ABSTRACT
Objective:To stablish of chemiluminescent Southern blot detection system for examining HBV DNA replication intermediates in HepG2.2.15,and analyse the inhibition of HBV replication with three kind of drugs with different targets.Methods:The HBV DNA replication intermediates were extracted and analyzed by Southern blot with HBV probe,which(pTHBV1047) was labelling with digoxigenin.The results of the hybridization were detected by chemiluminescent,and the condition of hybridization was optimized.After treated with lamivudine,Bay41-4109,?-Galcer in different concentration,the HBV DNA from the HepG2215 cells were detected with the system.Results:the sensitivity of the system was 1pg of pTHBV1047,and HBV specific positive signals was detected with the DNA from HepG2.2.15.The three kinds of drugs can inhibit the HBV replication obviously with chemiluminescent Southern blot detection system,the IC50 were 1.53?mol/L,0.41?mol/L,0.01?mol/L.Conclusion:The HBV replication intermediates from the cell of HepG2.2.15 can reflect the antiviral effect accurately with different targets drugs and this mothod would be used in the study of Chinese-midicine.
ABSTRACT
Objective:To improve the genetic stability of HBV gene in transgenic mice.Methods:HBV transgenic mice were bred by backcross and double cross.The HBV gene expression and replication were studied with real-time PCR,ELISA and chemiluminescence.Results:The HBV transgenic mice have stably bred to 23rd generation.The serum HBsAg level is 4122.31?2044.74IU/ml;The rate of HBV transgenic mice whose serum HBV DNA reach 104~106copies/ml was 93.93%.The HBV replication and expression were improved markedly.There is no difference between male and female mice about serum HBsAg level.Conclusion:After breeding the HBV gene was expressed stably with high-level in transgenic mice.
ABSTRACT
Objective: To screen candidate antigen for therapeutic HBV vaccine with a novel adjuvant DC-Chol. Methods: BALB/c mice were injected with DC-Chol liposome and HBsAg prepared from CHO and Yeast respectively. One week later, IL-4, IL-2, IFN-?were measured by ELISA or ELISPOT. Results: The levels of IL-2, IFN-?of HBsAg from Yeast with DC-Chol liposome were 20 and 119 times higher respectively than those of HBsAg from CHO with DC-Chol liposome. ELISPOT assay showed that the counts of spot-forming cells of IL-4 and IFN-?of HBsAg from Yeast with DC-Chol liposome were 2.8 and 46.3 times higher respectively than those of HBsAg from Yeast with Al(OH)3. Conclusion: HBsAg prepared from Yeast together with DC-Chol liposome may be an appropriate candidate for therapeutic HBV vaccine .