Abstract:

【Objective】 A strain of replication deficient recombinant adenovirus encoding HA of H3N2 subtype swine influenza virus (SIV) was generated and the immune efficacy was evaluated in mice. 【Method】 To construct a recombinant adenovirus shuttle plasmid pDC315-H3HA-EGFP, HA gene of A/Swine/Guangdong/9/2005(H3N2) amplified by PCR from the recombinant plasmid pMD18-HA was sub-cloned into pIRES2-EGFP. The gene fragment containing HA and EGFP was then inserted into adenovirus shuttle plasmid pDC315. A replication-defective recombinant adenovirus expressing HA gene (rAd-H3HA-EGFP) was generated by cotransfecting the recombinant shuttle plasmid pDC315-HA-EGFP and the backbone plasmid pBHGlox△E1, E3Cre in HEK293 cells. The recombinant adenovirus was screened by the typical cytopathic effect and expression of EGFP gene in HEK293 cells. The immunogenicity of the recombinant adenovirus was evaluated by inoculating 6-week-old BALB/c mice, through detecting specific antibody titer and protection against the virus challenge. 【Result】 HA gene was recombined into the genome of the recombinant adenovirus rAd-H3HA-EGFP, and HA protein could be efficiently expressed in vitro. The TCID50 of the rAd-H3HA-EGFP was evaluated as 1.58×1010•mL-1 after propagation and purification. Mice inoculated with 108 TCID50 were protected against the challenge with H3 subtype SIV and accompanied with high titer of antibodies. 【Conclusion】 A strain of replication-defective adenovirus rAd-H3HA-EGFP with good immunogenicity was constructed, which would lay a foundation for the development of the engineered live virus vectored vaccine of H3 subtype of SIV.

Key words: swine influenza virus')">swine influenza virus, HA gene, recombinant adenovirus, mice, immunogenicity