Studies On The Immunoprotection Of H.Parasuis HAPS_RS00465 Protein And The Biological Characteristics Of The Gene Deletion Strains

Disease caused by Haemophilus parasuis is characterized of polyserositis, arthritis, and meningitis, seriously affected the development of the pig industry. Peptide ABC transporter substrate-binding proteins of bacteria not only can transport small peptide into cells to provide nutrients, but also involved in many important cellular activities,such as the uptake of cell wall peptides, biofilm formation, cell-tocell communication, pathogenicity. Additionally, studies found that some peptide ABC transporter substrate-binding proteins had good immunoprotection. HAPS_RS00465 is a gene of Haemophilus parasuis, which encodes a peptide ABC transporter substrate-binding protein. Cloning and expression of the HAPS_RS00465 gene and the immunoprotection of the recombinant protein in a mouse model were carried out to assess the immunoprotection of H. parasuis HAPS_RS00465 protein. Simultaneously, in order to initially reveal the influences of the HAPS_RS00465 gene to some biological characteristics of H.parasuis, we constructed a HAPS_RS00465 deletion mutant of H. parasuis and researched its biological characteristics. The results are as follows:1.Cloning and expression of H.parasuis HAPS_RS00465 geneThe HAPS_RS00465 gene were PCR amplified with the genome of Hps M-3 strain and cloned into pMD19-T simple vector. Then the cloned gene were digested with XhoI and ScaI, purified, and cloned into pET-39b to construct a pET-39b-HAPS_RS00465 recombinant expression vector,which was identified by PCR, enzyme digestion and sequencing. The recombinant expression vector was transferred into BL21(DE3) to express, separation and purification of the recombinant protein were carried out to get the protein, which molecular weight was about 70 kDa and was the same with expected. The result of western blot showed that the HAPS_RS00465 recombinant protein had good reactogenicity, could react with serum of the rabbit which immuned with Hps M-3 strain.2.Immunoprotection of H.parasuis HAPS_RS00465 recombinant proteinHAPS_RS00465 recombinant protein(50?g) was thoroughly mixed and emulsified with adjuvant. Multi-point injection by subcutaneous to immunize each mouse, the second immunization was carried out after 14days. The antibody levels was detected in the 7th day after the second immunization. The result showed that the mice immuned with the HAPS_RS00465 recombinant protein produced a higher level of antibody, indicated that the protein had good immunogenicity.5×LD50 (6×109 CFU) Hps M-3 were used to attack the mice by intraperitoneal injection in the 14th day after the second immunization. The result showed that the survival rate of the HAPS_RS00465 protein group was 70%(7/10), indicated that the protein had good immunoprotection, the immune protection rate was 70%, and had the potentiality to become a vaccine candidate antigen. This study confirmed a new immune protective antigen of H.parasuis, and laid a foundation for the development of subunit vaccine.3.Construction of H. parasuis HAPS_RS00465 gene deletion strainsA upstream homologous fragment(875bp) and a downstream homologous fragment (865bp) of the HAPS_RS00465 were PCR amplified from the genome of Hps EP8 strain. A kanamycin resistant (kanR) cassette(935bp) was PCR amplified from pKD4. All these fragments were integrated by fusion PCR. Then digested with EcoRI and BamHI, purified, and cloned into pkl8mobsacB to construct a UKD-pK18 recombinant plasmid, which was identified by PCR, enzyme digestion and sequencing. Finally, the UKD-pK18 recombinant plasmid was introduced into EP8 by natural transformation following a previously described method to construct a HAPS_RS00465 gene deletion strains, which was identified by PCR, RT-PCR, sequencing and polarity effect detection.4.Biological characteristics of H.parasuis HAPS_RS00465 gene deletion strainsBacteria were stained with the gram stain solution and the shape of bacteria was observed with microscope. A variety of shapes were observed in the mutant strains, long rod-shaped and short rod-shaped were the main shapes. There were no significant differences in the shape between the wild-type strains and the mutant strains, indicated that the HAPS_RS00465 gene had no influence on the shape of H.parasuis. The OD600-time curve reflected that the growth rate of the mutant strains was slower compared with the wild strains, indicated that deletion of the HAPS_RS00465 gene had influence on the growth of H.parasuis. The biofilm production of the mutant strains (OD630=0.522) was significantly lower than that of the wild-type strains (OD630=0.930) in the biofilm formation assay, indicated that the HAPS_RS00465 gene of H.parasuis play a role in biofilm formation. The bacteria were treated with 50% serum of healthy pig, and the survival rate of the mutant strains was 5.16%, which was not significantly different from that of the wild-type strains(5.47%), indicated that the deletion of the HAPS_RS00465 gene had no influence on the serum resistance of H.parasuis. The median lethal dose of the HAPS_RS00465 gene deletion strains and the wild strains was determinated. Mice were used as animal model of the acute toxicity experiment. The result showed that the median lethal dose of the HAPS_RS00465 gene deletion strains was 7.03×108 CFU and the median lethal dose of the wild strains was 6.14×108 CFU. The virulence of the HAPS_RS00465 gene deletion strains was decreased. These results demonstrated that the deletion of the HAPS_RS00465 gene had influences on the biological characteristics of Hps EP8 strain, particularly significant on the formation of biofilm.This study provided basic data for exploring the role of HAPS_RS00465 in the growth and pathogenic process of Haemophilus parasuis.