Expression Of Cholera Toxin B Subunit And Hepatitis B Virus PreS2-S Fusion Gene In Bombyx Mori Nuclear Polyhedrosis Virus Expression Vector System And Its Activity Asssays In Vitro

Current hepatitis B virus (HBV) vaccines consist of preparations of recombinant HBV major surface antigen and are not protective in approximately 10% of vaccinated subjects. In improved vaccines,the frequency of nonresponders to the classical vaccine could be reduced by including additional epitopes from the preS2-domain of the middle surface antigen.Cholera toxin B subunit(CTB),with very strong immunogenicity,might help to stimulate mucosal IgA and serum IgG, it has more and more important role in study of immunoloical adjuvant,oral vaccine and vaccine derived from polypeptide.Cholera toxin B subunit and hepatitis B virus PreS2-S fusion gene was expressed by Nuclear Polyhedrosis Virus(BmNPV) expression vector system in this paper in order to obtain the fusion protein which can induce immune response and can be taken by oral administration.CTB-PreS2-S gene is inserted into BmNPV expression transfer vector pBacPAK8 and co-transfected with lineared DNA of Bm-PAK6 virus into BmN cells. The homologous recombination occurred inside the cells, and the recombinant virus what is named BamPAK-CTB-HBM is obtained. It was identified by Southern blotting and Western blotting analysis. The BmN culture cells and the pupae were infected by recombinant baculovirus BamPAK-CTB-HBM with 10 pfu/cell. Fusion protein expressed in Bombyx mori culture cells and pupae was analyzed with SDS-PAGE and one anticipative band was detected by Western blotting corrensponding tomolecular weights of approximately 43kD.The antigenicity of the recombinant fusion protein was detemined by ELISA.The immunogenicity of recombinant fusion protein was confirmed after immunized in Balb/c mice. After prime oral administration for 6 weeks,specific serum antibody(anti-Hbs) was detected 4 weeks later in 60% mice in fusion protein group and 20% mice in middle protein group by ELISA;Boost oral taken for 4 weeks since P/N lower than 5.0 after prime oral administration with yeast-derived vaccine,antibody response was boosted distinctly in all mice in both group.