Construction And Coexpression In Vitro Of Recombinant Expression Vector Encoding Hbvpres2s-Rhgm-Csf Fusion Gene

A cytokine GM-CSF can act as an effective adjuvant through enhancing activation and maturation of antigen presenting cells. On the other hand, HBV PreS2 protein plays an important role in adsorption to hepatocytes for HBV. Furthermore, the T cell epitopes on PreS2 antigen can potentiate the immune response against HBsAg. To investigate further the immune enhancement of GM-CSF and PreS2 as hepatitis B DNA vaccine,the eukaryotic coexpression plasmid PcDNA3.1-S2S-rhGM-CSF was constructed by means of PCR,T-A clone and directional cloning techniques, and the recombinant plasmid expressed in HepG2 cell line. The results were as follows:1．Recombinant plasmids PcDNA3.1-S2S-GM-CSF, PcDNA3.1-S2Sc, PcDNA3.1-S-GM-CSF and PcDNA3.1-Sc were successfully constructed respectively, and identified by restriction enzyme analysis, PCR amplification and/or DNA sequencing.2．Recombinant plasmids PcDNA 3.1-S2S-GM-CSF,PcDNA3.1-S2Sc, PcDNA3.1-S-GM-CSF and PcDNA3.1-Sc expressed in HepG2 cells respectively, and the fusion proteins specifically reacted with the monoclonal antibody, anti-HBs, anti-PreS2 and anti-GM-CSF respectively. The results indicated that the plasmids encoding fusion gene expressed successfully in vitro.The study laid the foundation of preparation of HBV DNA vaccine.