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
1995-06-06
2001-06-19
Swartz, Rodney P. (Department: 1641)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C424S263100, C435S069100, C435S069700, C435S071100, C435S320100, C536S022100, C536S023100, C536S023700
Reexamination Certificate
active
06248563
ABSTRACT:
INVENTION FIELD
This invention refers to the PCTD plasmid isolated from
Chlamydia trachomatis
serotype D, cloned and sequenced and to the genes present in said plasmid, to the proteins expressed by said genes, to the expression vectors containing said genes and to the microrganisms transformed by said vectors. The invention further refers to the process for the preparation of genes and of said vectors and to the use of said proteins as antigens for the preparation of polyclonal and monoclonal antibodies apt to recognize
Chlamydia trachomatis
and hence useful for the preparation of vaccines capable of imparting a protective immunity against infections caused by
Chlamydia trachomatis
and pathologic conditions deriving from said infections and for the development of diagnostic methods for the search of specific antibodies produced following
C. trachomatis
infections.
PRIOR ART
Chlamydias are gram-negative bacteria, obligate intracellular parasites of eukariotic cells. Chlamydias show an extracellular infective and metabolically practically inert form, called elemental body (EB), and intracellular replicative forms called reticular bodies (RB).
The reticular bodies, after multiplication by binary fission, are transformed into elemental bodies which come out of the host cell and infect new cells.
The masses or mini-colonies of reticular and elemental bodies inside an infected cell constitute the characteristic “inclusions” visible at the optical microscope.
Chlamydia trachomatis
(
C. trachomatis
or
CT
), a bacterial species pathogenic to man, is the etiological agent of venereal lymphogranuloma (VLG), of various inflammatory patologies of the genital male and female apparatus and of trachoma, a chronic disease which affects 500 million people and can lead to blindness. In the technical literature ca. 15
CT
serotypes pathogenic to man were described and divided in two groups which differ both as to virulence and tissular tropism.
Twelve serotypes of the trachoma group (biovar) are identified as A to K and infect, in general, epithelial tissues, such as the ocular (trachoma) and uro-genital (cervicitis and urethritis) mucous membranes, and show a low virulence.
The venereal lymphogranuloma (VLG) serotypes (L
1
, L
2
and L
3
) cause instead an infection of the reticulo-endothelial tissue, mainly of the inguinal and femoral lymphonodi, and are highly invasive. Urethritis and cervicitis induced by
CT
(A to K serotypes) when not precociously diagnosed and treated by adequate therapy, may led to a variety of chronic inflammations, such as, e.g., vaginitis, salpingities and pelvic inflammation which may resolve in sterility and extrauterine pregnancy.
Furthermore the new born from infected mothers may contract pulmonary and/or ocular infections during delivery.
For said reason it is necessary to possess adequate diagnostic methods for determining
CT
and formulating effective vaccines against said bacterium.
As known, factors which determine the bacterial virulence are often encoded by genes present on plasmids.
In the literature, the presence is reported, in all 15 serotypes and in the clinical isolates examined up to now, of a plasmid of ca. 7.5 Kb referred to in the present invention as pCT followed by the denomination of the bacterial serotype concerned. For example: pCTD for the plasmid isolated from serotype D, etc.
Up to now, however, no specific function or products encoded by it were associated with said plasmid.
DETAILED DESCRIPTION OF THE INVENTION
A variant of the plasmid, corresponding to serotype D, was now isolated, indicated in what follows a pCTD, which comprises at least eight genes encoding for new proteins.
FIG. 1
a
shows the nucleotidic sequence of said plasmid and 7 of the 8 protein structures expressed by said sequence. The eighth protein structure, encoded on the DNA chain complemental to the one of
FIG. 1
a
, is shown in
FIG. 1
b.
Object of the present invention are thus: the cloned and sequenced PCTD plasmid, the nucleotide sequences encoding for the above named proteins, the expression vectors containing one of said sequences or fragments thereof.
Further object of the present invention are the pCTD proteins or fragments of them having immunogenic properties.
Still another object of the present invention are the fusion polypeptides comprising one of said proteins or its fragments suitable as antigens.
The present invention further refers to the preparation of said proteins and of their fragments possessing immunogenic activity or of fused polypeptides comprising said proteins.
Said proteins, their fragments or fusion polypeptides comprising said proteins or their fragments, according to the invention may be employed to determine the
CT
produced infections in biological samples.
Said proteins, their fragments or fusion polypeptides comprising the protein or its fragments may further be employed, according to the invention, as antigens useful in the formulation of vaccines against infections due to
CT.
According to the invention, said proteins, their fragments or fusion polypeptides may be used furthermore as antigens for the preparation of poly- or mono-clonal antibodies to be used in diagnostics.
In particular, the present invention relates to the pgp 3D protein encoded by the gene of the pCTD plasmid identified as ORF3D having the nucleotide sequence reported in
FIG. 2
, and characterized by a molecular weight of 27,802 and by the aminoacid sequence reported in FIG.
2
.
According to the present invention, plasmid pCTD is obtained from the
C. trachomatis
G0/86 strain isolated from the urethra of a patient with non-gonococcic urethritis, and successively identified as serotype D by the immunofluorescence method described by Wang, S. P. and Grayston, J. T. [Am. J. Ophtalmol. 70; 367-374 (1970)].
The ORF3D gene may be isolated from the PCTD plasmid employing one of the known methods such as, e.g., the in vitro amplification method [Saiki, A. K. et al. Science, 239:487-491 (1988)] using as primers synthetic oligonucleotides having a primary structure suitably derived from the sequence data shown in
FIGS. 1
a
and
1
b
. The thus emplified gene is then cloned in a vector placing it under the control of sequences regulating its expression.
One can similarly proceed for the other seven genes the nucleotide sequences of which are reported in
FIGS. 1
a
and
1
b.
The proteins encoded by said genes are represented by the aminoacid sequences also reported in
FIGS. 1
a
and
1
b.
Vectors suitable for the ends of the present invention may be plasmids with expression in host cells selected among the ones known and available commercially or at authorized collection centers. The cells transformed by said vectors are then cultivated in a suitable culture medium in the presence of carbon-, nitrogen- and mineral salts sources, possibly in induction conditions, at a temperature and time period selected in order to obtain the production of the desired protein.
Said protein, obtainable also as fused polypeptide, constituted by a polypeptide produced by the vector fused with the protein itself, is then separated and purified from the culture medium or from the cell lysate.
According to one embodiment of the present invention, the ORF3D gene is cloned in the plasmidic
E. coli
pEX34a vector, a derivative of pEX29 and pEX31 described by Strebel et al. [J.Virol., 57:983-991 (1986)], following the description by Nicosia et al. in Infect. Imm. 1987, Vol.55, 963-967.
The results show the presence in the bacterial extracts of a polypeptide, indicated as MS2-pgp3D, the sequence of which is shown in
FIG. 3
, with a mol. weight of ca. 39 Kd, consisting i.e. of a RNA-polymerase fragment of bacteriofage MS2, produced by the expression system of ca. 11 Kd and by the protein encoded by the ORF3D gene of ca. 28 Kd.
Said polypeptide employed as antigen in a Western-Blot assay, or in immunologic assays, is recognized by antibodies present in the serum of patients with
CT
infection and may further be employed for the production, in lab
Comanducci Maurizio
Giuliani Marzia M.
Ratti Giulio
Tecce Mario F.
Blackburn Robert P.
Harbin Alisa
Scalvo SpA
Swartz Rodney P.
Woodcock Washburn Kurtz Mackiewicz & Norris LLP
LandOfFree
Chlamydia trachomatis serotype D genes does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Chlamydia trachomatis serotype D genes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Chlamydia trachomatis serotype D genes will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2503774