Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1999-10-22
2001-03-20
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C424S094400, C424S185100, C424S248100
Reexamination Certificate
active
06204038
ABSTRACT:
BACKGROUND OF THE INVENTION
Tuberculosis and leprosy, caused by the bacilli from the
Mycobacterium tuberculosis
complex and
M. leprae
respectively are the two major mycobacterial diseases. Pathogenic mycobacteria have the ability to survive within host phagocytic cells. From the interactions between the host and the bacteria results the pathology of the tuberculosis infection through the damages the host immune response causes on tissues (Andersen & Brennan, 1994). Alternatively, the protection of the host is also dependent on its interactions with mycobacteria.
Identification of the bacterial antigens involved in these interactions with the immune system is essential for the understanding of the pathogenic mechanisms of mycobacteria and the host immunological response in relation to the evolution of the disease. It is also of great importance for the improvement of the strategies for mycobacterial disease control through vaccination and immunodiagnosis.
Through the years, various strategies have been followed for identifying mycobacterial antigens. Biochemical tools for fractionating and analysing bacterial proteins permitted the isolation of antigenic proteins selected on their capacity to elicit B or T cell responses (Romain et al., 1993; Sorensen et al., 1995). The recent development of molecular genetic methods for mycobacteria (Jacobs et al., 1991; Snapper et al., 1990; Hatful, 1993; Young et al., 1985) allowed the construction of DNA expression libraries of both
M. tuberculosis
and
M. leprae
in the &lgr;gtl1 vector and their expression in
E. coli.
The screening of these recombinant libraries using murine polyclonal or monoclonal antibodies and patient sera led to the identification of numerous antigens (Braibant et al., 1994; Hermans et al., 1995; Thole & van der Zee, 1990). However, most of them turned out to belong to the group of highly conserved heat shock proteins (Thole & van der Zee, 1990; Young et al., 1990).
The observation in animal models that specific protection against tuberculosis was conferred only by administration of live BCG vaccine, suggested that mycobacterial secreted proteins might play a major role in inducing protective immunity. These proteins were shown to induce cell mediated immune responses and protective immunity in guinea pig or mice model of tuberculosis (Pal & Horwitz, 1992; Andersen, 1994; Haslov et al., 1995). Recently, a genetic methodology for the identification of exported proteins based on PhoA gene fusions was adapted to mycobacteria by Lim et al. (1995). It permitted the isolation of
M. tuberculosis
DNA fragments encoding exported proteins. Among them, the already known 19 kDa lipoprotein (Lee et al., 1992) and the ERP protein similar to the
M. leprae
28 kDa antigen (Berthet et al., 1995).
SUMMARY OF THE INVENTION
We have characterized a new
M. tuberculosis
exported protein named DES identified by using the PhoA gene fusion methodology. The des gene, which seems conserved among mycobacterial species, encodes an antigenic protein highly recognized by human sera from both tuberculosis and leprosy patients but not by sera from tuberculous cattle. The amino acid sequence of the DES protein contains two sets of motifs that are characteristical of the active sites of enzymes from the class II diiron-oxo protein family. Among this family, the DES protein presents significant homologies to soluble stearoyl-ACP desaturases.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention.
Bacteria, media and growth conditions
The bacterial strains and plasmids used in this study are listed in
FIG. 8
E. coli
DH5&agr; or BL21(DE3)pLysS cultures were routinely grown in Luria B medium (Difco) at 37° C. Mycobacterium cultures were grown in Middlebrook 7H9 medium (Difco) supplemented with Tween 0.05%, glycerol (0.2%) and ADC (glucose, 0.2% BSA fraction V, 0.5%; and NaCl, 0.085%) at 37° C. Antibiotics when required were added at the following concentrations: ampicillin (100 &mgr;g/ml), kanamycin (20 &mgr;g/ml).
Human and cattle sera
Serum specimens from 20 individuals with pulmonary or extra-pulmonary tuberculosis (
M. tuberculosis
infected) were obtained from the Bligny sanatorium (France), 6 sera from
M. bovis
infected human tuberculous patients and 24 sera from BCG-vaccinated patients suffering from other pathologies were respectively obtained from Institut Pasteur, (Madagascar), and the Centre de Biologie Médicale spécialisée (CBMS) (Institut Pasteur, Paris). Sera from tuberculous cattle (
M. bovis
infected) were obtained from CNEVA, (Maison Alfort).
Subcloning procedures
Restriction enzymes and T4 DNA ligase were purchased from Gibco/BRL, Boehringer Mannheim and New England Biolabs. All enzymes were used in accordance with the manufacturer's recommendations. A 1-kb ladder of DNA molecular mass markers was from Gibco/BRL. DNA fragments used in the cloning procedures were gel purified using the Geneclean II kit (BIO 101 Inc., La Jolla, Calif.). Cosmids and plasmids were isolated by alkaline lysis (Sambrook et al., 1989). Bacterial strains were transformed by electroporation using the Gene Pulser unit (Bio-Rad Laboratories, Richmond, Calif.).
REFERENCES:
Philipp et al. “An integrated map of the genome of the tubercle bacillus,Mycobacterium tuberculosisH37Rv, . . . ” Proc. Natl. Acad. Sci. U. S. A. 93, 3132-3137, Apr. 1996.*
Jackson et al. “Mycobacterium tuberculosisDes protein: an immunodominant . . . ” Infection and Immunity 65, 2883-2889, Apr. 1996.*
Lim et al., “Identification ofMycobacterium tuberculosisDNA Sequences Encoding Exported Proteins by Using phoA Gene Fusions”,J. Bacteriology, vol. 177, No. 1, pp 59-65 (Jan. 1995).
Gicquel Brigitte
Jackson Mary
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Nashed Nashaat T.
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