Construction Of HhdA-defective Mutants Of Haemophilus Parasuis And Antigenicity Analysis Of Its Extracellular Serine Protease

Haemophilus parasuis (HPS) is a commensal organism of the upper respiratory tract of healthy pigs. Under appropriate conditions, this bacteria cause Gl?sser's disease, characterized by fibrinous polyserositis, arthritis and meningitis. Gl?sser's disease has recently emerged as one of the typical bacterial diseases in swine industry in the worldwide, which has increasingly attented by people. Although significant studies on HPS have been reported as well as several virulence factors have been screened, the pathogenesis is yet to understand.In this study, the overlapping PCR method was used to construct the Hup-Kan-Hdown gene, an exogenous gene targeting gene of hhdA in HPS. When the recombinant gene was inserted into pDS132, the targeting plasmid, pDS-HPhhdA-Kan, was constructed, which was stable duplicated in E. coli harboringλpir gene. Electroporation of pDS-HPhhdA-Kan into HPS, through the screening with kanamycin resistance and 5% sucrose, HPS hhdA gene deletion strain (HPS△hhdA) was selected that could grow on plates with kanamycin resistance but not on plates containing 5% sucrose. Biochemical tests showed that the biochemical characteristics of the mutant strain did not change as well as the NAD-dependent growth. The growth curve showed that mutant strains growed little slower than standard strains. The virulence of the mutant strains did also not change significantly preformed by the guinea pig infection experiment. All those results showed that hhdA gene does not affect the growth of HPS and its reproduction. There was no significant correlation between virulence and hhdA gene. This established HPS mutation system opens the possibility for the functional analysis of genes involved in the infectious process of this animal pathogen.The HPS esp was then sequenced, analysis and expression. The sequence analysis showed that the first 23 aa was signal peptide. The longest conserved sequence, E(M/V)NNLNKRMG(E/D)LR(G/D), was compared with the C-terminal of the serine protease family. The validation of 3-D structure of the C terminal translocator domain was showed the monomericβ-barrel containing 12β-strands and anα-helix in the N-terminal. Many distinct antigenic epitopes in the passger domain of HPS esp were identified by computation : 50～57, 95～108, 120～130, 185～196, 230～241 and 421～425. When the esp gene was inserted into pET30a, the pET-esp was constructed. After induced with Isopropylβ-D-1-Thiogalactopyranoside (IPTG) and optimized the conditions of expression, the esp fusion protein was highly expressed at 0.6 mmol/L IPTG, 32℃, 6 h. SDS-PAGE showed the molecular weight of about 82 kDa. To evaluate the immunogenicity, were immunized with esp vaccine contained equal volume 206 adjuvant. Then guinea pigs were challenged with 5×10~9 CFU per guinea pigs of HPS and partial protection. This result indicates that esp is good candidate immunogens that could be used to improve HPS vaccines.