Functional Study On The AT1 Autotransporter Protein Of Haemophilus Parasuis

Haemophilus parasuis is a small, pleomorphic rod-shaped bacterial in the family Pasteurellaceae, and colonizes in the upper respiratory tract of conventional pigs. However, H. parasuis can also invade into body and cause Glasser's disease characterized by polyserositis, meningitis and arthritis under appropriate conditions. Currently, H. parasuis has emerged one of the most significant bacterial pathogens that grievously threaten pig industry worldwide, but the pathogenesis of the H. parasuis infection and its virulent fators were poorly understood. As a commensal organism of the upper respiratory tract, adherence and invasion are especially critical for the initiation of infection. Recent years, several researches demonstrated that autotransporter protein is one of the potential virulence factrors of H. parasuis. Moreover, the complete genome sequence analysis of local strain SH0165 predicted that there were 4 open reading frames including there functional genes named AT1, AT2 and AT3 respectively and the other one was pseudogene.Autotransporter proteins are a family of outer membrane/secreted proteins with unique structural properties that have been identified in a wide range of Gram-negative bacteria. The structure contain there motifs, N-terminal cleavable signal sequence, passenger domain in the middle, as known as a domain, and C-terminal translocator domain namelyβdomain. The signal peptide initiates translocation across the IM. Subsequently, the (3 domain is integrated into the OM and mediates the translocation of passenger domain across the OM. Autotransporter proteins possess diverse functions which are often associated with bacterial virulence such as adhesion, invasion, toxicity, biofilm formation and seroresistance.AT1 in H. parasuis belongs to type Va secretion system, of which translocator domain has 27% similarity with B. pertussis Pertactin. Consequently, this study targets on the function of AT1. The main contents include:1. Cloning and sequencing of at] and at1-pdat1 was amplified using genome DNA of SH0165 as template, AT1F and AT1R as upstream and downstream primers, the target fragement size is about 2577 bp. The amplication of at1-pd also use genome DNA of SH0165 as template, AT1PF and AT1PR as primers, and the amplified gene size was 1566 bp. GeneBank accession number is NC 011852.2. Prokaryotic expression of AT1 and AT1-PDat] and at1-pd gene was inserted into pET-30a(+) and pET-28a(+) prokaryotic expression vectors respectively, then the recombinant plasmids were transformed into E.coli BL21(DE3) strain followed by IPTG induction. SDS-PAGE showed that the expressing protein of ATI was in the sediment and the molecular weight was about 100 kDa, and AT1-PD fusion protein was about 85 kDa. Besides, analysis on different inducing time estimated that both proteins reached a peak at the 4th hour after IPTG induction. Moreover, AT1-PD fusion protein mainly existed in soluble form and could be purified by affinity chromatography with Ni SepharoseTM 6 Fast Flow chromatographic column.3. Preparation of monoclonal antibody anginst AT1-PD4 to 6 week-old femal Balb/c mice were hypodermically immunized at an interval of two weeks with 100μg of the emulsified His-tagged ATI-PD purified protein in Freund's adjuvant. A booster injection was given intraperitoneally with 100 to 300μg of ATI-PD protein before fusion. The supernatant of the hybridomas was detected for secreted antibodies by indirect-ELISA. Positive hybridomas were cloned by limiting dilution for at least three rounds and ascitic fluids were produced by intraperitoneally injection of mice. We made fusion twice in this study and 9 positive hybridomas were acquired, each of which was used for ascites production. The ELISA titer of the ascitics of one strain reached 100×29. The specificity of the monoclonal antibody was detected by Western-blot and the result showed high specificity.4. Preparation of procine primary tracheal epithelial cellsThe animal in this study was suckling pig free of colostrum. The primary cells were obtained after cold-digestion of the trachea. The aquired cells were proved to be useful in the following experiment and could be passaged for no more than six generation.5. The location of AT1Both the outermembrane protein and secretion protein of pET-30a-AT1/BL21 and SH0165 were extracted. And then the location of ATI was determined by Western-blot, the result indicated that AT1 was located on the outermembrane both in pET-30a-AT1/BL21 and SH0165.6. Study on the function of AT1 in vitroSince ATI was proved to be membrane protein, the interaction of SJPL, PIEC and procine primary tracheal epithelial cells with pET-30a-AT1/BL21 which displayed ATI on the sueface was estimated by adhesion assay and indirect immunofluorescence assay. The result manifested that AT1 may have some role in H. parasuis adherence.