Construction, identification and pharmacodynamics research on a minicircle DNA recombinant plasmid of HBV

RAO Gui-rong; ZHANG Huan-jing; HUANG Bin; CHEN Guang-ming; WANG Hong-min; YANG Fu-qiang

Abstract：Objective To develop a therapeutic minicircle DNA plasmid of HBV and investigate their immune activity in vitro and in vivo. Methods Two genes were amplified, which separately encoded HBV middle envelope protein (preS2.S) as a vaccine and human interleukin (IL)-2/interferon (IFN)γfusion protein as an adjuvant by PCR from plasmids pcNDA3.1/preS2.S and pcDNA3.1/hIL-2-IFNγ. Then two genes were cloned into eukaryotic vector ZY781 and the parent plasmid preS2.S/ZY781and hIL-2-IFN γ/ZY781 were obtained respectively. The new plasmids were analyzed by restriction endonuclease and DNA sequencing. The parent plasmids were induced to produce minicircle pMCS2.S and pMCIIF by arabinose. The minicircle plasmids were transfected into COS-7 cells in vitro with LipofectamineTM 2000. At 24, 48 and 72 h after transfection, the supernatants were quantified by ELISA. The two minicircle plasmids were injected into BALB/c mice by electroporation in situ together. When the mice were killed 4 weeks later, specific humoral and cellular immune responses were examined. Results The gene segments and inserting direction of pMCS2.S and pMCIIF were correct by genetic analysis. At 48 h after transfection, the concentration of HBsAg, IL-2 and IFNγwere 60.3±7.8 ng/ml, 17.7±1.9 ng/ml and 10.3 ±0.9 ng/ml, respectively, which were the peak values. Protective antibodies of HBsAb were produced, and co-injection of minicircle pMCS2.S together with adjuvant pMCIIF resulted in more evident immune responses. There was strongly specific humoral and cell immunity by only 5μg plasmid per mice. Conclusions The minicircle pMCS2.S and pMCIIF are constructed successfully, and in-duce strongly specific immune activity in vitro and in vivo.