Research Of Sap A Gene Identificantion And Biologycal Features In Haemophilus Parasuis

Haemophilus parasuis(HPS), belonging to the hemophilus genera of Pasteur bacillus, is a Gram-negative bacteria. Research shows that sensitive of antimicrobial peptide gene sap A plays a significant role in resisting host antimicrobial peptide among various Gramnegative bacteria. It has been confirmed to be a virulence factor in the same genera bacteria with HPS, such as Haemophilus influenza, Haemophilus ducreyi. However, no any reports have publiced before about the situation of this gene in HPS.This study cloned and sequenced the sap A gene of HPS isolates H31(virulent stains of serotype 12) isolated from the lung tissue of a sick swine, and then predict the sap A gene encoded protein by using biological information software, indicated the sap A protein contains abundant secondary structures and high antigenicity. 66 strains of HPS were indentified and analysised, results shows that sap A gene existed in 29 strains, speculated the sap A gene maybe exist in the virulent strains and mesogen strains only, but further study is needed to prove it, due to the limited number of strains that we used. The obtained 29 sap A gene of different strains were sequenced and analysised, indicated sap A gene has highly homology in different strains.A prokaryotic expression vector p ET32a(sap A) was recombined and successfully explored and optimized the expression condition, then a batch of sap A fusion protein(protein concentration 195μg/m L) was expressed and purified. Purified sap A fusion protein was used to make rabbit hyperimmune serum and indirect ELISA was established to detect serum antibodies. Indirect ELISA, western blot test found sap A protein can recognize the whole bacterial serum, confirmed its good immunogenicity. Indirect immunofluorescence, western blot experiment found the extracted bacteria cytoplasm protein got an objective reaction with sap A protein serum, indicateded that sap A protein exit in cytoplasm component. Serum bactericidal assay found the sap A protein serum increased bacterial survival rate obviously, indicateded that sap A protein antibody promote the growth of bacteria.Sap A protein resistant to antimicrobial peptide assay was tested in vitro, result showed sap A protein can interdict the bactericidal effect of antimicrobial peptides remarkably, as well as increase the bacterial survival rate, speculated sap A protein inactivate antimicrobial peptides by combining and then disintegrate them into amino acid which was used to help the promoting the growth of bacteria. Guinea pig immunizations and bacterial artificial infection experiment found the high serum antibody aggravate guinea pig death, verified the hyperimmune serum bactericidal assay previously and proved that sap A gene is a virulence factor.This study identified and studied HPS sap A gene for the first time. The sap A gene sequence and its encoded protein features were analysised and predicted, and successfully obtained the purified sap A fusion expression protein through prokaryotic expression. Then the immunogenicity of the protein and the subcellular location of the protein were identified, the hyperimmune serum bactericidal effect and the sap A protein resistant to antimicrobialpeptide bactericidal effect assay were verified in vitro. At the same time, guinea pig as a animal model wers used to verify sap A protein as an important toxic protein in HPS, which provided reference for the detailed study of sap A gene in the future.