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Pasteurellosis Branch (Pasteurellaceae) including the Pasteurella genus (Pasteurella), put mast genus (Actinobacillus) and Haemophilus (for Haemophilus). This bacteria Division contains a large number of animal disease pathogens, they can make a lot of livestock pathogenic or even death, has brought huge economic losses to the aquaculture industry around the world this has been increasing concern about the pathogen bacteria Division. In order to study the pathogenic mechanism of pathogens, to study its gene's function is a must. The study of gene function, the conventional method is to build the gene mutant strain. Gene mutation methods currently used, such as natural transformation, conjugation transfer and electroporated into the suicide plasmid transformation efficiency of double-swap and the occurrence of homologous recombination efficiency is relatively low, and therefore the establishment of an efficient mutation system is very necessary. Because the replication of temperature-sensitive plasmid at a specific temperature can be automatically lost, enabling rapid identification of the occurrence of homologous recombination, the double-swap mutant, and the system can act as expression vectors and mediated double exchange cloning vector in PCR directed mutation, in recombinant enzyme the aid, the probability of double exchange will greatly improve, so replication temperature-sensitive plasmid can greatly improve the the mutation system's efficiency. This study sequenced and analyzed the plasmid pD70KanR Pasteur bacilli bacterial origin, and found that this plasmid having a kanamycin resistance gene, a streptomycin resistance gene and a sulfonamide resistance gene, also contains a in the conjugal transfer the essential mobA gene and the restriction sites of this plasmid using DNAMAN software analysis, some single restriction sites, can be used for cloning. Subsequently, the verification pD70KanR in APP and Pm are shuttle plasmid, was constructed by PCR site-directed mutagenesis, replication in PM and APP Thermosensitive Plasmid pGA318 and PSK-GA318, and analysis of these two plasmids in the Pm and APP different passage time and replication properties, i.e. the culture temperature at 30 ℃ normal replication, passaged, lost in the case of higher than 41 ℃. pGA318 and pSK-GA318, in Pm, 42 ℃ third passage can be lost; in the APP, pGA318 pSK-GA318, 42 ℃ want to pass nine generations to be lost. These results lay the foundation for the building of Pm and APP of pasteurellosis Division bacteria efficient mutation system. Finally, the copy of a temperature-sensitive vector pGA318 was applied to the APP urease C gene deletion screening, coming urea C enzyme and gene Preparation homology arms, and then cloned into pGA318 by homologous recombination principle attempts urease C gene, but deletion of APP mutants could not be obtained, probably because the the pGA318 kanamycin resistance expression cassette under high temperature environment in the APP expression, is not conducive to screening the mutant. Also, verify that the plasmid from Bordetella pJN105 and pD70kanR of the APP can co-exist, and the operating tool to be able to simultaneously express two different proteins in APP gene.
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