Objective: To explore the cleaving and inhibitory activity of hepatitis C virus ( HCV) -specific deoxyri-bozymes (DRz) at both molecular and transgeneic cellular levels. Methods: According to the secondary structure o...Objective: To explore the cleaving and inhibitory activity of hepatitis C virus ( HCV) -specific deoxyri-bozymes (DRz) at both molecular and transgeneic cellular levels. Methods: According to the secondary structure of HCV 5'-noncoding region (5'-NCR) and the sites characterized with 5'…Y ↓ R…3'(Y = A/G,R = U/C) , HCV-spe-cific naive deoxyribozymes were designed and named DRz-232, DRz-127, DRz-84, DRz1, and the phosphorothioate deoxyribozymes (PSDRz) and mutated phosphorothioate deoxyribozymes (MPSDRz) were also designed. HCV RNA 5'-NCR was transcribed in vitro from linearized plasmid pHCV-neo and radiolabelled at its 5'-end. DRz, PSDRz or MPSDRz was respectively mixed with the substrate RNA and incubated under appropriate conditions, the cleaved products were displayed by 8% denaturated polyacrylamide gel electrophoresis (PAGE) and autoradiography, and the optical density of each band was measured to calculate cleavage rates. After that, every kind of DRz was added respectively to the cultured transgeneic HepG2 cells containing luciferase gene controlled by HCV 5'-NCR. The cells were lysed at intended time points and the activity of luciferase was measured with chemiluminescence method for calculating inhibition rates. Results: After incubated for 90 min in vitro, the cleavage rates of DRz-127, PSDRz-127, DRz1 and PS-DRz1 reached 32.6% , 30. 8% , 24. 3% and 21. 5% , respectively. No cleavage product was observed in any MPSDRz. DRz-127, PSDRz-127, DRz1 and PSDRzl had an inhibitory rate of 53. 2% , 50. 6% , 44. 7% and 43. 3% respectively in transgeneic HepG2 cells in the first 24 h when the final dose of the DRz was 0. 5μmol/L, higher than that of the corresponding MPSDRz. There was no significant difference between the inhibitory effect of each DRz and its PSDRz in HepG2 cells, but the inhibitory rate of DRz decreased more rapidly than that of the latter with the elapse of time. The results from transfection groups were significantly better than those of non-transfection groups. Conclusion: Rationally-designed HCV-specific deoxyribozymes are able to cleave target RNA at molecular level in vitro, and efficiently inhibit the expression of luciferase gene controlled by HCV 5'-NCR in transgeneic cells. Appropriate PSDRz may be more stable, and thus more suitable than the naive DRz in the application to cells. Introduction of the deoxyribozymes with transfection is more efficient than with direct delivering ways.展开更多
HBV Pre S1 sequence is supposed to play an important role in the infection of HBV. Presence of Pre S1 /anti-Pre S1 in serum has valuble clinical imphations. In order to improve the study of Pre S1, Pre S1 sequence was...HBV Pre S1 sequence is supposed to play an important role in the infection of HBV. Presence of Pre S1 /anti-Pre S1 in serum has valuble clinical imphations. In order to improve the study of Pre S1, Pre S1 sequence was overexpressed in E. coli as a fusion protein with MBP (Maltose- binding Protein), and anti-Pre S1 antiserum was elicited in rabbits by Pre S1 MBP purified by affinity chromatography. The recombinant plasmid constructed from PMAL-cRI expressed the 106aa Pre S1 sequence at the C terminal of MBP by tac promoter. The resulting Protein is about 54kD in size. Western-blot analysis confirmed its reactivity with antiserum derived from synthetic Pre S1 peptide and serum from patients with acute hepatitis B (AHB). ELISA showed that Pre S1-MBP and Dane Particles purified from AHB patient’s serum reacted with antiserum against synthetic Pre S1 peptlde,and this reaction was specifically inhibited by synthetic Pre S1 peptide. ELISA also demonstrated that antiserum against Pre S1-MBP reacted with synthetic Pre S1 peptide, but not with synthetic HCV peptide or HEV peptide.展开更多
文摘Objective: To explore the cleaving and inhibitory activity of hepatitis C virus ( HCV) -specific deoxyri-bozymes (DRz) at both molecular and transgeneic cellular levels. Methods: According to the secondary structure of HCV 5'-noncoding region (5'-NCR) and the sites characterized with 5'…Y ↓ R…3'(Y = A/G,R = U/C) , HCV-spe-cific naive deoxyribozymes were designed and named DRz-232, DRz-127, DRz-84, DRz1, and the phosphorothioate deoxyribozymes (PSDRz) and mutated phosphorothioate deoxyribozymes (MPSDRz) were also designed. HCV RNA 5'-NCR was transcribed in vitro from linearized plasmid pHCV-neo and radiolabelled at its 5'-end. DRz, PSDRz or MPSDRz was respectively mixed with the substrate RNA and incubated under appropriate conditions, the cleaved products were displayed by 8% denaturated polyacrylamide gel electrophoresis (PAGE) and autoradiography, and the optical density of each band was measured to calculate cleavage rates. After that, every kind of DRz was added respectively to the cultured transgeneic HepG2 cells containing luciferase gene controlled by HCV 5'-NCR. The cells were lysed at intended time points and the activity of luciferase was measured with chemiluminescence method for calculating inhibition rates. Results: After incubated for 90 min in vitro, the cleavage rates of DRz-127, PSDRz-127, DRz1 and PS-DRz1 reached 32.6% , 30. 8% , 24. 3% and 21. 5% , respectively. No cleavage product was observed in any MPSDRz. DRz-127, PSDRz-127, DRz1 and PSDRzl had an inhibitory rate of 53. 2% , 50. 6% , 44. 7% and 43. 3% respectively in transgeneic HepG2 cells in the first 24 h when the final dose of the DRz was 0. 5μmol/L, higher than that of the corresponding MPSDRz. There was no significant difference between the inhibitory effect of each DRz and its PSDRz in HepG2 cells, but the inhibitory rate of DRz decreased more rapidly than that of the latter with the elapse of time. The results from transfection groups were significantly better than those of non-transfection groups. Conclusion: Rationally-designed HCV-specific deoxyribozymes are able to cleave target RNA at molecular level in vitro, and efficiently inhibit the expression of luciferase gene controlled by HCV 5'-NCR in transgeneic cells. Appropriate PSDRz may be more stable, and thus more suitable than the naive DRz in the application to cells. Introduction of the deoxyribozymes with transfection is more efficient than with direct delivering ways.
文摘HBV Pre S1 sequence is supposed to play an important role in the infection of HBV. Presence of Pre S1 /anti-Pre S1 in serum has valuble clinical imphations. In order to improve the study of Pre S1, Pre S1 sequence was overexpressed in E. coli as a fusion protein with MBP (Maltose- binding Protein), and anti-Pre S1 antiserum was elicited in rabbits by Pre S1 MBP purified by affinity chromatography. The recombinant plasmid constructed from PMAL-cRI expressed the 106aa Pre S1 sequence at the C terminal of MBP by tac promoter. The resulting Protein is about 54kD in size. Western-blot analysis confirmed its reactivity with antiserum derived from synthetic Pre S1 peptide and serum from patients with acute hepatitis B (AHB). ELISA showed that Pre S1-MBP and Dane Particles purified from AHB patient’s serum reacted with antiserum against synthetic Pre S1 peptlde,and this reaction was specifically inhibited by synthetic Pre S1 peptide. ELISA also demonstrated that antiserum against Pre S1-MBP reacted with synthetic Pre S1 peptide, but not with synthetic HCV peptide or HEV peptide.
