Synthesis Of A Novel Anti-HBV Compound And Primary Study Of Its Mechanism Of Action

Objective: To synthesize a novel nucleoside analogue β-L-D4A and explore its inhibition effect on hepatitis B virus (HBV) in 2.2.15 cells.Methods: β -L-D4A was stereo-controlled synthesized from D-glutamic acid, and the structure was identified by IR, MS and NMR. 2.2.15 cells were used and treated with various concentrations of β -L-D4A. Then the upper mediums were used for the estimation of HBsAg and HBeAg by enzyme linked immunosorbent assay. At the same time, intracellular DNA was extracted and subjected to Southern blot, hybridized with a ³²P-labeled HBV probe and autoradiographed. The intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED₅₀ was calculated. Compound's cytotoxicity of different concentrations was examined by MTT method, then ID50 was calculated.Results: β -L-D4A was successfully synthesized and confirmed by IR, NMR and MS. Autoradiographic bands showed similar for supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. EC50 of 3 -L-D4A was 0.2 μM. The experiment of cytotoxicity gained its IC₅₀ 200 μM.Conclusion: 3 -L-D4A has been synthesized successfully. 3 -L-D4A possesses a potent inhibitory effect on the replication of HBV in vitro with little cytotoxicity, TI value is 1000. It is expected to be developed into a new anti-HBV drug. Objective: To investigate four expression vectors carrying enhanced green fluorescent protein (EGFP) gene, which under the control of different HBV promoters, detect and compare their expressions in different cell lines, in order to establish a suitable cell model for the study of anti-HBV drug's mechanism of action.Methods: Four HBV promoters (including enhancers) were amplified by PCR, subcloned into the expression vector pEGFP-1. The four recombinants controlled by HBV promoters were analyzed and identified by restriction enzymes and sequencing. After transfection into human hepatoma cell lines and non-liver cells, transfection efficiency was measured by the expression of EGFP through fluorescence microscopy and analyzed with fluorescence activated cell sorter (FACS).Results: All target fragments were separately got and successfully cloned into the expression vector. The transfection results showed that in hepatoma cells, the expression of EGFP was more obvious than it was in non-hepatic cells. Among the four promoters, S gene promoter presented the strongest transfection efficiency. After screening with G418, only the three hepatoma cell lines transfected by pEGFP-Sp and pEGFP-Xp got the stable clones.Conclusion: Our findings indicate that HBV promoters lead the highest expression in hepatoma cells. The three hepatoma cell lines transfected by pEGFP-Sp and pEGFP-Xp can be used for the study of the drug's mechanism of action, which indicates that hepatoma cells might be the best cell model for the study of HBV's cis-acting elements.