Cloning and expression of neuraminidase gene of Haemophilus parasuis and immunogenicity of its expressed product

The full-length Neu gene was amplified from Haemophilus parasuis(HPS) serotype 5 SP strain by PCR with a pair of primers based on the whole genome shotgun sequence(Accession No.:NZ-ABKM01000005) in GenBank.Sequencing showed that the amplified Neu gene was successfully cloned into pMD18-T vector and the complete open reading frame with an ATG initiation codon comprised 2187 nucleotides and encoded a protein of 805 amino acids.It shared 99.0% identity with that of the reference sequence.The HPS Neu gene fragment was amplified by PCR from pMD-Neu,and cloned into the prokaryotic expression vector pET-28a(+) to obtain the recombinant prokaryotic expression vector pET-Neu.The pET-Neu was transformed into Escherichia coli Rosetta(DE3) and expressed optimally.The recombinant protein Neu of approximately 52ku was detected by SDS-PAGE and existed mainly in the form of inclusion bodies.Western-blotting showed that the recombinant protein Neu could specifically react with the antibodies in positive sera well.Kunming mice were immunized with the purified recombinant protein Neu,and challenged with HPS post-immunization.The results showed that the Al(OH)3 group got a poor protective(10%),but the Freund's had a higher protection(60%).