Study On The Functional Domains Of ToxA Gene Of Porcine Toxigenic Pasteurella Multocida

Toxigenie Pasteurella multocida (T⁺Pm) is a major pathogen of progressive atrophicrhinitis (PAR). T⁺Pm secretes P. multocida toxin (PMT), which is a dermonecrotic toxinabout 146 kDa, and PMT was encoded by toxA gene. PMT is one of the most importantvirulence factors and protective antigens of Toxigenic Pasteurella multocida. In order toanalyses and accredits the structural domains of virulence and immunoprotection on toxAgene, The projects are as follows:1. cloning and expressing of toxA gene and its truncated segments: T⁺Pm, which wasisolated and preserved by our lab, named HN-13. First, according to the toxA gene whichwas published in GenBank, compare their gene sequence, and find the conservation one.Three pairs of palmers were designed to clone three fragments of toxA gene with thePyrobest^TM DNA polymerase. Connect with ligase, and procured the whole toxA gene.The whole toxA gene is 3858bp. The compared result shows that the length of the toxAgene was same to the toxA gene, which was published in GenBank Z25388. Andaccording to the literature, Absenced the whole toxA gene, procured nine fragments, andgot the nine prokaryote recombinant expressive vectors. Prokaryote recombinantexpressive vectors were transformed into E.coli BL21(DE3) competent cells and theninduced to express. Cleansing five of them, procured inclusion body. Purifiedrecombination protein. And the result of Western-blot shows that the five recombinationprotein can specific reaction with polyclonal antibody of HN-13.2. Immunogenicity analysis of recombination protein: using the Freund's adjuvant,emulsifying five of the recombination proten, immunize KUN-Ming mice of each group,immunize the KUN-Ming mice again after two weeks. Using the PMT-ELISA todetermine the level of the antibody of each group before and after immunize. Fourteendays after second time immunize, Inject of toxigenic Pasteurella multocida(HN-13) toeach group, observe the survival percentage of each group at 24h and 48h. Take liver andlung of each group, analysis in histopathology. This work shows that theimmunoprotection of recombination protein from C- fragment to N- fragment isattenuated, and the full length fragment have good immunoprotection.3. Cytotoxicity analysis of recombination protein: using the collagenoblast ofmice(NIH-3T3 cell), appraisal cytotoxicity of five of the recombination protein. Observethe death and pathological changes of the cell. The result shows that compare with thenatural PMT, toxicity of each recombination protein is to lower. In summary, in this study, we study the funtional of toxigenic Pasteurella multocida,cloned the whole toxA gene from toxigenic Pasteurella multocida, named HN-13, andexpressed it successfully. Authenticate immunoprotection and virulence with the animalexperiment and cell experiment. Experiment shows that the recombination protein havegood immunoprotection. This was useful for further primary study of T⁺Pm and theproduction of vaccine.