Preparation of hemagglutinin antiserum against H9 subtype avian influenza virus

To prepare hemagglutinin(HA) antiserum against H9 subtype avian influenza virus(AIV),the HA gene of 6 different clades of H9 subtype AIV were optimized with chicken's codon and chemically synthesized.The codon optimized HA genes were inserted into the eukaryotic expression vector pCAGGS to construct recombinant expression plasmid,which were transfected into 293T cells.The expressed HA proteins were verified by indirect immunofluorescence assay on 48 hours post transfection.One-month-old SPF chickens were immunized with the recombinant plasmid intramuscularly at a dose of 200 μg.The antiserum were collected on weeks 6 post inoculation.The results of cross hemagglutinin inhibition(HI) test demonstrated that the antiserum titer reached from 8 log2 to 12 log2,and no interaction with other AIV subtypes antigen.The antiserum was proved to be high specificity to AIV subtype H9 and could be used as subtype-specific reference antiserum.