KR101564751B1 - Polypeptide for detecting Sox2 and uses thereof - Google Patents

Abstract

The present invention relates to a polypeptide for detecting Sox2, which comprises a polypeptide for detecting Sox2 (SRY (Sex-determining Region Y) -box2) comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOS: 5 to 9, Thereby providing a cancer diagnosis system using the same. The polypeptide of the present invention can be used for the treatment of cancer stem cells, Sox2 can be specifically recognized so that it can be used as a super sensitive peptide peptide group which overcomes the limitations of the stability and sensitivity of the existing antigen-antibody diagnostic system and can be used as a therapeutic agent for a disease caused by the detection of a trace amount of disease- There is an effect of suggesting possibility of early diagnosis and improvement of treatment efficiency through this.

Description

Polypeptides for detecting Sox2 and uses thereof [0002]

The present invention relates to polypeptides for detecting Sox2, and more particularly to polypeptides that specifically bind to Sox2 protein and uses thereof.

Cancer arises from the accumulation of genetic mutations within the human body, which is caused by the exposure to various chemicals inside or outside the body and the interaction of various factors within the body. Genetic mutations affect important signaling elements inside the body and cause a disability of certain functions. If functional disorders induced by these genetic mutations persist for long periods, abnormal colonies are formed in the cells, which accumulate and cause cancer.

Radiation therapy used to treat cancer is divided into radiation therapy, in which relatively low doses of radiation are repeatedly administered to reduce the adverse effects of high dose radiation on normal cells. However, it is known that split radiation therapy induces adaptive radioresistance in cancer cells to induce regeneration and malignancy of cancer cells.

On the other hand, malignant tumor tissues contain cancer stem cells that regenerate and maintain cancer tissues similar to normal stem cells. These cancer stem cells infiltrate into the blood, invade / migrate into the metastatic target tissue, , And it has been reported to induce metastatic cancer by differentiating into a specific cancer. In addition, the claim that cancer stem cells are the main cause of acquisition of adaptive radiation resistance in split radiation treatment is attracting attention.

Conventional chemotherapy including split radiation therapy is performed with primary tumor cells that are actively dividing in the primary tumor tissue, and it has a therapeutic effect on the primary cancer. However, it has a relatively new proliferative activity, It has been described that cancer stem cells that acquire malignant characteristics do not effectively remove, thereby increasing the chance of metastatic cancer formation from the blood to the target tissue.

Recent research on cancer stem cells has shown that Sox2 is a representative cancer stem cell marker that is expressed in various types of cancer tissues such as glioma, breast cancer, and stomach cancer. In the case of cancer cells, overexpression of Sox2 has been reported to regulate gene activity by various Sox2 proteins and increase the expression of various proteins. These results suggest that early detection of Sox2 leads to early diagnosis of cancer stem cells and high efficiency in the treatment of cancer.

Accurate recognition of specific proteins within the cell, as well as imaging technology and imaging with imaging and chemotherapeutic agents, provides an opportunity to improve treatment efficiency and develop potential diagnostic tools for early detection of cancer. In order to develop a probe for the early diagnosis and treatment of cancer, a probe that finds cancer cells with high affinity and specificity is identified and used for early diagnosis of cancer. According to the prior art, PCR (Polymerase Chain Reaction) is the most common method for diagnosing a specific diagnostic target. This method is a method of analyzing a small amount of a diagnostic object by analyzing a unique gene of a diagnostic object and amplifying it in vitro . This method has been extended to a wide range of genetic information databases through the recent Human Genome Project. In addition, the PCR method uses single-strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) methods, which confirm the genetic variation of the amplified genes using various probes To provide useful information. However, there are limitations in the diagnostic system due to problems such as difficulty in pretreatment of the sample and adaptability to the field.

In this respect, ELISA (Enzyme-Linked Immunosorption Assay) has been mainly used as a method for determining increase and change in the expression level of a specific protein. This is an antibody that specifically binds to a specific protein to be used as a diagnostic probe. It effectively separates a diagnostic target from a biological sample such as blood or urine, and determines whether or not a specific protein exists in the sample through an enzyme- do. However, the low sensitivity of (10 ^-9 ) nano-mol levels also has a disadvantage in that it is difficult to diagnose low-level markers present in actual diagnostic specimens and has a lower sensitivity than PCR diagnosis methods.

In addition, drug screening has the advantage that a large number of potential anticancer drugs can be simultaneously administered to cells to confirm the results in a short time. However, these new drug candidate substances have toxicity problems in the human body And when used as an anticancer agent, resistance to cancer cells may occur.

Therefore, the development of a low molecular probe capable of diagnosing a very small amount of cancer stem cells will prevent recurrence of cancer, and at the same time, improve the efficiency of cancer treatment by early diagnosis of cancer.

An object of the present invention is to provide a low molecular probe capable of detecting Sox2, which is a cancer diagnostic marker, and a cancer diagnosis system using the same.

In order to accomplish the above object, one aspect of the present invention provides a method for detecting Sox2 (sex-determining region Y) -box2 (SRY) comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: Lt; / RTI > polypeptide.

Another aspect of the present invention provides a cancer diagnostic composition comprising the polypeptide.

In addition, another aspect of the present invention provides a cancer diagnostic kit comprising the polypeptide.

The polypeptide of the present invention can be used for the treatment of cancer stem cells, Sox2 can be specifically recognized so that it can be used as a super sensitive peptide peptide group which overcomes the limitations of the stability and sensitivity of the existing antigen-antibody diagnostic system and can be used as a therapeutic agent for a disease caused by the detection of a trace amount of disease- There is an effect of suggesting possibility of early diagnosis and improvement of treatment efficiency through this.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

1, (M: molecular weight marker, 1: inducible, 2: inducible, 2: inducible) was obtained by SDS / PAGE, and the purified plasmid of Sox2 protein was transformed into Escherichia coli and induced with 0.5 mM IPTG , 3: soluble protein, 4: insoluble protein, F1-F7; Sox2 protein).
FIG. 2 is a diagram showing a plan of M13 phage screening for screening phage peptides binding to Sox2 protein.
FIG. 3 is a graph showing the binding potency of Sox2 protein analyzed by measuring concentration after amplifying the five kinds of polypeptide expression phages identified in the present invention (SBP1 to SBP5 are the polypeptides of SEQ ID NOS: 5 to 9, respectively).
FIGS. 4 (a) to 4 (c) are graphs showing binding affinities of the single polypeptides of SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, respectively, synthesized with fluorescence to Sox2 protein (SBP1, SBP3 alc SBP5 SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, respectively).

Hereinafter, terms used in the present invention will be described.

The term "diagnosis" as used herein means to identify the presence or characteristic of a disease associated with the expression of the protein by measuring the presence or absence of the polypeptide for detection of Sox2 of the present invention in a biological sample or a tissue sample.

Hereinafter, the present invention will be described in detail.

One. Sox2 (SRY (Sex-determining Region Y) -box2)

One aspect of the present invention provides a polypeptide for detecting Sox2 (Sex-determining Region Y) -box2.

Preferably, the polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9, and the polypeptide comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: And most preferably the polypeptide has the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9. However, the polypeptide is not limited thereto, but may be selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9 within a range that has an activity of specifically binding to Sox2 expressed in cells such as cancer cells and cancer stem cells Polypeptides having at least 50%, preferably at least 60%, 70%, 80% or more, more preferably 90% or more, and most preferably 95% or more homology with a polypeptide having any one or more amino acid sequences .

The polypeptide may be derived from nature, and may be synthesized using a known peptide synthesis method.

The Sox2 is preferably derived from human, and Sox2 preferably comprises a polypeptide having the amino acid sequence of SEQ ID NO: 3. The polypeptide specifically binds to the amino acid sequence of Sox2.

The polypeptide may be tagged with a detector. The detection member is for easily identifying, detecting and quantifying whether or not the polypeptide specifically binds to the Sox2. The detection member includes a chromogenic enzyme (peroxidase, alkaline phosphatase, etc.), a fluorescent substance FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots), chromophore and radioactive isotope ¹²⁴ I, ¹²⁵ I, ¹¹¹ In, ^99m Tc, ³² P, ³⁵ S, etc.). When the detector is tagged to the polypeptide of the present invention, it is preferable that the detector is attached so as not to affect the specificity or selectivity for the target in the polypeptide. To this end, the detector is linked (covalently bonded or bridged) to the peptide . The tagging position of the detector as described above can be easily determined by a person skilled in the art through repeated experiments.

In a specific embodiment of the present invention, the present inventors screened peptides binding to Sox2 protein to discover polypeptides as probes that effectively recognize Sox2, an intracellular signal regulatory protein active in the differentiation process of cancer stem cells, Diagnostic peptides were selected. Specifically, a peptide library was screened by a phage display technique. Specifically, a phage library expressing peptides composed of 12 amino acids was added to the surface of M13 phage and the Sox2 protein was immobilized on the plate well. Only the phage peptides that specifically bind to the Sox2 protein were finally selected by extracting the binding phage peptides. By performing biopanning in which the round was repeated several times in a round, a polypeptide binding to Sox2 was obtained (see Table 1 and FIG. 2). It is preferable to carry out at least four rounds at the time of bio-panning in order to select a phage peptide having a stronger binding force. As a result of analyzing the amino acid sequence of the peptide thus obtained, it was confirmed that the polypeptide was composed of the amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 9 (see Table 2). The selected polypeptides showed excellent binding to Sox2 protein (see FIG. 3), confirming that the polypeptide of the present invention can act as a novel probe for recognizing Sox2. In addition, even when the polypeptide was synthesized by attaching a fluorescent substance as a detection target, it was confirmed that Sox2 protein binds effectively, so that it was confirmed that a polypeptide including a detection substance can be usefully used in diagnosis (see FIG. 4). Therefore, the polypeptide specifically binding to Sox2 of the present invention can be usefully used as a low molecular diagnostic probe for detecting Sox2 protein.

2. Composition for cancer diagnosis

Another aspect of the present invention provides a composition for cancer diagnosis comprising a polypeptide for Sox2 detection comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO:

Sox2 polypeptide for the detection is for the detection of the Sox2 specifically, the same as described in "Sox2 1. (SRY (Sex determining Region Y-) -box2) for detecting a polypeptide" section. Therefore, for the Sox2 detection polypeptide, the description of the item " 1. Sox2 (Sexy determining Region Y) -box2 detection polypeptide " is used and the detailed description will be omitted. Hereinafter, Only a specific configuration will be described.

The cancer is a disease associated with cell death control, which refers to a disease caused by excessive cell proliferation when a normal cell death balance is broken. These abnormal hyperproliferative cells sometimes invade surrounding tissues and organs to form a mass, which destroys or transforms normal structures in the body. Such a condition is called cancer. Such cancers may be selected from the group consisting of brain tumor, lung cancer, pancreatic cancer, liver cancer, prostate cancer, colon cancer, breast cancer, stomach cancer and rectal cancer. It is known that Sox2 is overexpressed in these cancer cells. Therefore, when it is detected that the intracellular Sox2 is overexpressed, the individual from which the cell is derived can be diagnosed as having a high risk of being or suffering from cancer.

When the composition is administered to a subject, the polypeptide in the composition specifically and selectively binds to the Sox2 protein expressed in the cancer stem cells, and the polypeptide is bound to the target The position or expression level of the bound polypeptide can be determined. The detector may be any one of a coloring enzyme (peroxidase, alkaline phosphatase, etc.), a fluorescent material (FITC, RITC, rhodamine, Texas Red, fluorescein, Phycoerythrin, quantum dots), chromophore and radioactive isotopes ( ¹²⁴ I, ¹²⁵ I, ¹¹¹ In, ^{99 m} Tc, ³² P, ³⁵ S, etc.) Or at least one of them. That is, when a large amount of Sox2 is present in the tissues of the test sample when a reaction of the test specimen occurs in tissues or cells of the test subject, the test subject can be diagnosed early that the cancer is caught. In addition, the composition can be non-invasively identified in the subject by administering the composition in vivo.

In a specific example of the present invention, a peptide binding to Sox2 protein overexpressed in cancer stem cells was screened, and it was confirmed that the selected peptide could specifically bind to Sox2 protein (see FIGS. 3 and 4) , The composition comprising the polypeptide for detecting Sox2 of the present invention can be usefully used for diagnosis of cancer.

3. Cancer Diagnostic Kits

Yet another aspect of the present invention provides a cancer diagnostic kit comprising a polypeptide for Sox2 detection comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO:

The cancer diagnostic kit of the present invention may include one or more other component compositions or devices suitable for the method of assaying a polypeptide for measuring the level of expression of Sox2. For example, in addition to a polypeptide specifically binding to Sox2 of the present invention, a cancer diagnostic kit for measuring the level of Sox2 can be used without limitation in a kit using known methods used for measuring protein expression levels.

The above-mentioned "measurement of protein expression level" refers to a process of confirming the presence and expression level of a protein expressed in a cancer marker gene in a biological sample to diagnose cancer, using a polypeptide specifically binding to the Sox2 protein The amount of protein can be confirmed. A detection method according to a detection body is well known in the art, but can be performed, for example, by the following method. If a fluorescent substance is used as a detection body, immunofluorescence staining can be used. For example, the polypeptide of the present invention labeled with a fluorescent substance can be reacted with a sample, and unbound or non-specific binding products can be removed, and fluorescence by the peptide under a fluorescence microscope can be observed. In the case of using an enzyme as a detection body, the absorbance can be measured by a color development reaction of a substrate through an enzyme reaction, and in the case of a radiation substance, a radiation emission amount can be measured.

The kit of the present invention may further comprise components necessary for carrying out this assay. For example, a polypeptide represented by any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOS: 5 to 9 A coated reactor or a wash solution to be used for each reaction step, and the like.

Unlike the composition of the above item 2. " Composition for diagnosing cancer ", the kit is not intended to be administered to a subject but may be administered to a subject such as a tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine To diagnose the presence or absence of cancer cells in a subject using a biological sample. Therefore, it is intended to detect the protein contained in the biological sample, and in particular, the kit of the present invention can be used to detect the Sox2 protein contained in the biological sample.

In a specific example of the present invention, peptides binding to Sox2 protein were screened by a phage display technique to confirm that the selected peptides could specifically bind to Sox2 protein (see FIGS. 3 and 4) A kit containing a polypeptide for detection can be usefully used for diagnosis of cancer.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are provided only for illustrating the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Mass production and isolation of Sox2 protein

A polymerase chain reaction (PCR) was performed with forward and reverse primers having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 to amplify the Sox2 gene having the nucleotide sequence of SEQ ID NO: 4, pET-28a (Novagen, USA). The SEQ ID NO: nucleotide sequence is inserted in an expression vector for the expression host E. coli of 4 as shown in (E. Coli) BL21 (DE3 ) was transformed to the cells, and incubated for 12 hours in a 0.5 mM IPTG, 37 ℃ conditions SEQ ID NO: Gt; Sox2 < / RTI > protein having the amino acid sequence of SEQ ID NO: 3. Then, the E. coli cells were obtained by centrifugation, and the solubility of the proteins in the E. coli cells was confirmed to confirm that most of the proteins were insoluble.

Only the intracellular insoluble protein was obtained, and the protein was isolated and purified under denaturing conditions. Specifically, the insoluble protein was dissolved under denaturation conditions of 6M Guanidium-HCl and passed through a Ni-Sepharose column to bind the Sox2 protein. Subsequently, to refold the denatured Sox2 protein, the Sox2 protein was refolded with a linear gradient (gradually decreasing urea concentration) while being bound to the column, and the protein was refolded to 500 mM imidazole linear gradient. The protein thus obtained was dialyzed in a dialysis buffer solution (50 mM Tris, 2 mM? -Mercaptoethanol) to remove excess imidazole. Purification of the protein obtained through the above procedure was confirmed by SDS / PAGE (FIG. 1) and the amount was confirmed using Bradford reagent.

As a result, Sox2 protein having the amino acid sequence of SEQ ID NO: 3 was obtained at a concentration of 0.8 mg / l.

Screening-Phage Display of Polypeptides Binding to Sox2 Protein

Using the Example 1, SEQ ID NO: 3 Sox2 protein and peptide libraries obtained in the ^{Ph.D TM (phage display peptide library kit} , New England Biolabs (NEB))), Republic of Korea Laid-Open Patent Publication No. 10-2008-0083807 No. And 10 < RTI ID = 0.0 > 020132455 < / RTI > were screened for polypeptides that specifically bind to the Sox2 protein. More specifically, referring to FIG.

Sox2 protein of SEQ ID NO: 3 obtained in Example 1 was immobilized on a 96-well polystyrene plate (Qiagen, USA) in order to screen a polypeptide specifically binding to Sox2 protein which is a cancer stem cell mark marker, and M13 phage peptide library were added to ^TM Ph.D (phage display peptide library kit, New England Biolabs (NEB))) it induced the binding between the protein and any polypeptide Sox2. Thereafter, the bio-panning procedure was repeated four times to select a phage having a stronger binding force by repeating washing conditions, binding times and elution conditions as shown in Table 1 below.

round Washing condition Phage peptide binding time Dissolution One 50 mM Tris, 150 mM NaCl,
0.1% Tween-20 60 minutes Chemical elution
(0.2 M glycine pH 2.2,
1 mg / ml BSA) 2 50 mM Tris, 150 mM NaCl,
0.3% Tween-20 40 minutes 3 50 mM Tris, 150 mM NaCl,
0.5% Tween-20 20 minutes 4 50 mM Tris, 150 mM NaCl,
0.5% Tween-20 10 minutes Competitive leaching
(More than 10 mole Sox2)

The thus-obtained phage was infected with Escherichia coli ER2738 cells as host cells, and the cells were cultured and amplified in LB medium. After amplification, 100 phage plaques were selected to separate and purify M13 phage genomic DNA (single stranded circular DNA) (Gp3 minor coat protein) and revealed a nucleotide sequence of a polynucleotide encoding a part of the polypeptide that binds to the Sox2 protein. Based on the nucleotide sequence thus identified, the amino acid sequence of the polypeptide binding to the Sox2 protein was identified.

As a result, five polypeptides having the amino acid sequences of SEQ ID NOS: 5 to 9 binding to the Sox2 protein of SEQ ID NO: 3 were identified as shown in Table 2 below (Table 2).

SEQ ID NO: Peptide sequence Loss rate (%) 5 IPKRIIPHSRHI 91 6 SPTSIHGNGKMH 1.8 7 PPKRSHHLRPMR 1.8 8 QSHEQNNYNQRS 1.8 9 NVPTVWDNTFWY 3.6

Selected polypeptides and Sox2 Analysis of binding force of protein

In order to analyze the binding ability of the selected polypeptides in Example 2, phage expressing the polypeptides of SEQ ID NOS: 5 to 9 were amplified, respectively, and the concentration of the phage solution was determined by titering. Then, the binding force of the polypeptide was measured by measuring the amount of the phage bound by adding the phage to the plate to which the Sox2 protein of SEQ ID NO: 3 was bound.

Specifically, the phage expressing the polypeptide of SEQ ID NO: 5 to SEQ ID NO: 9 was added to the plate to which the Sox2 protein of SEQ ID NO: 3 was bound, respectively, and the binding reaction was induced for a certain period of time and then washed. Then, ELISA using horse-radish peroxidase (HRP) and TMB solution was performed, and ELISA signals corresponding to respective concentrations were collected. The dissociation constant (K _D ) The binding ability of the polypeptide of SEQ ID NO: 5 to SEQ ID NO: 9 to the Sox2 protein of SEQ ID NO: 3 was confirmed.

As a result, as shown in Fig. 3, the polypeptides of SEQ ID NO: 5 to SEQ ID NO: 9 had K _D values of 4.1 ρM, 129 ρM, 7.3 ρM, 23.5 ρM and 6.5 ρM, It was confirmed that all of the five kinds of the obtained polypeptides had excellent binding force at the level of picomolar concentration (rho M) (Fig. 3).

Analysis of Binding Power between Single Polypeptide and Sox2 Protein

(FITC: fluorescein isothiocyanate) was attached to the polypeptide sequence of SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, which were confirmed to have high binding ability to the Sox2 protein of SEQ ID NO: 3 in the phage- A polypeptide of the following sequence was synthesized.

Polypeptide of SEQ ID NO: 5 -FITC: Ac-IPKRIIPHSRHIGCGK-FITC- NH 2

Polypeptide of SEQ ID NO: 7 -FITC: Ac-PPKRSHHLRPMRGCGK-FITC -NH 2

Polypeptide of SEQ ID NO: 9 -FITC: Ac-NVPTVWDNTFWYGCGK-FITC- NH 2

The N-terminal of the polypeptide of SEQ ID NO: 5 and SEQ ID NO: 9 was acetylated to remove the reactivity of the amino terminal and the amino acid lysine (K) was added to attach the FITC to the C-terminal. The GCG sequence was further added to the C-terminus of the polypeptide of SEQ ID NO: 5 and SEQ ID NO: 9, which is the polypeptide-FITC of SEQ ID NO: 5, the polypeptide-FITC of SEQ ID NO: 7, 9 < / RTI > polypeptide-FITC '.

In order to analyze the binding ability of the polypeptide-FITC of SEQ ID NO: 5, the polypeptide FITC of SEQ ID NO: 7 and the polypeptide FITC of SEQ ID NO: 9 to the Sox2 protein of SEQ ID NO: 3, FITC 'of SEQ ID NO: 7 and the polypeptide-FITC' polypeptide of SEQ ID NO: 9 were diluted by concentration to the plate on which the Sox2 protein of SEQ ID NO: 3 was immobilized. Each well was then washed and the dissociation constant (K _D ) of the polypeptide of SEQ ID NO: 7 and SEQ ID NO: 9 was analyzed by measuring the absorbance of FITC linked to the polypeptide by means of a fluorophotometer.

As a result, the polypeptides of SEQ ID NO: 5 and SEQ ID NO: 9 had K _D values of 100 nM, 8.9 nM and 1.3 nM, respectively, and the polypeptides of SEQ ID NO: 5 and SEQ ID NO: It was confirmed that the Sox2 protein of SEQ ID NO: 3 had a high binding force at a nano-mole concentration (nM) level (Fig. 4).

Analysis of binding force of Sox2 in cells using synthesized peptide bound to Sox2

In order to diagnose Sox2 protein present in cells, Sox2 protein, which is present in cells, was diagnosed using U87 glioma cell line, which is an overexpressed cancer cell U87 glioma cell line using Immunocytochemistry. In addition, Sox2 overexpression in real glioma cells was treated by dividing radiation into glioma cells, and the expression of Sox2 in normal cells was compared when radiation was not treated.

  As a control, Sox2 antibody was used to compare peptides binding to Sox2 protein, and the experimental method was as follows.

U87 cells were plated on a 60 mm cell culture plate with DMEM medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin, 100 μg / ml streptomycin and incubated at 37 ° C , ≪ / _RTI > 5% CO2. The grown cells were exposed to 3.81 Gy / min gamma-ray (gamma-ray) for some split radiation treatment. In order to quantify the prepared cells (cells treated with U87 cells and split radiation), each cell was taken to destroy the cell wall, and proteins present in the cells were separated and separated by SDS-PAGE electrophoresis. Immunoassay Immunoblot assay) was carried out on a nitrocellulose membrane. The transferred membrane was blocked using 5% skim milk and the Sox2 antibody was bound at 4 ° C for 12 hours. After 12 hours, the secondary antibody was bound and finally the protein was quantitated by chemiluminescence. β-actin was analyzed together for use as a control. It was confirmed that Sox2 was overexpressed by treatment with split radiation (see Fig. 5).

In order to identify quantified proteins and to diagnose actual intracellular Sox2, the above-mentioned two types of U87 cells were fixed with 4% paraformaldehyde containing 0.1% Triton-X and NP40 for 1 hour . After blocking with 1% BSA (bovine serum albumin), Sox2 binding peptide / Sox2 binding antibody was bound for 2 hours and DAPI (4 ', 6-Diamidino-2-phenylindole) The reaction was allowed to proceed for 5 minutes. Lastly, fluorescence microscopy confirmed the presence of peptides and antibodies binding to Sox2 present in the cells. Sox2 antibody was used as a control, and it was confirmed that diagnostic signal was increased compared to antibody when peptide was treated relatively. In addition, intracellular Sox2 protein using peptides was confirmed due to overexpression of Sox2 in the cells treated with split radiation (see Fig. 6). 6 (a) shows DAPI, (b) shows FITC, and (c) shows merge.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the invention is not limited to the disclosed exemplary embodiments. It will be possible to change it appropriately.

<110> Industry-University Cooperation Foundation Hanyang University <120> Polypeptide for detecting Sox2 and uses thereof <130> PN130451 <150> 10-2012-0149167 <151> 2012-12-20 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Sox2 <400> 1 aaggatccat gtacaacatg atggagacgg ag 32 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Sox2 <400> 2 aactcgagtt atcacatgtg tgagaggggc ag 32 <210> 3 <211> 317 <212> PRT <213> Homo sapiens <400> 3 Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln   1 5 10 15 Thr Ser Gly Gly Gly Gly Gly Asn Ser Thr Ala Ala Ala Ala Gly Gly              20 25 30 Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn Ala Phe          35 40 45 Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu Asn Pro      50 55 60 Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu Trp Lys  65 70 75 80 Leu Leu Ser Glu Thr Glu Lys Arg Pro Phe Ile Asp Glu Ala Lys Arg                  85 90 95 Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr Arg Pro             100 105 110 Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr Leu Pro         115 120 125 Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Ala Ser Gly Val Gly     130 135 140 Val Gly Ala Gly Leu Gly Ala Gly Val Asn Gln Arg Met Asp Ser Tyr 145 150 155 160 Ala His Met Asn Gly Trp Ser Asn Gly Ser Ser Ser Met Met Gln Asp                 165 170 175 Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly Ala Ala             180 185 190 Gln Met Gln Pro Met His Arg Tyr Asp Val Ser Ala Leu Gln Tyr Asn         195 200 205 Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr Tyr Ser     210 215 220 Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly Ser Met 225 230 235 240 Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val Val Thr                 245 250 255 Ser Ser Ser Ser Ser Ala Pro Cys Gln Ala Gly Asp Leu Arg Asp             260 265 270 Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro Ala Ala         275 280 285 Pro Ser Arg Leu His Met Ser Gln His Tyr Gln Ser Gly Pro Val Pro     290 295 300 Gly Thr Ala Ile Asn Gly Thr Leu Pro Leu Ser His Met 305 310 315 <210> 4 <211> 954 <212> DNA <213> Homo sapiens <400> 4 atgtacaaca tgatggagac ggagctgaag ccgccgggcc cgcagcaaac ttcggggggc 60 ggcggcggca actccaccgc ggcggcggcc ggcggcaacc agaaaaacag cccggaccgc 120 gtcaagcggc ccatgaatgc cttcatggtg tggtcccgcg ggcagcggcg caagatggcc 180 caggagaacc ccaagatgca caactcggag atcagcaagc gcctgggcgc cgagtggaaa 240 cttttgtcgg agacggagaa gcggccgttc atcgacgagg ctaagcggct gcgagcgctg 300 cacatgaagg agcacccgga ttataaatac cggccccggc ggaaaaccaa gacgctcatg 360 aagaaggata agtacacgct gcccggcggg ctgctggccc ccggcggcaa tagcatggcg 420 agcggggtcg gggtgggcgc cggcctgggc gcgggcgtga accagcgcat ggacagttac 480 gcgcacatga acggctggag caacggcagc tacagcatga tgcaggacca gctgggctac 540 ccgcagcacc cgggcctcaa tgcgcacggc gcagcgcaga tgcagcccat gcaccgctac 600 gacgtgagcg ccctgcagta caactccatg accagctcgc agacctacat gaacggctcg 660 cccacctaca gcatgtccta ctcgcagcag ggcacccctg gcatggctct tggctccatg 720 ggttcggtgg tcaagtccga ggccagctcc agcccccctg tggttacctc ttcctcccac 780 tccagggcgc cctgccaggc cggggacctc cgggacatga tcagcatgta tctccccggc 840 gccgaggtgc cggaacccgc cgcccccagc agacttcaca tgtcccagca ctaccagagc 900 ggcccggtgc ccggcacggc cattaacggc acactgcccc tctcacacat gtga 954 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide # 1 <400> 5 Ile Pro Lys Arg Ile Ile Pro His Ser Arg His Ile   1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide # 2 <400> 6 Ser Pro Thr Ser Ile His Gly Asn Gly Lys Met His   1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide # 3 <400> 7 Pro Pro Lys Arg Ser His His Leu Arg Pro Met Arg   1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide # 4 <400> 8 Gln Ser His Glu Gln Asn Asn Tyr Asn Gln Arg Ser   1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide # 5 <400> 9 Asn Val Pro Thr Val Trp Asp Asn Thr Phe Trp Tyr   1 5 10

Claims (11)

A polypeptide for detecting Sox2 (Sexy determining Region Y) -box2 (SRY) comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: delete The polypeptide for detecting Sox2 according to claim 1, wherein the polypeptide is tagged with at least one detection element selected from the group consisting of a chromogenic enzyme, a fluorescent substance and a radioactive isotope. The polypeptide for detecting Sox2 according to claim 1, wherein the Sox2 is derived from human. 5. The polypeptide for detecting Sox2 according to claim 4, wherein the Sox2 comprises the amino acid sequence of SEQ ID NO: 3. A cancer diagnostic composition comprising the polypeptide of claim 1. delete The cancer diagnostic composition according to claim 6, wherein the cancer is at least one selected from the group consisting of brain tumor, lung cancer, pancreatic cancer, liver cancer, prostate cancer, colon cancer, breast cancer, stomach cancer and rectal cancer. A cancer diagnostic kit comprising the polypeptide of claim 1. delete [Claim 11] The cancer diagnostic kit according to claim 9, wherein the cancer is at least one selected from the group consisting of brain tumor, lung cancer, pancreatic cancer, liver cancer, prostate cancer, colon cancer, breast cancer, stomach cancer and rectal cancer.

Images