| Haemophilus parasuis is a species of gram-negative bacteria belonging to Pasteurellaceae, Pasteurellales. It is a commensal bacterium in upper respiratory tract of pigs, but under certain condition, it can invade and cause meningitis. The pilus of many Gram-negative bacteria has been observed distributing on the bacterial surface. As a kind of pilus, type Ⅳ pilus plays a main role in adhesion and invation, DNA uptaking, and promoting bacteria through blood brain barrier.The type Ⅳ pilus operon of HPS SH0165and17reference strains have been cloned and analysed. Comparing these sequnces, we can learn about the conservatism and homology of structural genes. In addition, we also looked for the conditions which make HPS produce pilus. Finally, we find HPS produces pilus when it is incubated in vivo. Western blot also confirms this. At last, we try to delete the pilin gene pilA, which is for more profound researches on pathogenesis of HPS.The main contents of this project are as following:1. Cloning and analysing the type Ⅳ pilus operonAccording to complete genome sequence of SH0165, a pair of primers was designed to clone the type Ⅳ pilus operon. The analyses of these sequences show that the operon includes four structural genes, namely pilA, pilB, pilC and pilD, which encode170aa,462aa,399aa and223aa, respectively. Gene sequencing indicates the homology of gene pilA from17reference stains are above85percent, and the latter partial sequence of pilA are more conserved. In addition, the homology of gene pilB, pilC, and pilD between these strains is40-50percent. The alignment of amino acid sequence of pilA showed a high homology between the close species. We also find the C-terminus of PilA possesses cysteine residues that are conserved in type Ⅳ pilus of many species, and it forms a disulphide bond that produces a loop, which is important for the function of pilus and also comprises a major antigenic determinant.2. The prokaryotic expression and polyclonal antibodies production of PilA proteinPilA fragment was amplified via PCR by using genome DNA of SH0165as template, and was ligated into pET-28a. The ligation product was transformed into E.coli/DH5α, and the correct plasmid confirmed by sequencing was then transformed into E.coli/BL21for prokaryotic expression. The protein was purified by dialysis and used to immunize BALB/c mice. Polyclonal antibodies of PilA protein was obtained and indirect ELISA and Western-blot analysis showed a nice specificity of the antibodies. The highest titre was1:102400and the antibodies can be used for further studies.3. The expression of pilus in different situation.We attempted to use other media to achieve the expression of pilus in vitro instead. In this study, we used LB, chocolate agar medium, blood agar medium and chemically defined media to culture HPS, and no presence of pilus was observed under electron microscope. The expression of HPS pilus was induced by using co-culture of cells and bacteria, The growth of pilus was not seen, either. In addition, by using HPS passaged in chick embryo chorioallantoic membrane, we still could not observe the presence of pilus. Healthy pigs were infected with HPS of high dose to produce the disease and atypical symptoms of HPS infection. Sera and infected tissues were collected to isolate HPS. The specimen was prepared for electron microscope observation and finally the potential pilus structure was observed.4. PilA was identified to be expressed in vivoPurified PilA protein was subjected to Western blot assay using recovery swine pig serum, immunized pig serum and healthy pig serum as the first antibody. When recovery swine pig serum was used as the first antibody, a band of20kD can be observed, but the band was absent when other serums were used. Also the Western blot showed that the pilA gene of HPS cultured in the lab was not expressed. Therefore, PilA protein was identified to be expressed in vivo.5. Construction of pilA-deleted mutantsUsing temperature-sensitive shuttle plasmid pSHK3TS as the backbone, we cloned the upstream and downstream homologous arms of pilA gene from SH0165genome, and ligated it to the plasmid backbone, generating recombinant plasmid pSHK3TS-UD, which was then transformed into SH0165strain via electroporation to screen deleted mutants by temperature sensitivity and homologous recombination. The recombinant plasmid was correctly constructed by digestion confirmation and can be successfully electroporated into SH0165. But we failed to obtain deleted mutants after several attempts of screening. |
