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WO1998032862A9 - L-alanine deshydrogenase de mycobacterium marinum - Google Patents

L-alanine deshydrogenase de mycobacterium marinum

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Publication number
WO1998032862A9
WO1998032862A9 PCT/EP1998/000484 EP9800484W WO9832862A9 WO 1998032862 A9 WO1998032862 A9 WO 1998032862A9 EP 9800484 W EP9800484 W EP 9800484W WO 9832862 A9 WO9832862 A9 WO 9832862A9
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WIPO (PCT)
Prior art keywords
tuberculosis
aladh
mar3
strains
dna sequence
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PCT/EP1998/000484
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German (de)
English (en)
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WO1998032862A2 (fr
WO1998032862A3 (fr
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Priority to AU60979/98A priority Critical patent/AU6097998A/en
Publication of WO1998032862A2 publication Critical patent/WO1998032862A2/fr
Publication of WO1998032862A3 publication Critical patent/WO1998032862A3/fr
Publication of WO1998032862A9 publication Critical patent/WO1998032862A9/fr

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Definitions

  • the 40 kD antigen treated in this paper is in many ways an interesting object for in-depth studies.
  • the antigen had already been cloned into an expression vector for Esche ⁇ chia coli (Konrad & Singh, unpublished). Therefore, the expression and purification of the recombinant protein should be optimized. With a homogeneous protein fraction then the crucial biochemical parameters of the enzyme should be determined. From such data, experience has shown that conclusions can be drawn about the physiological function of an enzyme. The question was whether the hypothetical role of the enzyme in cell wall biosynthesis can be underlined or disproved. In case of refutation, other possible functions should be identified.
  • biochemical starting points for a targeted influencing of the enzyme in vivo can result.
  • the physiological function is the key point of all efforts. If the antigen plays an essential role in the bacterium, targeted efforts to eliminate the gene or protein could provide opportunities to prevent the tuberculosis pathogen from growing at a defined point. The protein would then be an ideal drug target. If, as postulated (Delforge et al., 1993), the 40 kD antigen also represents a virulence factor, then such activities could influence the natural virulence of the bacterium. Therefore, this point should be checked by different approaches. The ability to discriminate against the strains of M.
  • tuberculosis and M.bovis BCG by means of mAb HBT-10 makes it possible to develop methods that can distinguish an infection from a vaccine. This is not possible with the conventional screening methods, the PPD or the Mantoux test (Bass Jr. et al., 1990, Huebner et al., 1993).
  • the basis should be laid for enabling rational process development for such a test.
  • the strain Escherichia coli was used to optimize the expression of the recombinant 40 kD antigen (Table 2.1). In addition, it has already overproduced cloned mycobacterial antigens (Table 2.2).
  • E. coli POP (pKA 2101) J. van Embden 70 kD antigen, Ap M. tuberculosis heat
  • E.CO./TB1 (pKAM1101) di Guan ef a /. (1987) MBP-36 kD antigen, Ap heat Maina ef a /. (1988) M.leprae Thole ef a /. (1990) Tab. 2.2 (2/2): Producers of mycobacterial antigens and their characteristics
  • E.CO./TB1 (pKAM4101) J. van Embden MBP-2nd 65kD-Ap Heat Antigen, M.leprae
  • tuberculosis H37R V H37R V M. tuberculosis H37R V , RiV
  • tuberculosis 163 Tub163
  • tuberculosis 925 patient isolate No. 32, INH> 1, StrR, RifS, EthS
  • mice were used macrophages cell line J774.
  • This cell line was originally established from a tumor of a female BALB / c mouse (Ralph & Nakoinz, 1975).
  • J774 is used for phagocytosis assays, IL-1 production, and for a variety of biochemical studies. It has receptors for immunoglobulins and complement.
  • J774 produces lysozyme in large quantities and constitutively secretes IL-1 (Ralph & Nakoinz, 1976; Snyderman et al., 1977). Bacteria are absorbed by phagocytosis. Direct cytolysis of foreign organisms is relatively rare.
  • Fig. 2.1 The plasmid pJLA604Not and its relevant functional sections
  • the translational reading frame starts with the ATG codon of the Sp ⁇ I site. Transcription starts at lambda promoters P R and P, but is effectively repressed at temperatures of 28-30 ° C by the cl ts857 gene product. Induction is achieved by raising the temperature to 42 ° C. At this temperature, the temperature-sensitive lambda repressor becomes inactive and can no longer repress transcription. Transcription ends at the fd terminator.
  • the vector has the atpE translation initiation region (TIR) of E. coli.
  • the plasmid has the ⁇ -lactamase gene coding for ampicillin resistance.
  • pJLA603 As a negative control plasmid pJLA603 was also used, which is identical to pJLA604 except for a few bases in the cloning site.
  • Fig. 2.2 The plasmid pMSK12 and its relevant functional sections This is a derivative of the plasmid pJLA604Not, in which between the Sph - and the
  • oligonucleotides (Table 2.5) were prepared by Ms. Astrid Hans (GBF, Braunschweig) on a 394 DNA / RNA synthesizer (Applied Biosystems). The oligonucleotides were purified with an oligonucleotide purification cartridge (Applied Biosystems).
  • the localization of the oligos on the AlaDH gene is schematized in Fig. 2.3.
  • Fig. 2.3 The ve i v / ended oligos and their location on the AlaDH gene
  • Antibiotics were added to the liquid media just before use from stock solutions. When making media, the addition was allowed to go until the solution was lukewarm after autoclaving. The antibiotics listed in Tab. 2.6 were used. Tab. 2.6: Antibiotics used and concentrations used
  • TAE 40 mM Tris-acetate, 1 mM EDTA, pH 8.0, autoclave
  • TBE 89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.0, autoclave
  • TBS 50 mM Tris base, 137 mM NaCl, 3 mM KCl, pH 7.4, autoclave
  • TBS-TWEEN TBS + 0.05% Tween-20
  • PBS 137 mM NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.0, autoclave 2.6.4 Alanine Dehydrogenase Assays
  • Fig. 2.4 Principle of the alanine dehydrogenase assay
  • the violet end product can be seen very well with the naked eye. This assay was used to rapidly screen FPLC fractions and to demonstrate AlaDH activity in native protein gels.
  • This assay is based on a reaction mixture consisting of 1/2 vol. 0.5 M glycine-KOH, pH 10.2 and 1/8 vol. 0.5 M L-alanine, 6.25 mM NAD + , 2.4 mM NBT and 0.64 mM PMS.
  • the substrate mix was mixed 1: 1 with the solution to be tested. Native gels were incubated directly in 10 ml of substrate mix after electrophoresis.
  • This assay was used to study AlaDH activities in mycobacteria.
  • the mycobacteria were grown on Löwenstein's medium. Using the loop, bacteria were removed from the slant agar tubes, resuspended in water, and adjusted for turbidity equivalent to a McFarland Standard # 5. For the separation of cell aggregates, the suspensions were treated for 10 min in an ultrasonic bath.
  • the cells were then mixed 1: 1 with a reaction mixture (see 2.6.4.1) and incubated at RT for 10 min. After centrifugation at 20,000 g for 2 minutes, the absorbance of the supernatant against the blank was measured.
  • the reference measurement used was an approach to which no L-alanine was added.
  • An absorbance change of one unit per minute in this test corresponds approximately to an absorbance change of three units per minute in the quantitative assay (measurement at 340 nm, see 2.6.4.3).
  • the Standardre 'action approaches had a volume of 1 ml.
  • the composition is shown in Tab. 2.7.
  • the absorbance was monitored for 10 minutes at 37 ° C and 340 nm.
  • the extinction coefficient ⁇ of NADH at 340 nm is 6.22 ⁇ 10 6 cm 2 / mol.
  • AlaDH unit is defined as the amount of enzyme that catalyses the formation of 1 ⁇ mol NADH in oxidative deamination in one minute.
  • composition of the reaction mixture for the oxidative deamination is shown on the left, which is shown for the reductive amination on the right.
  • Densitometry was used to quantify the expression level of AlaDH. Furthermore, the signals of epitope mapping were quantified and correlated with each other.
  • the implementation was carried out with a Personal Densitometer (Molecular Dynamics) with the software Image Quant (Molecular Dynamics).
  • the strains studied can be divided into three groups.
  • the first group is that of the strong positive strains (Table 3.11).
  • This group comprises the strains which have an AlaDH activity of more than 0.5 ⁇ A 5 95 units in the test system used.
  • the second group that of the moderately positive strains, comprises those with an activity between 0.1 and 0.5 ⁇ Asgs units (Table 3.3).
  • M.smegmatis In this group, except M.smegmatis, only pathogenic clinical isolates of M. tuberculosis and other mycobacteria are found. However, both M.smegmatis strains tested also show very high NAD + -reducing activities in the absence of L-alanine. It is important to mention here that the strain M. smegmatis 1-2c (a derivative of M. smegmatis mc 2 6, Zhang et al., 1991, Garbe et al., 1994, by Dr. Peadar 0 Gaora , St.Mary's Hospital, London), a strain for genetic work in mycobacteria, shows no AlaDH activity but also has high background activity. Finally, in the last group all strains are listed which were found to be negative for AlaDH-activity, ie have an activity of less than OJ ⁇ A 5S5 units (Table 3J 3).
  • FIG. 3J6 A graphical representation of the AlaDH activities is shown in Fig. 3J6 ordered by phylogenetic aspects.
  • Fig. 3.16 AlaDH activity in the kingdom of mycobacteria
  • AlaDH activity within the world of mycobacteria.
  • ⁇ M. tuberculosis can be differentiated from the vaccine strain M.bovis BCG by AlaDH activity.
  • the lack of measurable activity can be explained to some extent by the fact that not all strains were in exactly the same growth phase, as it is very difficult to attract all strains in parallel, at the same stage. But the lack of activity could also be one of the reasons why genetic changes affect the expression of the gene. These changes could have occurred in the coding or regulatory area.
  • AlaDH gene from various strains was tried to be amplified in whole or in part by PCR.
  • oligonucleotides based on the sequence of M. tuberculosis H37R V (Andersen et al., 1992, see Section 2.2.2, Table 2.5).
  • Fig. 3.17 PCR of different strains with the primer pair Annabel.
  • Lane 1 M. tuberculosis H37R V lane 6: M.bovis BCG 4 lane 2: M. tuberculosis H37R a Eahn 7: M.africanum 1 lane 3: M. tuberculosis 1 Eahn 8: M.microti 1 lane 4: M. bovis 3 Train 9: M.marinum 3 Train 5: M.bovis BCG 2 Train 10: M.chelonae 7
  • this second fragment was also part of the AlaDH gene, which was formed by attachment of the primer AlaDH-RM at a more C-terminal point.
  • this second fragment could be suppressed (see Fig. 3.18, lanes 2 and 3).
  • Fig. 3.18 PCR products of strain M.tuberculosis H37R V
  • the area amplified by all strains of the M. tuberculosis complex comprises 1260 bp. It contains the complete coding section for the AlaDH, as well as further 75 bp ⁇ pstream and 63 bp downstream. This area was completely sequenced by all strains of the M. tuberculosis complex (Fig. 3J 9). Only in the last about 20 bases inaccuracies creep in. The complete remaining region ', however, is protected by multiple sequencing.
  • H37Ra 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AATTCCG GGTGGCCATC 60
  • mice 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AATTCCG GGTGGCCATC 60
  • mice Micl 61 ACCCCGGCCG GCGTCGCGGA ACTAACCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120
  • H37Rv 181 GGCACCGCCG ACCAGGTGTG C-GCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240
  • H37Ra 181 GGCACCGCCG ACCAGGTGTG GGCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240
  • Bov3 181 GGCACCGCCG ACCAGGTGTG GGCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240
  • mice 181 GGCACCGCCG ACCAGGTGTG C-GCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240
  • Fig. 3.19 (1/5): Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex
  • Tubl 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG CAGATCTTGT TCACGTTCTT GCATTTGGCC 3C0
  • H37Rv 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG CAGATCTTGT TCACGTTCTT GCATTTGGCC 300
  • H37Ra 241 GCGGCGGAAT ACGGCCGCCT GCGACACGGG CAGATCTTGT TCACGTTCTT GCATTTGGCC 300
  • 3CG4 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG C-GATCTTGT TCACGTTCTT GCATTTGGCC 300
  • 3ov3 2 1 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG C-GATCTTGT TCACGTTCTT GCATTTGGCC 300
  • Afrl 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG CAGATCTTGT TCACGTTCTT GCATTTGGCC 300
  • mice 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG CAGATCTTGT TCACGTTCTT GCATTTGGCC 300
  • mice Micl 301 GCGTCACGTG CTTGCACCGA TGCGTTGTTG GATTCCGGCA CCACGTCAAT TGCCTACGAG 360
  • mice 361 ACCGTCCAGA CCGCCGACGG CGCACTACCC CTGCTTGCCC CGATGAGCGA AGTCGCCGGT 420
  • ECG4 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTG.ATGCGAA CCCAAGGGGG CCGCGGTGTG 480
  • 3ov3 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTGATGCGAA CCCAAGGGGG CCGCGGTGTG 460
  • mice 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTGATGCGAA CCCAAGGGGG CCGCGGTGTG 480
  • Fig. 3.19 (2/5): Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex
  • Eov3 541 GCCGGCTACA ACGCAGCCCG CA.TCGCCAAC GGCATGGGCG CGA.CCGTTAC GGTTCTAGAC 600
  • mice 541 GCCGGCTACA ACGCAGCCCG CATCGCCAAC GGCATGGGCG CGACCGTTAC GGTTCTAGAC 600
  • 4CkD 601 ATCAACATCG ACAAACTTCG C-CAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660
  • Tubl 601 ATCAACATCG ACAAA.CTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660
  • H37Rv 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660
  • BCG4 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660
  • BCG2 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660
  • Bov3 601 ATCAACATCG ACAAACTTCG C-CAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660
  • Afrl 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGGAT CCACACTCGC 660
  • mice 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGC-AT CCACACTCGC 660
  • Tubl 651 TACTCATCGG CCTA.CGAGCT CGAGG TGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720
  • H37Ra 661 TACTCATCGG CCTA.CGAGCT CGAC-GGTGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720
  • Bov3 661 TACTCATCGG CCTACGAGCT CGAGC-GTGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720
  • Tubl 781 AAACCAGC-TG CGGTACTGGT C-GATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840
  • H37Rv 781 AAACCAGGTG CGGTACTGGT C-GATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840
  • Afrl 781 AAACCAGGTG CGGTACTGGT GG TATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840
  • Fig. 3.19 (3/5): Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex
  • Bov3 6 1 CGACCGACCA CCTACGACCA CCCGACGTTC GCCGTGCACG ACACGCTGTT TTA.CTGCGTG 900
  • mice 841 CGACCGACCA CCTACGACCA CCCGACGTTC GCCGTGCACG ACACGCTGTT TTA.CTGCGTG 900
  • H37Ra S61 CCGTATGTGC TCGAGCTTGC CGACCATC-GC TGGCGGGCGG CGTGCCGGTC GAATCCGGCA 1020
  • 3CG4 1021 CTAGCCAAAG GTCTTTCGAC C-CACGAAGGG GCGTTACTGT CCG.AA.CGGGT GGCCACCGAC 1080
  • 3CG2 1021 CTAGCCAAAG GTCTTTCGAC GCACGAAC-GG GCGTTACTGT CCGAACGGGT GGCCACCGAC 1080
  • Fig. 3.19 (4/5): Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex
  • Tubl 11 1 GCCGAGCACA CNTCGGGAGT AANG-GAAGCG ATGATGTCGN C 1185
  • H37Rv 11 1 GCCGAGCA.CA CGTCGGGAGT AAC-G AAGCG ATGATGTCGG CCG 1185
  • H37Ra 11 1 GCCGAGCACA CGTCGGGAGT AAGC-G.AAGCG ATGA 11S5
  • Bov3 11 1 GCCGAGCACA CGTCGGGAGT AAC-C-GAAGCG ATGATGTCGG CC 1185
  • Fig. 3.19 Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex
  • the third distinct site is Base 272.
  • M.bovis and two strains of M.bovis BCG this base is deleted. This deletion results in a frameshift that affects the entire following portion of the resulting protein. By this reading frame shift occurs at the bases 404 to 406 an opa / stop signal.
  • the product of this gene is only about one-third the size of the functional AlaDH of the other strains.
  • M.bovis and M.bovis BCG are the only strains of M. tuberculosis complex that show no activity. All other strains were classified as moderate or strong positive. The observed deletion is therefore the reason for the lack of a functional AlaDH.
  • HBT-10 mAb can not detect the truncated protein (the epitope of HBT-10 is in the range before the frameshift), it can be assumed that the truncated protein does not occur at all, or only in very small amounts. with the mAb HBT-10 undetectable amounts is produced.
  • H37RV TTCCGACCGAGACCA-AAAACAACGA ⁇ TTCCAATTCCGGGTGGCCATCACCCCGGCCGGCG
  • H37RV GCCGCCTGCGACACGGGCAGATCTTGTTCACGTTCTTGCATTTGGCCGCGTCACGTGCTT
  • MIIMIMI II II I MIMMMIMM II IMIIMMII II IIIMM
  • H37RV GCACCGATGCGTTGTTGGA-TTCCGGCA-CCACGTCAATTGCCTACGAGACCGTCCAGACCG
  • Fig.3.20 AlaDH gene alignment of M.marinum and M. tuberculosis H37R V
  • H37RV CAGCCCGCATCGCCAACGGCATGG3CGCGACCGTTACGGTTCTAGACATCAACATCGACA
  • Fig. 3.20 (2/2): AlaDH gene alignment of M.marinum and M. tuberculosis H37R V
  • Fig. 3.20 A total of 682 bases of this gene could be determined (Fig. 3.20), which represents about 60% of the complete gene. These 682 bases encode the N-terminal region of the protein. Only the first 35 bases after the start codon are missing.
  • the sequenced section of the AlaDH from M.marinum shows 80.4% identity with the corresponding section of M. tuberculosis H37R V.
  • the sequence differences are distributed relatively uniformly over the entire section.
  • the deduced peptide of the M.marinum gene sequence ( Figure 3.21) shows 85.3% identity and 92.0% similarity to the M. tuberculosis H37R V protein. Homologies to other proteins of the Swiss Prot database are given in Tab. 3J5 (as of 9/1996, Release 33). For comparison, it should be noted that the AlaDH of M.tuberculosis '' has 53% identity to the enzymes of B.sphaericus and B.stearothermophilus (Andersen et al., 1992).
  • Fig.3.21 AllaDH's alignment of M.marinum and M. tuberculosis H37R V
  • the Swiss Prot database was searched using the FASTA software (http: / ⁇ w ⁇ V.embl- ebi.sc.uk) (as of 9/1996, Release 33).
  • amino acid residues Gly165, Gly167, Gly170 and Asp198 are considered to be essential for the binding of the cofactor of AlaDH of M. tuberculosis H37R V. These four amino acid residues are characteristic of the ⁇ secondary structure of NAD (H) binding sites (Wierenga et al, 1986; Bork & Grunwald, 1990). In M.marinum's enzyme, as in all other AlaDH's in Table 3.14, these four residues are conserved.
  • the corresponding section of the mAb HBT-10 mAb epitope of M.marinum AlaDH has the sequence AISDADFKAAG, thus differing only in third place from the M. tuberculosis H37R V sequence. Based on the consensus epitope of the antibody determined in this work (Table 3.9), it would also have to react with the AlaDH of M.marinum. In fact, this strain is also the only one with which a cross-reaction could be observed (Andersen et al., 1992). The determined sequence is also consistent with this observation.
  • AlaDH activity in mycobacteria The measured AlaDH activities allow some interesting observations regarding the lifestyle of the organisms with positive activity.
  • strains with high activity are all pathogenic.
  • two of the four strains that belong to this group are pathogenic for fish (Austin & Austin, 1987).
  • M.marinum and M.chelonae can also be humans
  • M.chelonae is a comparatively fast-growing, non-chromogenic bacterium. Infections in humans often occur as secondary wound infections after surgery (Cooper et al, 1989). M.marinum is a slow-growing organism that forms a yellow pigment when grown in light. In more than 50 warm-blooded species (reptiles, amphibians, fish) infections with M.marinum were detected (Clark & Shepard, 1963). In humans, the bacterium usually manifests itself in the elbow or knee area.
  • the other two strains with strong-positive AlaDH activity are representatives of the M. tuberculosis complex. These are the tuberculosis reference strain M.tuberculosis H37R V , as well as the strain M.microti, which is considered to be a phylogenetic link between M.tuberculosis and M.bovis.
  • M. smegmatis Apart from M. smegmatis, all strains classified as moderately positive are also pathogenic. The majority of these strains include clinical isolates of M. tuberculosis. Pathogenic variants of tuberculosis strains thus generally have AlaDH activity. However, two isolates were found that do not have AlaDH activity. The only non-pathogenic organism with AlaDH activity is the rapidly growing M.smegmatis strain. However, M.smegmatis has an unusually strong background NAD + -reducing activity and is therefore very easily distinguishable from all other strains with AlaDH activity. Furthermore, in the strain M.smegmaits 1-2c, a mycobacterial expression strain, no AlaDH activity was found. Within the 44 strains of mycobacteria tested, and this is by far the majority of all known strains, the conclusion is therefore:
  • the AlaDH gene in mycobacteria was identified in all strains investigated in the M.tuberculosis complex and in the M.marinum strain.
  • AlaDH gene that of M.marinum, is significantly different at the DNA level from the genes of the M.tuberculosis complex. However, four out of five bases (80.4%) are on average still identical when comparing these sequences. This value is even higher at the protein level (85.3 o identity, 92.0% similarity). However, since AlaDH activity was also found in a number of other species, it can be assumed that the corresponding genes, lacking homology to the primers used, can not be amplified under the conditions used. A more detailed study on this point would have to find these genes as well. A comparison of all these sequences could allow further conclusions about the role of the enzyme.
  • assays based on AlaDH have been described for the enzymes dipeptidase (Ito et al, 1984), ⁇ -glutamyltransferase (Kondo et al, 1992), and ⁇ -glutamyl cyclotransferase (Takahashi et al, 1987). All three of these enzymes are found in various diseases in altered urine, serum or blood concentrations.
  • PCR assay would also have to be established for the M.marinum strain, which differs considerably at the gene level from the M. tuberculosis complex.
  • a PCR assay based on the amplification of a portion of the gene sequence encoding the J ⁇ S rRNA has heretofore been used (Knibb et al, 1993). This is of great importance given the increasing number of M.marinum infections in fish farms in recent years (Knibb et al., 1993). Also
  • the disclosure of the present application also includes the disclosure of the attached EP 97 101 33g. e, in particular the literature cited therein.
  • the disclosure also includes all conceivable combinations of disclosed individual features.

Abstract

La tuberculose est une maladie infectieuse qui tue plus de 3 millions de personnes par an. Il existe un vaccin et différents procédés de diganostic et de thérapie mais vu le nombre croissant de victimes, l'efficacité de toutes ces mesures nécessite une amélioration urgente. La recherche se concentre sur la caractérisation d'antigènes qui sont sécrétés de façon précoce lors d'une infection puisque ces antigènes établissent le premier contact du système immunitaire avec l'agent infectieux. L'antigène de 40 kD dont il est question dans ce document se présente in vivo sous forme d'hexamère et, malgré un poids moléculaire élevé et l'absence d'une séquence-signal, on le trouve de façon extracellulaire déjà après quelques jours de croissance. Il constitue fonctionnellement une L-alanine déshydrogénase et réagit avec l'anticorps HBT-10 monoclonal dirigé contre cette protéine. HBT-10 fut le premier anticorps connu spécifique de la protéine de M. tuberculosis, qui ne produise pas de réaction croisée avec la souche vaccinale M. bovis BCG.
PCT/EP1998/000484 1997-01-29 1998-01-29 L-alanine deshydrogenase de mycobacterium marinum WO1998032862A2 (fr)

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AU744325B2 (en) * 1997-01-29 2002-02-21 Lionex Gmbh Test kit for tuberculosis diagnosis or the like
US6316205B1 (en) 2000-01-28 2001-11-13 Genelabs Diagnostics Pte Ltd. Assay devices and methods of analyte detection
JP4233458B2 (ja) * 2002-04-16 2009-03-04 リウ、ジュン アラニンデヒドロゲナーゼ、セリンデヒドラターゼおよび／またはグルタミンシンテターゼを発現する結核ワクチンとしてのリコンビナントｂｃｇ株

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