| PATENT ASSIGNEE'S COUNTRY | Italy |
| UPDATE | 08.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 29.08.00 |
| PATENT TITLE |
Recombinant Chlamydia trachomatis pgp3 fusion protein |
| PATENT ABSTRACT |
A plasmid isolated from Chlamydia trachomatis is described, which comprises 8 genes encoding proteins useful in the formation of vaccines or diagnostic test for determining the bacterium or specific antibodies generated during C. trachomatis infections. In particular, the recombinant fusion protein MS2-pgp3D is described, which comprises polypeptide sequences encoded by pCT and is immunogenic in the course of infections in man. A method for preparing the recombinant fusion protein MS2-pgp3D in E. coli is also described. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 18.05.95 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Klinkert et al Molecular & Biochemical Parasitology 27: 233-240, 1988. Database Search of Sequence in Plasmid: 23: 149-154 1990. Palmen et al Plasmid 16: 52-62 1986. Maniatis et al Molecular Cloning A Laboratory Manual. Caldwell, H. et al., "Purification and Partial Characterization of the Major Outer Membrane Protein of Chlamydia trachomatis", Infection and Immunity 1981, 31 (3), 1161-1176. Cevenini, R. et al., "Effects of Penicillin on the Synthesis of Membrane Proteins of Chlamydia trachomatis LGV2 Serotype", FEMS Microbiol. Letters 1988, 56, 41-46. Davis, L.G., "Basic Methods in Molecular Biology", Elsevier Edit., New York, 1986. Mandel, M. and Higa, "Calcium-Dependent Bacteriophage DNA Infection", J. Mol. Biol. 1970, 53, 159-162. Nicosia, A. et al., "Expression and Immunological Properties of the Five Subunits of Pertussis Toxin", Infection and Immunity 1987, 55 (4), 963-967. Remaut, E. et al., "Improved Plasmid Vectors with a Thermoinducible Expression and Temperature-Regulated Runaway Replication", Gene 1983, 22, 103-113. Saiki, R. et al., "Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase", Science 1988, 239, 487-491. Sanger, F. et al., "DNA Sequencing with Chain-Terminating Inhibitors", PNAS USA 1977, 74(12), 5463-5467. Strebel, K. et al., "Characterization of Fott-and-Mouth Disease Virus Gene Products with Antisera Against Bacterially Synthesized Fusion Proteins", J. of Virology 1986, 57(3), 983-991. Wang, S. and Grayston, "Immunologic Relationship Between Genital Tric, Lymphogranuloma Venereum, and Related Organisms in a New Microtiter Indirect Immunofluorescence Test", Am. J. of Ophthamology 1970, 70(3), 367-374. Comanducci, M. et al., "Identification and Characterization of a 28-Kd Protein of C. trachomatis Encoded by the 7.5-Kb Common Plasmid", in "International Symposium on Human Chlamydial Infections", Jun. 24-29, 1990, pp. 121-124. Comanducci, M. et al., "The Structure of a Plasmid of Chlamydia trachomatis Believed to be Required for Growth Within Mammalian Cells", Mol. Microb. 1988, 2(4), 531-538. Comanducci, m. et al., "Diversity of the Chlamydia trachomatis Common Plasmid in Biovars with Different Pathogenicity", Plasmid 1990, 23, 149-154. Morrison, R. et al., "Immunology of Chlamydia trachomatis Infections", in Sexually Transmitted Diseases, Quinn, T., ed., Raven Press, Ltd., New York, 1992, pp. 57-84. Zhang, Y.-X. et al., "Protective Monoclonal Antibodies to Chlamydia trachomatis Serovar--and Serogroup-Specific Major Outer Membrane Protein Determinants", Infection and Immunity 1989, 57(2), 636-638. Maniatis et al., "Molecular Cloning A Laboratory Manual", 1982. Palmer et al., "A Common Plasmid of Chlamydia trachomatis", Plasmid 1986, 16, 52-62. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. An isolated and purified recombinant pgp3D fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 12. 2. The recombinant pgp3D fusion protein of claim 1, wherein said fusion protein is MS2-pgp3D consisting of the amino acid sequence set forth in SEQ ID NO: 23. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION 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. BACKGROUND OF THE INVENTION 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 elemtal 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.sub.1, L.sub.2 and L.sub.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. SUMMARY OF THE INVENTION The present invention relates to a recombinant MS2-pgp3 fusion protein and methods of producing the same. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B shows the nucleotide and amino acid sequences of pCTD. FIG. 1A shows open reading frames (ORF) 1-7, whereas FIG. 1B shows ORF8. FIG. 2 shows the nucleotide sequence of ORF3 and the amino acid sequence of the pgp3D protein encoded thereby. FIG. 3 shows the amino acid sequence of the fusion protein MS2-pgp3D. 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. 1a 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. 1a, is shown in FIG. 1b. 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 GO/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. 1a and 1b. 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. 1a and 1b. The proteins encoded by said genes are represented by the aminoacid sequences also reported in FIGS. 1a and 1b. 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 laboratory animals, of mono- and poly-clonal antibodies which recognize the--and react with the corresponding pgp3 protein, in all its variants, of C. trachomatis. In accordance with the present invention the pCTD and p03/60/MCI plasmids were deposited as ATCC N.sup.o 68314 and ATCC N.sup.o 68315 respectively. The experimental examples that follow are illustrative and non limitative of the invention. |
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