Desaturase antigen of Mycobacterium tuberculosis

Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase

Reexamination Certificate

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C424S185100, C424S248100, C424S094400

Reexamination Certificate

active

06551808

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;gt11 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 base 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 se a 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 description, serve to explain the principles of the invention.


REFERENCES:
Cahoon et al. “Expression of a coriander desaturase results in petroselinic acid . . . ” Proc. Natl. Acad. Sci USA 89, 11184-11188 (1992).*
Lim et al., “Identification ofMycobaterium tuberulosisDNA Sequences Encoding Exported Proteins by Using phoA Gene Fusions”,J. Bacteriology, vol. 177, No. 1, pp. 59-65 (Jan. 1995).
Jackson et al.,Infection and Immunity, vol. 65, pp. 3882-2889 (Jul. 1997).
Philipp et al.,Proc. Nat'l. Acad. Sci. U.S.A., vol. 93, pp. 3132-3137 (Apr. 1996).

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