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Construction and Characterization of a Salmonella Choleraesuis C78-1 △crp Deletion Mutant

Author: YuChuan
Tutor: ChengXiangChao
School: Henan University of Science and Technology
Course: Basic Veterinary Science
Keywords: Salmonella choleraesuis Gene deletion Recombinant suicide plasmid Conjugal transfer Deletion mutant ( crpC78 )
CLC: S852.61
Type: Master's thesis
Year: 2010
Downloads: 104
Quote: 0
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Abstract


Salmonella choleraesuis (Salmonella Choleraesuis) is one of the main pathogens causing piglet paratyphoid weaned piglets, can cause a large number of disease, often associated with secondary infection of other diseases or not treated a large number of deaths, causing heavy losses to the pig industry . The animals alive infection or food contamination, food poisoning can occur, also has a very important significance in the public health. Vaccination is the most appropriate strategy for the prevention and control of piglet paratyphoid. In China, the development of more successful piglet paratyphoid live vaccine experts antigenic Salmonella choleraesuis virulent strain C78-1 vaccination containing thallium acetate medium for hundreds of generations to elect a good immunogenicity the attenuated strain C500. The strain was passaged attenuated by chemical methods, the genetic background of its debilitating and cause immune protection mechanism is still unclear. In addition, the vaccine strain having the a certain residual vestiges force of, and the risk of reversion of virulence. The purpose of this study is to develop safe and effective Salmonella choleraesuis vaccine attenuated strain, and for the further development of oral vaccine live vector suitable for mucosal immunity to lay a certain foundation, CRP (cAMP receptor protein, CRP) gene as Salmonella choleraesuis the C78-1 strains attenuated gene, the use of recombinant suicide plasmid-mediated bacterial allelic exchange technology of two-step screening Salmonella choleraesuis C78-1 strains the crp genetically engineered deletion mutant, and its biological characteristics were studied. The main contents include: gene cloning and sequence analysis of 1.crp reference Salmonella typhimurium LT2 strain (GenBank No.: AE008859) crp gene sequences from Salmonella choleraesuis C78-1 genome amplified crp gene. Through the sequencing and comparative analysis of the and C500 strains crp gene sequences are identical, log on Genbank other complete sequence of the three known Salmonella crp gene is highly conserved. 2. Reorganization the suicide plasmid pREΔcrp the build the Salmonella choleraesuis C78-1 genome as a template, were amplified fragment of crp Preparation and is cloned into the suicide plasmid pRE112 on, transformed into E. coli χ7213 Building with Missing 320bp crp gene recombinant suicide plasmid pREΔcrp. Conjugative transfer and deletion mutant ΔcrpC78-1 screening and identification of to transformation the restructuring suicide plasmid pREΔcrp in the E. coli χ7213 donor strain C78-1 receptor bacteria joined metastasis. The restructuring suicide plasmid-mediated bacterial gene allelic exchange technology two-step screening CRP gene deletion mutants, and PCR and sequencing to determine the deletion mutant ΔcrpC78-1 was successfully constructed. 4 deletion mutant ΔcrpC78-1 biological characteristics of the deletion mutant ΔcrpC78-1 phenotype, genetic stability, growth characteristics and biological characteristics of virulence in mice identified and found: deletion mutant? CrpC78-1 lost the ability of the carbon source such as maltose, sucrose, fructose and mannitol, and not the decomposition of H2S, but retained the ability to utilize glucose; serotype with the parental strain C78-1, and can be stably genetic deletion of the crp gene; mouse The fatal results showed that its virulence is about 750 times lower than the C78-1.

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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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