| Actinobacillus pleuropneumoniae(APP) is the etiological agent of Porcine contagious pleuropneurnonia(PCP),a severe,contagious pulmonary disease of pigs,that causes important economic losses in pig industry worldwide.Until now,15 serotypes have been described.The main virulence factors are RTX-toxins,Lipopolysaccharide, capsule,outer membrane proteins,protease and so on.ThpB is an important virulence factor of APP and used for acquiring iron which found in all serotypes.Zmp can be expressed in host which is predicted as a virulence factor of APP.ArcA-ArcB tow-component system is anaerobic respiration control system which can regulate nearly all of genes belong to anaerobic respiration system and plays a important role in metabolism of APP.This study contains the following 3 parts of works:1.Cloning and expression of tbpB gene of APP and development of an indirect ELISATransferrin binding proteins(TbpA and TbpB) are two major outer membrane proteins of APP.TbpB is located on the surface of the bacteria and plays important role in the pathogenesis and immune response of the bacteria infection.The complete coding sequence(1,644bp) of the tbpB gene was amplified from the genome of APP serotype 1, and highly expressed in E.coli.The recombinant TbpB protein was applied for coating antigens to develop an indirect ELISA,whose specificity,sensitivity were evaluated and compared with indirect haemagglutination assay(IHA).A total of 980 clinical sera were screened using TbpB-ELISA method.The results showed that the TbpB-ELISA method has very high sensitivity but specificity is not so high.compared with the indirect haemagglutination assay(IHA),TbpB-ELISA was more sensitive than IHA.The positive ratio of samples was 53.57%and partly reflected the epidemic of PCP which can be used as reference.2.The study of zmp gene of A.pleuropneumoniaeThe complete coding sequence(2,610bp) of the zmp gene was amplified from the genome of A.pleuropneumoniae serotype 1 which was known,and highly expressed in E. coli.The protease activity of Zmp recombinant protein was identified by special protein substrate.Three methods were tried to construct zmp gene deleted mutant,finally we constrcuted a partial zmp gene deleted mutant of APP serotype 1 and studied the growth characters of mutant whose virulence was compared with parent.The results showed that the protease activity of Zmp recombinant protein can be identified by special protein substrate like casein.The partial zmp gene deleted mutant showed the same growth characters with parent so we can obtain that the zmp gene has little effect of growth of APP in vitro.Virulence compare of mutant with parent showed that virulence of mutant decreased 15-20 times than parent which certified that zmp gene is contributed to the virulence of APE3.The study of ArcA-ArcB two-component system ofA.pleuropneumoniaeFive two-component systems were identified through comparing the known genomes of APP with E.coli genome and other bacterial genomes.ArcA-ArcB is anaerobic respiration control system which was chosen to study.The main goal is to construct arcA-arcB single gene deleted mutants and arcA-areB double genes deleted mutant which will be analysised by DNA chip to find genes which are controled by ArcA-ArcB system,then to study the effect of APP with host in anaerobic respiration condition.In this work,we mainly focused on prophase analysis and the construction the vectors of homologous recombination mutant.The homologous recombination vectors were constructed propotional which is helpful for further study of ArcA-ArcB two component system.
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