Construction and identification of a eukaryotic expression vector of fusion protein containing avian H5N1 influenza virus hemagglutinin antigen and the cholera toxin B subunit / 中华传染病杂志

ABSTRACT

Objective To construct a eukaryotic vector which contains avian H5N1 influenza virus hemagglutinin (HA) antigen and the cholera toxin B subunit (CTB) and to investigate its expression in COS7 cells,and the ability to induce specific immune responses in vivo in different periods.Methods After cloned by polymerase chain reaction (PCR),CTB and HA genes were digested with BamH Ⅰ and connected into CTB-HA gene with T4 ligase.The connected gene was referred to as CH.After double digestion,CH gene was inserted into a eukaryotic recombinant plasmid pCI-neo.The pCI-CH plasmid was then transfected into COS7 cells.Western blot was used to detect the expression of HA antigen.After New Zealand white rabbits were immunized,the titer of HA antigen-specific antibody in serum and its specificity with other strains such as H1N1,H9N1,H3N2 and influenza B virus were determined by indirect enzyme-linked immunosorbent assay.Results The pCI-CH vector (DNA vaccine) was successfully constructed,which could be efficiently expressed in COS7 cells and induce specific antibodies against pCI-CH in rabbits.Cross reactions indicated that DNA vaccine pCI-CH specific antisera could not only react with H5N1 strain (P/N＞2.1),but also H1N1,H9N1 and H3N2 strains,but did not cross react with influenza B virus.Conclusion The newly constructed avian H5N1 influenza virus nucleic acid vaccine has good immunogenicity.