WO2000044905A1 - Slow growing acid-fast bacterium polypeptide - Google Patents

Abstract

A polypeptide represented by SEQ ID NO: 2 (having 205 amino acids), optionally having substitution, addition or deletion of one or more amino acids, which has an immunogenicity against pathogenic acid-fast bacteria; a DNA encoding this polypeptide; a vector and a transformant containing this DNA; a process for producing the polypeptide; a vaccine; and a method for diagnosing diseases caused by pathogenic acid-fast bacteria.

Description

 Specification

 Slow-growing acid-fast bacterium polypeptide

 Technical field

 The present invention relates to a polypeptide produced by a slow-growing acid-fast bacterium (mycobacterium) and a derivative thereof, a DNA encoding the polypeptide, and a vaccine containing the polypeptide or DNA. The present invention also relates to a vector containing the DNA, and a transformant containing the vector. Furthermore, the present invention relates to a method for diagnosing pathogenic mycobacteriosis.

 Background art

 Mycobacterium tuberculosis (Mycobacterium leprae) and other pathogenic mycobacteria (mycobacterium) are extremely slow-growing bacteria that infect one-third of humans. Will be Slow growth allows intracellular parasitism and confers resistance to drugs.

 An object of the present invention is to elucidate the mechanism of slow growth of pathogenic mycobacteria, and to provide a new diagnostic prevention, vaccine and therapeutic agent for pathogenic mycobacteria such as tuberculosis.

 BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Identification of MA binding protein and purification of MDP1. In FIG. 1 (A), identification of the DNA binding protein was performed by Southwestern blotting. After SDS-PAGE, 20 ^ g of protein derived from BCG lysate (12.5% polyacrylamide gel) was transferred. The film [α- ³² P] labeled p BSKS + and Inkyubeto, protein - was visualized MA complex in autoradiographs. MDP1 is indicated by an arrow. The molecular weight marker is kilodalton (kDa). Figure 1

(B) shows the separation of acid-soluble proteins through a column of Hitrap CM Sepharose (Pharmacia). For elution, a linear gradient of 200 mM phosphate buffer (pH 6.8) and 0-5% GdnCl was used. Bars indicate fractions containing MDP1. Figure 1

(C) shows separation of MDP1 through a Hiload Superdex 200p column (Pharmacia). Bars indicate fractions containing MDP1. FIG. 1 (D) shows a CBB_stained SDS-12.5% polyacrylamide gel showing proteins obtained from each purification step. Lane 1 is total protein from BCG lysate; lane 2 is total protein from BCG lysate. Acid-soluble protein obtained by treatment with 25N HCl; Lane 3 is an MDP1-rich fraction after ion exchange chromatography; Lane 4 is purified MDP1 passed through a gel filtration column. The MDP1 protein band in the cell lysate is indicated by the arrowhead. The molecular weight marker is kDa on the left.

 Figure 2. MDP1 nucleotide and amino acid sequences and amino acid sequence homology. FIG. 2 (A) shows the nucleotide sequence and deduced amino acid sequence of the MDP1 gene. Numbers corresponding to nucleotides are placed every 60 on the right. The hypothetical SD sequence is underlined and the asterisk indicates the termination codon. The peptide sequence in the box is determined by microsequencing of the purified MDP1, and the bold underlined peptide sequence is the DNA binding motif observed by Yuka Naka et al. ((1984) Nature, 310, 376-381). is there. Arrowheads indicate one possible terminator. The nucleotide sequence data is deposited in the EMBL / GenBank / DDBJ nucleotide sequence data library under accession number AB013441. FIG. 2 (B) shows BCG MDPKBCG-MDP), tubercWosis (Mt-MDP) (nucleotides 274 to 918 in Y349 cosmid library, accession number; Z83018), and f. Ieprae (MI-MDP) (B637). MDP1 homologue from nucleotides 38644 to 39246 in the cosmid library, accession number; Z99263), E. coli HU2 (Ec-HU2) (Kano et al., 1987 Mol Gen Genet 209, 408-410, accession number; X05994) And an alignment of amino acid sequences allowing a gap (-) between human histone H1 (h-Hl) (Albig, et al., 1991, Genomics 10, 940-948, accession number; M60748). The dot shows the same amino acid as BCG MDP1.

Figure 3. MDP1 binding to nucleic acid. FIG. 3 (A) is an autoradiograph showing the binding of radiolabeled pBSKS + to recombinant MDP1 (GST-MDP1, indicated by arrow) expressed in £ .coJi. Protein expression is 0. ImM. It was induced by culturing in LB medium containing IPTG (lanes 3 and 5). Protein from parental E. coli (lane 1) and transformants with PGEX4T-3 (lanes 2 and 3) or pGEXMDPl (lanes 4 and 5) was subjected to 12.5% SDS-PAGE and transferred to a membrane. Was reacted. Molecular weight markers are displayed on the left side as kDa. Figures 3 (B)-(D) are in vitro analyzes of the binding activity of native MDP1 to circular (B) or linear (C) DNA and RM (D) by gel retardation assay. C, 0 and L are super coils respectively) NA, Nick DM, Hi It is a linear form of DNA cleaved by ndIII. Different molar amounts of purified native MDP1 (indicated by M on the lane) were incubated with pBSKS + (B), HindIII-cleaved pBSKSKC) and MS2 phage RNA (D) at 37 ° C for 10 minutes. The samples were subjected to 0.8% agarose gel electrophoresis. Nucleic acids were visualized under UV light after EtBr treatment. 7 ^ 1 shows /] £ / 111 cleavage; 10 ^.

 Figure 4. MDP1 localization in cells. FIG. 4 (A) shows the localization of the MDP1 homologue in s / segisaiis by an immobilized electron microscope. FIG. 4 (B) In panel a: the separated proteins in each subcellular fraction and the liposome subnet were visualized by SDS-PAGE (12.5% gel) followed by staining with CBB. Arrowheads indicate estimated MDP trends. Lanes are: molecular weight marker (lane 0), secreted protein (lane 1), protein in cell lysate (lane 2), cell wall (lane 3), membrane (lane 4), cytoplasm (lane 5), ribosome (Lane 6) and 3OS (lane 7) and 50S (lane 8) ribosome subunits. The MDP render is indicated by an arrowhead. Fig. 4 (B) In panel b: the protein of each fraction was blotted on the membrane and the reaction with the anti-MDP1 antibody was visualized. The samples on each lane are identical to those on panel a. Molecular weight markers are indicated by kDa on the left side of both panels. Figure 4 (C) Panel a: on a YMC-GEL C4 column equilibrated with 30% acetonitrile in 0.1% TFA and eluted with a linear gradient of 30-70% acetonitrile at room temperature at 0.6 ml / min. Of the 50S ribosomal protein from BCG in the laboratory. Figure 4 (C) Panel b: SDS-PAGEC 12.5% gel of proteins contained in each fraction. The gel was stained with CBB. The first lane is the molecular weight marker (MWM); the second lane is the protein in the ribosome fraction (RF). The number of fractions is indicated on the lane. The 28 kDa N-terminal amino acid sequence in fraction 27 is the same as that of MDP1. Fig. 4 (C) Panel c: detection of 27 kDa and 28 kDa (MDPl) proteins recognized by anti-MDP1 antibody in 50S ribosomal subunit.

Figure 5. Effect of MDP1 on macromolecular biosynthesis in vitro. Figure 5 (A), In vitro effect of MDP1 on DNA synthesis by Klenow fragment after annealing of type I single-stranded DM and primers. DNA synthesis was performed at 22 ° C. for 8 minutes in a suitable medium containing different molar amounts (indicated by / M below the bar) of MDP1 (see Examples). e- is a negative control for type I DNA minus. TCA insoluble [α- ³² Ρ] - dTTP incorporation (cpm; vertical axis) was quantified and displayed. Figure 5 (B), Effect of MDPl on transcription by T7 RNA polymerase in vitro. The transfer was performed at 37 ° C. for 30 minutes with appropriate conditions (see Examples) containing different molar amounts (M below the bar) of MDP1. The reaction was stopped by EDTA. DNA- indicates a negative control without type I DNA. Counted Bok and and displayed in TCA - insoluble [a- ³² P] -dTTP incorporation scintillator one Chillon counter (vertical axis). Figure 5 (C), Effect of MDPl on translation in vitro. Different molar amounts of MDP1 (M below the bar) were mixed with MS2 phage RNA, E. coli S30 in vitro translated Atsemix (Promega) and [ ³⁵ S] -methionine. Translation was performed at 37 ° C for 30 minutes. Details are described in the examples. RN A— is a negative control for type I DM minus. Ly indicates chicken white egg lysozyme. The amount of TCA-insoluble [ ³⁵ S] -methionine (vertical axis) determined by scintillation counting is shown.

 Figure 6. Effect of MDP1 on bacterial growth rate. Figure 6 (A), Effect of MDPl expression on growth of M. smegmatis. M. smegmatis transformed with empty plasmid (panel a) or pSOMDPl (panel b) were harvested for 7 days at 37 ° C on 7H10 agar (see Examples). Figure 6 (B), Effect of MDPl on .co? I growth. CoJi transformed with the ET22M DPI (panels a and c) was treated with 0.5 mM IPTG (panel d) or without (panel.), Carpenicillin (50 g / ml), chloramphenicol (34 / zg / ml) and harvested at 37 ° C for 18 days.

 Figure 7, MDP1 expression by different species of mycobacterium in exponential phase (indicated by "EX" above the number of lanes) or stationary phase (indicated by "ST"). Figure 7 (A), BCG,

SDS-PAGE analysis of proteins in lysates from M. tuberculosis (Mt), M. lepare (Ml), M. smegmatis (Ms) and £ coii (Ec). The gel (12.5%) was stained with CBB. The protein band presumed to be MDP1 or its homologue is indicated by an arrowhead. FIG. 7 (B), Western blotting of each cell lysate (shown above the lanes as in FIG. 7 (A)) was analyzed with anti-MDP1 antibody. Molecular weight markers are indicated in kDa on the left side of both panels.

Figure 8. BCG Tokyo strain cultured in Sauton's medium, cells (cell protein) and culture filter The location of MDP1 was identified by Western Blotting into liquids (containing secretory proteins). As a result, as shown in Fig. 8, a protein reacting with the anti-MDP1 antibody was observed only in the protein prepared from the cells, indicating that MDP1 is a protein distributed in the cells or on the surface of the cells. all right.

Figure 9. It was confirmed that the antigenicity (antibody-inducing activity) of MDP1 was enhanced in the presence of DNA (derived from BCG). Mice were immunized with BCG (10 ⁶ cells / mouse), MDPl (5 / g / mouse), DNA (500 ng / mouse) and [MDP1 (5 ^ g) + DNA (500 ng)) / mouse. As a result of confirming the induction of antibodies against MDP1, antibody production was confirmed by Western Blotting only in the BCG and MDP1 + DNA administration groups, and it was found that the antigenicity of MDP1 was enhanced in the presence of DNA. Was.

 Fig. 10-: I 3. 5 g each of histone HI (Fig. 10), H2A (Fig. 11), H3 (Fig. 12) and MDP1 (Fig. 13) derived from pesticide Alternatively, as a result of immunization of mice mixed with DNA (0.5 ^ g), it was confirmed that the antibody titer of MDP 1 was increased in the presence of DNA, and that of histone protein was not affected by the presence or absence of DNA. No antibody production was confirmed.

 Figure 14. After SDS-PAGE of MDP1 in one lane, Western Blotting was performed to determine the presence of anti-MDP1 antibody in serum dilutions from leprosy patients (Ll-3) and tuberculosis patients (Tl-4) The presence or absence was checked. As a result, it was confirmed that an antibody against MDP1 was present in the patient's serum.

 Figure 1 5. In the protective effect experiment, whether or not antibodies were induced before infecting the mice with M. tuberculosis was confirmed by the ELISA method using purified MDP1 and purified Ag85. As a result, it was confirmed that antibodies were induced to both MDP1 and Ag85 antigens.

1 6. By Lymphocytes were prepared from mice infected with M. tuberculosis and stimulated in MDP1, Ag85, H37Ra及Beauty PPD (Bberukurin), measures the uptake of ³ H-Thymidine proliferative activity of lymphocytes confirmed. As a result, the proliferation activity of lymphocytes was confirmed in MDP1, Mycobacterium tuberculosis H37Ra and PPD. The activity of MDP 1 was the strongest.

Figure 1 7. The expression of recombinant BCG (pMDPlMutant-3PYBM rBCG, pMDPl Mutant-3PYBC rBCG) protein was confirmed. PS0246 rBCG and control Using pS0246MDP1Mutant rBCG and pS0246MDP1Mutant rBCG, a protein was prepared from the cells, and after SDS-PAGE, expression of the recombinant protein of MDP1-3PYB was confirmed. Mutant-3PYBM, in which the amino acids at positions 146 to 165 of the MDP1 protein were deleted and 3PYB was introduced instead, was stained with an anti-NANP antibody at the migration position of MDP1 as shown in lane 3 in Figure 17b. A band was obtained. Since the pMDP mutant Mutant-3PYBC bound to the C-terminus has an added molecular weight of 20 amino acids, a band stained with the anti-MDP1 antibody can be confirmed between MW 28000 MDP1 and MW 32500 (Fig. 1). Lane 4 in 7a) and anti-NANP antibody stained in the same place (lane 4 in FIG. 17b). The MDP1 band was confirmed by running 1 g of purified MDP in lane 5 and staining with an anti-MDP1 antibody. The fusion protein with MDP1 confirmed to be expressed by each recombinant BCG is indicated by an arrowhead.

 Figure 18. The 3PYB gene, a malaria antigen B-cell epitope, was ligated into MDP1 and transformed into BCG. Mice were immunized with 3PYBC and induced antibodies against 3PYB.As a result, no antibody induction was confirmed with the antigen peptide (3PYB) alone, but in the group immunized with rBCG, increased antibody titers were confirmed in both groups. Was done.

 Disclosure of the invention

The present inventors have conducted intensive studies in view of the above problems, and as a result, isolated a polypeptide represented by SEQ ID NO: 2 (hereinafter, abbreviated as MDP 1 (hereinafter abbreviated as Mycobacterium DNA Binding Protein)) from BCG Tokyo strain, Was found to be a cause of delayed growth _{c. Further,} it was found that administration of the polypeptide exhibited a protective effect against M. tuberculosis infection and was useful as a vaccine.

 Furthermore, they have found that a fusion protein in which MDP1 and a heterologous antigen are bound can enhance the antigenicity of a heterologous antigen due to the high antigenicity of MDP1.

 That is, the present invention provides the following items 1 to 9.

Item 1.1 A polypeptide having immunogenicity against a pathogenic acid-fast bacterium represented by SEQ ID NO: 2 (205 amino acids) in which one or more amino acids may be substituted, added or deleted. Puchido.

Item 2. The phosphorylated polypeptide of Item 1.

Item 3. A DNA encoding the polypeptide of Item 1. Item 4. A vector containing the DNA described in Item 3

Item 5. A transformant containing the vector according to Item 4.

Item 6. The method for producing a polypeptide according to Item 1, wherein the transformant according to Item 5 is cultured.

Item 7. A peptide comprising the polypeptide according to Item 1 or the DNA according to Item 3. Item 8. A diagnostic method for pathogenic mycobacteriosis, comprising detecting an antibody against the polypeptide according to Item 1.

Item 9. The diagnostic method according to Item 8, wherein the pathogenic mycobacteriosis is selected from the group consisting of tuberculosis, MAC (Mycobacterium avium-intracellulare complex), and Hansen's disease.

 The polypeptides, particularly the phosphorylated MDP1, and the DNA encoding the polypeptides are useful for diagnosing tuberculosis and producing vaccines. The polypeptide may be modified with a sugar chain.

 The polypeptide of the present invention can be obtained, for example, from the BCG Tokyo strain, but is not limited to this strain, and may be a protein obtained from another BCG strain or a Mycobacterium bacterium such as Mycobacterium tuberculosis or Mycobacterium leprae. Even if they are included in the present invention.

 In the polypeptide of the present invention consisting of 205 amino acids, one or more amino acids, preferably one to several amino acids are substituted at a specific position or randomly, as long as they have immunogenicity against pathogenic mycobacteria. May be added or deleted. Further, the polypeptide of the present invention is preferably phosphorylated. The term "immunogenic to pathogenic mycobacteria" means that an antibody against pathogenic mycobacteria is produced when the polypeptide is administered to a mammal in combination with other proteins as necessary. It has the ability to induce.

 One or more, preferably one to several, nucleobases are specified for the DNA encoding the amino acid as long as the polypeptide encoded by the DNA has immunogenicity against pathogenic mycobacteria. May be substituted, added or deleted at random positions. The DNA of the present invention includes a DNA that can hybridize with the DNA of SEQ ID NO: 1 under stringent conditions.

Conventionally known methods such as point mutation and deletion / insertion using PCR are widely used as methods for substituting, adding or deleting specific amino acids. Used.

 In the present specification, “stringent conditions” means conditions usually used in a hybridization method, and such conditions can be easily understood by those skilled in the art.

 The polypeptide of the present invention is characterized in that, for example, a vector incorporating a DNA encoding the polypeptide is introduced into cells to obtain a transformant, and the transformant is cultured in a medium. Produced by the method for producing the polypeptide represented by

 The cell transformed with the vector incorporating the DNA encoding the above polypeptide is not particularly limited, and conventionally known cells for transformation are widely used, for example, Escherichia coli, BCG bacteria, etc. And cultivated cells of various mammals such as mouse, rat, hamster, human and the like, preferably bacteria or yeast.

 The introduction of the vector into a host cell such as E. coli can also be performed according to a known method.ο

 The vector used for the production of the polypeptide of the present invention is not particularly limited as long as it has a promoter necessary for translation of the DNA encoding the polypeptide of the present invention.Examples include pBluescript pGEX and the like. Is mentioned.

 The present invention also relates to a transformant in which a vector incorporating DNA encoding the polypeptide is incorporated in the cell.

 The medium in which the transformant is cultured depends on the type of cells to be transformed. For example, in the case of a microorganism such as Escherichia coli, a carbon source (such as glucose), a nitrogen source (such as ammonium sulfate), A medium containing an inorganic substance (sodium phosphate, iron sulfate, manganese sulfate, etc.) can be mentioned. Culture conditions such as temperature, pH, and time are the same as those used for normal culture of various cells.

When the polypeptide of the present invention is used for diagnosis of tuberculosis or mycobacteria, several to several tens of peptides contained in the polypeptide or polypeptide of the present invention are used as antigens, and the antigen is used as the antigen. A biological sample (such as serum) containing the antibody in vitro, and then detecting the resulting antigen-antibody complex to diagnose tuberculosis or mycobacteria. It can be performed.

 Application of MD P 1 to vaccine

 Protective antigens for various intractable diseases (protozoal infections such as malaria, bacterial infections such as pneumococci and chlamydia, and viral infections such as HIV difluenza) have been identified, and they have been effective at the level of animal experiments. However, when applied to humans, a safe and effective adjuvant has not been developed, so that a sufficient infection protective effect cannot be obtained, and it has not yet been used as a vectin.

 The present inventors have found that the DNA encoding MDP1 consisting of the amino acid sequence of SEQ ID NO: 2 enhances the immunogenicity of the protective antigen by binding to the above-described DNA encoding the heterologous protective antigen. And proved that the antibody titer against the heterologous protective antigen was raised. By linking the above protective antigen to MDP1 or a derivative thereof and forming a fusion protein, a safe and effective vaccine without infectivity can be produced. In particular, when the adjuvant activity is not sufficient, the fusion protein can be expressed in BCG bacteria as a host cell for transformation and used as a vaccine. BCG is a live vaccine with the highest adjuvant activity, is effective, and is still used as a safe vaccine.

 In one embodiment of the use of MDP 1 as a vaccine,

 1. Use a fusion protein expressing a heterologous antigen in MDP 1 together with DNA as a vaccine;

 2. Use the fusion protein as a vaccine with DNA and existing adjuvants;

 3. expressing the fusion protein in BCG bacteria and using it as a live vaccine;

 Furthermore, a vaccine against tuberculosis can be obtained by administering the polypeptide of claim 1 or the DNA of claim 2 as it is or together with an appropriate carrier. The protective effect of the vaccine against tuberculosis infection has been confirmed in Examples. The dose of the polypeptide described in claim 1 or the DNA described in claim 2 per vaccination is about 5 to 1,000 g for an adult.

According to the present invention, it has a slow growth property against bacteria such as mycobacterium and Escherichia coli. We isolated a novel polypeptide and elucidated its structure.

 The polypeptide can be easily mass-produced by a genetic engineering method, and can be applied to the development of an antigen for diagnosing atypical mycobacteriosis such as tuberculosis and leprosy, vaccine development, and the like.

 The polypeptide of the present invention binds to DNA, RNA or ribosome to delay proliferation, and can be used for treating infectious diseases or cancer.

 BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in more detail using specific embodiments.

Production Example 1

 (1) Bacterial strain, plasmid, culture solution

 BCG Tokyo strain, M. tuberculosis H37Rv and .smegmat is ATCC 606 are Middlebrook 7H9 broth (Difco Laboratories, Detroit, Inc.) supplemented with Soton's medium or 10% ADC concentrate and 0.05% Tween 80. , USA) at 37 ° C. The M. leprae 53 Thai strain was obtained from Pine Talent Dr. Masanori. M. smegmatis was used as a host for plasmid pSO246 and its derivatives. Recombinant M. smegmatis clones are: 10% 0 ADC concentrate (Difco Laboratories), 0.5% glycerol, penicillin 400 units Zm I, cycloheximide 100 g / m I 7 H 1 Selection was performed by culturing on Middlebrook 7H10 agar (Difco Laboratories) supplemented with 0 agar. The E. coli strain XU-Blue is a plasmid pBluescript SK (I) (pBS SK +) (Stratagene Cloning 'System, California, USA) or pGEX4T-3 (Pharmacia Biotech, Tokyo, Japan) And its derivatives. The large intestine strain BL21 (DE3) pLysE was used as a host for pET22b (+) (Novagen Madison, USA); iMSOElox (Amersham) and its derivatives. All E. coli strains were grown in LB broth.

 (2) Purification of MDP1 from BCG

_C- Nocteria, in which the acid-soluble protein was precipitated by some of the modifications described above (Jhon et al., 1967), was 50 ml TMN SH (10 mM Tris-HC 1 H = 7.5, 1 0 mM Mg C 1 2, 6 0mM NH 4 C l, and resuspended in 6 mM 2-mercaptoethanol one Le), and disrupted by sonication. Centrifuge at 300 g for 2 hours The pellet was resuspended in 0.25 N HC1 by stirring at 4 ° C. overnight and centrifuged at 20000 g for 20 minutes. 0.1 volume of 100% (w / v) TCA was added to the supernatant with vigorous stirring. The precipitate formed by standing at 4 ° C for 4 hours is collected by centrifugation, washed once with acetone (20 ml) and acidic acetone containing 0.01 ml of concentrated hydrochloric acid, and washed twice with acetone. And dried in a vacuum desiccator. The dried precipitate was resuspended in 0.2 M sodium phosphate buffer (pH 6.8). Next, the acid-soluble protein was purified by a linear gradient of granidine hydrochloride G dn C 1 in 0.2 M sodium phosphate buffer (pH 6.8) using a fast-mouth column (bed volume 5 m 1; (Pharmacia) Chromatography on Hi trap CM Sepharose at room temperature for fractionation. The gradient was performed on a gradient apparatus filled with 15 ml of 0 and 5% GdnCl solution. The flow rate was maintained at 1 ml Zmin, and each fraction of lm was collected. The fraction containing the purified MDP1 was dialyzed against 0.2 M phosphate buffer containing 5% GdnCl and concentrated. Finally, it was further purified by gel filtration on a Hi load Seperdex 200 pg column (Pharmacia). Protein purity was monitored by measuring absorbance at 22011111 or by analysis with 303-86E.

 (3) Determination of amino acid sequence

 A sample for amino acid analysis was obtained by excising a protein band from a PVDF membrane (Millipore, Mass., USA). 1 2.5% polyacrylamide gel contains 0.05% SDS protein 3- [cyclohexylamino] -1-propanesulfonic acid (CAPS; Sigma, St. Louis, Missouri) — NaOH buffer The solution (pH 11) was electrophoretically blocked on a PVDF membrane. After staining with CBB, the protein spots were subjected to amino-terminal sequencing by automated Edman degradation on an Applied Biosystems 477 A gas phenol-Applied Biosystems (Applied Biosystems).

 (4) Gene cloning, DNA sequencing and computer analysis

The BCG genomic DNA is fragmented by repeatedly passing the injection needle until the main part of the DNA becomes 1 to 5 kbp, and the cDNA is excised using Amersham's cDNA rapid adapter ligation module. 0 Connect the RI adapter And then! ^ 03 £ 10 inserted into the £ (; 01 ^ 1 site. Colony hybridization was performed as previously described (Matsuo et al., 1988), with two sets of [α_ homologous to the N-terminal amino acid sequence. ³² [rho] using labeled oligonucleotide probes row ivy probe 1, 2 sequences, 5 '-. ATGAACAAGGC (〇 or 0) (corresponding to Met- l~Val- 9) G AGCT (〇 or 6) ATCGACGT And 5'-GA CGT (C or G) (T or C) T (C or G) AC (C or G) C AG A AG (T or C) T (C or G) GG (Asp-8 to Gly -Equivalent to-15. The inserted DNA fragment containing the MDP1 gene was used for the tack 'die' primer 'cycle sequencing kit and the 373A system (Applied Biosystems). The search for sequence homology was performed using the DDBJ (Shizuoka) database using the fasta program (Pearson and Lippman, 1988). I went through a tab.

 (5) Expression of MDP1 as a fusion protein with GST in Escherichia coli

 Primers were synthesized for amplification of the MDP1 gene. The DNA sequence of Primer-1A was 5'GGggatccGGGAGGGTTGGGATGAACAAAGCAG (sense strand), and the DNA sequence of Primer-1B was 5'GGGGggatccAGCACGTGGGTGTTGTCGTTG (antisense strand). Lowercase letters indicate added restriction enzyme sites. The products amplified by the primers A and B were digested with Hindlll and BamHI and introduced into the same site of pGEX4T_3. The final construct was named pGEXMDP1. E. coli was transformed with this plasmid. GST-MDP1 expression was performed according to the manufacturer's instructions (Falman).

 (6) Gel delay test

 l OO ng of pBSKS +, Hindlll-digested pBSKS +, or 240 ng of MS2 phage RNA in various amounts of MDP1 (final concentration) in PBS61 containing 5% glycerol. 20, 10, 5, 2.5, 1.25, 0, M). Samples were preincubated at 37 ° C for 10 minutes, analyzed in a 0.8% (w / v) agarose gel by electrophoresis using TAE buffer, and treated with ethidium bromide (EtBr). Stained. Nucleic acids were visualized under UV light.

(7) Immunoelectron microscopy test Immunoelectron microscopy studies were performed as known (Ferreira et al., 1992).

(8) Preparation of BCG fraction

 BCG cultured at 37 ° C. in soton medium was disrupted by ultrasonication in a TM NSH buffer. The crushed material was centrifuged twice at 1000 g at 4 ° C for 5 minutes to remove unbroken cells. Then, the cell wall, cell membrane, ribosome, and cytoplasmic fraction were prepared by a known method (Ohara et al., 1997). Secreted proteins were prepared as previously described (Matsumoto et al., 1991b).

(9) Purification of ribosomes derived from 50S ribosome subunit of BCG and MDP1 Preparation of ribosomes derived from BCG and separation of ribosome subunits have already been described (Yamada et al., 1987) ). Ribosomal protein, as is known (Hin Den'natsuha et al., 1 9 7 1) was extracted from 5 0 S Sabuyuni' Bok with 0. 1 M Mg C 1 ₂ in the presence a 6 6% acetic acid. The extract was then dialyzed against 5% acetic acid and lyophilized. The extracted protein, reverse phase using a Hitachi L- 6 0 0 0 HPLC system using a C ₄ column - were separated by HP LC. 2 mg of 50S total protein was chromatographed using a linear gradient of 30-70% acetonitrile in 0.1% TFA at a flow rate of 0.6 ml Zin in for 90 minutes. . The eluate was monitored by measuring the absorbance at 220 nm or analysis by SDS-PAGE.

 (10) Immunization method

 Antisera to MDP1 were obtained by intraperitoneal immunization of female BALB / c mice twice with 20 μg of purified MDP1 in Freund's incomplete adjuvant.ス タ ン Estanblot analysis was performed as known (Matsumoto et al., 1991b :).

 (11) Inhibition of DNA synthesis in vitro by MDP1.

In vivo synthesis of DNA was performed using Klenow fragment (Takara) as previously described (Sambruk et al., 1989). Single stranded DNA was prepared from E. coli XL1-B1ue with pBSKS + by infection of the f1 phage. Prior to the reaction, incubate single-stranded DNA (1 μg) and 5 pmo 1 of the M13 phage in 124 4 primers (Nippon Gene) at 85 ° C for 10 minutes, and place at 22 ° C. It was cooled down slowly and annealed. Extension reaction of DN A synthesis, 5 0 mM tris (p H 7. 5), 5 0 mM N a C l, 1 OmM Mg C 1 2, 1 2 4 4 primers one Aniru DNA was铸型0. 6 g, dATP of 0. 9 mM, dGTP and d TTP, 50 Ji 1 [shed one ^32?] D CTP, 8 Yuni' Klenow fragment of Bok, in DTT in 1 0 mM, MDP 1 or in the presence or absence of BSA. The final volume was 301. The extension reaction was performed at 22 ° C for 8 minutes. The reaction was stopped by adding 30 1 of 0.5 M EDTA (pH 8.0). The sample was then incubated at 68 to inactivate the Klenow fragment. 5 μl of each sample was spotted on a glass fiber filter (Toy 0 filter paper). The filter paper was washed three times with 5% TCA containing 20 mM sodium pyrophosphate. After drying the filter paper, the TCA-insoluble [α- ³² P] d CTP in each sample was measured using a scintillation counter.

 (12) In vitro transcription analysis

In vitro transcription analysis was performed using a modification of the previously described method (Bederson et al., 1994). Various amounts (20, 10, 5, 2.5, 1.25 and O ^ M) of purified MDP 1 and BSA in PBS were adjusted to 0.1 I pmol (200 ng) pBSKS + and the reaction volume to 20 I. And mixed. After incubation at 37 ° C for 10 minutes, an equal volume of the following solution was added: 0.02% (wZv) DE PC; 8 OmM Tris-HCI (pH 8.0); 16 mM M g C12; 4 mM spermidine; 10 mM DTΤ; 1.8 mM ATP, CTP, GTP and UTP; 1.84 UZm1 RNase inhibitor; 1.42 UZm1 T7 RNA Polymerase (Takara) and [α- ³² P] UTP of 0.36 mC i Zml. In vitro transcription was performed at 37 ° C for 30 minutes. Next, 51 samples were spotted on a glass fiber filter. The filter paper was washed three times with 5% TCA containing 20 mM sodium pyrophosphate. After drying the Gurasufa I bar filter foremost, the TCA insoluble [α- ³² P] UTP in each sample was determined by scintillation counting.

 (13) In vitro translation analysis

In vitro translation analysis was performed using the E. coli S30 coupled transcription and translation system (Promega). MDP 1 or egg white lysozyme in each amount (final concentration; 20, 10, 5, 2.5, 1.25 and 0 //), S 30 extract of 71, 50 nC i [ ³⁵ S] —methionine, free of methionine 1 0 1 Premix (Promega), 4 g (3.33 MS 2 RN A (base one ringer one Manheim pmo 1), Tokyo, Japan) were mixed _c final reaction volume was 34 1. Translation was performed at 37 ° C for 1 hour. To assess the uptake of radiolabeled methylate Onin, according to the manufacturer's instructions, taking a sample of 5 n 1, and dissolved in 245 mu 1 of 1 MN a ΟΗ, _c TCA treated precipitated with a final 20% TCA The sample was trapped on a glass fiber filter and then washed three times with 5% TCA. The membrane was dried and the radioactivity was measured.

 (14) Detection of phosphorylation of MDP 1

Purification MD P 1 1 g of have use bacteria al force Rihosufata Ichize the (BAP), 5 OmM Tris - solution containing HC 1 (p H 9. 0) and _{1 mM M g C 1 2,} 6 5 Treated at ° C for 1 hour. Antibodies to MDP1, treated with or without BAP, against phosphonothreon, phosphoserine, phosphotyrosine, phospholipidin, phosphoserine, phosphotyrosine, as described in the manufacturer's instructions (Transduction Laboratories, Kentucky, USA ) Was used to perform the stamp lot method.

 (15) MDP1 expression in fast-growing bacteria

For expression of MDP1 (BCG) in M. smea (J's, primer C for the sense strand was synthesized. The oligonucleotide sequence was 5 'GGGaagcttTTTGA GGGTGCGTGCGCGTAC. The gene encoding the MDP1 structural gene and its upstream region amplified by primers B and C was digested with both Hindlll and BaMHI, and the same site of pBSKS + (named pBMDP1). Was purchased. pBMDP1 was digested with HindiII and BamHI, and a 1 kbp DNA fragment containing the MDP1 gene was inserted into PS0246 (Matsumoto et al., 996a) at the same site. It was named p S OMD P1. M. smegmatis was transformed with pS OMD P1 by electoral poration as previously described (Matsumoto et al., 1996b). The following primers were newly synthesized to express the unfused form of MDP1 in E. coli. Primer D for the sense strand was CcatatgAACAAAGCAGA GCTCATTGAC and Primer E was CaagcttCTATTTGCGACCCCGCCGAGCGG. The amplified DNA containing the structural gene for MDP1 was cut with both Ndel and Hindlll and inserted into the same site in pET22b (+). This plasmid was named pET22MDP1. E. coli strain BL 2 1 (DE 3) p Lys E, p ET 2 Transformed with 2MD P1. The transformed cells contained 50 ^ g / m1 of carbenicillin, 34 g / m1 of clonal ramjunicol, and LB agar with or without 0.5 mM IPTG. Grown on top. result

 (1) Analysis of the most abundant proteins with DNA binding ability

To identify DNA binding proteins in BCG, cell lysates were subjected to SDS-PAGE and blotted onto PVDF membrane. The membrane was then reacted with [α- ³² P] -labeled pBlues cript KS + (pBSKS +) to visualize the protein-DNA interaction with an autoradiograph. As shown in FIG. 1A, a strong response was observed at 28 kDa. This 28 kDa DNA binding protein was named MDP1.

 SD S-PAGE analysis indicated that MDP1 was the most abundant protein (indicated by the arrow in lane 1 in FIG. 1D). The present inventors purified MDP1 as described above. First, a sample rich in MDP1 was prepared by treating BCG lysate with 0.25N-HC1 (lane 2 in Figure 1D) and then purifying it on an ion exchange column. FIG. 1B shows the chromatographic profile, with the proteins in the major fractions visualized in lane 3 of FIG. 1D. The protein in this fraction was further purified through a gel filtration column (Fig. 1C) to yield highly purified MDP1 (lane 4, Fig. 1D). In the final step, MDP 1 was eluted as a 195 kDa protein (FIG. 1D). This indicates that MDP1 forms a multimer. The final yield of MDP 1 was about 5 mg from a fresh wet weight of 100 g BCG.

The N-terminus of the purified amino acid sequence of MDP1 was identified as MNKAELIDVLYQKLG-D by the amino acid sequencer. To clone the gene encoding MDP1, colony hybridization was performed using two [α- ³² P] -labeled sets of oligonucleotide probes corresponding to the N-terminal amino acid sequence (see Examples). -I went there with a bus. DNA fragments hybridized with the two probes were obtained and sequenced. FIG. 2A shows the nucleic acid sequence and the deduced amino acid sequence. The DNA fragment contains an open reading frame (ORF) beginning with ATG at position 265 and ending with a TAG stop codon at position 882. N-terminal amino acid of MDP 1 The acid sequence (boxed in FIG. 2A) was found to be completely consistent with this ORF. As expected, MDP1 is extremely basic (isoelectric point (PI) force, '12 .4) and contains high amounts of alanine, arginine, lysine, proline and threonine. A potential Shine-Dalgarno (SD) sequence was observed 7 nucleotides upstream of the start codon (underlined in Figure 2A). In bacteria, the DNA binding motifs observed in some chromosome-binding proteins were at positions 46 to 65 (bold underline in FIG. 2A). This region is predicted as the DNA binding site of MDP1.

A computer search showing alignments of some protein and amino acid sequences homologous to MDP1 is shown in FIG. 2B. High homology encoded by the two ORFs was observed in the DNA sequences of genomic cosmid libraries from M. tuberculosis and M. Jeprae. MDP1 has 95% homology with M. tuberculosis and 83% homology with Ueprae. Computer analysis shows that the N-terminal region of MDP1 has partial homology to HU from bacteria and the C-terminal region has partial homology to the histone H1 class in eukaryotic cells. Was. As a typical example, the best § Line Instruments of _c initial 9 0 amino acids showing a comparison of MD P 1 and HU 2 and human Bok histone H 1 of E. coli in Figure 2 B is the MD P 1 and HU 2 Between 4 1%

(Kano et al., 1989), and 25% (MDg et al., 1991) between MDP1 and human histone H1.

 (2) Recognition of nucleic acid conformation by MDP1

The DNA binding ability of MDP1 was confirmed as follows. MDP1 was expressed as a fusion protein with Schistosom a japonicum glutathione S_transferase (GST) (GST-MDP1). All proteins of E. coli-expressed GST-MDP1 were transcribed to the membrane. Film reacts with [α _ ³² Ρ] labeled p BSKS +, its auto Rajiogurafu is shown in Figure 3 Alpha. Additional bands were observed in lanes 4 and 5 (indicated by arrows on Figure 3A) and recognized by anti-MDP1 antibody (data not shown) (data not shown) . This confirms the DNA binding ability of the product encoded by the gene. Degradation products were observed, indicating that the fusion protein was not stable in E. coli. To analyze the nucleic acid binding activity of MDP1 in more detail Next, a gel delay assay was performed. After incubation of various concentrations of purified MDP1 with cyclic, linear, or RNA, the complexes were analyzed by agarose gel electrophoresis (Figures 3B, 3C, and 3D). The data showed that MDP1 binds to both DNA and RNA, as nucleotides are delayed in slots depending on the concentration of MDP1. The binding abilities to DNA (FIG. 3B), linear form DNA (FIG. 3C) and RNA (FIG. 3D) having a nick of MDP1 were almost the same. On the other hand, preferential inhibition of supercoiled DNA migration to the gel over the others was observed at 10-5 M MDP1 (FIGS. 3C, 3C). These indicate that MDΡ1 recognizes the conformation of nucleic acids.

 (3) Localization of MD Ρ 1 in cells

To determine the localization of MDΡ1, we first performed an immunoelectron microscopy study on BCG. The results showed that the MDP1 homolog localized to the cell wall, cell membrane, ribosome region, and chromosome DN DN region (Figure 4A, panel a). Second, each subcellular fraction from BCG was prepared. These samples were transcribed onto a membrane and reacted with anti-MDP1 antibody (Figure 4B, panel b). A strong response was observed for the 28 kDa protein in the cell wall, cell membrane, and ribosome fraction, but not for secreted or cytoplasmic proteins. At the same time, the protein was visualized by gel staining after SDS-PAGE, and the putative MDP1 band is indicated by the arrow in panel a of FIG. 4B. These biochemical observations are consistent with the results of immunoelectron microscopy studies on BCG. Interestingly, some of the anti-MDP1 antibodies appear to react directly with liposomal particles in immunoelectron microscopy analysis, and they react strongly with the liposome fraction (Figure 4B, c. Flannel b). These indicate that MDP1 may bind to the ribosome. To elucidate this point, ribosomal particles (30 S and 50 S subunits) were separated by sucrose density gradient centrifugation. Proteins derived from the 30 S or 50 S subnet were reacted with anti-MDP1 antibody on the transferred membrane after SDS-PAGE. The reaction was observed in the 28 kDa and 27 kDa proteins of the 50 S subunit but not the 3 OS subunit, as shown in panel b of FIG. 4B. To confirm this result, 50 S subunit protein was replaced with RP- Separated by HP LC (Figure 4C, panel a). The protein in each fraction was visualized by SDS-PAGE stained with CBB (Fig. 4C, panel b), or these were reacted with anti-MDP1 antibody after blotting on a membrane. . The results showed that the 28 kDa and 27 kDa proteins eluted at a concentration of about 47% (fraction 27 of panel a in FIG. 4C) and 40% (fractions 18, 19), respectively, of acetonitrile. Sequencing of the N-terminal amino acid by the amino acid sequencer indicates that the 28 kDa protein is MDP1. This result indicates that the 50 S ribosomal submission contains MDP1. However, the 27 kDa N-terminal amino acid has not been sequenced. Therefore, it is not clear whether the 27 kDa protein is a modified amino acid at the N-terminus or a homolog of a gene encoding a different gene.

 (4) Inhibition of MDP 1 replication, transcription and translation at the in-vitro mouth

 Macromolecular synthesis at the mouth of in vivo was studied to elucidate the molecular processes of MDP1. First, the effect of MDP1 on the function of DNA polymerase I was examined to see the elongation of DNA synthesis (FIG. 5A). DNA synthesis was dose-dependently inhibited by MDP1. Up to 97% inhibition was observed with 1.25 MDP 1.

Second, the inventors evaluated the effect of MDP1 on the transcription of Τ7RNΑ polymerase (FIG. 5B). Transcription, 2. _c 3 was almost completely inhibited in the 5 M MDP 1, examined the effect of MDP 1 relating to the translation in vitro. Translation analysis at the in-vitro mouth was performed using an E. coli S30 extract (FIG. 5C). After a 30 minute incubation, protein synthesis was observed even without the type 2 of MS2 phage RNA. This may be due to the endogenous native mRNA of the E. coli S30 extract. Inhibition of protein synthesis by MDP1 was observed depending on the concentration of MDP1. At 10, MDP 1 almost completely inhibited translation.

 (5) MDP 1 delays bacterial growth

From the above results, MDP1 may delay growth by inhibiting macromolecular biosynthesis depending on its binding to DNA, RNA and ribosomes. The present inventors investigated this hypothesis by expressing MDP1 in rapidly growing bacteria. To express a non-chimeric form of MDP1, p SOMDP 1 and p ETMD P1 was constructed (see Example). M. smegmatis and E. coli were transformed with each plasmid and collected on a plate. As shown in FIG. 6, the growth rate of both bacteria was dramatically reduced by the expression of MDP1, indicating that MDP1 reduces the growth rate of the bacteria.

 (6) Threonine phosphorylation of MDP1 protein

 Phosphorylation and dephosphorylation are one of the major mechanisms controlling cell growth in eukaryotic cells. The present inventors examined whether MDP1 was a phosphorylated protein or not. The purified MDP1 was reacted with a monoclonal antibody against phosphoserine, phosphotyrosine or phosphothreonine. Only anti-phosphothreonine antibody reacted with MDPI and, as expected, this reaction was abolished by treatment of MDP1 with bacterial alkaline phosphatase (BAP) prior to the reaction (data not shown). These indicate that MDP1 is a threonine phosphorylated protein.

(7) Comparison of MDP1 expression between logarithmic and stationary growth phases between fast and slow growing mycobacteria

The expression of MDP1 in various mycobacterial species was examined by SDS-PAGE (FIG. 7A) and Western blot analysis (FIG. 7B). Antibodies include a 28 kDa protein of BCG (lanes 1 and 2 in FIG. 7B), a 30 kDa protein of M. tuberculosis (lanes 3 and 4), and a 26 kDa protein of M. leprae. It reacted strongly with the protein a (lane 5) and the 31 kDa protein of M. smegma iis (lanes 6 and 7), but did not react with the E. coli protein at all. Abundant protein bands in lysates from BCG and tuberculosis in the logarithmic and stationary growth phases are identical to the anti-MDP1 serum reaction bands. leprae is the slowest growing bacteria and has not been successfully cultured in vitro. Ueprae, which grows in the footpad of nude mice, expresses a large amount of MDP1 as shown in FIG. On the other hand, in M. smegmatis, a protein that recognizes anti-MDP1 serum is highly expressed only in the stationary phase. The present inventors have confirmed that these MDP1 homologs have the ability to bind to DNA by Southwestern blot analysis (data not shown). These results indicate that MDP1 is conserved in a wide range of mycobacteria, but expression levels of MDP1 differ between slow-growing and fast-growing bacteria. Slowly growing bacteria have large amounts of MD regardless of the growth phase It expresses P1, but fast-growing bacteria are mainly expressed in the steady state and not in the logarithmic growth phase.

Example 1

 Four mice (C3H / He, C57BL / 6, A / J, BALB / c) at the age of 6 to 10 weeks were treated with MDP 1 twice with 5 g of Freund's incomplete adjuvant (second time after 3 weeks After immunization (the first time was subcutaneous and the second time was intraperitoneal), blood was collected 4 weeks later, and the serum was diluted 100-fold (diluent: 1% BSA in PBS), and used as a primary antibody in a sterile blot. As the antigen, a lysate of BCG was used. Only BALB / c mice produced antibodies against MDP1.

 Next, the results of similarly reacting the sera of three kinds of mice to which BCG bacteria were administered (C3H / He, C57BL / 6, A / J; first blood, second intraperitoneal; BCG106 CFU) However, production of an antibody against MDP1 was confirmed in all mice. The antibody reacted most strongly with MDP1 among BCG antigens.

 The results of this experiment revealed that immunization with bacterial cells strongly induced antibodies against MDP1.

Example 2: Reaction with patient serum

 The serum of three patients with Hansen's disease, four patients with tuberculosis, and healthy individuals were diluted 100-fold. The purified MDP1 was transferred to a membrane and reacted with the diluted antibody. As a result, the presence of an antibody recognizing MDP1 was revealed in the sera of all mycobacteriosis patients. On the other hand, the presence of the antibody was not observed in the samples of healthy subjects. When reacted with secreted proteins (including 85 complexes) in the same Western blot system, they reacted with the sera of Hansen's disease patients but did not react with the sera of tuberculosis patients. This result indicates that MDP1 is more antigenic in mycobacteriosis than Ag85.

After culturing the BCG Tokyo strain in Sorton's medium, it was passed through a 0.45 m (Millipore) filter to separate the cells from secreted proteins. The cells were dissolved in SDS_sample buffer, disrupted by sonication, and boiled at 100 ° C for 5 minutes. Secretory protein is added to the culture filtrate by adding ammonium sulfate to a final concentration of 80% to precipitate protein. It was dialyzed against PBS (pH 7.2). SDS-sample buffer was added to the obtained secretory protein, and the mixture was boiled at 100 ° C for 5 minutes. After centrifuging each sample, the supernatant (2 O ^ g of the evening protein) was electrophoresed by SDS-PAGE (21.5%), and the protein was electrically transferred to polyvinylidene difluoride. (PVDF membrane; Millipore). The PVDF membrane was immersed in PBS containing 3% BSA for 30 minutes to perform blocking. Preparation of antibody 800 Tokyo strain 1 0 ⁷ 0? 11 administered to C 3 HZH e vein, one month after the same amount of cells were administered intraperitoneally, serum was collected after one month,? It was diluted 100-fold with 83 + 1% 83-8. The prepared antibody was reacted with BSA-blocked PVDF membrane overnight at 4 ° C, washed with PBS containing 0.05% Nonidet P40, and washed with PBS. After reacting with a peroxidase-labeled anti-mouse antibody diluted 1 000 in 83, washing with the above washing solution, 25 mg of 3,3-diaminobenzidine'tetrahydrochloride was added to 2 OmM Tris (pH 7.5) 10 The PVDF membrane was dissolved in OmI and immersed in a solution to which 291 H ₂ O ₂ was added, to detect antibodies. Fig. 8 shows the results. As shown in FIG. 8, in the bacterial cell protein, a protein having a molecular weight corresponding to MDP1 and an antibody against a protein of 45 to 47 kDa were detected. On the other hand, an antibody against a secreted protein was not detected under these conditions, and it was considered that MDP1 had an antigenicity higher than that of Ag85, which is said to have the strongest antigenicity.

Experiment 2

The BCG Tokyo strain ^{(1 0 7 CFU), AZj} , BALB / c, was administered intraperitoneally C 3 H / H e, C 5 7 BL / 6, taken 1 month after serum, PB S + 1% B SA Diluted 1: 100. The purified MDP1 was subjected to SDS-PAGE at 1 g / lane, transferred to a PVDF membrane, and reacted with the diluted serum in the same manner as above to detect an antibody against MDP1. As a result, it was found that anti-MDP1 antibodies were produced in the sera of all four mice.

Experiment 3

The BCG bacteria (1 0 ⁸ C FU), mice. (C 3 HZH e: s 1 c) to Kumishi throw the tail vein, MDP 1 (5 g) / RAS ( Ribi adjuvant system), DNA (M tuberculosis DNA 0.5 ^ g) / RAS, MD P 1 (5 // g) + DNA (0.5 g) ZRAS was administered subcutaneously to mice (C3HZHe: s1c), 3 weeks later, the same dose was intraperitoneally administered, 4 weeks later, serum was collected, and the collected serum was 100 times with PBS + 1% BSA. Diluted. The purified MDP1 was subjected to SDS-PAGE / lane, transferred to a PVDF membrane, and reacted with the diluted serum. As a result (Fig. 9), antibodies were produced only in mice immunized with BCG and MDP1 + DNA. No antibody production was observed with MDP1 alone or DNA alone, and MDP1 bound to DNA. It was confirmed that the immunogenicity was enhanced and the antibody titer was increased.

Experiment 4

5 g each of MDP1, histone H1, H2, H3 (from Behringa-Mannheim, cattle), alone or mixed with Mycobacterium tuberculosis DNA (0.5 ^ g), and mixed with Libya adjuvant ( C3 HZHe mice (at the time of the first immunization, 7 weeks of age) were immunized using RIBI i Marunouchi Research, Inc. Hamilton, MT). Samples were diluted in PBS and adjusted to 100 1 per mouse. The immunization was performed by subcutaneous administration of the primary immunization and booster immunization three weeks later by intraperitoneal administration. One week after the booster, blood was collected from the mice, and serum components were obtained by centrifugation. The detection of antibodies against the respective antigens was performed by ELISA. That is, each antigen was dissolved in a 96-well ELISA plate for ELISA (Sumitomo Beclite Co., Ltd.) with 0.05M carbonate buffer (pH 9.6) to 2 g / ml, One aliquot was dispensed into each well and allowed to stand at room temperature for 2 hours to layer the antigen. For blocking, wash the wells once with BBS (pH 8.0; 10.33 gZL of boric acid, 7.83 gZL of NaCl), then divide the BBS containing 3% BSA into 300 ^ 1 aliquots. It was poured, left at room temperature for 2 hours, and washed once with BBS. Each serum sample was diluted with BBS solution containing 1% BSA to 50 to 102400 times, added to each gel 100 1 each, and reacted at 37 ° C for 30 minutes. HBBS (pH 8.0; 10.33 g of boric acid, 29.22 g / L of NaCl) was prepared as a reaction plate, and each pellet was washed 300 × 1 seven times. Thereafter, the peroxidase anti-mouse antibody was diluted 2000-fold with a 883 solution containing 1% 83, dispensed 1001 into each well, and cultured at 37 ° C for 30 minutes. After that, the same washing is performed with the above washing solution, and the concentration of ortho-phenylenediamine dihydrochloride is adjusted to 0.4 mg / ml. As described above, color development was performed using a detection solution obtained by adding H ₂ O ₂ to 8 O mM citrate monophosphate buffer and adding 41 H ₂ O ₂ to the solution. After the reaction for 3-5 minutes, the reaction was stopped by adding an equal volume of 1N sulfuric acid, and the absorbance was measured at 492 nm. As a result, as shown in FIGS. 10 to 13, MDP1 and DNA were mixed and immunized, and an increase in absorbance was observed only in the obtained sample, and the same DNA-binding protein, histone H1 No increase in absorbance was observed in any of H2, H3. This revealed that the antigenicity was specific for MDP1.

Experiment 5

 The purified MDP1 was transferred on a PVDF membrane after SDS-PAGE in one lane, and the patients (L1, L2, L3) and tuberculosis patients (Tl, Τ2, Τ3,反 応 The antibody was reacted with the serum diluted solution of 4) in the same manner as described above, and an antibody against MDP1 was detected. As a result (Fig. 14), antibodies to MDP1 were detected in the serum of all patients tested. From these results, MDP1 is considered to be applied as a diagnostic agent for tuberculosis and leprosy patients.

 Application of MPΡ1 to diagnostics

 The above experiment confirmed that MD-1 was a protein specific to acid-fast bacteria and had strong antigenicity. In particular, the results of Experiment 1 revealed that the antigenicity is the strongest at present and has stronger antigenicity than the mycobacterial-specific antigen Ag85 complex, which is being studied as a diagnostic drug. Therefore, by identifying an antibody against MDP1, the presence or absence of infection by tuberculosis can be diagnosed (Experiment 5). In addition, since the sequence of MDP1 differs depending on the species of the acid-fast bacterium, it is possible to identify the infecting bacterium by using a species-specific antigen or peptide. Therefore, MDP1 of various mycobacteria including Mycobacterium tuberculosis and peptides contained in the MDP1 can be used as antigens of diagnostic agents.

Experiment 6: Protection against M. tuberculosis infection in mice

Group A vein 1 (5 μg) + DNA (5 ng, derived from BCG) -PBS / RIB, Group B DNA (5 ng, derived from BCG) / RIB, Group C α antigen (Ag85: 5 ^ g) / RIB, Group D PBS / RIB, group E PBS were prepared and 9 to 10 mice were immunized by subcutaneous administration first. After three weeks, the compound was again intraperitoneally administered, and three weeks later, the appearance of antibodies against MDP1 and Ag85 was confirmed. Thereafter, the M. tuberculosis Kuro no strain per animal from the mouse tail vein, were infected with about 10 ⁴ CFU. Evaluation 2 weeks after infection After 4 weeks, the mice were dissected, the lungs were aseptically removed, homogenized with a glass homogenizer, and the number of endophytic bacteria in the lung was measured using 7H11 agar medium. The vaccine effect was determined by comparison with the control group (Group D and E) using the Turkey test (significance level: 5%, two-tailed test).

 A) Confirmation of appearance of antibody after immunization

 Antibodies to MDP1 and Ag85 were detected by ELISA. As shown in FIG. 15, antibodies against MDP1 and Ag85 were detected.

 B) Results of judgment 2 weeks after infection

 As shown in Table 1, the number of bacteria in the lung 2 weeks after infection was higher in the MDP1 + DNA / RIB group than in the PBS and PBS / RIB groups, as compared to 5.57 and 5.45. In, the number of bacteria in the lung was significantly lower (5.17). However, for Ag85B / RIB, a significant difference of 5.60 was not obtained.

 C) Judgment results 4 weeks after infection

 As shown in Table 2, similar results were obtained after two weeks. That is, the number of bacteria in the lungs was significantly reduced in the MDP1 + DM / RIB administration group, but no significant difference was obtained in the Ag85B / RIB and other administration groups compared to the PBS administration group. Protective effect of Mycobacterium tuberculosis infection 2 weeks after infection

 Group A Group B. Group_ Group D Group E Pulmonary bacteria count 5.17 soil 0.15 * 5.56 + 0.15 5.60 + 0.17 5.45 + 0.18 5.57 + 0.05 0.05 Table 2. Tuberculosis Protective effect 4 weeks after bacterial infection

 A group B group C D group E

Number of bacteria in the lung 6.26 0.13 * 6.98 + 0.43 6.70 + 0.21 6.65 + 0.26 7.01 + 0.28 In Tables 1 and 2, * indicates P <0.05 indicates that it is significant to the control group (D group and E group).

Experiment 7: Lymphocyte activation of MDP1

Mycobacterium tuberculosis is transmitted to ICR mice and lymph nodes are removed 4 weeks later to prepare lymphocytes Afterwards, PBS ヽ MDPKO. 1, 1, 10 g / ml), Ag85B (0.1, 1, 10 ^ g / ml). H37Ra (0.1, 1, 10 g / ml), PPD (0.1 , 1, 10 g / ml) for 5 days, ³ H-thymidine was added, and the radioactivity in the cells was measured 24 hours later. Figure 16 shows the results. MDP1, H37Ra, the PPD stimulation, but uptake of ³ H- thymidine was observed, the Ag85 was observed.

 Ag85 is the only protein antigen derived from Mycobacterium tuberculosis and has been confirmed to be protective against Mycobacterium tuberculosis in guinea pigs. A vaccine trial has been conducted as an MA vaccine (Proc. Natl. Acad. Sci, 92 (1995) 1530-1534) . The data obtained revealed that MDP1 exhibited a 60-82% protective effect on the bacterial count in the lungs, despite the fact that the protective effect of Ag85 could not be confirmed. In other words, the results show that MDP1 is a novel antigen capable of achieving a protective effect in mice with a protein antigen, and that it is useful as a clinically novel vaccine.

In tables 1 and 2, PBS (5. 57) , MDP1 (5. 17) are logarithmic value, each original number of bacteria, 3. 7 x l0 ⁵ cells eight ung, 1. 5 x 10 ⁵ cells / lung. The following formula can be used to calculate the protective effect of the MDP1 vaccine group when PBS was set at 100% infection.

{(PBS count 3.7 x 10 ⁵ cells / lung— MDP1 count 1.5 x 10 ⁵ cells / lung) / (PBS count 3.7 x eel Is Aung)} x 100 = MDP1 Similarly, the number of bacteria protected by the effect (%) = 60% Similarly, the PBS group: 1.0 x 10 ⁷ cells / lung. The MDP1 group: 1.8 x 10 ⁶ cells at the 4-week value. Using the above formula, the infection protection rate is 82%.

It is becoming clear that the protective effect of tuberculosis cannot be obtained by humoral immunity, but can be obtained by cell-mediated immunity (Thl dominance). However, the detailed mechanism is unknown, and extensive research is being conducted on the search for new antigenic proteins, Mycobacterium tuberculosis-specific glycolipid antigens, DNA vaccines, and rBCG. Tuberculosis patients are predominantly Th2, and it is not clear whether this has resulted in the onset of the disease as a result of its natural constitution, or whether it has led to a Th2 predominance. However, it has been reported that the administration of IFNa kills M. tuberculosis bacterium and makes it harder for Thl-dominant healthcare workers to get sick. In the above Experiment 7, it was confirmed that the antibody could be induced. When the subclass of this antibody was measured, IgG2a was produced more than IgGl. IgG2a is known to be a subclass that is frequently induced in the presence of IFNa. These data show that mice are induced to Thl dominance and activate cell-mediated immunity. Indeed, it has been demonstrated that MDP1 can induce M. tuberculosis-specific cellular immunity by activating lymphocytes obtained from M. tuberculosis-infected mice. PPD, H37Ra and the like are known as antigens capable of inducing such M. tuberculosis-specific cell-mediated immunity, but it is known that these antigens cannot obtain a protective effect against infection. In addition, the same results were obtained in experiments using BALB / c mice for the same protective effect against infection and production of IgG2a-dominant antibodies. The same effect was confirmed in two different strains of mice, indicating the usefulness of MDP1 as a new vaccine. By using the DNA vaccine, which has been shown to have a protective effect as a protein antigen, and more durable, or using rBCG (live vaccine) with high expression of MPD, the BCG up to now can be used. It is expected to be applied to the realization of short-term chemotherapy in combination with a chemotherapeutic agent, as well as a longer lasting protective effect against infection, disease prevention, or combined use with chemotherapeutic agents.

 Ag85 complex is said to have high antigenicity, and many possibilities have been reported as diagnostic antigens and vaccine antigens (Infect. Immu. 167 (1999) 6187-6190). The newly discovered MDP1 has been confirmed to have higher antigenicity than Ag85 (FIG. 8). The major difference between these proteins is that Ag85 is a secreted protein, MDP1 is a superficial • endogenous protein, and there are differences in the localization sites of both proteins. One of the reasons for the high antigenicity of MDP1 is that it is localized near BCG, which has a safe and reliable adjuvant effect. By making use of the properties of MDP1, a protein with high antigenicity, and creating a fusion protein of MDP1 and a heterologous antigen, a new protein can be produced. An example of the method will be described below.

MSP1, NANP (3PYB), and SERA are known as malaria vaccine candidates, and it has been confirmed that the induction of antibodies against them can provide a protective effect against malaria infection. However, adjuvants that can be used in animal experiments cannot be applied to humans, and complete vaccination has not been achieved. Therefore, we attempted to develop a new vaccine by expressing these antigens in BCG as fusion proteins with MDP1. The vector used here used a mutannt MDP1 expression vector (pSOMDPlMutant) in which the fourth base from the N-terminus was substituted with adenine for guanine, and the growth inhibitory activity of BCG was weakened. Experiment 8: Construction of fusion protein expression vector

The recombinant vector was amplified with PiuTurbo ^T "DNA polymerase (STRATAGENE, CA, USA) using 1 ng of pSOMDPlMutant and 鍀 -type MDP1-A and MDP1-B, respectively. Was treated with phenol Z-cloth form Z isoamyl alcohol, precipitated with ethanol, treated with Mlu I, agarose gel electrophoresed, and purified using the EZN A Gel Extraction Kit (0MEGA BI OTEK, GA, USA). The sporozoite surface B cell epitope of mouse malaria, which is an insert fragment, takes 2 nmol of each of 3PYB-A and 3PYB-B, and performs an annealing reaction buffer (20 mM Tris_HCl (pH 7.5), 10 mM MgCh, 50 mM NaCl). After holding at 85 ° C for 5 minutes in the medium, annealing was performed by slow cooling, and CH ₃ C00Na having a final concentration of 300 mM was added to the reaction solution, and ethanol precipitation was performed. Treated with phenol, Alcohol treatment and ethanol precipitation were carried out using the thus prepared 0.03 pmol recombinant vector and 0.3prao 1 insert fragment in DNA Ligation Kit Ver. 2 (TAKARA SUZ0 CO., LTD., Otsu, Japa This plasmid was transformed into Escherichia coli DH5a strain, and pSOMDPlMutant was transformed into p-type using MDP1-C and MDP1-D. PMDPlMutant-3PYBM was constructed by ligating the amplified vector and the insulator fragment.

On the other hand, in order to bind to the C-terminus of MDP1Mutant and express various antigens, a Sea I recognition sequence was introduced into the portion encoding the MDP1 C-terminus of pSOMDP1Mutant plasmid. Specifically, pSOMDPlMutant was treated with i / 2i / III and BamHI, a lOObp fragment containing the MDPlMutant gene was purified, and introduced into pKF19 DNA also treated with indU1 and BamHI. Thereafter, a PCR reaction was performed using pKF19-MDP1Mu as a template using a primer containing a Sea I recognition sequence. The PCR product was treated with Phenol Z-Kokuguchi Form / Isoamyl alcohol, precipitated with ethanol, and then treated with Dpn KNEW ENGLAND Biolabs, Inc., MA, USA). Then, pKF19-MDPlMuScaI was constructed using DNA Ligation Kit Ver. 2 CTAKARA SUZO CO., LTD., Otsu, Japan). This plasmid transformed the E. coli DH5a strain. The transformed Escherichia coli was seeded on an LB plate containing kanamycin at a concentration of 30 g / ml. After the grown cells were cultured in LB broth, plasmid was extracted and pKFl9-MDPlMuScaI was selected. This enables the MDPlMutant gene PKF19-MDP1MuScaI was prepared by introducing a Sea I recognition sequence into the 3'-end-encoding region of the gene. This plasmid was treated with Hind III and feiz / HI, the lOOObp fragment containing the mutated MDPlmutant gene was purified, and introduced into a PS0246 vector treated with Hind Ui and Bam HI. This resulted in pSOMDPlmutantScaI. This pSOM DPlmutantSca I was treated with Sea I and dephosphorylated at the 5 'end with Calf intestine Alkaline Phosphatase.

The following insert sequence was introduced into the vector thus prepared. First, the NANP sequence, a sporozoid surface B cell epitope of mouse malaria, anneals 3PYB-c and 3PYB-d, precipitates with ethanol, and then uses T4 Polynucleotide Kinase (TAKARA SU Z0 CO., LTD., 0tsu, Japan). The 5 'end was phosphorylated. The MA fragment was introduced into the Sea I site of pSOMDPlmutantScaI to construct pMDPlMutant-3PYBC. MSP1 (Merozoite Surface Protein 1), which is a major antigen of human malaria parasite (Plasmodium falciparum) in the melozoid stage, is amplified with PfuTurbo ^T "DNA polymerase using went.

 For each of the amplified fragments, pMDPlMutant-PfMSPl and pMDPlMutant-PyMSPl were constructed using TaKaRa BKL Kit (TAKARA SUZO CO., LTD., Otsu, Japan). Escherichia coli containing these plasmids were selected on an LB plate containing 30 g / ml Kanamycin, cultured in an LB broth containing 30 j «g / ml Kanamycin, and then purified using Aldrich SDS method.

Experiment 9: Transformation into BCG

 The constructed plasmid DNA was transduced into the recombinant BCG prepared by the following method.

First, the M. bovisBCG Tokyo strain was transformed into a Middlebrook 7H9 medium (Becton Dickinson Microbiology Systems, MD, USA) containing albumin dextroleum complex (OADC, Becton Dickinson Microbiology Systems, MD, USA) and 0.05% Tween80. The cells were cultured with shaking at 37 ° C in Oml. OD _{6 6.} When the concentration reached 0.2, the cells were centrifuged at 7, OOO g for 15 minutes to collect the cells. The recovered cells were suspended in 50 ml of ice-cooled 10% glycerol solution, and centrifuged at 7,000 g for 15 minutes to recover the cells. Further, glycerol treatment was performed 5 times while reducing the 10% glycerol solution by 10 ml. Finally 5 ml of 10% glyce And stored at -80 ° C.

 One hundred ng of plasmid DNA was added to 100% of the BCG suspension solution, and the mixture was placed in an electrification cuvette and allowed to stand on ice for 30 minutes. After that, electroporation was performed at 2.5 KV using Electroporre. The cuvette was again allowed to stand in ice for 30 minutes, 9001 of 7H9 was added, and the cells were cultured at 37 ° C for 18 hours. This was added to a 7H11 medium (Becton Dickinson Microbiology Systems) supplemented with albumin dextrose complex (0ADC, Becton Dickinson Microbiology Systems, MD, USA), 0.5% glycerol and kanamycin at 30 g / ml. , MD, USA) and cultured for 20 days to obtain a transformant.

Experiment 10: Antigen expression on recombinant BCG

 ロ ッ Stamp lotting was performed to confirm the expression of malaria antigen. First, recombinant BCG was isolated and seeded on a 7H11 medium supplemented with 0.5% glycerol and kanamycin at 30 g / ml. After 14 days from the culture, the cells were recovered using a protease. The collected cells were suspended in water and collected by centrifugation. The recovered cells were weighed, and 5 // I of sample buffer per mg was added. The cells were treated with an ultrasonic homogenizer for 60 seconds and centrifuged again. The supernatant was collected and treated at 100 ° C. for 8 minutes to obtain a sample for electrophoresis.

Electrophoresis was performed using 15% separation gel and 3.9% stacking gel. The separated proteins were transferred to PVDF transfer membrane I obilon ^T "-P (Millipore Corporation, A, USA) using a protein blotting device, TRANS BLOT SD (BioRad Laboratories, CA, USA). Specifically, the transfer membrane was treated with MeOH for 20 seconds, and then immersed in a solution of 48 mM Tris-HCl, 39 mM Glycin (pH 9.2) in Blotting Buffer containing MeOH to 20% for 10 minutes. Similarly, the gel that had been subjected to electrophoresis was immersed in Blotting Buffer and held for 10 minutes.This gel was overlaid on the transfer membrane, and treated with TRANS BLOT SD at 10 V for 30 minutes. (137mM NaCl, 2. 7mM KC1, 4. 3mM Na 2 HP0 4 - 7H 2 0, 1. 4m KH 2 P 0 4) in 0. 05% Tween20 for 10 minutes shaken in Washing buffer was added, Block Ace After washing for 3 minutes for 10 minutes with the washing buffer, the primary antibody and membrane were added to a washing buffer containing albumin so that the concentration became 0.5%. Placed, with anti MDP1 antibody, anti-NANP antibodies as overnight standing were. Primary antibodies 4 ° C. 1 Tsugiko The transfer membrane treated with the body was washed 3 times for 10 minutes with Washing buffer, and then added to the Washing buffer to which 0.5% of casein had been added in a peroxidase conjugate anti-mouse IgG antibody or anti-Egret IgG antibody. And a membrane, and kept at 4 ° C for 1 hour. Further, the cells were washed three times with a washing buffer for 10 minutes, and then washed with PBS for 10 minutes. Then, the expression of the recombinant protein was detected using Konica Immunstein HRP-1000, and the expression of the recombinant protein was confirmed. The results are shown in FIG.

Experiment 1 1: Immunization of mice with recombinant BCG

Cells were collected from the recombinant BCG obtained above using 7H11 medium supplemented with 0.5% glycerol and 30 μg / ml of kanamycin, and 14 days after culturing. did. The collected cells were suspended in water and treated using a glass homogenizer so as to be uniform. Its cells were prepared to be 10 ^8/1111, injected bacteria body fluid 200] subcutaneously C3H / the He, were sensitized. 3PYB was also sensitized subcutaneously to 50 g / mouse. Three weeks after the initial sensitization, similar cells or antigenic peptides were intraperitoneally administered, and three weeks later, serum was collected.

Experiment 12: Detection of protective antibodies in the serum of the sensitized mice

 The serum obtained above was diluted serially appropriately, and the above-mentioned diluted serum was dispensed in 100 1 portions each into a 96-well plate coated with antigen, and reacted at room temperature for 2 hours.Washing buffer (400 // l / well) 5 times, add 100 ^ 1 of peroxidase conjugated anti-mouse IgG antibody, react at room temperature for 30 minutes, wash 5 times with Washing buffer, and wash with Peroxidase Color Kit (Sumitomo). (Bec Client Co.) was used to confirm the presence of protective antibodies. Figure 18 shows the results. That is, in the immunization group (No. 1 to No. 10) alone with the antigen (3PYB), no increase in the antibody was observed, but rBCG in which 3PYB was expressed in MDP1 In the group immunized with pMDPlutant-3PYBC, an increase in antibody titer was confirmed for No. II to No. 20, and No. 21 to No. 30, respectively.

 The sequences of the primers used in the experiment are shown below.

3PYB-3

5 '-GGG ACG CGT CCG CAA GGC CCG GGT GCT CCG CAA GGC CCG GGT GCT CCG CAA G GC CCG GGT GCT CCG-3' 3PYB-b

 5 '-CGG AGC ACC CGG GCC TTG CGG AGC ACC CGG GCC TTG CGG AGC ACC CGG GCC T

TG CGG ACG CGT CCC-3 '

 3PYB-C

 5 '-TCC GCA AGG CCC GGG TGC TCC GCA AGG CCC GGG TGC TCC GCA AGG CCC GGG T

GC TCC GTA G-3 '

 3PYB-d

 5 '-CTA CGG AGC ACC CGG GCC TTG CGG AGC ACC CGG GCC TTG CGG AGC ACC CGG G

CC TTG CGG A-3 '

 DPl-f

 5 '-GGGaagcttTTTGAGGGTGCGTGCGCGTAC-3'

MDPl-r

 5 '-GGGggatccAGCACGTGGGTGTTGTCGTTG-3'

MDPl-a

 5 '-GGG ACG CGT CAT CCC AAC CCT CCG AAA CC-3'

MDPl-b

 5 '-GAC AAA GCA GAG CTC ATT GAC G- 3'

 DPl-c

 5 '-GGG ACG CGT GGC GGG CGC CTT GGT CGC-3'

MDPl-d

 5 '-ACC GCG GTC AAG GCA TCG GTG CG- 3'

MSPlF-f

 5 '-GATACGAAAAAAGATATGCTTGGCAAATTAC-3'

MSPlF-r

 5 '-TTATCCTAAGAAGTTAGAGGAACTGCAG-3'

MSPlY-f

 5 '-CACATAGCCTCAATAGCTTTAAACAACTTA- 3'

MSPlY-r

5 '-TTATCCCATAAAGCTGGAAGAACTACAG-3' From the above results, it was proved that a malaria vaccine could be prepared by expressing the malaria antigen as a fusion protein with MDP1 in BCG. Also, by using the same method, a new safe and persistent pectin against other diseases can be produced.

Claims

The scope of the claims

 1. A polypeptide having immunogenicity against pathogenic mycobacteria represented by SEQ ID NO: 2 (205 amino acids), in which one or more amino acids may be substituted, added or deleted.

 2. The phosphorylated polypeptide of claim 1.

 3. A DNA encoding the polypeptide of claim 1.

 4. One day containing DNA according to claim 3

 5. A transformant containing the vector according to claim 4.

 6. The method for producing a polypeptide according to claim 1, wherein the transformant according to claim 5 is cultured.

 7. A vaccine comprising the polypeptide of claim 1 or the DNA of claim 3.

 8. A method for diagnosing pathogenic mycobacteriosis, comprising detecting an antibody against the polypeptide according to claim 1.

 9. The diagnostic method according to claim 8, wherein the pathogenic mycobacteriosis is selected from the group consisting of Mycobacterium avium-intracellulare complex (M. tuberculosis) and Hansen's disease.