DNA Engineering And Hepatitis B Virus Replication

Purpose:Recombinant DNA technology is a vital method in human hepatitis B virus(HBV),producing reporter viruses or vectors for genetransferring.Researchers have engineered several genes into the HBV genome for different purposes;however,a systematical analysis of recombinant strategy is lacking.Here,we systematically analysed the recombinant strategy of HBV,focusing on the relationship between exogenous gene recombination and the DNA replication of recombinant Hepatitis B virus.Method:First,employing a 500-bp deletion strategy,we scanned the HBV genome to identify suitable regions for recombination;Second,Ten exogenous genes(Pac,BSD,Neo,Gluc,Nluc,Cop GFP,m Cherry,Una G,e GFP and t TA1)were inserted into HBV genome via two different recombinant strategies(Recombinant strategy I and II),respectively,and the influence of exogenous DNA sequences and recombinant strategy on the DNA replication of recombinant virus were analyzed,respectively;Finally,the DNA sequences of exogenous genes were optimized to improve their compatibility with HBV,thus improving the DNA replication of rebombinant viruses.Result:1.Deletion variants D2118-2617,D2217-2723,D99-596 and D300-800 formed more RC DNA;2.The pg RNA splicing rate of D1919-2416,D2018-2515 and D2899-197 was nearly 100%;3.Recombinant strategy I allowed 4 out of 10 exogenous genes(Pac,BSD,Gluc and Nluc)to form RC DNA;4.Recombinant strategy II allowed 9 out of 10 exogenous genes(Pac,BSD,Neo,Gluc,Nluc,Cop GFP,Una G,e GFP and t TA1)to form RC DNA;5.The DNA-sequence optimization of Una G gene allowed RC DNA formation;6.The DNA-sequence optimization of Pac gene promoted SS DNA completion.Conclusion:1.Region I(nt 2118-2723)and Region II(nt 99-800)are the most suitable regions of HBV for recombination;2.Regions from nt 1919 to 2515 and from nt 2899 to 197 are both important to protect authentic pg RNA from splicing;3.Due to its less impact on the function of h M,Recombinant strategy II holds more exogenous genes than Recombinant strategy I to form RC DNA;4.When the recombinant strategy is fixed,DNA sequence of exogenous gene determinates its compatibility with HBV;5.DNA-sequence optimization of exogenous gene could partially improve its compatibility with HBV.