Synthesis of recombinant HBV cccDNA
Recombinant HBV cccDNA was generated utilizing minicircle technology offered by System Biosciences. In short, the genetic material of HBV genotype D was inserted into a minicircle-producing plasmid equipped with attB and attP recombination sites, known as mini-HBV. The engineered mini-HBV construct was introduced into E. coli strain ZYCY10P3S2T (System Biosciences) and specific clones were selected, followed by a 4 h incubation at 37°C in kanamycin-supplemented Luria broth (LB). The resulting cell suspension was transferred into 200 mL of TB media and incubated overnight. This culture was then combined with 200 mL of induction media (composed of 1 N NaOH and 0.2% L-arabinose in LB), and incubated for 3 h at 30°C, followed by an additional 1 h incubation at 37°C. To isolate HBV rcccDNA, the resulting bacterial pellet was processed using the QIAGEN Plasmid Maxi Kit (Qiagen). Methylation of rcccDNA was performed using M.SssI CpG methyltransferase (SibEnzyme) as described previously 7,14,23.