The Research On Immunogenicity Of Haemophilus Parasuis Autotransporter Proteins And Their Interaction With Host Cells

Haemophilus parasuis is a species of commensal bacteria in swine upper respiratory tract that can invade and cause systemic disease under condition. Recently, the disease has led to a considerable economic loss for global pig industry and drawn concern from the researchers. At present, in spite of lots of documents describing the candidate virulence genes of Haemophilus parasuis, our understanding on its pathogenesis is still rather limited.Type V protein secretion system of Gram-negative bacteria is also called Autotransporter protein. The protein mainly consists of signal peptide, passenger domain at N-terminal and P domain at C-terminal, among which passenger domain is the functional domain. Researchers found that the protein has been found involved in the adhesion and invasion of bacteria and related to cytotoxicity. It can also mediate the formation of biofilm and the generation of serum resistance. Thus it was considered an important virulence factor of microbial pathogen.To investigate the potential role that Autotransporter protein plays in the pathogenesis of Haemophilus parasuis, this thesis targeted two Autotransporter genes discovered in Haemophilus parasuis SH0165 strain, at2 and at3, and carried out studies by using PCR, prokaryotic expression, monoclonal antibody, protein immunoblotting, etc.The main results are as following:1. The cloning and prokaryotic expression of at2,at3,at2-passenger domain (at2-pd) and at3- passenger domain (at3-pd)Four fragments, at2,at3,at2-pd and at3-pd, which were 2336bp,2443bp,1368bp, 1392bp in size, and encoded 771,780,450,458 amino acids respectively, were amplified by PCR from the template of SH0165 genomic DNA. at2 and at3 were ligated into pET-30a while at2-pd and at3-pd were ligated into pET-28a. E.coli/DH5a was transformed with the ligation products and then E.coli/BL21(DE3) was transformed with correct recombinant plasmids after sequencing for prokaryotic expression. The 4 fragments above were successfully cloned and corresponding prokaryotic expression vectors were constructed. The prokaryotic expression of the 4 fragments was achieved in E.coli/BL21 (DE3) strain and the 4 proteins were designated as AT2, AT3, AT2-PD and AT3-PD.2. The purification of AT2-PD and AT3-PD and the preparation of monoclonal antibodies AT2-PD and AT3-PD proteins expressed in E.coli/BL21 (DE3) were present in soluble form and we purified the two proteins by using histidine tag expressed in fusion. The purified proteins were used to immunize BALB/c mice for the production of monoclonal antibodies.5 strains of hybridoma generating monoclonal antibody against AT2-PD were obtained, named 1E4,3C9,3D8,3E8 and 4B10. While other 5 strains of hybridoma generating monoclonal antibody against AT3-PD were obtained, named 2E6, 3H8,4C8,4F12 and 4H10. Indirect ELISA and Western blot analysis indicated that the antibodies were highly specific. The titer of 4B10 reached 1:102400 and that of 4H10 was 1:25600. These two strains of monoclonal antibodies were used in subsequent experiments.3. The localization analysis of AT2 and AT3 protein on E.coli/BL21(DE3) and SH0165The localization of AT2 and AT3 protein on E.coli/BL21 (DE3) and SH0165 was analyzed by using monoclonal antibodies combined with Western blot assay. The cellular total proteins, membrane total proteins and secretive proteins of the two bacteria were extracted respectively. The results showed that AT2 and AT3 were present in the cellular total proteins, membrane total proteins and secretive proteins of E.coli/BL21(DE3) strain that expressed each protein. AT2 was present in cellular total proteins and membrane total proteins of SH0165 while AT3 was present in cellular total proteins, membrane total proteins and secretive proteins of SH0165.4. The immunogenicity analysis of AT2 and AT3Since AT2 and AT3 are membrane protein and/or secretive protein in SH0165, we conducted a analysis regarding their immunogenicity. First we investigated their distribution among 15 HPS reference serotype strains. The results indicated at2 gene can be amplified from all strains except for reference serotype 4 and at3 gene can be amplified from all 15 reference strains. In the indirect ELISA, purified AT2-PD and AT3-PD were used as antigen to cover the plate and the 7 infected sera were harvested from pigs challenged with strains of different serotypes. The results exhibited positive reaction. Meanwhile, AT2-PD and AT3-PD and the infected sera were also analyzed by Western blot and the results gave obvious positive reaction as well.5. The host cell interaction of AT2 and AT3 proteinAdhesion and adhesion repression assay was performed by using E.coli/BL21(DE3) strains expressing AT2 and AT3 with PIEC, SJPL, and pig trachea epithelial primary cells. The results showed no significant adhesion effect between the two bacteria and the three cell lines. In the binding assay, we used purified AT2-PD and AT3-PD and observed via indirect immunofluorescence. Both the two proteins can bind onto cell surface.Summarizing the results above, we conclude that the two Autotransporter proteins are secretive proteins and have nice immunogenicity. They can bind many pig-derived cell lines cultured in vitro and we speculate that they are potentially valuable for the application as subunit vaccine and diagnostic antigen. This is the first study on monomer Autotransporters in Haemophilus parasuis, which confirmed their localization and preliminarily demonstrated their functions. However, more intensive functional studies are required with further experiments.