JPH09502095A - DNA molecule encoding the cellular uptake of Mycobacterium tuberculosis - Google Patents

Abstract

(57) Summary The present invention relates to a DNA molecule that confers on Mycobacterium tuberculosis the ability to enter mammalian cells and survive in macrophages. The protein encoded by this gene fragment is useful as a vaccine for preventing infection by Mycobacterium tuberculosis, while the antibody raised against this protein is the one already infected by the organism. Can be used to passively immunize. Both of these proteins and antibodies can be used in diagnostic assays to detect Mycobacterium tuberculosis in tissues or body fluids. The proteins of the invention can be associated with a variety of other therapeutic agents to achieve uptake of these agents by such cells for administration to mammals, particularly humans.

Description

DETAILED DESCRIPTION OF THE INVENTION DNA Molecules Encoding Cellular Uptake of Mycobacterium tuberculosis FIELD OF THE INVENTION The present invention encodes uptake of Mycobacterium tuberculosis. DNA molecules and their use in medicine, vaccines and diagnostic tests. BACKGROUND OF THE INVENTION Tuberculosis is the leading cause of death worldwide, with an estimated 9 million new cases of tuberculosis with an estimated 2.9 million deaths each year from the disease. In the United States, a steady decrease in tuberculosis incidence has reversed since 1985. This problem is further complicated by the increasing incidence of drug-resistant strains of Mycobacterium tuberculosis. The dramatic outbreak of tuberculosis in recent years has involved situations where large numbers of HIV-infected individuals live in close proximity (eg, AIDS co-operative rooms in hospitals, correctional facilities and hospice). Transmission of tuberculosis to health care workers occurs with these dramatic outbreaks; 18-50% of such workers show a conversion in their skin tests ((cited herein by reference). See F. Laraque et al., “Tuberculosis in HIV-Infected Patients,” The AIDS Leader, September / October 1992. ). After infection with Mycobacterium tuberculosis, there are two basic clinical patterns. In most cases, inhaled tuberculosis pathogens phagocytosed by phagocytic alveolar macrophages are either killed directly or grow intracellularly to a limited extent in local lesions called tuberculosis nodules. It is either. Very rarely, in children and immunocompromised individuals, there is premature hematopoietic dissemination with the formation of small chestnut (millet-like) lesions or life-threatening meningitis. More generally, cellular immunity occurs within 2-6 weeks post-infection, often without recognition of symptoms by infected individuals, causing immune lymphocytes and activated macrophages to infiltrate the lesion, As a result, most pathogens are killed and this primary infection is shielded. The skin-test reactivity to the purified protein derivative of M. tuberculosis ("PPD") and in some cases the X-ray evidence of a healed and calcified lesion provides only the fact of infection. Nonetheless, to the extent that it remains unknown, there are still dormant but viable Mycobacterium tuberculosis pathogens. The second pattern is the development or weakening of the infection into an active disease. Individuals infected with Mycobacterium tuberculosis have a 10% lifetime risk of developing the disease. In any case, the pathogen spreads from the site of initial infection of the lungs through the lymph glands or blood to other parts of the body, the apex of the lung, which is the preferred site, and regional lymph nodes. Extrapulmonary tuberculosis of the pleura, lymph nodes, bones, genitourinary system, meninges, peritoneum or skin occurs in about 15% of tuberculosis patients. Although many pathogens die, a large proportion of invasive phagocytic cells and lung parenchyma cells also die, producing the characteristic solid, solid butty (cheese-like) necrosis in which Mycobacterium tuberculosis grows. You can live without it. If the protective immune response is predominant, the lesion can be prevented, even if there is some residual damage to the lungs or other tissues. When the necrotic reaction invades the bronchus and spreads, a cavity is created in the lungs, and a large number of pathogens spread out by coughing. In the worst case, hydrolases released by inflammatory cells probably lyse the solid necrosis, which causes the pathogen to grow (probably 10 ⁹ It can produce a nutrient-rich medium (up to / ml). Pathological and inflammatory processes cause weakness, fever, chest pain, coughing, and bloody sputum when blood vessels are eroded. The lack of understanding of the molecular basis of pathogenicity and virulence is crucial. Need to establish molecular evidence for non-pathogenic strains, identify and clone putative pathogenic genes of pathogens, and prove that these genes can lead pathogenic strains to non-pathogenic strains Is shown. Although non-pathogenic strains of Mycobacterium tuberculosis exist, the nature of the mutant strain is unknown. No single gene involved in the pathogenicity of tuberculosis has been defined in the prior art. The molecular basis of host cell invasion, intracellular survival, proliferation, spread, or histocompatibility is also unknown. No existing drug targets have been characterized at the molecular level and no mechanism of resistance to any drug has been identified; new TB targets for drug development have been completely characterized for 20 years. It has not been done. There are many prescribed treatment modalities for tuberculosis. The mode recommended by the US Department of Public Health and the American Thoracic Society is to administer a combination of isoniazid, rifampicin and pyrazinamide for 2 months, followed by isoniazid and rifampicin. It is to be administered for another 4 months. Treatment of isoniazid and rifampicin will continue for an additional 7 months in individuals with HIV infection. This treatment, called short-term chemotherapy, results in a treatment rate of 90% or more for patients who complete it. For the treatment of multidrug-resistant tuberculosis, ethambutol and / or streptomycin or kanamycin, amikacin, capreomycin, ethionamide, cyclcoserine, PAS and clofazimin in the early mode are used. ), It is necessary to add a second type of drug. New agents such as ciprofloxacin and ofloxacin can also be used. For individuals infected with conventional Mycobacterium tuberculosis and showing PPD positive symptoms, chemoprevention with isoniazid was about 90% effective in treating the disease. For tuberculosis and its treatment (cited by reference herein) B. Bloom et al., “Tuberculosis: Commentary on a Reeme. rgent Killer "Science 257: 1055-64 (1992);" Control of Tuberculosis in the United States ", American Thoracic Society. 146: 1623-33 (1992); City Health Information, Vol. 11 (1992). Although the currently used treatments for tuberculosis have a relatively high level of success, there remains a need to improve the success rate of treating this disease. Moreover, given the ever increasing levels of Mycobacterium tuberculosis strains that are resistant to conventional treatment modalities, new types of treatments must be developed. In highly endemic areas both in the United States and abroad, an increasing number of such resistant strains are present. SUMMARY OF THE INVENTION The present invention encodes an isolated DNA molecule that confers on Mycobacterium tuberculosis the ability to enter mammalian cells and survive in macrophages, as well as encoded by the isolated DNA molecule. Isolated protein or polypeptide. The molecule can be inserted as exogenous DNA into an expression vector forming a recombinant DNA expression system that produces the protein or peptide. Similarly, foreign DNA that is usually inserted into an expression vector to form a recombinant DNA expression system can be incorporated into cells to achieve this end. To prevent infection by Mycobacterium tuberculosis, the isolated protein or polypeptide of the invention may be combined with a pharmaceutically acceptable carrier to form a vaccine for administration to mammals, particularly humans. , Or can be used alone. Alternatively, a protein or polypeptide of the invention can be used to raise an antibody or binding site thereof. Mammals, which have already been exposed to Mycobacterium tuberculosis to induce passive immunity in order to prevent the development of disease, using an antibody or its binding site alone or in combination with a pharmaceutically acceptable carrier, especially Humans can be treated. The protein or polypeptide of the present invention, or the antibody or its binding site generated against them can also be used in a method for detecting Mycobacterium tuberculosis in a tissue or body fluid sample. When utilizing the protein or polypeptide, these are provided as antigens. Either reaction with the antigen or antibody is detected using an assay system that indicates the presence of Mycobacterium tuberculosis in the sample. Alternatively, a nucleotide sequence of a gene or a fragment thereof that confers on Mycobacterium tuberculosis the ability to invade mammalian cells and survive in macrophages can be analyzed by nucleic acid hybridization assay or gene amplification detection Mycobacterium tuberculosis in such a sample can be detected by providing it as a probe (for example, using the polymerase chain reaction method). Both reactions are detected with the probe, indicating the presence of Mycobacterium tuberculosis in the sample. The proteins or polypeptides of the invention may also be used for purposes unrelated to the treatment or detection of Mycobacterium tuberculosis. More specifically, the ability of a protein or polypeptide to confer the ability to enter into mammalian cells to Mycobacterium tuberculosis can be utilized to take up other substances into such cells. This can be accomplished with a product that includes a substance that is taken up by mammalian cells and a protein or polypeptide of the invention associated with that substance. The isolation of the DNA molecule of the present invention constitutes a significant advance in the treatment and detection of such bacteria. It also provides the basis for vaccines that prevent infection by Mycobacterium tuberculosis, and pharmaceutical agents for passive immunization of those exposed to Mycobacterium tuberculosis. Proteins for use in vaccines or for producing pharmaceutical agents can be produced at high levels using recombinant DNA technology. In diagnostic applications, the proteins or polypeptides of the present invention and the antibodies to them and their binding sites can be used to quickly determine whether a particular individual is infected with Mycobacterium tuberculosis. Furthermore, such detection can be performed without the need for testing of individuals testing for antibody responsiveness. Aside from the development of therapeutic and diagnostic instruments for Mycobacterium tuberculosis, the ability of the present invention to confer entry of such organisms into mammalian cells depends on the therapeutic treatment required to introduce the substance into cells, especially macrophages. Has great utility. By associating the protein or polypeptide of the present invention with a pharmaceutical agent, such agent can be rapidly introduced into cells for the treatment thereof. By improving the cellular uptake of such products, drug dosages can be reduced, thus reducing toxicity and cost. For example, in conventional cancer therapies, drug toxicity is a major problem due to the need to administer large doses; the present invention is capable of delivering comparable or higher drug levels intracellularly, It has the potential to reduce such high dose levels. Furthermore, the binding property of the protein or polypeptide of the present invention to a DNA fragment can be used in combination with a gene therapy method. In particular, the ability of the coding product of the DNA molecule of the present invention to increase macrophage uptake provides an opportunity for the gene to be specifically delivered to macrophages. By using such a system, not only humoral immunity but also cell-mediated immunity can be induced. BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A, 1B and 1C show a Mycobacterium tuberculosis strain containing H37Ra (ATCC 25177) (FIG. 1A) and an invasive recombinant E. coli XL1-Blue. 1 is a thin section electron micrograph of HeLa cells infected with the (pZX7) strain (FIGS. 1B and 1C). An electron permeable region surrounds the Mycobacterium tuberculosis organism (arrow in Figure 1A). Cells were incubated with Mycobacterium tuberculosis strain for 72 hours in FIG. 1A and XL1-Blue (pZX7) for 7.5 hours in FIGS. 1B and 1C. The presence of multiple organisms in FIG. 1C indicates bacterial growth inside the phagosome. The bar represents 0.5 μm. FIG. 2 shows unidirectional deletion subclones (pZX7.3, pZX7.4, pZX7.5, and pZX7.6) and BamHI-PstI (pZX7.1), PstI-HindIII from the original vector pZX7. (pZX7.2) as well as the production of BamHI-EcoRI (pZX7.7) subclones. Black bars represent Mycobacterium tuberculosis DNA sequences and white bars represent pBluescript sequences. Subclone vectors were transferred to E. coli XL1-Blue and these transformants were then incubated with HeLa cell monolayers for 6 hours. 3A, 3B and 3C show that the invasive recombinant E. coli clone XL1-Blue (pZX7) was compared to cells exposed to the non-pathogenic E. coli XL1-Blue (pBluescript) for 24 hours (FIG. 3C). 3D is a thin section electron micrograph of human macrophages exposed for 3 hours (FIG. 3A) and 24 hours (FIG. 3B). Bacteria are compartmentalized and surrounded by a layer of inner macrophage membrane (Fig. 3B). In the macrophages exposed to XL1-Blue (pBluescript), no bacteria were observed after 24 hours by electron microscopy. Bars represent 1 μm. FIG. 4 shows an SDS-polyacrylamide gel electropherogram of the acetone-precipitated soluble fraction of the treated bacterial cell sonication. Polypeptides were analyzed in a 9% gel (left): molecular weight marker (lane 1), carrying a vector (pZN7) containing an unrelated Mycobacterium tuberculosis DNA fragment between the BamHI-EcoRI pBluescript cloning sites. E. coli XL1-Blue (lane 2), and XL1-Blue (pZX7) (lane 3). Analysis in 8% gel (right side): Vector with a 2 base frameshift introduced 12 bases upstream from the BamHI cloning site in pZX 7 (lane 1) and XL1-Blue (pZX7) (lane 2) (pZX7.8). ) Containing XL1-Blue. The molecular weight is shown on the far right. We detected a 52-kD polypeptide in the soluble protein fraction of XL1-Blue (pZX7) (arrow). The approximately 50 kD protein is expressed by XL1-Blue containing pZX7.8. Expression of the 52-kD protein has always been associated with HeLa cell interaction of recombinant E. coli clones. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to isolated DNA molecules that confer on Mycobacterium tuberculosis the ability to enter mammalian cells and survive in macrophages. This DNA molecule has the following SEQ ID NO: 1: Corresponding to the nucleotide sequence. The DNA molecule encodes a polypeptide having a molecular weight of about 50 to about 55 kilodaltons, preferably 52 kilodaltons. The amino acid sequence predicted from the nucleotide sequence corresponding to SEQ ID NO: 1 represents a very hydrophobic protein having a hydrophobic region at its carboxy terminus. It can be a secreted protein, a cytoplasmic protein or a surface protein bound at its carboxy terminus to the outer membrane of an organism. This protein or polypeptide has the following SEQ ID NO: 2: It is believed to have a predicted amino acid sequence corresponding to In the immediately preceding sequence, Xaa represents the stop codon. The production of this isolated protein or peptide is preferably carried out using recombinant DNA technology. The protein or peptide is believed to have one or more antigenic determinants that confer on Mycobacterium tuberculosis the ability to enter mammalian cells and survive in macrophages. The protein or polypeptide of the invention is preferably produced in purified form by conventional techniques. Typically, the proteins of the invention are secreted into recombinant E. coli growth medium. To isolate the protein, E. coli host cells carrying the recombinant plasmid are grown, homogenized and the homogenate centrifuged to remove bacterial contaminants. The supernatant is then subjected to a series of ammonium sulfate precipitations. Fractions containing the protein of the invention are subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the protein. If desired, the protein fraction can be further purified by HPLC. DNA molecules that confer on Mycobacterium tuberculosis the ability to enter mammalian cells and survive in macrophages can be incorporated into cells using conventional recombinant DNA techniques. Generally, this involves inserting the DNA molecule into an expression system in which the DNA molecule is foreign (ie, normally absent). The exogenous DNA molecule is inserted into the expression system or vector in the proper orientation and correct reading frame. The vector contains the elements necessary for the transcription and translation of the sequence encoded by the inserted protein. U.S. Pat. No. 4,237,224 to Cohen and Boyer (which is hereby expressly incorporated as part of this specification) uses restriction enzyme digestion and ligation with DNA ligase. The production of expression systems in the form of recombinant plasmids has been described. This recombinant plasmid is then introduced by means of transformation and allowed to replicate in monolayer cell culture containing prokaryotic and eukaryotic cells grown in tissue culture. Alternatively, the recombinant gene can be introduced into a virus such as vaccinia virus. Recombinant virus can be produced by transfecting cells infected with the virus with the plasmid. Suitable vectors include, but are not limited to, lambda vector systems gt11, gtWES.tB, viral vectors such as Sharon 4, and pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pLG339, pR290, pKC37, pKC101, SV40, pBluescriptIISK +/- or KS +/- ((exclusively incorporated herein by reference) Stratagene, La Jolla, CA. "Stratagene Cloning Systems" catalog (see 1993), pQE, pIH821, pGEX, pET series ((cited explicitly as part of this specification) W Studio (FW Studier) , "Use of T7 RNA Polymerase for Direct Expression of Cloned Genes (Use of T7 RNA Polymerase to Direct Expression of Cloned Genes)" Gene Expression Technology (Vol. 185, 1990). Vectors, and their derivatives are included. Recombinant molecules can be introduced into cells via transformation, in particular transduction, conjugation, mobilization or electroporation. The DNA sequence may be found in Maniatis et al. (Molecular Cloning: A Laboratory Manual, incorporated herein by reference), Cold Clonings, NY. Clone into the vector using standard cloning techniques in the art, as described by the Cold Springs Harbor Laboratory, Cold Springs Harbor, New York. A variety of host-vector systems can be utilized to express the protein-encoding sequence (s). First, the vector system must be compatible with the host cell used. Host-vector systems include, but are not limited to, bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA; microorganisms such as yeast containing yeast vectors; viruses (eg, vaccinia virus). , Adenoviruses, etc.) and mammalian cell lines infected with viruses; insect cell lines infected with viruses (eg, baculovirus). The expression elements of these vectors differ in their strength and specificity. Any one of a number of suitable transcription and translation elements may be used, depending on the host-vector system utilized. Different genetic signals and processing events control many levels of gene expression, such as DNA transcription and messenger RNA (mRNA) translation. Transcription of DNA depends on the presence of a promoter, a DNA sequence which is bound by RNA polymerase and thereby promotes mRNA synthesis. The DNA sequence of a eukaryotic promoter differs from that of a prokaryotic promoter. Furthermore, eukaryotic promoters and their associated genetic signals cannot be recognized or function in prokaryotic systems, and prokaryotic promoters are not recognized or function in eukaryotic cells. Similarly, translation of mRNA in prokaryotes depends on the presence of the appropriate prokaryotic signals that differ from those of eukaryotes. A ribosome binding site called the Shine-Dalgarno (SD) sequence is required on the mRNA for efficient translation of mRNA in prokaryotes. This sequence is a short nucleotide sequence of mRNA located upstream of the start codon, usually the AUG, which encodes the amino-terminal methionine of the protein. The SD sequence is complementary to the 3'-end of the 16S rRNA (ribosomal RNA) and probably double-strands with the rRNA to place the ribosome in the correct position, thereby promoting binding of the mRNA to the ribosome. To do. For a review on maximizing gene expression, see Roberts and Lauer (cited by reference herein), Methods in Enzymology, Vol. 68: See page 473 (1979). Promoters differ in their "strength" (ie, their ability to promote transcription). For purposes of expressing a cloned gene, it is desirable to use a strong promoter to obtain high levels of transcription and thus expression of the gene. Depending on the host cell system utilized, any one of a number of suitable promoters can be used. For example, when cloning in E. coli, the bacteriophage or plasmid, or T7 phage promoter, including but not limited to lacUV5, ompF, bla, lpp, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, P of coliphage lambda _R And P _L Promoters, such as promoters, can be used to cause high levels of transcription of adjacent DNA segments. In addition, the hybrid tr p-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques can be used to transcribe the inserted gene. Bacterial host cell lines and expression vectors can be chosen to inhibit the action of the promoter without specific induction. For some operons, the addition of specific inducers is required for efficient expression of the inserted DNA. For example, the lac operon is induced by adding lactose or IPTG (isopropylthio-β-D-galactoside). Various other operons such as trp, pro, etc. are under different control. Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals can be varied in "strength" as measured by the amount of gene-specific messenger RNA and protein synthesized, respectively. A DNA expression vector containing a promoter can contain any combination of various "strong" transcription and / or translation initiation signals. For example, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5'to the start codon (ATG), which provides a ribosome binding site. Thus, any SD-ATG combination that can be utilized by the host cell ribosome can be used. Such combinations include, but are not limited to, SD-ATG combinations from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. . In addition, any SD-ATG combination produced by recombinant DNA or other techniques involved in the incorporation of synthetic nucleotides can be used. Once the isolated DNA molecule, which confers on Mycobacterium tuberculosis the ability to invade mammalian cells and survive in macrophages, has been cloned into an expression system, it is ready for incorporation into host cells. Such uptake can be achieved by the various transformation forms described above, depending on the vector / host cell system. Suitable host cells include, but are not limited to, bacteria, viruses, yeasts, mammalian cells, etc. Generally, the human immune system responds to infection by pathogenic bacteria by producing antibodies that bind to specific proteins or carbohydrates on the bacterial surface. The antibody is F of the antibody. _c It stimulates binding to macrophages that have receptors that bind to the region. Another serum protein, called complement, coats foreign particles and stimulates their digestion by binding to specific surface receptors on macrophages. When the particles bind to the surface of macrophages, continuous deposition of plasma membrane slices on the surface of the particles initiates a series of digestion processes. The surface receptors on the membrane then interact with the ligands, which are evenly distributed on the particle surface, to bind to the surface together. The macrophages encapsulating the particles are then delivered to lysosomes where they are digested. Certain organisms are digested (ie, taken up) by macrophages but not killed. Mycobacterium tuberculosis is among these. As a result, such organisms can survive indefinitely within macrophages, producing active tuberculosis when it escapes from macrophages. The present invention's determination of the nucleotide sequences that confer on Mycobacterium tuberculosis the ability to enter mammalian cells and survive in macrophages demonstrates the molecular basis of Mycobacterium tuberculosis uptake. Was done. With this information and the recombinant DNA technology described above, a wide array of therapeutic and / or prophylactic and diagnostic methods, respectively, for treating and detecting Mycobacterium tuberculosis can be developed. For example, the protein or polypeptide of the invention can be administered to a human in an effective amount to prevent infection by Mycobacterium tuberculosis, alone or as a vaccine in combination with a pharmaceutically acceptable carrier. . Alternatively, as passive immunization, an effective amount of an antibody against the protein or polypeptide or its binding site can be administered to an individual exposed to Mycobacterium tuberculosis. Such antibodies or binding sites thereof, administered alone or in combination with pharmaceutically acceptable carriers, provide short-term treatment of individuals who may have recently been exposed to Mycobacterium tuberculosis. Suitable antibodies for use in inducing passive immunity can be monoclonal or polyclonal antibodies. Monoclonal antibody production can be performed by techniques well known in the art. Basically, the process involves first immunizing a mammal previously immunized with the desired antigen (i.e., a protein or peptide of the invention), either in vivo or in vitro. It involves obtaining immune cells (lymphocytes) from the spleen of an animal (eg mouse). The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells capable of unlimited replication in cell culture, thereby creating an immortal immunoglobulin-secreting cell line. The resulting fused cells or hybridomas are cultured and the resulting colonies screened for production of the desired monoclonal antibody. Colonies that produce such antibodies are cloned and propagated in vivo or in vitro to produce large amounts of antibody. A description of the theoretical basis and practical methodologies for fusing such cells can be found in Kohler and Milstein, Nature 256, (cited by reference herein): P. 495 (1975). Mammalian lymphocytes are immunized by immunizing an animal (eg, mouse) with the protein or polypeptide of the present invention in vivo. Such immunization is repeated as often as necessary at intervals of up to several weeks to obtain a sufficient antibody titer. The virus is delivered in a suitable solution or adjuvant. Finally, after boosting with the antigen, the animal is killed and the spleen is removed. Fusions with mammalian myeloma cells or other fusion partners capable of unlimited replication in cell culture are performed by standard and well-known techniques, such as polyethylene glycol (PEG) or other fusion agents. Do ((cited as part of this specification) Milstein and Kohler European Journal of Immunology (Eur. J. Immunol.) Volume 6: 511 ( (1976)). This immortalized cell line, which is preferably murine but can be derived from cells of other mammals, including but not limited to rat and human, provides the enzymes necessary to utilize a particular nutrient. They are selected for their lack, their ability to grow rapidly, and their ability to fuse well. Many such cell lines are known to those of skill in the art, and others are regularly described. Methods for raising polyclonal antibodies are also well known. Typically, a protein or polypeptide of the invention will be raised subcutaneously in New Zealand white rabbits which have been bled and pre-immune serum collected to raise such antibodies. it can. The antigen can be injected at 6 different sites in a total volume of 100 μl per site. Each injectable material would include a synthetic activator adjuvant containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis, a pluronic polyol, or a preparative acrylamide gel. The rabbits are then bled 2 weeks after the first injection and boosted periodically with the same antigen 3 times every 6 weeks. Serum samples are then taken 10 days after each boost. Polyclonal antibodies are then collected from the sera by affinity chromatography with the corresponding antigen to capture the antibodies. Finally, the rabbits are euthanized with pentobarbitol 150 mg / Kg IV. This and other methods for raising polyclonal antibodies are described in "Antibodies: A Laboratory Manual," edited by E. Harlow et al. (Cited by reference herein). (Antibodies: A Laboratory Manual) "(1988). The vaccines and passive immunizing agents of the present invention can be administered orally, parenterally, for example, by subcutaneous, intravenous, intramuscular, intraperitoneal, or intranasal inhalation, or by application to mucosa, such as mucous membranes of the nose, throat and trachea. Can be administered locally. It can be administered alone or with a suitable pharmaceutical carrier and can be in solid or liquid form such as tablets, capsules, powders, solutions, suspensions or emulsions. The solid unit dosage form can be of the conventional type. Solid forms are of the usual gelatin type containing the protein or peptide of the invention or the antibody or binding site thereof of the invention and a carrier such as a lubricant and an inert filler such as lactose, sucrose or corn starch. It can be a custom capsule. In another embodiment, these compounds are combined with a binder such as acacia, corn starch or gelatin, a disintegrant such as corn starch, potato starch or alginic acid, and a lubricant such as stearic acid or magnesium stearate. Tablet with a conventional tablet base such as lactose, sucrose, or corn starch. The protein or polypeptide of the present invention or the antibody of the present invention or its binding site may also be administered at an injectable dose as a solution or suspension of these substances in a physiologically acceptable diluent with a pharmaceutical carrier. it can. Such carriers include sterile liquids such as water and oils, with or without the addition of active agents and other pharmaceutically acceptable adjuvants. Exemplary oils are of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly injectable solutions. When used as an aerosol, the protein or polypeptide of the invention or the antibody of the invention or its binding site in solution or suspension may be combined with a conventional adjuvant and a high pressure hydrocarbon, such as propane, butane, or isobutane. Compressed aerosol containers can be filled with a suitable high pressure gas, such as gas. The substances of the invention can also be administered in unpressurized form, such as in a nebulizer or atomizer. In yet another aspect of the invention, the protein or polypeptide of the invention can be used as an antigen in a diagnostic assay to detect Mycobacterium tuberculosis fluid. Alternatively, detection of the pathogen can be accomplished with a diagnostic assay that uses the antibody raised by such antigen or its binding site. Such techniques can detect Mycobacterium tuberculosis in samples of the following tissue or body fluids: blood, spinal fluid, sputum, pleural fluid, urine, bronchoalveolar lavage fluid, lymph nodes, bone marrow or other. Biopsy material. In one embodiment, the assay system has a sandwich format or a competition format. Examples of suitable assays include enzyme-linked immunosorbent assays, radioimmunoassays, gel diffusion precipitation assays, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays, or immunoelectrophoresis assays. In another diagnostic embodiment of the invention, the nucleotide sequences of isolated DNA molecules conferring on Mycobacterium tuberculosis the ability to enter mammalian cells and survive in macrophages vary. It can be used as a probe in a nucleic acid hybridization assay to detect Mycobacterium tuberculosis in patient body fluids. The nucleotide sequences of the invention include, but are not limited to, Southern blotting (Southern, Journal of Molecular Biology (J. Mol. Biol. 98: 508 (1975)); Northern blotting (Thomas et al., Proceedings of National Academy of Sciences in Youth Aye (Proc. Natl. Acad. Sci). .USA) 77: 5201-05 (1980)); Colony blotting (Grunstein et al., Proceedings of National Academy of Sciences in Youth Aye (Proc. Natl). .Acad.Sci.US A) 72: 3961-65 (1975)), any nucleic acid hybridise known in the art. It can also be used in Deployment assay system. Alternatively, the isolated DNA molecule of the present invention can be used in a gene amplification detection method (eg, polymerase chain reaction) (exhibited and regarded as part of the present specification). A. Erlich) et al., "Recent Advances in the Polymerase Chain Reaction," Science 252: 1643-51 (1991). More generally, the molecular basis of the uptake phenomenon achieved by Mycobacterium tuberculosis can also be used to uptake other substances into mammalian cells. This is accomplished by utilizing the protein or polypeptide of the present invention bound to such a substance for uptake by mammalian cells. This phenomenon can be used to introduce a wide variety of substances into such cells, including antibiotics, DNA fragments, anti-neoplastic agents and mixtures thereof. The opportunity for direct cell transfer of antibiotics constitutes a substantial advance. Because, it would be able to kill intracellular Mycobacterium tuberculosis. One approach to achieve such uptake is by impregnating the microspheres with an antibiotic and then coating the spheres with a protein or polypeptide of the invention to achieve such uptake. Alternatively, instead of utilizing microspheres to deliver antibodies, such therapeutic agents can be chemically linked to the proteins or polypeptides of the invention. This technique can be used to treat a wide variety of diseases caused by intracellular pathogens. Antibiotics for the treatment of tuberculosis, which have little cellular invasion by themselves, but have high activity against extracellular Mycobacterium tuberculosis when tested in vitro. The repertoire can be used in combination with the protein or polypeptide of the present invention. In the treatment of cancer, the intracellular delivery of anti-neoplastic agents can be significantly improved by coupling such agents to the proteins or polypeptides of the invention. This would reduce the dose of such agents and their resulting toxicity. Another aspect of the invention is the use of gene therapy or genetic methods in which a therapeutically or prophylactically useful piece of DNA is linked at its thymine residue to a protein or polypeptide of the invention via a linker arm. The use of the protein or polypeptide of the present invention in a vaccine. As a result, genetic material can be introduced into cells to correct genetic defects or to produce products that serve as the desired property or immunogen. Example Example 1 Preparation and Screening of HeLa Cell Invasive Clones To identify the Mycobacterium tuberculosis DNA sequence encoding mammalian cell invasiveness, recombinant invasive clones were constructed as follows: Mycobacterium The genome of the tuberculosis H37Ra strain (ATCC251 77) was digested with the restriction enzymes Sau3AI and EcoRI, and the DNA fragment was digested with the phagemid vector pBluescript II (Stratagene, LaJ olla, CA). It was ligated to the BamHI-EcoRI restriction site. This recombinant vector was introduced into E. coli EL1-Blue (Stratagene) by electroporation. The present inventors (R. Isberg and S. Falkow, Nature 317, (cited by reference herein). Recombinant strains were screened for HeLa cell-invasive clones by a method similar to that described by P.262 (1987). By a method of screening for consistent association with HeLa cells, a type of E. coli transformant XL 1-Blue having a plasmid (pZX7) containing an insert of 1535 bases at the BamHI-EcoRI restriction enzyme site of the pBluescript vector was selected. I found (pZX7). The invasion of this clone into HeLa cells was confirmed by transmission electron microscopy (Fig. 1). FIG. 1A shows He La cells infected with the Mycobacterium tuberculosis H37Ra strain (ATCC 25177), while the invasive recombinant E. coli XL1-Blue strain (pZ X7) is shown in FIGS. 1B and 1C. Show. The cells were incubated with the Mycobacterium tuberculosis strain for 72 hours in FIG. 1A and with XL1-Blue (pZX7) for 7.5 hours in FIGS. 1B and 1C. Internalization of this clone by HeLa cells was time-dependent (Fig. 1B), intracellular organisms being visible 3.5 hours post infection. Some phagosomes contained multiple organisms (Figure 1C), suggesting that the bacterium grew intracellularly. Certain internalizing pathogens were surrounded by different ETZs, as well as the appearance of the clear area surrounding Mycobacterium tuberculosis inside HeLa cells (FIG. 1A, arrow). This region is often found around other pathogenic intracellular Mycobacterium tuberculosis organisms, ETZ ((cited herein by reference) P. Draper and RJ W.・ RJW Lees Nature 228, 860 pages (1970); N. Rastogi, Research in Microbiology (Res. Microbiol.) 141, 217 (1990); T. Yamamoto, M. Nishimura, N. Harada, T. Imaeda, International Journal. -It is not clear whether it represents of Reprocy (Int. J. Lepr.) Vol. 26, p. 111 (1958)) or is an artifact of the preparation. The non-pathogenic E. coli XL1-Blue strain containing the vector pBluescript or another recombinant vector derived from pBluescript (pZN7) showed no association with HeLa cells after 7.5 hours. To demonstrate that the invasive phenotype is indeed encoded by the cloned Mycobacterium tuberculosis DNA fragment, we have examined other non-pathogenic E. coli strains, especially HB101, DH5α and NM522 was transformed with pZX7. The constructs HB101 (pZX7), DH5α (pZX7) and NM522 (pZX7) were invasive to HeLa cells. The spontaneous loss of pZX7 during long-term storage of XL1-Blue (pZX 7) was associated with loss of the invasive phenotype. Four exonuclease III unidirectional deletion subclones of pZX7 and subclones BamHI-PstI (pZX7.1), Pst-I-HindIII (pZX7.2), and BamHI-EcoRI (pZX7.7) were associated with HeLa cells. I used it for. Unidirectional deletion subclones of pZ X7 were generated using exonuclease III according to the manufacturer's instructions (Erase-a-Base System, Madison, WI). WI) Promega). The plasmid pZX7 was double-digested with HindIII and KpnI restriction enzymes downstream from the EcoRI site of the BamHI-EcoRI DNA insert to give a 5'protruding end adjacent to the insert and a 4-base 3'overhang adjacent to the insert. A 4-base 3'overhang was created at the end, as well as the opposite strand, to protect it from ExoIII digestion. The digested plasmid was mixed with 300 U of ExoIII at 37 ° C. and every 30 seconds an aliquot of 2.5 μl of ExoIII digest was transferred to a tube containing S1 nuclease to remove residual single-stranded tail. The S1 nuclease was neutralized and inactivated by heating at 70 ° C. for 10 minutes. Klenow DNA polymerase was added to create blunt ends, which were ligated to circularize the defect-containing vector. The ligation mixture was then used to transform competent E. coli XL1-Blue strains by electroporation. These transformants were incubated with HeLa monolayers for 6 hours. The results of this method are shown in FIG. Black bars represent Mycobacterium tuberculosis DNA sequences and white bars represent pBluescript sequences. As shown, the E. coli XL1-Blue strain carrying pZX7.3, pZX7.4 or pZX7.5 associates with HeLa cells in a pattern similar to that of E. coli ZL1-Blue (pZX7), while The other subclones were not. Example 2 Infection of human macrophages Macrophage monolayers infected with the E. coli recombinant clones of Example 1 were fixed on glass cover slips at the bottom of polystyrene wells. They were first infected with ~ 10 overnight grown bacteria per macrophage cell for 1 or 2 hours, followed by washing with phosphate buffer saline (pH 7.4) and further 1, 6 or 22. Incubated for hours. Culturing was carried out at 37 ° C. in RPMI-1640 medium (Gibco) supplemented with 2% AB heat-inactivated human serum containing gentamicin (10 μg / ml). Gentamicin was included to kill extracellular bacteria. The macrophage monolayer was washed again with sterile distilled water and then dissociated. The dissociated product was plated on soybean agar medium treated with trypsin, and colonies were counted. For microscopy, macrophage monolayers were fixed with 100% methanol, stained with 10% Giemsa stain and examined with a light microscope or processed for electron microscopy. Monolayers infected for only 1 hour were examined by light microscopy immediately after washing, fixing and staining them. The results of culture and light microscopy of the macrophage dissociate are shown in Table 1 below. The percentage of infected macrophages was calculated from counts on cover slip monolayers or from infected macrophages per 100-200 macrophage cells. Each E. coli strain was tested 4-6 times at each time point to determine the percentage of cells infected with the E. coli recombinant clones and the control strains XL1-Blue (pBluescript) and XL1-Blue (pZX7.3). Mean values were compared by T-test. FIG. 3 shows thin section electron micrographs of human macrophages exposed to invasive recombinant E. coli clone XL1-Blue (pZX7) for 3 hours (FIG. 3A) and 24 hours (FIG. 3B). In FIG. 3C, a thin section electron micrograph is of a human macrophage exposed to non-pathogenic E. coli XL1-Blue (pBluescript) for 24 hours. After 24 hours, the pathogen had become even more numerous inside the cells, compartmentalized and surrounded by a bilayer membrane (possibly of host origin) (Fig. 3B). No bacteria were found inside the macrophages 24 hours after infection with E. coli (pBluescript) (FIG. 3C). Table 1 shows human macrophage monolayer cells infected with HeLa cell invasive E. coli XL1-Blue (pZX7), subclone XL1-Blue (pZX7.3), and non-invasive XL1-Blue (pBluescript). Figure 4 shows the results obtained from this light microscopy and culture experiments. Colony forming units (CFU) were measured per ml of cell culture dissociate. As shown, 1 hour after infection, the percentage of cells infected with the recombinant clone (82 ± 8%) was that of cells infected with XL1-Blue (pBluescript) (15 ± 6%, P <0.001). More than five times. This finding suggests that the cloned Mycobacterium tuberculosis DNA sequence promotes bacterial uptake in amounts above the background phagocytic activity of macrophage cells. Twenty-four hours after infection, 12% (± 10%) of macrophages exposed to XL-Blue (pBluescript) and 60% (± 13%) of cells exposed to XL1-Blue (pZX7) were infected (P < 0.001). As demonstrated in Table 1, dissociation cultures of macrophages infected for 24 hours showed that the intracellular E. coli XL1-Blue (pZX7) strain was viable. Ability of XL1-Blue (pZX7), XL1-Blue (pBluescript) and one HeLa cell invasive defect derivative E. coli XL1-Blue (pZX7.3) at 1 hour infection with macrophages from Table 1. In comparison of E. coli XL 1-Blue (pZX7.3), the invasion ability was 4 times that of XL1-Blue (pBluescript) (P <0.001), but by 24 hours the difference was no longer present. It wasn't clear. Thus, DNA sequences associated with HeLa cell invasiveness are responsible for the increased uptake by macrophages, and sequences conferring viability within macrophages are located downstream of those required for mammalian cell invasion. Example 3 -Homology analysis BamHI-EcoRI DNA fragments are referred to (F. Sanger) et al. (Cited by reference herein) in "DNA Sequencing with Chain Termination Inhibitors". with Chain-Terminating Inhibitors ", Proc. Natl. Acad. Sci. USA, 74: 5463-67. It was sequenced by the method and was found to have 1535 base pairs [European Molecular Biology (EMBL) Accession No. X70901]. The sequence showed no homology with any DNA sequence in the GenBank (R72.0) or EMBL (R31.0) databases. No obvious prokaryotic promoter consensus sequences could be identified. Given that Mycobacterium tuberculosis uses a common prokaryotic stop codon sequence, we can identify amino acid sequence homologies. NH of the predicted sequence of one potential open reading frame ₂ -A region near the end is (i) NH of 80 residues of internalin, which is a protein encoded by Listeria monocytogenes related to mammalian cell invasion. ₂ -Terminal regions ((cited as part of this specification) A.B. Hartman, M. Venkatesan, E.V. Oaks (E.V. . Oaks), JM Buysse, J. Bacteriol. 172, 1905 (1990)) and 27% homology; (ii ) The 145-residue region of the IpaH gene product of the Shigella non-invasive plasmid ((excluded by reference herein) B. Anderson, J.I. A. McDonald, G. D. C. Jones, D. R. Regnery Infection and Immunity, Volume 58. , 2760 (1990)) and 20% homology; and (ii i) 176 residues of human β-adaptin, a plasma membrane protein that links clathrin to a receptor in the coated vacuole responsible for receptor-mediated endocytosis. Territory (S.Ponnam balam, (cited by reference herein), MS Robinson, A.P.Jackson ), L. Peiper, P. Parham, Journal of Biological Chemistry (J. Biol. Chem.) 265, 4814 (1990) and Jay L.・ Goldstein (JL Goldstein), MS Brown (MS Brown), R.G.W.Anderson (R.G.W.Anderson), D.W.Russell (D. W. Russel l), W Jay Schneider (WJ Schne) ider), Annual Review in Cell Biology (Annu. Rev. Cell Biol. Vol. 1, p. 1 (1985)), and it was found that there is 18% homology. When compared to the Yersinia pseudotuberculosis invasin protein, the region associated with cellular uptake is a region of 100 residues near the invasive COOH-terminus ((referenced herein). Partial) R. Isberg, D. L. Voorhis, S. Falkow, Cell, Volume 50, p.769. (1987)) and 19% homology. The functional importance in these sequences is not clear. Example 4 Functional analysis of the -52 kD polypeptide The protein fraction analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was prepared as follows: overnight in trypsinized soy broth containing ampicillin (100 μg / ml). 5 ml aliquots of grown bacteria (absorbance at 550 nm adjusted to optical density 600) were collected by centrifugation. Next, the present inventors established 5 mM MgCl. ₂ The bacterial pellet was sonicated in 1.5 ml of 10 mM Tris-HCl buffer (pH 8.0) containing. The sonicated product was centrifuged at 4 ° C. and 12,000 rpm for 25 minutes in a tabletop centrifuge (Eppendorf model 5415C). Acetone was added (60% v / v) to 600 μl of the supernatant in a fresh bench top centrifuge tube and the mixture was centrifuged at 14,000 rpm for 25 minutes at 4 ° C. The pellet was resuspended in 20 μl of distilled water and 20 μl of boiling buffer of Raemmli, heated on boiling water for 5 minutes and analyzed by SDS-PAGE. The bacterial debris containing the outer membrane fraction after the first centrifugation was treated with 100 μl of water, as well as 7.5 mM MgCl. ₂ And resuspended in 100 μl of 15 mM Tris-HCl buffer (pH 8.0) containing 3% (v / v) Triton X-100 and centrifuged at 14,000 rpm for 25 minutes. The pellet was resuspended in 25 μl water and 25 μl boiling buffer, it was boiled and a 20 μl aliquot of the sample was analyzed by SDS-PAGE. The soluble fraction of bacterial cell sonicates was precipitated by SDS-PAGE of acetone (ie, SDS-polyacrylamide gel electrophoresis). Polypeptides analyzed in 9% gel (left): molecular weight marker (lane 1), carrying vector (pZN) containing an unrelated Mycobacterium tuberculosis DNA fragment between BamHI-EcoRI pBluescript cloning sites. E. coli XL1-BBlue (lane 2), and XL1-Blue (pZX7) (lane 3). Analysis in 8% gel (right): XL1-Blue (lane 1) containing a vector (pZX7.8) into which a frameshift of 2 bases was introduced 12 bases upstream from the BamHI cloning site in pZX7, and XL1-. Blue (pZX7) (lane 2). The molecular weight is shown on the far right. We detected the 52-kD polypeptide in the soluble protein fraction of XL1-Blue (pZX7) (arrow). A protein of approximately 50 kD is expressed by XL1-Blue containing pZX7.8. Expression of the 52-kD protein was always associated with the HeLa cell interaction of recombinant E. coli clones. From the SDS-PAGE results in Figure 4, the soluble fraction of XL1-Blue (pZX7) sonicated bacterial cells carried a pBluescript-derived vector (pZN7) carrying an unrelated Mycobacterium tuberculosis DNA fragment. It can be concluded that it contained a 52-kD polypeptide that was not detected in the soluble fraction of XL1-Blue with. An plasmid containing this plasmid (pZX7.8) was introduced by a frameshift of 2 bases introduced by blunt end ligation after filling the 5'overhanging ends with Klenow DNA polymerase at the XbaI site 12 bases upstream from the BamHI cloning site in pZX7. -The association of E. coli XL1-Blue with HeLa cells was lost. This clone did not express the 52-kD protein, but a new low molecular weight polypeptide was detected in the soluble fraction. The spontaneous loss of the ability to associate with HeLa cells after long-term storage of XL1-Blue (pZX7) was accompanied by loss of the 52-kD protein. Therefore, this 52-kD protein appears to be the product expressed by the cloned Mycobacterium tuberculosis DNA fragment. No difference was observed in the bacterial outer membrane polypeptide fraction. Whether the cloned 1535-bp fragment has more than one open reading frame, or whether a single gene product mediates both cell invasiveness and survival in macrophages And not. Drugs designed to target Mycobacterium tuberculosis products that mediate macrophage viability, or vaccines against products that encode mammalian cell invasiveness, are strategies for controlling tuberculosis globally. Can contribute substantially to. Although the present invention has been described in detail for purposes of illustration, such details are for purposes of illustration only and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention as defined by the claims below. It is understood.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI C12Q 1/68 9453-4B C12Q 1/68 G01N 33/569 0276-2J G01N 33/569 F // (C12N 15 / 09 ZNA C12R 1:32) (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (1)

[Claims] 1. The ability of mycobacillus to invade mammalian cells and survive in macrophages An isolated DNA molecule imparted to T. tuberculosis. 2. The isolated DNA according to claim 1, comprising a nucleotide sequence corresponding to SEQ ID NO: 1. molecule. 3. Claim to encode a polypeptide having a molecular weight of about 50-55 kilodaltons. Item 1. The isolated DNA molecule according to Item 1. 4. The ability of mycobacillus to invade mammalian cells and survive in macrophages Encoded by a DNA molecule conferring on T. tuberculosis An isolated protein or polypeptide. 5. The DNA molecule comprises a nucleotide sequence corresponding to SEQ ID NO: 1. The isolated proteins or polypeptides listed above. 6. The isolated protein according to claim 5 having an amino acid sequence corresponding to SEQ ID NO: 2 or Is a polypeptide. 7. The isolated protein of claim 4 or having a molecular weight of about 50-55 kilodaltons. Is a polypeptide. 8. The isolated protein or polypeptide according to claim 4, which is a recombinant product. 9. The isolated protein or polypeptide according to claim 4, which is purified. 10. The ability of mycobacterium to invade mammalian cells and survive in macrophages Has one or more antigenic determinant sites conferred on Cterium tuberculosis The isolated protein or polypeptide according to claim 4. 11. Administration of an effective amount of the isolated protein or polypeptide of claim 4 to a mammal Infection by Mycobacterium tuberculosis characterized by In contrast, a method of vaccinating a mammal. 12. The administration is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or intranasal. The method according to claim 11, wherein 13. The ability of mycobacterium to invade mammalian cells and survive in macrophages Insert exogenous DNA imparted to Cterium tuberculosis into it A recombinant DNA expression system comprising the expressed vector. 14． The foreign DNA comprises a nucleotide sequence corresponding to SEQ ID NO: 1. 13. The recombinant DNA expression system according to 13. 15. The foreign DNA is inserted into the vector in the proper orientation and correct reading frame. The recombinant DNA expression system according to claim 13. 16. The ability of mycobacterium to invade mammalian cells and survive in macrophages Host that takes in foreign DNA conferred on Cterium tuberculosis cell. 17． The foreign DNA comprises a nucleotide sequence corresponding to SEQ ID NO: 1. 16. The host cell according to 16. 18. The foreign DNA was inserted into a recombinant DNA expression system consisting of an expression vector The host cell according to claim 16. 19. An isolated protein or polypeptide according to claim 4; and   Pharmaceutically acceptable carrier: Mycobacterium tuberculosis consisting of For preventing infectious diseases and illnesses in mammals caused by A. 20. The protein or polypeptide has a molecular weight of about 50-55 kilodaltons The vaccine according to claim 19, which is a polypeptide. 21. The vaccine according to claim 19, wherein the protein or polypeptide is purified. . 22. An effective amount of the vaccine according to claim 19 is administered to a mammal. A mammal against infection with Mycobacterium tuberculosis. How to treat cutin. 23. The administration is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or intranasal 23. The method of claim 22. 24. An isolated antibody against the protein or polypeptide according to claim 4, or a conjugate thereof. Joint part. 25. The protein or polypeptide has a molecular weight of about 50-55 kilodaltons 25. The isolated antibody or the binding site thereof according to claim 24, which is a polypeptide. 26. 25. The antibody is a monoclonal antibody or a polyclonal antibody. The isolated antibody or binding site thereof as described. 27. The ability of mycobacterium to invade mammalian cells and survive in macrophages Encoded by the gene fragment conferred on Cterium tuberculosis 25. The antibody is specific to an antigenic determinant site of a protein or polypeptide The isolated antibody or binding site thereof as described. 28. An effective amount of the antibody or its binding site according to claim 24 is Mycobacteria. M. tuberculosis, which is administered to a mammal infected with Those who passively immunize mammals infected with Icobacterium tuberculosis Law. 29. The administration is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or intranasal. 29. The method according to claim 28. 30. 25. The isolated antibody of claim 24 or a binding site thereof; and Sensitive to Mycobacterium tuberculosis consisting of a pharmaceutically acceptable carrier A composition for passive immunization of a dyed mammal. 31. 31. The antibody is a monoclonal antibody or a polyclonal antibody. The composition as described. 32. The ability of mycobacterium to invade mammalian cells and survive in macrophages Encoded by a gene fragment conferred on Cterium tuberculosis The antibody is specific to an antigenic determinant site of the protein or polypeptide 30. The composition according to 30. 33. Claim to a mammal infected with Mycobacterium tuberculosis 30. The mycobacterium, which comprises administering an effective amount of the composition according to item 30. A method of passively immunizing a mammal infected with tuberculosis. 34. The administration is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or intranasal. 34. The method according to claim 33. 35. Preparing the protein or polypeptide according to claim 4 as an antigen;   Contacting a sample of tissue or body fluid with the antigen;   Using the assay system, Mycobacterium tuberculosis was detected in the sample. Detect any reactions that are present: Detecting Mycobacterium tuberculosis in the sample characterized by how to. 36. The protein or polypeptide has a molecular weight of about 50-55 kilodaltons 36. The method according to claim 35, which is a polypeptide. 37. The assay system is an enzyme-linked immunosorbent assay, a radioimmunoassay. B, gel diffusion-precipitation assay, immunodiffusion assay, agglutination assay, fluorescent immunoassay A group consisting of essays, protein A immunoassays, and immunoelectrophoresis assays 36. The method of claim 35 selected from: 38. Preparing the antibody or binding site thereof according to claim 24;   Contacting a sample of tissue or body fluid with the antibody or binding site thereof;   Using the assay system, Mycobacterium tuberculosis was detected in the sample. Detect any reactions that are present: Detecting the Mycobacterium tuberculosis in the sample, characterized in that How to get out. 39. The protein or polypeptide has a molecular weight of about 50-55 kilodaltons 39. The method according to claim 38, which is a polypeptide. 40. The assay system is an enzyme-linked immunosorbent assay, a radioimmunoassay. Say, gel diffusion-precipitation assay, immunodiffusion assay, agglutination assay, fluorescent immunity Assay, protein A immunoassay, and immunoelectrophoresis assay 39. The method of claim 38 selected from 41. The nucleic acid hybridization of the nucleotide sequence of the DNA molecule according to claim 1. Prepared as a probe in the assay;   Contacting the probe with a sample of tissue or body fluid;   Shows the presence of Mycobacterium tuberculosis in the sample Detect both reactions: Detecting Mycobacterium tuberculosis in the sample characterized by how to. 42. A method for detecting a nucleotide sequence of a DNA molecule according to claim 1 in a gene amplification detection method Prepared as a probe   Contacting a sample of tissue or body fluid with the probe;   Indicates the presence of Mycobacterium tuberculosis in a sample Detect shift reaction: Detecting Mycobacterium tuberculosis in the sample characterized by how to. 43. Substances taken up by mammalian cells; and   Protein or polypeptide according to claim 4: The protein is associated with the substance, wherein the substance is incorporated into a mammalian cell. The product for you. 44. The protein or polypeptide has a molecular weight of about 50-55 kilodaltons 44. The product according to claim 43, which is a polypeptide. 45. 44. The product of claim 43, wherein the protein or polypeptide is purified. 46. The substance is an antibiotic, a DNA fragment, an anti-neoplastic agent, and mixtures thereof. 44. The product of claim 43 selected from the group consisting of: 47. Directing a substance to a mammalian cell with the protein or polypeptide of claim 4. A cellular uptake method characterized by the following. 48. The protein or polypeptide has a molecular weight of about 50-55 kilodaltons 48. The method of claim 47, wherein the method is a polypeptide. 49. 48. The method of claim 47, wherein the protein or polypeptide is purified. 50. The substance is an antibiotic, a DNA fragment, an anti-neoplastic agent, and a mixture thereof. 48. The method of claim 47 selected from the group consisting of: 51. 48. The method of claim 47, wherein the mammalian cells are macrophages. 52. 52. The method of claim 51, which induces cell-mediated immunity.