Exploration About Construction Of Fluorescence Reporting System For Replication Of Hepatitis B Virus In Vitro

Hepatitis B Virus(HBV)is a DNA virus with an envelope that is hepatotropic and can cause persistent viral infection in humans.Chronic HBV infection is one of the most serious public health problems worldwide,and is the major cause of cirrhosis and primary hepatocellular carcinoma(HCC).Although prophylactic vaccines are currently available to effectively prevent new-onset HBV infections,there are still about 248 million chronic HBV-infected people worldwide and they are difficult to be cured by existing treatments.Although the existing clinical treatment strategies based on interferon and nucleoside/nucleotide analogues and their combinations can control virus replication and alleviate disease progression,they cannot effectively achieve clinical cure(usually<5%),and there is still a risk of recurrence after withdrawal.A large number of studies have shown that the persistence of cccDNA,as a template for HBV replication,is the basis of the difficulty in curing hepatitis B infection.And for the fact that our existing drugs cannot effectively eliminate cccDNA,it is extremely urgent to develop more effective therapeutic drugs.Therefore,it is necessary to construct a cell model in vitro which can quickly detect or indicate HBV replication for high-throughput drug screening.The purpose of this study is to try to construct a cell model that uses fluorescence to indicate HBV replication,without any additional detection,but the effect of the drug can be judged simply by the change of fluorescence.Firstly,in order to have as little impact on HBV itself as possible,and considering the high degree of overlap of the HBV genome expression frames,the fragment we insert into the precore region of the HBV genome is GFP11 with only 16 amino acids,but both the fluorescence and HBV replication are weakened to a great extent.Then we optimize the fluorescence of the system.It has been reported that the split mNG system has a stronger fluorescence signal and lower background fluorescence,that is,higher signal-to-noise ratio,so we compare split GFP system with split mNG system and determine to use split mNG as the basis for subsequent improvements.Considering the inefficiency of multi-plasmid transfection,we construct a Dox-inducible "all in one"fluorescence reporting system.After preliminarily verifying the feasibility of the system,we explore to optimize the fluorescence and HBV replication efficiency of the system.In terms of fluorescence,through the comparison of different N11 variants and different numbers of N11 tandem,as well as the comparison of 2A self-cleaving peptide and 22-nt Rbm3 IRSE used for multi-cistron expression,we find that the combination of DN11-E2A was relatively better In terms of HBV replication efficiency,by replacing the pairing of A1901G mutation(CM type)with A18 52T/T1902A mutation(XM type),we find that the insertion of foreign gene has little effect on HBV replication efficiency in the case of XM type.In addition,we construct a stable integrated cell line,then,analyze the correlation between the fluorescence and HBV replication on the cell line.Finally,we compare 34 different genotypes of HBV(mainly B,C,D),and find the D-type HBV-Dtw,with higher replication level and infection ability than our current C-type S11.On the basis,the effect of exogenous gene insertion sites on HBV replication in the fluorescence reporting system is preliminarily studied on the Dtw skeleton,which reveals that the integrated HBV has a relatively stronger replication level when the insertion site is about 7 bp from the right side of the "TATAAA"-like module in the HBV poly(A)sequence.In summary,we have constructed a cell model in vitro-"all in one" fluorescence reporting system,which can indicate HBV replication by fluorescence to some extent,but the fluorescence intensity and replication level of the system are currently weak.The results of the exploration to improve the efficiency of HBV replication need to be further applied to the system and to be further verified.While in terms of fluorescence,the system needs to be further optimized.