Self-excising reporter gene in recombinant Fowlpox virus expressing H5 hemagglutinin gene of influenza A virus using Cre-loxp systerm

Abstract:

Hemagglutinin gene of subtype H5 avian influenza virus was amplified by polymerase chain reaction to construct expression cassette containing FPV early，late promoter and SV40 polyA tail. Then delivery vector was constructed by subcloning hemagglutinin gene of subtype H5 and GFP gene into fowlpox virus recombinant arm. The delivery vector and Lipid were transfected into CEF cells preinfected with FPV 282E4 strain virus. Recombinant fowlpox virus expressing the green fluorescence protein and hemagglutinin gene was screened and plaques were purified in CEF cell. After a second cotransfection with Cre recombinase plasmid，a recombinant virus only including hemagglutinin gene was gained. The immunofluorescent assay and replication efficiency of virus proved the recombinant could replicate steadily and express subtype H5 hemagglutinin gene. Two groups of 8-day-old SPF chickens were vaccinated with rFPVH5 by the wing-web method at the dosage of 10⁵PFU and 2×10⁵PFU respectively. After 28 days，antibodies titer was tested by HI. The results showed that the recombinant fowlpox virus could activate high antibody response.

Key words:Fowlpox virus; Avian influenza virus; subtype H5 hemagglutinin; recombinantion; Cre-loxP