AIM: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro. METHODS: HepG2 2.2.15 cells in cult...AIM: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro. METHODS: HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold over- length plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS: The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION: Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.展开更多
Using cotyledonary petioles as explants, the PAP cDNA controlled by wound-in-ducible promoter has been introduced into Brassica napus by coculture with Agrobacterium tumefaciens and laser microbeam puncture respective...Using cotyledonary petioles as explants, the PAP cDNA controlled by wound-in-ducible promoter has been introduced into Brassica napus by coculture with Agrobacterium tumefaciens and laser microbeam puncture respectively. Regenerated plants resistant to gen-tamycin have been selected out. PCR amplification and Southern blotting analysis indicated that PAP cDNA together with wound-inducible promoter had been integrated into Brassica genome with transformation frequencies of 2.0% and 1.7% for two transformation methods respectively. The test of virus challenge showed that these transgenic Brassica plants were resistant in different degrees to mechanically inoculated TuMV.展开更多
Crystals of pokeweed antiviral protein (PAP) from seeds of Phytolacca americana with high diffraction ability were grown from high protein concentration (100 mg/mL) solution at high temperature (33℃). The crystal str...Crystals of pokeweed antiviral protein (PAP) from seeds of Phytolacca americana with high diffraction ability were grown from high protein concentration (100 mg/mL) solution at high temperature (33℃). The crystal structure was solved by use of molecular replacement method and refined by use of molecular dynamic method at 0 25 nm to an R factor of 18.15% with standard deviations from standard geometry of 0.001 6 nm and 2.04° for bond lengths and bond angles, respectively. Comparison with two other PAPs revealed, near the active center, a sequence and structure variable region, consisting of the loop connecting the fifth β strand with the second α helix and including a proposed active residue, suggesting this loop probably to be related to difference in activity.展开更多
基金the National Natural Sciences Foundation ofChina, No. 30671860
文摘AIM: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro. METHODS: HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold over- length plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS: The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION: Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.
文摘Using cotyledonary petioles as explants, the PAP cDNA controlled by wound-in-ducible promoter has been introduced into Brassica napus by coculture with Agrobacterium tumefaciens and laser microbeam puncture respectively. Regenerated plants resistant to gen-tamycin have been selected out. PCR amplification and Southern blotting analysis indicated that PAP cDNA together with wound-inducible promoter had been integrated into Brassica genome with transformation frequencies of 2.0% and 1.7% for two transformation methods respectively. The test of virus challenge showed that these transgenic Brassica plants were resistant in different degrees to mechanically inoculated TuMV.
文摘Crystals of pokeweed antiviral protein (PAP) from seeds of Phytolacca americana with high diffraction ability were grown from high protein concentration (100 mg/mL) solution at high temperature (33℃). The crystal structure was solved by use of molecular replacement method and refined by use of molecular dynamic method at 0 25 nm to an R factor of 18.15% with standard deviations from standard geometry of 0.001 6 nm and 2.04° for bond lengths and bond angles, respectively. Comparison with two other PAPs revealed, near the active center, a sequence and structure variable region, consisting of the loop connecting the fifth β strand with the second α helix and including a proposed active residue, suggesting this loop probably to be related to difference in activity.
