The Molecular Epidemiology Of H. Parasuis, Cloning Expression And Immune Protective Efficacy Study Of HhdA Gene

HPS(Haemophilus parasuis) is a pathogen of Glasser’s disease which may cause the porcine polyserositis and arthritis symptom. With the development of the large-scale hoggery in recent years, this disease become the key reason of increasing Morbility and mortality.In this study, the serotype classification of HPS isolated strains in 2012 to 2014 were accomplished based on agar gel diffusion test,and we also finished the ERIC-PCR genotyping.On the basis of biological information forecast of HPS hhd A protein, the HPS hhd A gene was cloned, and then express hhd A protein in prokaryotic expression system.and identify the protein. This research may help further study on its immunogenicity.the results were as follows:1. ERIC-PCR fingerprints of Haemophilus parasuis field strains isolated from Jiangxi during 2012 and 2014Genotyping the 41 Jiangxi HPS isolated strain by comparing 15 reference strains of HPS that from different serotype. The total 56 strains were divided into 44 genotype.It should be noted that the undetermined eight strain were got fully ERIC-PCR geno typing,which supplement shortage of traditional serotype analyses method.2. Cloning and bioinformatics analysis of hhd A gene from Haemophilus parasuisIn this study, HPS hhd A genes were amplified by PCR,then successful get the full sequence which consist of 1797 base pair and encoding 599 amino acids,so we can deduce the molecular weight is 61.1k Da and may be a hydrophilic protein, the protein might have the transmembranes from inside-out and outside-in, The protein signal peptide analyses predicted hhd A protein have no signal peptide and it is a intracellular protein. The prediction and analyses of secondary structure of hhd A protein showed that hhd A protein contains lots α- helix, β- folding, β-turn and random coil. etc. and also have many scattering flexible regions, the results show nucleotide similarity was 99.9% and the amino acid similarity was 99.8% compare with the ZJ0906 strain(CP005384) published in Gen Bank, which can concluded that the hhd A gene of HPS is very conservative.3. Expression and identification of hhd A protein of Haemophilus parasuisThe amplified hhd A gene was cloned into p ET-32a(+) vector to construct a recombinant expression plasmid referred as p ET-32a(+)-hhd A. and then transform the vector into E.coli BL21(DE3), followed by hhd A protein expression induced with IPTG.SDS-PAGE and Western blot analyses can show us the gene expressivity and reactogenicity of recombination protein. Immunized mice with the recombination protein that was purified by Ni-chelating affinity chromatography and then detect the Ig G antibody level and protective rate on mice. SDS-PAGE result show that the protein molecular mass was about 82 k Da which was consistent with we expected. Western-blot result show that recombinant protein have good reactgenicity. And the animal immune test show that the recombinant hhd A protein can induce high level specific antibody, and provided almost 50% protection rate from HPS virulent strain. This all results explain that the hha A is the protective antigen of HPS.