Chemistry: molecular biology and microbiology – Vector – per se
Patent
1994-10-06
1999-10-19
Guzo, David
Chemistry: molecular biology and microbiology
Vector, per se
435 691, 435 701, 435 711, 4352523, 43525231, 43525233, 4352525, 536 231, 536 241, C12N 1563, C12N 121, C07H 2104, C12P 2102
Patent
active
059688157
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The object of the invention is a nucleotide sequence which makes it possible to done or express nucleotide sequences in a specific cell host.
A nucleotide sequence of the invention may be obtained from Mycobacterium paratuberculosis.
2. Description of the Related Art
Certain strains of mycobacteria are particularly well known, for example the bacillus of Calmette and Guerin (BCG) which is an avirulent strain of Mycobacterium bovis widely used throughout the world in the context of vaccination against tuberculosis. Its biological properties make it a useful candidate for the development of recombinant vaccines. The cell wall functions as a very effective adjuvant and a single inoculation can trigger long-lasting immunity. Serious side effects due to this bacillus are rare even on repeated immunizations.
The induction of specific immunity, following vaccination by BCG is initiated when T cells interact with macrophages presenting mycobacterial antigens in combination with products of the major histocompatibility complex (abbreviated to MHC). Clones of sensitized T cells proliferate and produce lymphokines which in turn activate macrophages in order to eliminate the bacilli non-specifically. In addition, helper T cells induce the proliferation of B cell clones which lead to the production of antibodies.
Attempts have already been made to carry out the cloning and expression of heterologous genes in BCG, in particular by using available know-how relating to replicative or integrative vectors. Thus an epitope of the gag protein of HIV-1 has been cloned in the form of a fusion polypeptide with the alpha antigen, this antigen being one of the major proteins exported by mycobacteria, in particular the BCG or Mycobacterium kansasii and resistance genes for antibiotics have been expressed under the control of their own regulatory region. In order to optimize the expression of heterologous antigens in BCG recombinants, the inventors have directed their researches towards the characterization of the gene regulatory units which are functional in the mycobacteria
The inventors have thus described the isolation and characterization from Mycobacterium paratuberculosis of nucleotide sequences which make possible the expression of given nucleic acids in mycobacteria or in other cell hosts.
By nucleic acid is meant any nucleotide sequence capable of being cloned and/or expressed whatever its composition, length or origin (synthetic or obtained by extraction).
Various experiments have been performed on M. paratuberculosis (also designated hereafter by Mptb) and Green et al. (Nucleic Acids Research Vol. 17 (22) 1989, pages 9063-9072) in particular have characterized and sequenced an insertion element of this mycobacteria, an element which has been called IS900. According to Green et al., this insertion element contains an open reading frame called ORF1197 which codes for a protein of 399 amino acids.
SUMMARY OF THE INVENTION
The inventors have investigated specific sequences of the species Mycobacterium paratuberculosis by screening a lambda gt11 genomic library by performing hybridization assays with the DNA of strains of other mycobacteria, in particular M. phlei described in Murray A. et al. New Zealand Veterinary Journal 37: 47-50. On this occasion they were interested in a specific DNA sequence which contained a fragment adjacent to the element IS900 described by Green et al.
They determined the presence of a sequence adjacent to the 5' part of the reverse sequence complementary to the open reading frame which codes for a potential transposase contained in the insertion element IS900; this novel sequence is capable of having promoter functions and of containing important signals for the regulation of transcription and translation.
A nucleotide sequence according to the invention which can be used for the cloning and/or expression of a nucleic acid is characterized in that it comprises a sequence (I) selected from: sequence likely to be implicated in the expression
REFERENCES:
patent: 5330754 (1994-07-01), Kapoor et al.
Fox "No Winners Against AIDS" Biotechnology vol. 12 Feb. 1994 p. 128.
Kunze etal. "IS901, A New Member of a Widespread Class of Stypleal Insertion Seqences . . . " Mol. Microbiol. 5(9)2265-2272 1991.
Gheorghiu Marina
Gicquel Brigitte
Murray Alan
Degen Nancy J.
Guzo David
Institut Pasteur
Massey University
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