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• • A—HUMAN NECESSITIES
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Definitions

• the invention is in the field of clinical medicine, molecular biology and genetic engineering. More particularly, it relates to the molecular methods of tuberculosis diagnosis using newly identified DNA sequences which can be used as probes for DNA hybridization and or for DNA amplification leading to the identification of pathogenic mycobacteria causing disease in humans and animals.
• Tuberculosis an infectious disease mainly caused by respiratory infection with Mycobacterium tuberculosis, represents an important subject of multidisciplinary investigation owing to the urgent need for rapid and reliable diagnostic tests and effective vaccines for disease control.
• PCR polymersase chain reaction
• the present invention is based on novel DNA sequences cloned from the genome of Mycobacterium tuberculosis, which can be used for strain differentiation and for the diagnosis of tuberculosis.
• DNA sequences of the cloned fragments is an aspect of the invention.
• the cloned DNA fragments are found to code for at least 7 proteins of about 9kDa, 15kDa, 17kDa,31kDa, 55kDa, 74kDa and 77kDa, the sequences of which are another aspect of the invention.
• the use of the cloned DNA or of the proteins coded by the cloned DNA for the purpose of serology, skin testing, vaccine development or drug design is another aspect of the invention.
• the invention concerns a DNA according to (c) or (e) , its single strands being hybridizable with those of the DNA according to (a) , (b) , (d) and (f) , respectively, at a temperature of at least 25 °C and at a concentration of NaCl of 1 M.
• RNA being a transcript of a DNA according to the invention.
• the invention concerns a protein being encoded by a DNA according to the invention.
• the invention concerns a protein having the amino a. d sequence (II) according to figure 13.
• the protein according to the invention can be an about 74 kDa protein. Further the invention concerns a protein having the amino acid sequence (III) according to figure 14.
• the protein according to the invention can be an about 77 kDa protein.
• the invention concerns a protein having the amino acid sequence (IV) according to figure 15.
• the protein according to the invention can be an about 9 kDa protein.
• the invention concerns a protein having the amino acid sequence (V) according to figure 16.
• the protein according to the invention can be an about 55 kDa protein.
• the protein according to the invention can be a recombinant protein, especially a protein produced by means of a bacterial strain, a yeast strain, a fungal strain or a cell line of a higher eucaryote.
• the protein according to the invention can be encoded by a DNA sequence according to the first embodiment of the invention and can be recovered by a method comprising the following steps: (i) subjecting proteins encoded by said DNA sequence to a usual test for diagnosis of tuberculosis,
• the invention concerns a DNA, RNA or protein according to the invention which can be used for
• the invention concerns a use of a DNA according to the invention for the identification of mycobacteria in media samples.
• the foregoing use can comprise the steps of (i) isolating the mycobacterium, (ii) preparing crude or purified genomic DNA,
• the invention concerns a use, wherein as samples clinical samples are used.
• the invention concerns a use of a DNA according to the invention or of a protein according to the invention for
• the invention concerns a use of a DNA according to the invention or of a protein according to the invention for the development of drugs useful for combating mycobacterial infections of humans or animals, especially tuberculosis, especially for testing and recovering of substances inhibiting mycobacterial infections in humans and animals, especially tuberculosis.
• the invention concerns a DNA sequence according to the invention or of a protein according to the invention for the development of drugs useful for combating mycobacterial infections of humans or animals, especially tuberculosis, especially for testing and recovering of substances inhibiting mycobacterial infections in humans and animals, especially tuberculosis.
• the invention concerns a DNA
• the invention concerns a DNA according to (c) or (e) , its single strands being hybridizable with those of the DNA according to (a) , (b) , (d) and (f) , respectively, at a temperature of at least 25 °C and at a concentration of NaCl of
• RNA being a transcript of a DNA according to the invention.
• the invention concerns a protein being encoded by a DNA according to the invention.
• the invention concerns a protein having the amino acid sequence (VII) according to figure 5.
• the protein according to the invention can be an about 15 kDa protein.
• the invention concerns a protein having the amino acid sequence (VIII) according to figure 6.
• the protein according to the invention can be an about 31 kDa protein.
• the protein according to the invention can be a recombinant protein, especially a protein produced by means of a bacterial strain, a yeast strain, a fungal strain or a cell line of a higher eucaryote .
• the protein according to the invention can be a encoded by a DNA sequence according to the second embodiment of the invention and can be recovered by a method comprising the following steps: (i) subjecting proteins encoded by said DNA sequence to a usual test for diagnosis of tuberculosis,
• the invention concerns a DNA, RNA or protein according to the invention which can be used for
• the invention concerns a use of a DNA according to the invention for the identification of mycobacteria in media samples .
• the invention concerns a use, wherein as samples clinical samples are used.
• the invention concerns a use of a DNA according to the invention or of a protein according to the invention for
• the invention concerns a use of a DNA according to the invention or of a protein according to the invention for the development of drugs useful for combating mycobacterial infections of humans or animals, especially tuberculosis, especially for testing and recovering of substances inhibiting mycobacterial infections in humans and animals, especially tuberculosis.
• the invention concerns a DNA according to (c) or (e) , its single strands being hybridizable with those of the DNA according to (a) , (b) , (d) and (f) , respectively, at a temperature of at least 25 °C and at a concentration of NaCl of 1 M.
• RNA being a transcript of a DNA according to the invention.
• the invention concerns a protein being encoded by a DNA according to the invention.
• the invention concerns a protein having the amino acid sequence (X) according to figure 7.
• the protein according to the invention can be an about 17 kDa protein.
• the protein according to the invention can be a recombinant protein, especially a protein produced by means of a bacterial strain, a yeast strain, a fungicidal strain or a cell line of a higher eucaryote.
• the protein according to the invention can be encoded by a DNA sequence according to the third embodiment of the invention and can be recovered by a method comprising the following steps: (i) subjecting proteins encoded by said DNA sequence to a usual test for diagnosis of tuberculosis,
• the invention concerns a DNA, RNA or protein according to the invention which can be used for
• the invention concerns a use of a DNA according to the invention for the identification of mycobacteria in media samples.
• the invention concerns a use, wherein as samples clinical samples are used.
• the invention concerns a use of a DNA according to the invention or of a protein according to the invention for
• the invention concerns a use of a DNA according to the invention or of a protein according to the invention for the development of drugs useful for combating mycobacterial infections of humans or animals, especially tuberculosis, especially for testing and recovering of substances inhibiting mycobacterial infections in humans and animals, especially tuberculosis.
• Fig. 1 shows a restriction endonuclease map of the 7.2 kb M. tuberculosis chromosomal region
• Fig. 2. shows a 2253 bp M. tuberculosis chromosomal region including BamHI, EcoRI and Kpnl restriction sites and oligonucleotides for screening the lambda gt 11 M. tuberculosis library (Primer 1 and Primer 2 underlined) and for amplification of the 377 bp region (377 bp region in bold, Primer 3 and Primer 4 underlined) ; amino acid sequences of the about 15 kDa and the about 31 kDa proteins are shown above the DNA sequences and are marked with arrows (small arrow about 15 kDa ORF 1, strong arrow about 31 kDa ORF 2) ;
• Fig. 3. shows a DNA sequence of the 440 bp M. tuberculosis chromosomal region including the 380 bp region (in bold) used in PCR experiments and the amino acid sequence of the ORF 3 shown below the complementary DNA strand ( ⁇ ORF 3) ;
• Fig. 4 is an overview of the isolated lambda gtll-clone C9-2; 7.2 kb insert fragment, sequenced chromosomal regions and ORF 1, ORF 2 and ORF 3 marked with arrows;
• Fig. 5 shows the amino acid sequence of the about 15 kDa protein (ORF 1) ;
• Fig. 6 shows the amino acid sequence of the about 31 kDa protein (ORF 2) ;
• Fig. 7 shows the amino acid sequence of the about 17 kDa protein
• Fig. 8 A shows SDS-PAGE of the insoluble pellet fraction (lane 1) and the purified about 15 kDa recombinant antigen (lane 2)
• lane A3 shows protein molecular weight standards (2.850 to 43.000 molecular weight range);
• Fig 8 B shows SDS-PAGE of the purified about 15 kDa thioredoxin fusion protein (lane 1) and the two protein bands obtained after enterokinase cleavage (lane 1) ;
• Fig. 9 shows a DNA sequence of M. tuberculosis
• Fig. 10 is a schematic drawing of the clone Mtub-Clara-Klon; the open reading frames of about 9 kDa (bp 3536 to bp 3829) , 55 kDa (bp 2111 to bp 3829) , 74 kDa (bp 1538 to bp 3829) and 77 kDa (bp 2698 to bp 2 on the complimentary strand) proteins are shown by arrows and the corresponding coding regions are numbered;
• Fig. 11 A shows are southern hybridization with genomic DNA from different mycobacteria digested with PvuII (1: M. tuberculosis H37Rv; 2: M. avium; 3: M. kanssasi ; 4: M. necroti ; 5: M. fortui tum; 6: M. phlei ; 1 : M. smegma tis ; 8: M. vaccae) ;
• Fig. 11 B shows a finger-print obtained using the DNA (BamHI digest) of (1) M. tuberculsosis H37 RV, (2) M. tuberculosis H37 Ra, (3) M. bovis BCG, and (4) M. tuberculosis H37Rv digested with Sail;
• Fig. 12 shows a finger-print with DNA from different M. tuberculosis clinical isolates (numbered 1 to 12) digested with PvuII restriction enzyme; the 4 kb Sal I fragment (Mtub-Klar- Klon) was used as probe;
• Fig. 13 shows an amino acid sequence of the protein of about 74 kDa (molecular weight 74999, length 764)
• Fig. 14 shows a glycine rich protein of about 77 kDa (molecular weight 77056, length 899);
• Fig. 15 shows the amino acid sequence of the about 9 kDa proline rich protein (molecular weight 9356, length 98) ;
• Fig. 16 shows the proline rich protein of about 55 kDa (molecular weight 55982, length 573) .
• M. tuberculosis-ONA containing putative IS-element (Insertion Element) and repeat sequences, e.g., PGRS-elements (Polymorphic GC-Rich-Sequences) and the use of the as yet unreported DNA sequences for strain identification and diagnosis of tuberculosis.
• putative IS-element Insertion Element
• repeat sequences e.g., PGRS-elements (Polymorphic GC-Rich-Sequences)
• PGRS-elements Polymorphic GC-Rich-Sequences
• Escherichia coli strains, phages and plasmids The Escherichia coli K12 strain
• Y1090r - (Huynh et al., 1985) was used to propagate the ⁇ gtl 1 library and the E. coli K12 strain GI724 (Invitrogen, Leek, The Netherlands) was the host for the production of the ca. 15kDa protein fused to thioredoxin.
• the recombinant DNA library of M. tuberculosis genomic DNA in the ⁇ gtl 1 ex ⁇ pression vector was constructed by Young et al. (1985).
• the plasmid vector pTrxFus (Invitrogen, Leek, The Netherlands) was used to make an in-frame fusion with thioredoxin as an amino-terminal fusion partner.
• Mycobacterial strains and preparation of cell extracts The mycobacterial strains used in this study are shown in Table 1 (Results and Discussion). All organisms
• DNA sequencing analysis was performed by the dideoxynucleotide- chain termination method using a PCR sequencing kit (ABI PRISMTM £>ye Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elmer, Warrington, Great Britain) on a 373A DNA Sequencer (Applied Biosystems, Warrington. Great Britain). DNA sequences were determined for both strands by primer walking.
• Digoxigenin-labeling of the oligodeoxyribonucleotide primers was carried out using a DIG Oligonucleotide 3'-End Labeling Kit (Boehringer Mannheim, Germany). Hybridzation was done at 45°C in hybridization buffer (Boehringer Mannheim, Germany) overnight. Then the membranes were washed under stringent conditions for 5 min twice in 2 x SSC, 0.1% SDS and for 15 min twice at 37°C in 0.1 x SSC, 0.1% SDS. Chemi ⁇ luminescent detection was carried out with the help of a DIG Luminescent Detection Kit (Boehringer Mannheim, Germany). Plaques were purified by three rounds of plating to obtain single plaques. Phage DNA was isolated using a Nucleobond AX L50 Kit (Machery-Nagel, D ⁇ ren, Germany) and restriction mapping of the selected clone was performed by standard procedures (Maniatis et al., 1982).
• the complete ORF of the ca. 15kDa protein is located on the 2253-bp fragment coded by a 408-bp fragment, corresponding to a coding capacity of 136 amino acids.
• the ca. 15kDa protein (Fig.5) is a novel product showing limited homology in the N-terminus of a 34kDa ORF reported by Mariani et al. (1993).
• the C-terminus of the ca. 3 lkDa protein showed homology to a 34kDa ORF (Mariani et al., 1993). We have not used the DNA sequence showing homology to the sequence reported by Mariani et al. (1993) as far as the claims of this patent application are concerned.
• An ORF (ORF 3, Fig. 3 and Fig.7) on the complementary strand to the 3'-end of the insert fragment of the recombinant ⁇ -clone C9-2 was identified, which had not been reported earlier. This sequence showed homology to a family of transcription regulators in microorganism.
• the ⁇ gtl 1 clone C9-2 (Fig. 4) was used as template to amplify a PCR fragment of 951- bp (Fig. 2, sequence position 451-1378) including the ORF for the ca. 15kDa protein (Fig. 5) and cleavage sites for the restriction endonucleases Smal and Sail at the 5 1 - and 3'-ends. Amplification of the Smal-Sall mycobacterial DNA fragment for insertion into pTrxFus (Invitrogen.
• oligonucleotide primers with the sequence 5'-TCTAGACATATGACGCGAGTGGGTGTGATTTCG-3' (PRIMER 7, forward) and 5'-CATATGGTCGACCTAGGGCGTGTCTCCCAA-3' (PRIMER 8, reverse) corresponding to sequence positions 451-474 and 1378-1361 (Fig. 2).
• Composition of the reaction mix was the same as described above with 400 ng phage DNA as template.
• the probe was amplified in 30 cycles consisting of the same conditions as described. Cleavage sites were introduced by appropriate primers. After digestion with both restriction endonucleases the product was inserted in pTrxFus (Invitrogen, Leek, The Netherlands) to form the plasmid pCH3-8.
• the E. coli strain GI724 was electroporated with the plasmid pCH3-8.
• Bacterial cultures 200 ml of Induction Medium (Invitrogen, Leek, The Netherlands) supplemented with 100 ⁇ g/ml ampicillin) grown at 30°C were induced to synthesize the fusion protein by tryptophan addition (lOO ⁇ g/ml) and temperature shift to 37°C. Cells were collected after 4 hours (10 000 x g, 5 min.
• the recombinant fusion protein was refolded by dialysing against 50 mM Tris/HCl (pH 8.0). Anion exchange chromatography was done with the help of a BioCAD perfusion system (Perseptive Biosystems) on a Poros column HQ/M (Perseptive Biosystems).
• SUBSTITUTE SHEET (RULE 261 Elution was performed using a linear NaCl gradient (0-1M). The fusion protein concentration was dete ⁇ nined with the BioRad Protein Assay Kit (BioRad, Kunststoff, Germany). Purity was assessed by densitometry (Molecular Dynamics, Software Image Quant) and analytical SDS- PAGE and coomassie staining.
• the ca. 15kDa protein fused to thioredoxin was refolded as described above. Further purification of the ca. 15kDa protein fused to thioredoxin was carried out by anion exchange chromatography (Fig. 8, A lane 3 and B lanel). After enterokinase cleavage of the purified ca. 15kDa protein fused to thioredoxin two protein bands were detectable on SDS-PAGE (Fig. 8, lane 2). By western blotting with a thioredoxin monoclonal antibody the lower 1 IkDa band was identified to be thioredoxin. The upper band corresponds to the ca. 15kDa recombinant protein of M. tuberculosis. This is the first report of expression and purification of the ca. 15kDa protein of M. tuberculosis in E. coli.
• oligodeoxyribonucleotide primers with the sequence 5'-GTCCATGTGCCGCCG CTG-3' (PRIMER 3, forward) and 5'-CTGCGCGGCTCCCGGCA-3' (PRIMER 4, reverse), specific for the DNA regions of the 2253-bp M. tuberculosis chromosomal region shown in Fig. 2 were used in PCR experiments to amplify a 377-bp fragment.
• the oligodeoxyribonucleotide primers with the sequences 5'-CGAGGCTGAACGGCT TTG-3' (PRIMER 5, forward) and 5'-TCAACGTCCGCGGCAAGC-3' (PRIMER 6, reverse) corresponding to the DNA region shown in Fig. 3 were used.
• Amplifications were performed in 0.2 ml Micro Amp Reaction Tubes (Perkin Elmer, Norwalk, Connecticut, USA) in a final volume of 100 ⁇ l using a GeneAmp® PCR Kit (Perkin Elmer, Branchburg, New Jersey, USA).
• Reaction mixtures contained 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 200 ⁇ M dNTP, 0.1 ⁇ M Primer, 30-100 ng chromosomal DNA from mycobacterial cell extracts (Table
• the 377-bp region (Fig.2) of the isolated and sequenced 2253-bp M. tuberculosis chromosomal fragment and the 380-bp region (Fig.3) of the identified 440-bp chromosomal fragment were examined for their suitability for strain differentiation (Table 1 ).
• a PCR-product of the predicted size and a 100% DNA sequence homology in the 377-bp region was detected only in the members of the M. tuberculosis complex. No amplification product was obtained from other mycobacteria (Table 1). Therefore, the PCR primers of the 377-bp region are useful for the rapid discrimination of M. tuberculosis complex (M. tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, Mycobacterium africanum and Mycobacterium microti) from other mycobacteria.
• a predominant amplification product of correct size of the 380-bp region was obtained from the chromosomal DNA samples of the M. tuberculosis complex including the vaccine strain M. tuberculosis BCG, the tuberculosis isolate Tubl 18 and the mycobacterial species M. asiaticum. M. gastri, M. gordonae and M. kansasii.
• this fragment can be used for the identification of above mycobacterial species, since no amplification product was obtained from other mycobacterial species (Table 1).
• C/GGCC/GGCC/GGGC/GACC/GGGC/GGGC/GGCCGGCTCC/GGG- 3' which was designed in such a way that it contained GC rich regions as well as it coded for a putative proline rich polypeptide.
• Colony hybridization using labelled oligonucleotide was performed using standard procedures (Maniatis et al.1982). Filters were prehybridized and probed at 42°C overnight in a solution containing 6xSSC, 1 mM Sodium phosphate, ImM EDTA, 0.05% skimmed milk, 0.5%SDS. Filters were washed twice in 2xSSC;0,3%SDS for 15 min at 65°C.
• First screeinig yielded six positive clones which were recheked by hybridization with the oligonucleotide. Three clones gave strong signal and restriction mapping of the clones showed identical restriction pattern. Further restriction mapping and Southern hybridization of one of the clones called identified an about 4kb Sail fragment that hybridized strongly to the oligonucleotide.
• the amino acid sequence showed restricted homology to Mucein like proteins from eucaryotes.
• the 77kDa protein is highly rich in amino acid glycine and may be a cell wall protein of Mycobacterium tuberculosis. Such proteins have not been reported from M. tuberculosis.
• the ca. 4kb Sail fragment was used to probe (Southern hybridization) genomic DNA of different mycobacteria digested by PvuII (Fig. 1 1). The results show that each strain showed a characteristic pattern making the differentiation of M. tuberculosis-Rv, M. tuberculosis-Ra, M. bovis and the M. tuberculosis Erdman strain.
• the ca. 4kb Sail fragment is also suitable for finger printing of clinical isolates, since hybridization of the probe to the genomic DNA of clinical isolates from tuberculosis patients also yielded strain specific finger print (Fig. 12). No hybridization to the genomic DNA of M. smegmatis, M. vaccae, M. avium. M. chelonie, M. fortituim, M. phlei was observed.
• the ribosomal intergenic spacer region a target for the PCR based diagnosis of tuberculosis. Tuber. Lung Dis. 75 (1994) 353-360
• tuberculosis M. tuberculosis H37Rv + + complex M. tuberculosis H37Ra + +

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