Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1996-07-10
2002-09-03
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S070100, C435S071100, C435S071200, C435S252300, C435S254110, C435S320100, C435S325000, C514S04400A, C536S023700
Reexamination Certificate
active
06444444
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of molecular biology and is particularly concerned with genes encoding mycobacterial proteins associated with cell binding and cell entry and uses thereof.
BACKGROUND OF THE INVENTION
Tuberculosis (TB) is a major cause of mortality throughout the world, particularly in developing countries. There are about 8 to 9 million new cases of clinical disease reported every year and the number of deaths is estimated to be about 3 million. In the U.S. the trend of steady decline in TB has reversed and the problem is compounded by increasing numbers of drug-resistant strains. The tuberculosis complex is a group of four mycobacterial species that are genetically closely related. The three most important members are
Mycobacterium tuberculosis,
the major cause of human TB;
Mycobacterium africanum,
a major human pathogen in some populations; and
Mycobacterium bovis,
the cause of bovine TB. None of these mycobacteria is restricted in being pathogenic for a single host species.
In addition to being an important human disease, TB is also a major veterinary problem in many countries. Infection of cattle with
M. bovis
results in bovine TB and all animals showing any signs of infection are systematically slaughtered. The economic losses are thus extensive, and furthermore, cattle can serve as a reservoir for human disease.
In a majority of cases of infection, inhaled tubercle bacilli are ingested by phagocytic alveolar macrophages and are either killed or grow intracellularly to a limited extent in local lesions called tubercles. In this way the infection is limited and the primary sites of infection are walled off without any symptoms of disease being observed. Such individuals have a lifetime risk of about 10% for developing active disease. In a latter eventuality, bacilli spread from the site of infection in the lung, through the lung and via lymphatics or blood to other parts of the body producing characteristic solid caseous (cheese-like) necrosis in which bacilli survive. If the necrotic reaction expands breaking into a bronchus, or in the worst case, if the solid necrosis liquefy, a rapid proliferation of the bacilli occurs. The pathological and inflammatory processes set in motion then produce the characteristic weakness, fever, chest pain, cough and bloody sputum which are the hallmarks of active TB.
Effective treatment of TB with antibiotics exists. However, this is expensive and requires prolonged administration of a combination of drugs. There is a problem in compliance with the drug administration regime because of the extended time periods involved and this has contributed to the appearance of drug resistant strains. There is a recognized vaccine for TB which is an attenuated form of
M. bovis,
known as BCG (bacilla Calmette Guérin). This strain was developed in 1921 and the basis for its attenuation is still not known (ref. 1—throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure). The efficacy of BCG as a TB vaccine is a subject of controversy and has been estimated in various trials to be anywhere between 0 and 70%.
The molecular basis for the virulence and pathogenesis of
M. tuberculosis
have not been extensively described. Some virulence factors, particularly those related to the sigma factors have been recently identified (ref. 2).
M. tuberculosis
can enter non-phagocytic cells in culture, such as HeLa cells (ref. 3) and once inside can multiply and survive. Recently, a DNA molecule (1535 bp long) from a strain of
M. tuberculosis
(H37Ra) was reported to mediate the entry of the bacterium and its survival in mammalian cells (ref. 4). This DNA fragment when introduced into a non-pathogenic strain of
E. coli
is able to confer invasiveness to the bacterium, and survival for up to 24 hours in human macrophages. The mce gene (mycobacterial cell entry) gene was mapped to an Open Reading Frame (ORF) extending from position 182 to 810 on the 1535 bp DNA fragment mentioned above and encodes a protein of molecular weight between 22 and 27 kDa.
Mycobacterial infection may lead to serious disease. It would be advantageous to provide genes encoding proteins of mycobacteria associated with cell binding and cell entry for the provision of these proteins as antigens in immunogenic preparations including vaccines, carriers for other materials including antigens and the generation of diagnostic reagents. The genes encoding mycobacterial proteins associated with cell binding and cell entry are particularly desirable and useful in the specific identification and diagnosis of mycobacteria and for immunization against disease caused by mycobacterial infection.
SUMMARY OF INVENTION
In the present invention, the gene of
Mycobacterium bovis
encoding the protein associated with cell binding and cell entry has been isolated and cloned and found to encode a protein of molecular weight about 45 to about 60 kDa. Corresponding genes have been detected in other members of the tuberculosis complex, including
Mycobacterium tuberculosis.
Accordingly, in one aspect of the invention, there is provided an isolated nucleic acid molecule encoding a Mycobacterium protein having a molecular weight of about 45 to about 60 kDa associated with cell binding and cell entry of a Mycobacterium strain.
The Mycobacterium strain is a strain of the tuberculosis complex, which includes a strain of
M. tuberculosis
and
M. bovis
as well as the other members of the complex, including
M. africanum.
The protein associated with cell binding and cell entry encoded by the nucleic acid molecule may have an amino acid composition as shown in Table 2.
The nucleic acid molecule of the invention may have a restriction map as shown in FIG.
5
. This restriction map is for the gene from the
M. bovis
strain BCG. The nucleic acid molecule also may have the restriction map of the corresponding genes in other mycobacteria from the tuberculosis complex.
The nucleic acid molecule may have a DNA sequence as shown in
FIG. 6
(SEQ ID No: 2) for
M. bovis
strain BCG or the sequence of the corresponding gene in other mycobacteria from the tuberculosis complex.
The nucleic acid molecule also may encode an amino acid sequence as shown in
FIG. 7
(SEQ ID No: 3) for
M. bovis
strain BCG or the sequence encoding the corresponding protein in other mycobacteria from the tuberculosis complex.
In another aspect of the invention, there is provided an isolated DNA molecule amplifiable by polymerase chain reaction (PCR) by a pair of primers having the sequence of primers 4879 (SEQ ID No: 12) and 4882 (SEQ ID No: 15); or 4879 (SEQ ID No: 12) and 4865 (SEQ ID No: 11); or 4879 (SEQ ID No: 12) and 4812 (SEQ ID No: 10). The DNA molecule may be from a member of the tuberculosis complex, particularly
M. tuberculosis
and
M. bovis.
In an additional aspect, the present invention includes a vector adapted for transformation of a host, comprising a nucleic acid molecule or DNA molecule provided herein. The invention further includes a plasmid containing the gene of a strain of Mycobacterium encoding the protein associated with cell binding and cell entry having the identifying characteristics of plasmid pBCGcepX having ATCC Deposit No. 97511.
The vector may be adapted for expression of the encoded protein in a heterologous or homologous host, in either a lipidated or non-lipidated form. Accordingly, a further aspect of the present invention provides an expression vector adapted for transformation of a host comprising a nucleic acid molecule or DNA molecule as provided herein and expression sequences operatively coupled to the nucleic acid molecule or DNA molecule for expression by the host of the protein associated with cell binding and cell entry. The expression seque
Anand Naveen N.
Klein Michel H.
Aventis Pasteur Limited
Duffy Patricia A.
Sim & McBurney
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