KR20140056472A - Biomarkers indicative of leukemia and diagnosis using the same - Google Patents

Abstract

The present invention relates to a kit for leukemia diagnosis or prognosis analysis. Prohibitin (PHB) protein according to the present invention is a marker for leukemia having significantly improved accuracy and reliability. Moreover, according to the present invention, early leukemia diagnosis and prognostic determination can be enabled in a very specific manner through a biological sample (for example, blood or serum) using the PHB protein. The expression of PHB protein specifically increases only in the tissue of a leukemia patient.

Description

Biomarkers for Leukemia and Diagnosis of Leukemia Using the Biomarkers of Leukemia and Diagnosis Using the Same [

The present invention relates to a biomarker specific to leukemia and its use.

Leukemia (leukemia) is a generic term for the positive proliferation of leukemia. Leukemia is divided into myeloid leukemia and lymphoid leukemia according to leukocyte origin, and acute leukemia and chronic leukemia according to progression rate. The clinical manifestations of leukemia vary according to the type of disease and the nature of the affected cells. Lymphocytic leukemia is caused by lymphocytic hematopoietic cells, myeloid leukemia by myeloid hematopoietic cells, and chronic myelogenous leukemia by mature cells, and acute myelogenous leukemia is a bone marrow It is caused by disorder of the germ cell.

In particular, myeloid leukemia is a very heterogeneous condition of pathologic molecular expression, and there are no biomolecular markers that can be used for diagnosis, follow-up, and prognosis estimation of all leukemia patients.

Therefore, in order to overcome these limitations, the present invention focuses on mitochondria and their proteases, which play an important role in the development, progression and deterioration of diseases, and obtains high purity leukemia cells and leukemia stem cells using single cell separation techniques, Leukemia cell specific protein markers were identified by selectively separating mitochondrial capture and analyzing mitochondrial protein.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to find a novel biomarker capable of rapidly and accurately molecular diagnosis of leukemia. As a result, the present inventors have completed the present invention by confirming that the biomarkers uncovered are capable of diagnosing leukemia and determining the prognosis.

Accordingly, an object of the present invention is to provide a kit for the diagnosis or prognosis of leukemia.

It is another object of the present invention to provide a method for detecting a leukemia marker in order to provide information necessary for diagnosis or prognosis of leukemia.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides an antibody or an aptamer that specifically binds to a prohibitin (PHB) protein, a nucleotide sequence encoding a PHB protein, a sequence complementary to the nucleotide sequence, or a nucleotide sequence complementary to the nucleotide And a kit for analyzing a leukemia diagnosis or prognosis, which comprises a fragment.

According to another aspect of the present invention, the present invention provides a method for detecting the expression of a nucleotide sequence encoding a prohibitin (PHB) protein or a PHB protein in a human biological sample to provide information necessary for a leukemia diagnosis or prognosis analysis A method for detecting a leukemia diagnosis or prognostic analysis marker through a method for detecting a leukemia diagnosis or a prognosis analysis marker.

The present inventors have tried to find out a novel molecular marker capable of rapidly and accurately molecular diagnosis of leukemia. As a result, it has been found that the molecular markers described above can be used for early diagnosis of leukemia, Respectively. In particular, the markers of the present invention are markers with greatly improved accuracy and reliability as markers for leukemia.

Prohibitin (PHB) protein is a protein encoded by the human PHB gene. The PHB gene is present on human chromosome 17, and the mRNA and protein sequences are known as NM_002634 and NP_002625, respectively.

The term " biological sample " in the present invention means all samples obtained from human or mammals such as leukemia cells, leukemia tissues, bone marrow, urine, saliva, blood, plasma or serum samples.

According to a preferred embodiment of the present invention, the molecular marker PHB protein of the present invention is contained in a bone marrow, a blood, a plasma or a serum sample, more preferably a bone marrow, a blood or a serum sample, Or serum samples.

According to a preferred embodiment of the invention, the leukemia is myeloid leukemia.

The molecular marker of the present invention can be an indicator for the development and development of leukemia and can be used for diagnosis of the occurrence and development of leukemia.

According to a preferred embodiment of the present invention, the molecular markers of the present invention are used for highly accurate prediction or diagnosis of myeloid leukemia.

The term " diagnosing " in the context of the present invention includes determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, Determining the prognosis of an object that is caught in the subject, or therametrics (e.g., monitoring the status of the object to provide information about the treatment efficacy).

The term " prognosis " in the context of the present invention encompasses the course of disease progression, in particular disease progression, disease regeneration, disease recurrence, and prediction in terms of death potential. Preferably, the prognosis in the present invention means the possibility that the disease of the leukemia patient is cured.

According to a preferred embodiment of the present invention, the present invention can be carried out by an immunoassay method, that is, an antigen-antibody reaction method. In this case, an antibody or an aptamer that specifically binds to the leukemia marker of the present invention described above is used.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies can be prepared by methods routinely practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of (Clackson et al., Nature , 352: 624-628 (1991) and Marks et al., J. Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

When the method of the present invention is carried out using an antibody or an aptamer, the present invention can be carried out according to a conventional immunoassay to diagnose leukemia.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay format may include, but is not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay, capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, But are not limited to, fluorescent staining and immunoaffinity purification. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotope is an antibody labeled (e.g., C _14, I _125, P ₃₂ and S ₃₅₎ of detecting the marker molecules of the present invention .

When the method of the present invention is carried out by an ELISA method, a specific embodiment of the present invention comprises the steps of (i) coating the surface of a solid substrate with an unknown cell sample lysate to be analyzed; (Ii) reacting the cell lysate with an antibody to a marker as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) measuring the activity of the enzyme.

Suitable as said solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and most preferably microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P450. When alkaline phosphatase is used as an enzyme that binds to the secondary antibody, it is preferable to use, as a substrate, bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1 chromophore and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine ), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronin, glucose oxidase and nitroblue tetrazolium and m-PMS methosulfate) can be used. have.

When the method of the present invention is carried out in the Capture-ELISA mode, a specific embodiment of the present invention comprises the steps of (i) coating an antibody against the marker of the present invention as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody with the sample; (Iii) reacting the result of step (ii) with a detecting antibody which is labeled with a signal generating label and specifically reacts with a PHB protein; And (iv) measuring a signal originating from said label.

The detection antibody has a label that generates a detectable signal. Wherein the label is a chemical (e.g., biotin), an enzyme (alkaline phosphatase, β- galactosidase, horseradish peroxidase, and cytochrome P450), radioactive materials (e.g., ₁₄ C, ₁₂₅ I, ₃₂ P And S ₃₅ ), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). The various labels and labeling methods are described in Ed. Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

In the ELISA method and the capture-ELISA method, measurement of the activity of the final enzyme or measurement of the signal can be performed according to various methods known in the art. This detection of the signal enables a qualitative or quantitative analysis of the marker of the present invention. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

According to another variant of the invention, an aptamer which specifically binds to the marker of the invention can be used in place of the antibody. Aptamers are oligonucleic acid or peptide molecules, and the general contents of aptamers are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med . 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA . 95 (24): 142727 (1998).

By analyzing the intensity of the final signal by the above-described immunoassay, it is possible to diagnose leukemia. That is, when the protein of the marker of the present invention is highly expressed in the biological sample and the signal is stronger than the normal biological sample (for example, normal tissue, bone marrow, blood, plasma or serum), it is diagnosed as leukemia.

The kit of the present invention may further include other components in addition to the above components. For example, when the kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally comprise reagents necessary for PCR amplification, such as a buffer, a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus Thermostable DNA polymerases obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs. The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

According to a preferred embodiment of the present invention, the kit of the present invention may be a microarray.

According to a preferred embodiment of the present invention, the kit of the present invention is a gene amplification kit.

When the kit of the present invention is a microarray, the probe is immobilized on the solid-phase surface of the microarray. When the kit of the present invention is a gene amplification kit, it includes a primer.

The probe or primer used in the diagnostic kit of the present invention has a sequence complementary to the PHB nucleotide sequence. The term " complementary " as used herein means having complementarity enough to selectively hybridize to the nucleotide sequences described above under any particular hybridization or annealing conditions.

Thus, the term " complementary " has a different meaning from the term complementary, and the primer or probe of the present invention may include one or more mismatches mismatch < / RTI > sequence.

As used herein, the term " primer " refers to a primer that, under suitable conditions (i.e., four different nucleoside triphosphates and polymerization enzymes) in a suitable buffer at a suitable temperature, - means strand oligonucleotide. The suitable length of the primer is typically 15-30 nucleotides, although it varies with various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.

The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a perfectly complementary sequence to the above-mentioned nucleotide sequence, which is a template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. The design of such a primer can be easily carried out by a person skilled in the art with reference to the above-mentioned nucleotide sequence, for example, by using a program for primer design (for example, PRIMER 3 program).

As used herein, the term " probe " refers to a linear oligomer of natural or modified monomer or linkages and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, Present or artificially synthesized. The probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.

The nucleotide sequence of the marker of the present invention to be referred to in the preparation of the primer or probe can be confirmed in GenBank using the accession number of the PHB mentioned above, and the primer or the probe can be designed with reference to this sequence.

In the microarray of the present invention, the probe is used as a hybridizable array element and immobilized on a substrate. Preferred gases include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. The hybridization array elements are arranged and immobilized on the substrate. Such immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface. In addition, the hybridization array element may be coupled to the gas through a linker (e.g., ethylene glycol oligomer and diamine).

On the other hand, the sample DNA to be applied to the microarray of the present invention can be labeled and hybridized with the array elements on the microarray. Hybridization conditions can be varied. The detection and analysis of the hybridization degree can be variously carried out according to the labeling substance.

The leukemia diagnosis kit of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention described above is used.

Hybridization-based analysis may be performed using a probe hybridized to the nucleotide sequence of the marker of the present invention to determine whether leukemia is present.

The label of the probe may provide a signal to detect hybridization, which may be linked to an oligonucleotide. Suitable labels include fluorescent moieties (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent moieties, magnetic particles, (Such as P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), joins, substrates for enzymes, heavy metals such as gold and antibodies, streptavidin, biotin , Hapten having specific binding partners such as digoxigenin and chelating groups. Markers can be generated using a variety of methods routinely practiced in the art such as the nick translation method, the Multiprime DNA labeling systems booklet (Amersham, 1989) and the kaination method (Maxam & Gilbert, Methods in Enzymology, 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, colorimetry, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

The nucleic acid sample to be analyzed can be prepared using mRNA obtained from various biological samples (biosamples). Instead of the probe, the cDNA to be analyzed may be labeled and subjected to a hybridization reaction-based analysis.

When a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined by a series of procedures by an optimization procedure. This procedure is performed by a person skilled in the art in a series of procedures to establish a protocol for use in the laboratory. Conditions such as, for example, temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. The detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc .; NY (1999). For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO ₄ , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA, followed by addition of 0.1 x SSC / 0.1% SDS Lt; RTI ID = 0.0 > 68 C < / RTI >

Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

After the hybridization reaction, a hybridization signal generated through the hybridization reaction is detected. The hybridization signal can be carried out in various ways depending on, for example, the type of label attached to the probe. For example, when a probe is labeled with an enzyme, the substrate of the enzyme can be reacted with the result of hybridization reaction to confirm hybridization. Combinations of enzymes / substrates that may be used include, but are not limited to, peroxidases (such as horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis- Acetyl-3,7-dihydroxyphenox), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2) , 2'-Azine-di [3-ethyl benzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF substrate; alkaline phosphatase and bromochloroindoleyl phosphate; Glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by a silver staining method using silver nitrate. Therefore, when the method for detecting the leukemia marker of the present invention is carried out based on hybridization, specifically (i) a step of hybridizing a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention to a nucleic acid sample; (ii) detecting whether the hybridization reaction has occurred. By analyzing the intensity of the hybridization signal by the hybridization process, it is possible to judge whether leukemia is present or not.

That is, when the hybridization signal for the nucleotide sequence of the marker of the present invention in the sample is stronger than the normal sample, it is diagnosed as leukemia.

According to a preferred embodiment of the present invention, the human leukemia diagnostic kit of the present invention may be a gene amplification kit.

The term " amplification " as used herein refers to a reaction that amplifies a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (LCR) (see, for example, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822) 17,18), Gap-LCR (WO 90/01069), repair chain reaction (EP439,182), transcription-mediated amplification (TMA, WO 88/10315) (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (PCR), and the like. The term " amplified polynucleotide sequence " (CP-PCR), U.S. Patent No. 4,437,975) (AP-PCR), U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA), U.S. Patent No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21,22), and loop-mediated isothermal amplification. (LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications.

For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

When the diagnostic kit of the present invention is used as a primer, the gene amplification reaction is carried out to investigate the degree of expression of the nucleotide sequence of the marker of the present invention. Since the present invention analyzes the degree of expression of the nucleotide sequence of the marker of the present invention, the amount of mRNA of the nucleotide sequence of the marker of the present invention is examined in a sample (for example, bone marrow, blood, plasma or serum) To determine the degree of expression of the nucleotide sequence of the marker.

Therefore, in principle, the present invention uses a mRNA in a sample as a template and performs a gene amplification reaction using a primer that binds to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. The isolation of total RNA can be carried out according to conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere , C. et al., Plant Mol . Biol . Rep . , ≪ / RTI > 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology , John Willey & Sons (1987); And Chomczynski, P. et al., Anal . Biochem . 162: 156 (1987)). For example, TRIzol can be used to easily isolate total RNA in a cell. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from a human sample, it has a poly-A tail at the end of the mRNA, and the cDNA can be easily synthesized using the oligo dT primer and the reverse transcriptase using such a sequence characteristic ( See, for example, PNAS USA, 85: 8998 (1988); Libert F, et al., Science , 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning . A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)). Next, the synthesized cDNA is amplified through gene amplification reaction.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Nucleic acid hybridization conditions suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization < RTI ID = 0.0 > (" , A Practical Approach, IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used in the amplification of the present invention, including the " Clenow " fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) .

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. To provide joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{+ 2} to the reaction mixtures to have a desired degree of amplification can be achieved is required. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reaction without changing the conditions such as the addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

The cDNA of the nucleotide sequence of the thus amplified marker of the present invention is analyzed by a suitable method to investigate the degree of expression of the nucleotide sequence of the marker of the present invention. For example, the result of amplification reaction described above is subjected to gel electrophoresis, and the resulting band is observed and analyzed to examine the expression level of the nucleotide sequence of the marker of the present invention. Through this amplification reaction, leukemia is diagnosed when the expression of the nucleotide sequence of the marker of the present invention in the biological sample is higher than that of a normal sample (for example, normal cells, blood, plasma or serum).

Therefore, when the method for detecting a leukemia marker of the present invention is carried out based on an amplification reaction using cDNA, specifically (i) performing an amplification reaction using a primer annealed to a nucleotide sequence of a marker of the present invention; And (ii) analyzing the product of the amplification reaction to determine the degree of expression of the nucleotide sequence of the marker of the present invention.

The marker of the present invention is a biomolecule which is highly expressed in leukemia. High expression of these markers can be measured at the mRNA or protein level. As used herein, the term " high expression " means that the degree of expression of a nucleotide sequence to be a target in a sample to be investigated is higher than that of a normal sample. For example, expression analysis methods conventionally used in the art, such as RT-PCR or ELISA methods (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001) In the case of expression analysis, it means that the expression is analyzed to be high. For example, when the marker according to the present invention is highly expressed about 2 to 10 times as much as a normal cell, it is determined to be " highly expressed " in the present invention and determined to be leukemia.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a kit for leukemia diagnosis or prognosis analysis.

(Ii) The prohibitin (PHB) protein in the present invention is a marker with greatly improved accuracy and reliability as a marker for leukemia.

(Iii) Furthermore, the present invention can determine the early diagnosis and prognosis of leukemia very specifically from a biological sample (for example, blood or serum) using PHB protein whose expression is specifically increased only in a tissue of leukemia patient .

FIGS. 1A and 1B are graphs showing the results obtained by extracting mitochondrial fractions from myeloid leukemia patients and leukemia cell lines using a mitochondrial separation kit, performing secondary protein electrophoresis, and performing matrix-phase laser deionization (MALDI) and ion flight time- The results are shown in Fig.
FIGS. 2A and 2B are graphs showing the results of immunohistochemistry (FIG. 2A) and the results of immunohistochemistry using a primary antibody, Prohibitin (Primary Antibody) after preparing bone marrow and normal human bone marrow stromal leukemia The results of the immunofluorescence assay (Figure 2b) are shown. Figures 2A and 2 A are normal, and B is the result for a leukemia cell line.
FIG. 3 shows the results of protein electrophoresis after extracting mitochondrial protein fractions from a myeloid leukemia patient, a leukemia cell line, and a normal sample using a mitochondrial separation kit to identify the protein of the prohibitin.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

Example 1: Proteomics

To protein electrophoresis of prohibitin (PHB) proteins, mitochondrial fractions were extracted from myeloid leukemia patients and leukemia cell lines using a mitochondrial separation kit (Pierce biotechnology, Rockford, USA). The mitochondrial fraction was performed according to the manufacturer's instructions. Concentrations of isolated proteins were determined using the Bradford protein assay (BioRad, Calif., USA). The separated proteins were firstly precipitated using pH3-10NLIPG strips, followed by secondary precipitation using 9-16% SDS-PAGE and then subjected to secondary protein electrophoresis. Only the protein spots that were clearly distinguished were identified and identified using matrix-assisted laser desorption ionization (MALDI) and ion flight time-of-flight analyzer (TOF).

As a result, more than 30 mitochondrial proteins overexpressed in patients with myeloid leukemia were identified, and PHB (prohibitin), which is overexpressed only in leukemic cells, especially myeloid leukemia cells, was identified.

Example 2: Immunohistochemistry < RTI ID = 0.0 >

Bone marrow and normal human blood of bone marrow-derived leukemia patients were blotted and slides were prepared and then microwaved with citrate buffer for 40 minutes. After washing twice with washing buffer, Endoblocker was used to remove enzyme for 10 minutes and then washed twice with washing solution. Then, the primary antibody PHB (Santa Cruze, California, USA) was reacted at room temperature for 1 hour, washed twice with a washing solution, and then incubated with a secondary antibody (Santa Cruze ), ≪ / RTI > California, USA) for 30 minutes. After washing with DAB (3,3'-Diaminobenzidine), the cells were stained with hematoxylin for 2 minutes, washed, and sealed with liposoluble encapsulant.

Example 3: Immunofluorescence < RTI ID = 0.0 >

Bone marrow and normal human blood of bone marrow leukemia patients were blotted, slides were prepared, washed with washing solution, and then the washing solution except for the part smeared with a cotton swab was removed as much as possible. The primary antibody, PHB (Santa Cruze, CA, USA), was reacted in the cow at room temperature for 1 hour and 30 minutes, and the washes were washed by pouring the washing solution onto the smear. After reacting with secondary antibody at room temperature for 30 minutes in a dark place, the cells were washed twice and sealed with a liposoluble encapsulating agent.

Example 4: Western blotting analysis

In order to confirm the protein of prohibitin through protein electrophoresis, mitochondrial protein fractions were extracted from myeloid leukemia patients, leukemia cell lines and normal subjects using a mitochondrial separation kit (Pierce biotechnology, , United States of America). The mitochondrial protein fraction was performed according to the manufacturer's instructions. Concentrations of isolated proteins were determined using the Bradford protein assay (BioRad, Calif., USA). 20 μl of protein was dispensed into SDS-PAGE gel (lower gel, upper gel) and electrophoresed at 100 V for 1 hour 30 minutes. Transfer of the gel to the membrane was carried out at 100 V for 1 hour. The membrane was then blocked for 1 hour at room temperature using a blocking buffer. The primary antibody PHB (Santa Cruze, California, USA), β-actin (Santa Cruze, CA, USA) was reacted overnight at 4 ° C with a stirrer, The cells were washed five times for 5 minutes each with 1xTBST buffer and incubated with a secondary antibody Goat anti mouse IgG antibody (Jackson immunoresearch center, PA, USA) For 1 hour. Again 5 times for 5 minutes each, the protein was detected by chemiluminescence detection system (Amersham ECL system, London, UK).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

An antibody or an aptamer that specifically binds to a prohibitin (PHB) protein, a nucleotide sequence that encodes a PHB protein, a sequence complementary to the nucleotide sequence, or a fragment of the nucleotide, .
The kit according to claim 1, wherein the PHB protein is contained in bone marrow, blood, plasma or serum sample.
The kit according to claim 1, wherein the leukemia is myelocytic leukemia.
The kit according to claim 1, wherein the kit is an immunoassay kit.
The kit according to claim 1, wherein the kit is a microarray.
A leukemia diagnosis or prognostic analysis marker is detected through a method of detecting the expression of a nucleotide sequence encoding a prohibitin (PHB) protein or a PHB protein in a human biological sample to provide information necessary for diagnosis or prognosis of leukemia / RTI >
7. The method of claim 6, wherein the leukemia is myeloid leukemia.
7. The method of claim 6, wherein the biological sample is bone marrow, blood, plasma or serum.
7. The method of claim 6, wherein the method is performed in an antigen-antibody reaction mode.
7. The method of claim 6, wherein the method is performed in a microarray manner.
7. The method of claim 6, wherein the method is performed by gene amplification.

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