Construction And Identification Of Helper-dependent Adenoviral Vector Encoding Hemagglutinin Protein Of Swin-Origin Influenza Virus A H1N1

Objective:The newly emerged swine-origin influenza virus (S-OIV) A H1N1is responsible for a new pandemic disease in2009. Both vaccine and antiviral drugs are now available for its prevention and therapy. However, antiviral drugs of the neuraminidase inhibitor group and the traditional split influenza virus vaccine have some limitations in clinical uses, such as the resistance to the availalbe antiviral drugs and the limited capability to produce conventional inactivated vaccines. Influenza vaccines based on first generation replication deficient adenovirus vector (FGAd) have been developed successfully. In this study, the hemagglutinin (HA) gene of S-OIV A H1N1is obtained by gene synthesis method and the helper virus-dependent adenovirus vector (HDAd) system is exploited to construct recombinant HDAd encoding HA, which lays the foundation for further in vivo investigation on immune response and immune protection against S-OIV A H1N1infection.Methods:The HA gene with1701bp is synthesized according to the published sequence of S-OIV A H1N1, strain California (A/California/07/2009(H1N1)) in GenBank. Confirmed by the nucleic acid sequence analysis, the HA gene is cloned to HDAd shuttle vector pSCll-CMV. After restriction endonuclease analysis, the HA gene expression cassette was further cloned to the HDAd backbone plasmid pSC15B to construct recombinant adenoviral plasmid of pSC15B/HA and identified with multiple restriction enzymes.The prokaryotic replication elements and resistance genes of pSCl5B/HA is removed by Pme I digestion to obtain HDAd/HA DNA molecules. Then, the linear HDAd/HA DNA is transfected into293Cre4cells with calcium phosphate. The cells are infected with helper virus16hours after transfection. HDAd/HA is amplified by serial coinfection of293Cre4cells by helper virus and the crude lysates from previous passage until it reaches plateau of amplification. Subsequently293Cre4cells are coinfected by HDAd/HA and the helper virus for large scale of preparation of HDAd/HA. The resulting HDAd/HA is purified by CsCl gradient ultracentrifugation and observed morphologically under transmission electron microscope, and the expression of HA protein is analyzed by RT-PCR.Results:Recombinant HDAd/HA expressing HA protein was successfully constructed. HDAd/HA could be produed rapidly and in large scale, purified by using CsCl gradient ultracentrifugation and confirmed morphologically under transmisson electron microscope. After infecting293cells, the expression of HA gene could be confirmed by RT-PCR.Conclusion:The HA protein expression from the constructed HDAd/HA has been confirmed by RT-PCR, which provides the foundation for in vivo investigation on immunogenicity and efficacy against S-OIV AH1N1infection.