Screening And Research On Anti-HBV Drugs

Objective:(1)To screen samples for discovering anti-HBV lead samples by using HepG2.2.15 cell cultured system in vitro.(2)The primary research on anti-HBV mechanism of the samples were done: HBV DNA copy numbe;the inhibition rate of HBV DNA polymerase;the inhibition rate of HBsAg,HBeAg in nude which was iNoculated by HepG2.2.15 cells.(3)To establish a cell model of drug screening for HBX protein.Methods:Cytoxicity of compounds on HepG2.2.15 cell was detected using MTT, the levels of HBsAg or HBeAg in the culture supernant of HepG2.2.15 cell were tested by ELISA.Adefovir was selected as a positive anti-HBV compound.Total 162 samples were screened in the primary screening.Among the active samples found in the primary screening,two compounds flavoNoids were selected for further evaluation of anti-HBV mechanism:Using The content of total HBV DNA were quantified by Taqman probe real-time PCR;HBV DNA polymeras was detected by Isotope method; HBsAg and HBeAg in serum of nude mice iNoculated by HepG2.2.15 cells was detected by ELISA.Construction of plasmid pTAL-Enhâ… -SEAP:PCR amplification sequence HBV- Enhâ… was inserted into secreted alkaline phosphatase(SEAP) expression vector,pTAL-Enhâ… -SEAP and HBX were transfected collectively to HepG2 cell line to detect the effect of inhibition for HBX protein.Results:(1) 162 samples were screened and re-screened.Therapeutic index(TI): 21 samples between 1 and 2,19 samples between 2 and 10,2 samples ≥10.(2) FlavoNoids No.322:TC₅₀＞200μg/ml,therapeutic index of HBsAg were 5.5±0.7.No.322 have more better inhibition for HBsAg than Adefovi in the same concentration.No.223:TC₅₀＞200μg/ml,HBsAg was 4.3±0.7.(3) HBV DNA copy number were 0.7±0.6×10⁵ copy/ml and 1.7±0.2×10⁶copy/ml when FlavoNoids No.322 were at Nontoxic concentrations 50μg/ml,25μg/ml.HBV DNA copy number were 3.9±0.6×10⁵copy/ml and 6.2±0.7×10⁵copy/ml when FlavoNoids No.223 were at Nontoxic concentrations 50μg/ml and 25μg/ml.(4) The inhibition rate of HBV DNA polymerase was 39.6±7.1%when FlvoNoids No.322 was at 30μg/ml.(5) The inhibition rates of FlavoNoids No.322 at 90mg/kg group were 45.0±12.8%,10.0±4.2%on HBsAg and HBeAg of nude mice which was iNoculated by HepG2.2.15 cells,HBsAg inhibition rate was 18.8±8.6% at 30mg/kg group.(6) Gene Bank showed that recombinant plasmid pTAL-Enhâ… -SEAP was digested by restriction endonuclease Mluâ… ,Bglâ…¡,inserted sequence had a same sequence with HBV Enhâ… .Conclusions:(1) 162 samples screening were finished.42 effective samples were found to have anti-HBV activity in vitro.(2) FlavoNoids No.223,No.322 have anti-HBsAg activity in vitro.(3) FlavoNoids No.223,No.322 have inhibition effects on HBV DNA copy number in vitro.No.322 have an obvious inhibition effects on HBV DNA copy number in Non-toxic concentrations(50μg/ml).(4) FlavoNoids No.322 at 30μg/ml concentrations have obvious inhibition effects on HBV DNA polymerase.HBV DNA polymerase is the target of FlavoNoids No.322.(5) FlavoNoids No.322 have an obvious inhibition effects on HBsAg in serum of nude mice which was inoculated by HepG2.2.15 cells.(6) The cell model of drug screening for HBX protein was established.