Analysis Of The Complete Sequence Of Plasmid Isolated From Riemerella Anatipestifer Wide-type Strain And Construction Of DPP â…£ Gene Deletion

The Riemerlla anatipestifer infection is a communicable disease of domestic ducks, gose, turkeys, and various other birds, which is characterized by septicemia anserum exsudativa. Infected ducks have fibrinous pericarditis, perihepatitis and capsulitis, some have fibrinous meningitis, salpingitis and arthritis. It accounts for significant economic losses to the duck industry throughout the world. Presently,21 serotypes of R.anatipestifer have been identified and no significant cross-protection was reported. It lead to the difficulty of using vaccines to immunize duck against infection. R.anatipestifer infection has been a continued problem in many duck farms. Therefore, emphasis on the study of molecular biology, pathopoiesis and immunologic mechanism, which play a very important role in control and prevent the disease.In this study, we isolated the plasmid from R.anatipestifer wild type strain. Using the experiment of digestion single restriction endonuclease and the primer walking method, we obtained the complete sequence of the wild type plasmid. The bioinformatics analysis of the complete sequence laid a foundation for the reconstruction of the plasmid. Otherwise, using suicide vector, we obtained DPP IV deleted mutant from R.anatipestifer strain RA-YM. The mutant wil be helpful for the further study of DPPâ…£as a virulence factor. The research contents are summarized as follows:1. Isolation and analysis of the complete sequence of plasmid from R.anatipestifer wide type strainUsing alkaline lysis, we isolated the high purity plasmid from R.anatipestifer wild type strainRA-JX. The plasmid was digested with single restriction endonuclease Xba I, and ligated to the cloning vector pUC18 digested with the same enzyme. The recombinatant plasmid was isolated and sequenced. Squencing of the complete sequence of the plasmid used the primer walking method. The result showed that the plasmid was 8048 base pairs in length.The bioinfromatics analysis showed that there are some primary open reading frames were identified. These putative gene products showed significant homology to Rep-3, Transposase-11, COG2378 and Cas2.â… .â…¡-â…¢. BlastN analysis showed the minimal replication was located in the fragment containing Rep and its upstream sequence. It was 2449 base pairs in length. This result makes it possible to continue research R.anatipestifer in molecular lever.2. Construction of DPP IV deleted mutant from R.anatipestifer strain RA-YMAccoding to the sequence of DPP IV, the homologous arm gene were respectively amplified from RA-YM genome, added to the spectinomycin resistance gene, subcloned into suicide plasmid pRE112. The recombination suicide plasmids were designated as pRE-LSR. The DPP IV deleted mutant was constrcted first. The E.coli donor strain X7213 transformed with the suicide plasmid pRE-LSR was conjugated with the recipient strain, the R.anatipestifer strain RA-YM. After transconjugation, spectinomycin-resistant transconjugants in which the whole plasmid had been incorporated into the recipient chromosome were analyzed by PCR. This laid a foundation for the molecular pathopoiesis mechanism and provide a theoretical basis for the design and research genetically engineering vaccine of R.anatipestifer.