Construction Of Signature-tagged Mutagenesis Pools And Preliminary Study On TadD Gene Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae(APP) is the etiological agent of porcine contagious pleuropneumonia(PCP) and causes great economic losses in pig industry worldwide. Immunization is the main method to prevent and control this disease.Nevertheless the serotypes of APP are numerous,cross-protection among each serotype is in a low level,there are a great deal of difficulties in vaccination with traditional vaccines.Reseachers take many new methods such as inactivited vaccine and subunit vaccine to prevent and eradicate the disease,but the effect is unsatisfactory.According to the pathogenesis and prevalence of APP, it is urgent at present to prevent,control and eliminate this disease by using new generation of vaccines which are much safer and much high-performance.The overall identification of virulence factor is the basic to the research of pathogenesis and to the exploit of new product which can prevent and control the disease well.Based on above considerations,we did the following research:1.Construction of a signature-tagged mutagenesis bank of APP.Signature-tagged mutagenesis(STM) is a high throughput method to identify virulence genes from bacteria, which was first developed by Holden in 1995.It is a novel approach to study pathogenesis of pathogens and to screen virulence genes with high throughput in vivo,which is based on whole genome of pathogen.To improve our understanding of the pathogenesis of APP,a large-scale mutant bank was constructed by using signature-tagged mutagenesis(STM) approach for pick-up of infection and virulence associated genes.48 of mini-Tn10 based tag plasmids(pLOF/Tag1-48) were transformed into E.coli S17-1λpir,and the transformants were mated with strain 4074 of APP serotype 1.211 mutant strains have been obtained using 4 different tags(Tag3,Tag4,Tag13 and Tag14) by the biparental mating in this study.This work will contribute to the construction of a STM bank and functional genomics study of APP.2.Structural and functional research on tadD of Actinobacillus pleuropneumoniae,flp operon is present in the genomes of a wide variety of bacteria and archaea,flp operon has different functions in different bacteria.The genes of flp operon are essential for microcolony formation,pilus production and the ability to attach cells,tadD gene is an important gene of flp operon but little is known about the function of tadD gene of APP.According to the GenBank sequences,the two flanks of tadD gene were amplified and cloned from S4074 genome.The upstream and downstream of tadD gene were respectively subcloned into suicide plasmid pEMOC2,which contained a sucrose sensitive(sacB) gene.The recombination suicide plasmids were designated as pEMâ–³D which contained 753bp-deleted tadD.The E.coil donor strainβ2155 transformed with plasmid,pEMâ–³D,was conjugated with the recipient,S4074.After transconjugation,Chloramphenicol-resistant(Cm^R) transconjugants were analyzed for the presence of a first crossover event by PCR.This first step selected for clones in which the whole plasmid had been incorporated into the recipient chromosome.Colonies with the correct PCR profile were incubated in TNB.Aliquots were then plated on to TNA plates containing 10%(w/v) sucrose.After incubation for 24 h, sucrose-resistant(Suc^R) colonies exhibiting a non-mucoid phenotype were tested for the chloramphenicol sensitivity(Cm^S) phenotype,which was indicative of loss of plasmid vector sequences.Following this,Suc^R and Cm^S colonies were identified using PCR and RT-PCR to determine the presence of the second crossover.Cultures of the parent strain S4074 and mutant strain were grown with shaking at 37℃in TSB(supplemented with NAD and sterile bovine serum) overnight.The cultures were sub-inoculated into fresh supplemented TSB at a 1:1000 dilution.The OD₆₀₀ of the bacterial cultures was determined at intervals of 1 h.No obvious difference was observed in the in vitro growth curves of S4074 and mutant strain, indicating that deletion of the tadD gene had no significant influence on the growth of APP S4074.No obvious difference was observed in virulence in mice of S4074 and mutant strain, indicating that deletion of the tadD gene had no significant influence on the virulence of APP S4074.The research on pilus production,adherence and colonization of mutant strain will be done in this study.