Study On Recombination Bacterial Ghosts Of Pasteurella Multocida

Pasteurella multocida is an important pathogen of several animal infectious diseases. Five different capsular types and sixteen somatic types are identified recently. Strains causing haemorrhagic septicemia in cattle and carabao are B:2 and E:2. Strains causing fowl cholera are A:l and A:3. Strains of swine plague appearing in our conntry are capsular types B whil the strains are capsular types A in abroad. Capsular types A and B which could produce dermatonecrotic toxins are one of important pathogens of infectious atrophic rhinitis. Capsular types F strains cause disease of varying degree to turkeys. Of late years, the case of pneumonia by P. multocida types A increase gradually in our country. Immunoprophylaxis is efficiency mesans to control above these diseases. However, the all vaccines of P. multocida existence a problem recently that immune phase is short and Protective Rate is lowness. The immunogenic efficacy of live vaccines for same strains is higher than inactivated vaccines. Yet, in inactivated vaccine preparations these epitopes may be destroyed by the physical and chemical processing treatments. Persisting the natural antigen epitopes is effective means. For this reason, we obtained E gene fragment from pHH43 and then cloneed in plasmid pPBA1100 to established shuttle plasmid for expressing E lysis gene by genetic engineering method. Then the recombination bacterial ghost of P. multocida was constructed as sample of P. multocida causes great losses at present. The effect of E gene expressing was understood by chromometry, CFU and scanning electron microscopy method. Reliability and protectability of the bacterial ghost were estimated by the immunogenic text and challenge experiment. The result was as following:1. The plasmid pHH43 with E gene and temperiture sensitivity control system been carved by restriction endonuclease EcoR I . The result showed that there are five bands in plate of agarose electrophoresis. Molecullar weight of every band was accord with theoretic value and confirmed success for recombination plasmid.2. Two bands been detected by restriction endonuclease SacI. Expressing plasmid pPBA1100-E were constructed by which E-cassette hybridized with shuttle plasmid pPBA1100. recombination strains of P. multocida was constructed when Expressing plasmid tansform to cells of P. multocida. The result of the E-cassette sequencing indicated that the sequence included E gene sequence and the sequence of gene control system (λPRmut promotor and CI857 inhibitor). And recombination strains constructed was wonderfully successful by restriction endonuclease analysis.3. The result of recombination text showed that bacterial ghosts of P. multocid were produced when E gene was expressed by the growth curve analyzing and SEM observing.4. The result of reliability and protectability detection of the bacterial ghost indicated that lysis rat of recombination strains attained 98.6 per cent and that bacterial ghost of P. multocid could produced preferable immune protection in mouse, and immune protection rate reached 100 per cent.This study was theoretical and application significance to change traditional inactivation method, to relize new idea of genetic inactivation, and to open up new way of animal vaccine preparation.