In-vitro Experiment On HBV S Gene Express HbsAg Independently Without CccDNA

Objective:Nucleoside analogues(acid)(NAs)antiviral treatment's ultimate goal is to realize cccDNA clearance clinically,and quantitative detection of HBV cccDNA is judge antiviral efficacy even cure the ideal indicator of HBV infection.However,quantitative detection of cccDNA requires liver biopsy,which is risky and contraindicated.Therefore,HBsAg,the indirect product of cccDNA,was used as the alternative indicator in the major guidelines.In our long term follow up cohort of CHB patients treated with NAs antiviral therapy,we observed that about 80% CHB patients who obtained sustained viral response after a long time(at least 3 years)NAs antiviral therapy got a gradually decrease in HBsAg level,while another 20% patients' HBsAg maintained after declining to a certain high level,and a minority of them have no viral relapse for a long time even drug withdraw.This part of patients did not follow the rule that the levels of HBsAg is parallel to HBV DNA and cccDNA.According to this result,we could deduce that there are an bybass of HBsAg expression.If HBsAg does have an expression bypass that does not depend on cccDNA,then HBsAg has limitations as the only indicator of cccDNA transcriptionalactivity.Therefore,the main purpose of this study is to establish an in vitro model that can simulate independent expression pathways of HBV S gene without cccDNA.This model can be used to explore the existence of HBsAg transcription and integration pathway in human,and the HBV S gene fragment integrated into the host cell can independently express HBsAg.Method: In this study,by using gene recombination and eukaryotic gene expression technology,we extrcated the total DNA from the serum of chronic hepatitis B patients with HBV DNA positive.Using the total DNA as a template,HBV S gene was amplified by the HBV S gene specific primers.Ligating the HBV S gene fragment with the plasmid vector pcDNA3.1-GFP to construct recombinant HBsAg expression vector plasmid pcDNA3.1 – S-GFP.The recombinant plasmid was transfected into Huh7 and HepG2 cell lines that were not infected with HBV and then observe the expression of HBsAg after transfection.Results:(1)HBV S gene was successfully amplified by specific primers.(2)Recombinant HBsAg expression vector plasmid pcDNA3.1-S-GFP was successfully constructed.(3)The recombinant expression vector plasmid pcDNA3.1-S-GFP was transfected into HepG2 and Huh7 cells successfully.(4)Test results of supernatant cell culture after transfection: In the transfection of cell culture supernatant,the expression of HBsAg in the experimental group was tested positively by Elisa method and HBsAg expression was 0 in the blank group and the control group.Then,the quantitative test results of roche method are as follow: The detection amount of HBsAg in the blank group and the control group was 0.In the experimental group,the detection amount of HBsAg before transfection was 0.In the Huh7 cell line,the detection value of HBsAg was 0.535±0.137 IU/ml at 48 h after transfection,0.495±0.144 IU/ml at 72 h,and 0.457±0.172 IU/ml after 96 h;in the HepG2 cell line,after 48 h transfection the value of HBsAg was 0.488±0.137 IU/ml,it was0.453±0.134 IU/ml after 72 h,and 0.429±0.126 IU/ml after 96 h;(5)Within 96 h after transfection,the effect of transfection time on HBsAg expression was not statistically significant.(P>0.05);The effect of transfected cell line on the expression of HBsAg was not statistically significant(P>0.05).Conclusion:(1)Recombinant HBsAg expression plasmid vector construction was successful,and the in vitro model of HBV S gene independent on cccDNA expression HBsAg was constructed.(2)Indeed there may be a bypass pathway for HBsAg expression:expression bypass that does not rely on cccDNA transcription.