专利汇可以提供Reduction in the virulence of Mycobacterium tuberculosis and protection against tuberculosis by means of phoP gene inactivation专利检索,专利查询,专利分析的服务。并且The construction of a mutant in the phoP gene by means of homologous recombination from a clinically isolated Mycobacterium tuberculosis reduces the virulence thereof in mouse bone marrow macrophage. Moreover, the phoP mutant reduces the virulence thereof in the experimental mouse model. Said phoP mutant can persist without being eliminated both in the macrophage and in the mouse. Mice inoculated with the phoP mutant are protected against M. tuberculosis infection. The use of mutants of mycobacteria in which the phoP gene or the genes regulated by phoP have been inactivated are candidates for vaccines against human and animal tuberculosis as well as possible recombinant vaccines against other pathogens.,下面是Reduction in the virulence of Mycobacterium tuberculosis and protection against tuberculosis by means of phoP gene inactivation专利的具体信息内容。
The invention claimed is:
This application is a 371(c) National Stage of PCT/ES02/00381 filed Jul. 20, 2002, which claims priority to Spanish patent application P 200101824 filed Jul. 31, 2001, the entire disclosures of which are incorporated herein incorporated by reference.
This invention pertains to the field of microbiology.
Recent developments of genetic tools for manipulating the tuberculosis bacillus make possible the construction of mutants in specific genes.
In other intracellular pathogen microorganisms such as Salmonella it has been described that the phoP gene is involved in the virulence. Inactivation of this gene has permitted the construction of attenuated mutants, which have been studied as vaccines against Salmonellosis in humans.
Our preliminary results (University of Zaragoza) in strains of the M. tuberculosis complex isolated from humans have indicated that possibly the most virulent strains have an alteration of the gene annotated in the M. tuberculosis genome as phoP.
The gene was inactivated in clinical isolation for the purpose of studying the involvement of phoP in M. tuberculosis virulence and its multiplication ability and persistence in macrophage and mouse.
Vectors were constructed where the phoP gene of M tuberculosis was inactivated by the introduction of a gene resistant to kanamicin. The vectors were transformed into a clinical isolate of M tuberculosis and a strain with the inactivated phoP gene was obtained.
Work relating to these constructions was recorded in the 1998-1999 workbooks of the Genetics Mico-bacteria Group laboratories of the University of Zaragoza and the Unité de Génétique Mycoctérienne of the Pasteur Institute of Paris.
The phoP mutant of M. tuberculosis has been deposited in the culture collection of the Pasteur Institute of Paris (I-2622), 28, rue du Dr. Roux, 75724 Paris Cedex 15, France, on Nov. 27, 2000.
When M. tuberculosis was studied and compared with the mutant M. tuberculosis phoP strain for 7 days in mouse bone marrow macrophages, it was observed that the mutant was not able to replicate in mammal macrophage. By continuing with the culture of the mutant for 14 days, it was observed that it was able to persist.
After infecting a mouse with the mutant strain, it was observed that there is no multiplication of the bacillus at 3 weeks or at 6 weeks, but the number of bacteria in the spleen, the liver, and the lungs is not reduced as compared with the initial inoculation.
The results of experiments with the M. tuberculosis phoP in macrophage and mouse are contained in the workbooks of the Genetics Mico-bacteria Group laboratories of the University of Zaragoza and the Unité de Génétique Mycoctérienne of the Pasteur Institute of Paris for the year 2000.
The mouse protection studies discussed below under the Description of Figures should be considered with these results. The M. tuberculosis phoP mutant was inoculated subcutaneously. Eight weeks later, the mice were intravenously infected with M. tuberculosis H37Rv. Bacterial count 4 weeks after infection clearly shows that the phoP mutant protects against the multiplication of M. tuberculosis with an effectiveness comparable to the BCG vaccine control. The results of the experiments are contained in the workbooks of the Unité de Génétique Mycoctérienne of the Pasteur Institute of Paris for the years 2001-2002.
Balb/c mice were inoculated subcutaneously with 107 colony forming units (CFUs) either of BCG control vaccine against tuberculosis or of candidate for vaccine of the M. tuberculosis phoP mutant.
Eight weeks later, the mice were infected intravenously with M. tuberculosis H37Rv (105 CFUs). Four weeks after infection, the animals were sacrificed and CFUs were counted in the spleen (
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