Correcting a Mutant HA Gene of the Avian Influenza Virus (AIV) H5N1 Subtype by SOE and the High Expression of the Corrected Gene in E.coli and It’s Application

Abstract：Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank (Accession number：DQ023145), the HA gene of AIV H5N1 subtype with the signal peptide sequence was obtained by PCR from the recombinant plasmid pUC-HA, which contains the full length of HA gene. Sequence analysis showed that a single base, an A, was replaced by aT to generate a stop codon (TAA) at position 967 of HA gene. Splicing by overlap extension ( SOE) method was applied to correct this mutation with two overlapped mutation primers P3 and P4, and then the correct HA gene was inserted into the expression vector pET-32a (+) to geerate the recombinant plasmid pET-HA-2. The target gene was successfully expressed in the host cell E.coli BL21(DE3) in inclusion bodies when induced with 1.0 mmol/L IPTG. Western-blot analysis proved that the recombinant protein had good immunoreactivity to positive serum of H5 subtype AIV. With this recombinant HA protein, an indirect ELISA method (iHA-ELISA) to detect antibodies of H5 subtype AIV was established. Interestingly, this iHA-ELISA could distinguish H5 positive serum from that of H7 and H9 subtypes. This results provide baseline for the investigating new vaccine and the development of new method to detect AIV.