, the levels of the HBs mRNA and HBx mRNA were noticeably reduced to 0.237±0.028(p<0.05)and 0.110±0.022 (p<0.05),compared with control. siRNA4 have the most marked gene- silencing efficiencies.Conclusion:The system of screening and validating siRNA targeting for HBx mRNA was constructed successfully by use of the pSOS vector system.