Construction Of Mutant Strains And Function Of Two-Component Regulatory System QseB/QseC On Actinobacillus Pleuropneumoniae

Pleuropneumoniae (Porcine Contagious Pleuropneumonia, PCP) from Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, APP) mainly due to pneumonia and pleural hemorrhagic necrotizing pneumonia characterized by a highly contagious respiratory disease. Actinobacillus pleuropneumoniae serotype, virulence factors, genome structure and its complex regulatory network, its molecular pathogenesis is unclear. Many studies indicate that two-component signal transduction system, not only in basic life activities of bacteria and bacterial virulence and pathogenicity which are closely related.Two-component signal regulation system (TCS) which was widespread in Gram-positive and negative bacteria as a signal transduction mechanism involved in the regulation of bacteria with pathogenicity, growth and metabolism, drug resistance, virulence factor expression and other kinds of important biological functions, especially in terms of pathogenicity, that is becoming a hot research.The main contents included:1. Construction and identification of the double gene deletion mutantâ–³QseBCThe APP SLW01 genomic DNA as template, with reference to the QseB QseC Genbank gene sequence was amplified from the SLW01 strain and QseB QseC gene upstream and downstream fragments. Obtained by TA cloning the upstream and downstream segments, connected to the pEASY-T1, and digested, then connected to the suicide plasmid pEMOC2 on the two missing 2092bp recombinant suicide plasmid pEMâ–³QseBC. Recombinant suicide plasmid was transferred intoβ2155 as the donor bacteria, a strain of bacteria SLW01 conjugation for the receptor, based on chloramphenicol (Cm) a positive selection marker and sucrose (SacB) negative selection marker two-step homologous method of screening recombinant Cm-sensitive sucrose resistant clones, PCR identification, determine the occurrence of a second single exchange, suicide plasmid has been lost. And the deletion mutant validated is by DNA sequencing.. To successfully construct the double gene deletion mutantâ–³QseBC.2. The biological characteristics of gene deletion mutants of and explore the biological pathogenicityGene deletion mutantsâ–³QseBC analysis of genetic stability, gene deletion mutant at 20 on behalf of the subculture plate, PCR identification, the results showed that the three gene deletion mutants are able to stabilize the inheritance. Mutant and parent strain viable count growth curve, showing mutants and the growth of the parent strain and no significant difference. Gene deletion mutant strains of hemolytic activity with the parent analysis showed no significant difference in hemolytic activity. Gene deletion mutant and the parental strain phenotype analysis, negative staining electron microscopy results showed that compared with the parent strain, mutant strain were significantly different fimbriae. But the determination of gene deletion mutants and parent strains Balb/C mice, LD50, the results show that compared with the parental strain SLW01,â–³QseBC's no significant difference in virulence. To further analyze the possible causes, compared with the parental gene deletion mutant strain of the Organization of the carrier volume, the results show that the establishment of infection in the early stages of APP, QseB/C plays an important role.3. Differences of Gene deletion mutants and wild strain in gene expression analysis and screening of APP expression microarray analysisUsing the parental strains and the number of gene deletion mutants of the transcriptional profiling of microarray raw data statistical analysis to identify the genes mutant and parental strains some of the differentially expressed genes,27 up-regulated genes, down-regulated gene 25, and by qRT-PCR results validate the reliability of the chip. Done on the differential gene bioinformatics analysis to identify the three possible control sequence:APJL₀393 (glycerol kinase), pilM (Tfp pilus assembly protein), APJL₁066 (Putative heme iron utilization protein). Combination of gene deletion mutants and parental strains of pathogenic biological characteristics and biological experiments, suggesting a direct regulation of genes for the pilM.4. Validation of differentially expressed genes by EMSARegulated by bioinformatics method of prediction of three possible regulatory promoter sequences. Gel retardation assays (EMSA) results showed that:the regulation of protein phosphorylation QseB can not deal with the APJL 0393, APJL₁066 promoter binding or regulatory proteins prone to rapid phosphorylation. Regulation of protein phosphorylation QseB deal with pilM promoter binding, phosphorylation of regulatory proteins more stable, less prone to phosphorylation, is slow signal transduction.5. Construction of a new gene mutant strainâ–³pilM by SOE-PCR(overIap extension technology) and explore the biological characteristics of theâ–³pilM mutant strain.Genome from the SLW01 gene was amplified pilM downstream homologous arm, mixed DNA fragments upstream and downstream, because of overlap, duplication between the annealing fragment, extending into the heteroduplex, adding a third outer primer PCR, be constituted by the upstream and downstream homologous arm fusion gene. Quick Connect to pEASY-T1, ligated into recombinant suicide plasmid was pEMApilM. Recombinant suicide plasmid through conjugation, two homologous recombination, gene deletion mutant was constructedâ–³pilM. RT-PCR in RNA level, to further verify the gene knockout pilM.Mutant gene can be stably inherited in vitro, compared with the parent strain, growth, no significant difference in hemolytic activity. Negative staining electron microscopy (TEM) results, the parental strains can express a complete pili, did not express mutantâ–³pilM. Gene deletion mutant and the parental strain adhesion assay, result analysis, the adhesion of mutant dropped by 50%. Determination of gene deletion mutants and parental strains of the LD50, the results show that the decline in the virulence of mutants.