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Analysis of the Complete Sequence of Plasmid Isolated from Riemerella Anatipestifer Wide-type Strain and Construction of DPP Ⅳ Gene Deletion

Author: PengXiaoZuo
Tutor: HuSiShun
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Riemerella anatipestifer Wild-type plasmid Sequence analysis DPP Ⅳ gene Missing
CLC: S852.61
Type: Master's thesis
Year: 2011
Downloads: 40
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Abstract


The duck anatipestifer disease Bordetella (Riemerella anatipestifer, RA) Riemerella domestic ducks, geese, turkeys and a variety of birds caused by a septic exudation of infectious diseases as the main feature. Clinical necropsy symptoms mainly manifested as the cellulose pericarditis perihepatitis or the airbag inflammation, and in some cases a fibrinous meningitis, salpingitis or arthritis of. Disease caused huge economic losses to the world duck industry. Duck anatipestifer serotypes have been reported 21, the low rate of cross-protection between serotypes, makes vaccine prevention more difficult. In the the the most duck, Riemerella Bordetella infection event difficult to eradicate, showing persistent infection state. Therefore, strengthening the study of the the RA molecular biology, pathogenesis and immune mechanisms, fundamental prevention from the occurrence of the disease has important practical significance. In this study, the use of alkaline lysis method of extraction RA clinical isolates of wild-type plasmid by restriction analysis, primer extension method to obtain the full sequence of the plasmid, and bioinformatics analysis, to lay the foundation for plasmid transformation. In addition to the first successful suicide vector constructed strains of RA-YM DPP Ⅳ deletion mutant, and lay the foundation for further study of the DPP Ⅳ role in the pathogenesis of RA. The results obtained in this study are as follows: wild-type plasmid anatipestifer Riemerella separation sequence analysis applications alkali lysis method of the Bordetella Riemerella clinical isolates RA-JX plasmid extraction, to obtain high purity plasmid. Xba Ⅰ restriction enzyme digestion, gel purification digested fragments, and then connected with the same digested vector pUC18, and the resulting positive clones were sent for DNA sequencing, and then the primer extension method, and determination of the sequence of the entire plasmid. The analysis results show, RA-JX strains containing plasmid size to 8048 bp. Bioinformatic analysis, including a search of the full sequence of plasmid ORF, ORF homology comparison and analysis of the replicon plasmid pRA-JX. Plasmid pRA-JX encoding Rep.3, the Transposase 1 1, COG2378 and Cas2 Ⅰ Ⅱ Ⅲ ORF. Than the outside of the plasmid PRA-JX ORF sequences BlastN analysis, and speculated that the replicon of the plasmid pRA-JX, including the plasmid replication initiation protein as well as its upstream to a sequence, the size of the 2449 BP. This test provides technical support for further research from the molecular biology level the Riemerella anatipestifer. Serotype 1 the Yunmeng isolates DPP Ⅳ sequence. Riemerella Bordetella serum type 1 the Yunmeng isolates DPP Ⅳ deleted vaccine the reference Riemerella anatipestifer DPP Ⅳ homology arm fragment was amplified from the genome of the RA-YM spectinomycin-resistant gene is inserted between the homology arms, and connected to the suicide plasmid pRE112, constructing a recombinant suicide plasmid PRE-LSR. Recombinant suicide plasmid was transformed into E. coli X7213, E. coli X7213 positive clones donor strain and recipient strain RA-YM conjugal transfer coating spectinomycin (Spc) resistance plate screening Spc resistance zygote suicide plasmid and PCR identification reorganization has been integrated into the chromosome. The test for further in-depth study of the molecular pathogenic mechanism of RA to lay a genetically engineered vaccine of the basic Riemerella anatipestifer provide a theoretical basis for design and R \u0026 D.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Pathogenic bacteria
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