Construction Of A Potent Strain Of Actinobacillus Pleuropneumoniae PotD2 Gene And Its Biological Characteristics

Actinobacillus pleuropneumonia is the main pathogenic microorganism of porcine pleuropneumonia. Polyamine bindingprotein D (potD) which belongs to ABCD polyamine transport system and can specifically recognize and bind polyamines is widespread in eukaryocyte and prokaryocyte. Previous studies have found that potD mutation of some bacterias could induce a series of changes of physiological function and pathogenicity. In order to explore the connections between putative polyamine transporter coding genes potD2 and biological characteristics and virulence of APP, in this study, in the serotype 1 isolated APP as the parent strain, we constructed the potD2 gene deletion mutant strain and complementation strain by conjugative transfer. The main contents as follows:1 Construction and identification of potD2 mutant strain MS?potD2 and complementation strain C?potD2With reference to the genome sequence of APP serovar 1 str.4074, the parent strain MS0721 genome as a template, amplified the upstream and downstream fragments of potD.The homology arms linked by two-step overlap extension PCR, and connected to pEMOC2. After PCR, enzyme digestion and sequencing identification, we constructed the recombinant suicide plasmid pEMOC2-?potD2 successfully. The Donor strain ?2155 contained plasmid pEMOC2ApotD2 was co-cultured with APP MS0721 for the plasmid transfer.Then by positive selection, obtained a random colony which chloramphenicol resistance and sucrose sensibility. Picked out a single exchange strain subcultured in TSB in order to stimulate the second recombination. Ultimately,4 APP potD2 mutants that chloramphenicol sensibility and sucrose resistance were screened.In a similar way, MS0721 genome was used as a template, Amplified potD2 entire reading expression frame, connected to the shuttle vector pLS88 directionally. Then constructed the shuttle plasmid complementary pLS88-potD2. PCR and restriction endonuclease analysis were correct and gene sequence bases without mutations. The complementary pLS88-potD2 was transformed into MS?potD2.Incubated it in Kan TSB, and obtain Kan resistant APP colony identified by PCR. Finally successfully constructed Complement strains C?potD2. At last, with the potD2 antiserum as a probe, colored by rabbit anti-mouse secondary antibody HRP, the western blot idenfication of APP bacterial protein was carried out. The results showed that the PotD2 protein was isolated in MS0721 and CApotD2, while not found in MSApotD2. Thus, the potD2 mutant and Complement strain C?potD2 were constructed successfully.2 The biological characteristics and toxicity in mice of MS?potD2The stability study of MSApotD2 and Complement strain indicated that APP can normal growth and reproduction after deactivation of potD2, and potD2 gene cannot resume in mutant strain.When cultivated in TSB, their growth rate did not change significantly,while MS?potD2 grow slower in the early logarithmic growth. Bacterias were inoculated in fresh medium containing 5% goat's blood, and observed their haemolytic activity, the results showed that all the strains could complete hemolysis. Cultivated in 96-well culture plate and in vitro respectively to detect the formation of biofilms.However, three strains cannot form biofilm, estimated that the results may be associated with bacterial serotype. In order to assess the relationship between potD gene and the bacterial resistance to environmental stress, cultrued MS0721, MS ? potD2 and C? potD2 cells grown to the logarithmic phase and diluted in ddH2O, then plated on TSA with or without 300mM NaCl or KC1. We found that the survival rate of MS?potD2 in hypertonic environment is apparent lower than that of MS0721 and C? potD2. Furthermore, the study about the resistance of the strain to oxidation and heat showed that the survival rate of MS ? potD2 treated with 0.8mM H2O2 was 8.67%, which was much lower than that of MS0721 and C? potD2. Similar results were obtained in the heat shock assay. The above results showed that the PotD2 proteinwas involved in the tolerance of multiple stresses.At last, the results of virulence experiment in mice demonstrated that MS?potD2 virulence reduced slightly, and the difference is not obvious. In other ways, potD2 has little impact on the virulence of Actinobacillus pleuropneumonia.