Construction and identification of Hepatitis B Virus (genotype C) recombinant baculovirus / 中华传染病杂志

ABSTRACT

Objective To construct Hepatitis B Virus (HBV) Chinese-strain wild type and Lamivudine-resistant variant recombinant baculovirus. Methods 1.2-unit length HBV construct (adr serotype, C genotype) was cloned into the multiple cloning region of the pFastBac Ⅰ vector and then transformed into DH10Bac, in which homologous recombination occurred between the pFastBac vector and the inside bacmid with the help of helper plasmid. So HBV recombinant bacmid was produced and transfected into sf9 cells to generate a Recombinant HBV baculovirus called vAcHBVc. The viral titers were detected by immune plaque assay and TCID 50. Lamivudine resistance mutation (YVDD) was introduced by site-directed mutagenesis using continuous PCR. HBV YVDD recombinant baculovirus (vAcHBVcYVDD) was generated and titered as above. Meanwhile, the recombinant HBV baculovirus containing GFP reporter was constructed for evaluating the efficacy of transfection and titration of virus. Results Recombinant wild and YMDD variant HBV baculovirus were successfully developed and the titer was between 10~6～10~8pfu/ml through plaque assay. Conclusion Different kinds of recombinant HBV baculovirus could be generated rapidly and efficiently with Bac to Bac expression system. The developed recombinant HBV baculovirus could be further used in the study of anti-viral resistances.