Prokaryotic expression of outer membrane protein D15 gene of Haemophilus parasuis and analysis of immunogenicity of the expressed protein.

The full-length D15 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 complete open reading frame(ORF) with an ATG initiation codon comprised 2 418 nucleotides and encoded a protein of 805 amino acids.It shared 99.9% identity with the reference sequence.The deduced amino acid from HPS D15 gene had the typical topology structure of Omp85 protein family.The D15 gene was successfully cloned into pMD18-T vector,and then subsequently sub-cloned into expression vector pET-28a(+) and the recombinant plasmid was then transformed into competent Rosetta(DE3) bacteria.SDS-PAGE and Western-blot analyses revealed that the recombinant Rosetta(DE3) bacteria induced by IPTG were able to express the D15 protein with molecular weight of 94 ku.Kunming mice and guinea pigs were immunized with the recombinant D15 protein,the rusults showed that the protein could provide certain protective immunity against diseases caused by HPS.