KR20140081660A - Polypeptide for detecting PKC-delta and uses thereof - Google Patents

Abstract

The present invention relates to a polypeptide for detecting PKC-delta. Provided are a polypeptide for detecting PKC-delta comprising an amino acid sequence of sequence number 6 or sequence number 7, and a cancer diagnosis system using the same. The polypeptide in the present invention specifically recognizes a minute amount of PKC-delta which is an intracellular signal-regulating protein showing activity in a differentiation process of cancer stem cells and can combine with the PKC-delta. Therefore, the polypeptide can be used as an ultrahigh-sensitivity peptide diagnostic substance which overcomes the limitations of stability and sensitivity of an existing antigen-antibody diagnostic system, and has an effect of offering possibilities of early diagnosis of diseases due to detection of a minute amount of disease indicators and enhancement of treatment efficiency through the early diagnosis of diseases.

Description

Polypeptides for PKC-delta detection and uses thereof < RTI ID = 0.0 > (Polypeptide for < / RTI >

The present invention relates to a polypeptide for detecting PKC-delta, and more particularly to a polypeptide specifically binding to a PKC-delta catalytic domain protein and its use.

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 in order to reduce 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 cell research has shown that the activity of protein kinase C (PKC) -δ, an enzyme involved in the regulation of various signals in cells, is dramatically increased in split radiation treatment, and the activity of activated PKC-δ It has been reported that CD133 expression on the cell surface is increased by intracellular signal regulation. The CD133 is a representative cancer stem cell mark marker expressed in tissues such as brain tumor, lung cancer, pancreatic cancer, liver cancer, prostate cancer, and colon cancer. Previous studies have reported the linkage of PKC-δ to apoptosis of glioma cells (see Peter M. Blumberg et al., Cancer Res 2001; 61: 4612-4619) Has been reported to have an abrupt increase in the activity of PKC-δ and to directly affect the expression of CD133, a marker of cancer stem cells (Su-jae Lee, Journal of Cell Science 124, 30843094). These results suggest that the regulation of PKC-δ activity may regulate and treat cancer stem cells, and early diagnosis of cancer stem cells through early detection of activated PKC-δ may improve the efficiency of cancer treatment. . However, although PKC-δ is attracting attention as a main cause of acquiring the adaptive radiation resistance of cancer cells in the split-radiation treatment, research on the diagnostic method and treatment method using PKC-δ as a labeling substance is still weak, There is a need for further research as a practical diagnosis and treatment.

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 difficulties 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.

It is an object of the present invention to provide a low molecular probe capable of detecting PKC-δ, which is a cancer diagnostic marker, and a cancer diagnosis system using the same.

In order to achieve the above object, one aspect of the present invention provides a polypeptide for detecting PKC-δ (Protein Kinase C-delta) comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO:

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, Since PKC-δ can be specifically recognized and bound, it can be used as a highly sensitive peptide conjugate that overcomes the limitations of stability and sensitivity of existing antigen-antibody diagnostic systems, and can be used for detecting a trace amount of disease- It is possible to prove the possibility of early diagnosis of disease and increase 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, BI: pre-inducible, pre-inducible, pre-inducible) was obtained by transforming Escherichia coli into an expression plasmid for PKC-δ, and purifying the purified PKC-δ protein in cells induced with 0.5 mM IPTG. AI: fraction after induction, So1: soluble protein, InS: insoluble protein, 1-7; PKC-delta catalytic domain protein).
FIG. 2 is a diagram showing a plan view of M13 phage screening for screening a phage peptide binding to a PKC-delta catalytic domain protein. FIG.
FIG. 3 is a graph showing the binding potency of PKC-delta catalytic domain protein analyzed by measuring concentration after amplifying two kinds of polypeptide expression phage identified in the present invention (Peptide # 1 and Peptide # 2 are SEQ ID NO: 6 and SEQ ID NO: No. 7 polypeptide).
FIG. 4 is a graph showing the binding ability of PKC-8 catalytic domain protein to a single polypeptide synthesized by fluorescence.

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

As used herein, the term "diagnosis" means identifying the presence or characteristic of a disease associated with the expression of the protein by measuring the presence or absence of a polypeptide for detecting PKC-delta of the present invention in a biological sample or a tissue sample.

Hereinafter, the present invention will be described in detail.

One. A polypeptide for detection of PKC-δ (Protein Kinase C-delta)

One aspect of the present invention provides a polypeptide for detecting PKC-delta (Protein Kinase C-delta).

Preferably, the polypeptide comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, more preferably the amino acid sequence of SEQ ID NO: 6, and the polypeptide has the amino acid sequence of SEQ ID NO: desirable. However, the polypeptide is not limited thereto, but may be a polypeptide having the activity of specifically binding to the catalytic domain of PKC-delta, particularly PKC-delta expressed in cells such as cancer cells and cancer stem cells, Or a polypeptide 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 the amino acid sequence of SEQ ID NO: 7 . ≪ / RTI >

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

It is preferable that PKC-delta is derived from a human, and PKC-delta comprises a polypeptide having an amino acid sequence of SEQ ID NO: 3. Among the amino acids of SEQ ID NO: 3, the amino acid sequence of the portion corresponding to the amino acid sequence of SEQ ID NO: 4 is the catalytic domain of PKC-delta, and the polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 is specifically It is the part to be combined. That is, the polypeptide specifically binds to a catalytic domain having the amino acid sequence of SEQ ID NO: 4 in the amino acid sequence of PKC-δ.

The polypeptide may be tagged with a detector. The detection element is for easily identifying, detecting, and quantifying whether the polypeptide specifically binds to PKC-δ. The detection element includes a chromogenic enzyme (peroxidase, alkaline phosphatase, etc.), fluorescence (FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots), chromophore and radioactive isotopes ( 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 inventors of the present invention have found that in order to discover a polypeptide as a probe that effectively recognizes PKC-delta, an intracellular signal regulatory protein active in the differentiation process of cancer stem cells, a peptide binding to PKC- Were screened to select low molecular diagnostic peptides. Specifically, a PKC-δ catalytic domain protein was immobilized on plate wells and a phage library in which peptides composed of 12 amino acids were expressed on the surface of M13 phage was added to the peptide library. , Only the phage peptides that specifically bind to the PKC-delta catalytic domain protein were finally selected by extracting the phage peptides binding to the PKC-delta catalytic domain protein. By performing biopanning in which the round was repeated several times in a round, the polypeptide binding to PKC-δ was obtained (see Table 1 and FIG. 2). It is preferable to perform three or more 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: 6 or SEQ ID NO: 7 (see Table 2). The polypeptide selected by the above method has an excellent binding ability to the PKC-delta catalytic domain protein (see FIG. 3), and thus the polypeptide of the present invention can act as a novel probe for recognizing PKC-delta Respectively. In addition, even when the polypeptide is synthesized by attaching a fluorescent substance as a detection substance, it has been confirmed that the polypeptide including the detection element can be effectively used for diagnosis by confirming that it binds effectively to the PKC-delta catalytic domain protein (see FIG. 4 ). Therefore, the polypeptide specifically binding to PKC-delta of the present invention can be usefully used as a low molecular diagnostic probe for detecting PKC-delta protein.

2. Composition for cancer diagnosis

Another aspect of the present invention provides a cancer diagnostic composition comprising a polypeptide for detecting PKC-delta comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

The polypeptide for PKC-delta detection is specifically for detecting the PKC-delta , and is the same as that described in the item " 1. PKC-delta (Protein Kinase C-delta) detection polypeptide ". Therefore, the above description of the polypeptide for PKC-delta detection will be omitted because of the description of the item " 1. PKC-δ (polypeptide for detecting protein kinase C-delta) " and the description will be omitted. Hereinafter, Only the 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 CD133, whose expression level is increased through intracellular signaling of activated PKC-δ, is overexpressed in the tissues of these cancers. Therefore, when an intracellular PKC-delta is detected to be overexpressed, the individual from which the cell is derived can be diagnosed as having a high risk of acquiring or contracting cancer.

When the composition is administered to a subject, the polypeptide in the composition specifically and selectively binds to a PKC-delta catalytic domain protein expressed in cancer stem cells, The position or expression level of the polypeptide bound to the target can be measured. 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 the reaction of the detection substance occurs in the tissue or cells of the subject, PKC-δ is present in excess in the tissue of the subject, and the subject can be diagnosed early in the cancer . 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, peptides binding to the PKC-delta catalytic domain protein overexpressed in cancer stem cells were screened, and it was confirmed that the thus selected peptides could specifically bind to the PKC-delta catalytic domain protein (See Figs. 3 and 4), the composition comprising the polypeptide for detecting PKC-delta 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 detecting PKC-delta comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

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

The above-mentioned "measurement of protein expression level" is a process for confirming the presence and the expression level of a protein expressed in a cancer marker gene in a biological sample to diagnose cancer, wherein a polypeptide specifically binding to the PKC- Can be used to confirm the amount of protein. 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 reactor coated with the polypeptide of SEQ ID NO: 6 or SEQ ID NO: 7, or a wash solution to be used in each reaction step .

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. Accordingly, the kit for detecting a protein contained in the biological sample, in particular, the kit of the present invention can be used for detecting a PKC-delta catalytic domain protein contained in the biological sample.

In a specific example of the present invention, peptides binding to PKC-delta catalytic domain protein were screened by phage display technique and it was confirmed that the selected peptides could specifically bind to PKC-delta catalytic domain protein 4), the kit comprising the polypeptide for detecting PKC-delta of the present invention 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 PKC-δ catalytic domain protein

A PKC-δ catalytic domain gene having the nucleotide sequence of SEQ ID NO: 5 was amplified by performing a polymerase chain reaction (PCR) with forward and reverse primers having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, And inserted into the bacterial expression vector pET-28a (Novagen, USA). Was transformed with SEQ ID NO: 5, the nucleotide sequence is inserted into expression vector expressed in a host E. coli (E. Coli) Rosetta cells as described above, 0.5 mM IPTG, and cultured in the 37 ℃ conditions having an amino acid sequence of SEQ ID NO: 4 Lt; RTI ID = 0.0 > PKC-8 < / RTI > catalytic domain. 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 PKC-8 catalytic domain protein. The PKC-δ catalytic domain was then slowly removed by linear gradient (decreasing urea concentration gradually) coupled to the column to refold the denatured PKC-δ catalytic domain, The domains were refolded and eluted under a 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, a PKC-8 catalytic domain having the amino acid sequence of SEQ ID NO: 4 was obtained at a concentration of 0.5 mg / l.

Selective-Phage Display of Polypeptides Binding to PKC-delta Catalytic Domain

Using the embodiment 1 of SEQ ID NO: 4 obtained in PKC-δ catalytic domain protein and peptide libraries ^{Ph.D TM (phage display peptide library kit} , New England Biolabs (NEB))), Republic of Korea-A-10 2008-0083807 and 10-2012-0132455, a polypeptide sequence that specifically binds to the PKC-8 catalytic domain protein was selected. More specifically, referring to FIG.

In order to screen for a polypeptide specifically binding to PKC-delta catalytic domain protein which is a cancer stem cell marking marker, the PKC-delta catalytic domain protein of SEQ ID NO: 4 obtained in Example 1 was introduced into a 96-well polystyrene plate (Qiagen, after fixing the US), M13 phage peptide library were added to ^TM Ph.D (phage display peptide library kit, New England Biolabs (NEB))) induced a bond with the PKC-δ catalytic domain protein and any polypeptide. Thereafter, the bio-panning procedure was repeated three times to select a phage having 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 Competitive leaching
(More than 10 moles of PKC-δ

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, about 45 phage plasmids were selected to separate and purify M13 phage genomic DNA (single stranded circular DNA) The nucleotide sequence of a polynucleotide encoding a part of a polypeptide which is expressed in a phage surface protein (gp3 minor coat protein) and binds to a PKC-delta catalytic domain protein is revealed. Based on the nucleotide sequence thus identified, the amino acid sequence of the polypeptide that binds to the PKC-delta catalytic domain protein was identified.

As a result, two polypeptides having the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 7, which bind to the PKC-delta catalytic domain protein of SEQ ID NO: 4, were identified as shown in Table 2 below (Table 2).

SEQ ID NO: Peptide sequence Loss rate (%) 6 LMNPNNHPRTPR 89% 7 HMSPNGHPTTEP 11%

Selected polypeptides and PKC-δ Binding force analysis of catalytic domain protein

In order to analyze the binding ability of the polypeptide selected in Example 2, the phage expressing the polypeptide of SEQ ID NO: 6 and SEQ ID NO: 7 was amplified, respectively, and the concentration of the phage solution was determined by titering. Then, the binding strength of the polypeptide was measured by measuring the amount of the phage bound by adding the phage to the plate to which the PKC-δ catalytic domain protein of SEQ ID NO: 4 was bound.

Specifically, the phage expressing the polypeptide of SEQ ID NO: 6 and SEQ ID NO: 7 was added to the plate to which the PKC-δ catalytic domain protein of SEQ ID NO: 4 was bound, respectively, to induce the binding reaction for a predetermined 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 ) To confirm the binding ability of the polypeptide of SEQ ID NO: 6 and SEQ ID NO: 7 to the PKC-delta catalytic domain protein of SEQ ID NO: 4.

As a result, as shown in Fig. 3, the polypeptide of SEQ ID NO: 6 and the polypeptide of SEQ ID NO: 7 had K _D values of 28.16? 2.96? M and 59.4? 8.6? M, All of the polypeptides had excellent binding force at the level of picomolar concentration (rho M) (Fig. 3).

Analysis of Binding Capacity of Single Polypeptide and PKC-δ Catalytic Domain Protein

(FITC: fluorescein isothiocyanate) was attached to the polypeptide sequence of SEQ ID NO: 6, which was confirmed to have high binding ability to the PKC-delta catalytic domain protein of SEQ ID NO: 4 in the phage, Were synthesized.

Of SEQ ID NO: 6 polypeptide -FITC: Ac-LMNPNNHPRTPRGCGK-FITC- NH 2

The N-terminal of the polypeptide of SEQ ID NO: 6 was acetylated to remove the reactivity of the amino terminal, and the amino acid lysine (Lysine, K) was added to attach the FITC to the C-terminal. The GCG sequence was further added to the C-terminal of the polypeptide of SEQ ID NO: 6 in order to maximize the sensitivity of the polypeptide of SEQ ID NO: 6-FITC.

In order to analyze the binding ability of the 'polypeptide-FITC' of SEQ ID NO: 6 to the PKC-δ catalytic domain protein of SEQ ID NO: 4, the polypeptide-FITC 'polypeptide of SEQ ID NO: 6 was substituted with the PKC- The domain protein was diluted by concentration on a fixed plate. Each well was then washed and the dissociation constant (K _D ) of the polypeptide of SEQ ID NO: 6 was analyzed by measuring the absorbance of FITC linked to the polypeptide by means of a fluorescence photometer.

As a result, the polypeptide of SEQ ID NO: 6 has a K _D value of 74.9 16.1 nM, so that the polypeptide of SEQ ID NO: 6 has a high nano-molar concentration (nM) level relative to the PKC-8 catalytic domain protein of SEQ ID NO: (Figure 4).

Test for specificity of FITC-labeled peptides with actual cell lines

The novel peptide sequences selected in Example 2 and tested for binding in Example 3 were subjected to Immunocytochemistry test for confirming specificity and binding force with PKC- ICC test). The cell line used in this example is a U87 glycomag cell line, which is a Glioma cell line. Before and after the fractionated radiation treatment, the cell lines were treated with the same concentration of peptides before and after each split radiation treatment to compare the amount of PKC catalytic domain expression. Cells grown for a period of time were treated with 800 nM FITC-peptide for one day and then washed through PBS buffer solution. Then, 2% BSA was used to block the nonspecific reaction. Finally, to confirm the result of fluorescence microscopy, intracellular nuclear staining was carried out using 4,6-diamidino-2-phenylindole (DAPI) staining.

It was confirmed that the intensity of green fluorescence can be visually distinguished through the peptide labeled with FITC, and the fluorescence intensity after the split radiation treatment is stronger than that before the split radiation treatment (see FIG. 5 (1)). This means that the peptide has a specific binding depending on the degree of protein expression.

In order to select the control group for the specificity test, the experiment was carried out under the same conditions through a peptide having any scramble peptide not selected from the peptide selected in Example 2. As shown in Fig. 5 (2), it can be seen that a peptide having an arbitrary sequence does not bind to a target protein or other substance.

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> Polypeptides for detecting PKC-delta and uses thereof <130> PN130257P <150> 12/0149138 <151> 2012-12-20 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PKC-delta catalytic domain <400> 1 gaattctgga tgccgttc 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PKC-delta catalytic domain <400> 2 ggattcatct tccaggag 18 <210> 3 <211> 676 <212> PRT <213> Homo sapiens <400> 3 Met Ala Pro Phe Leu Arg Ile Ala Phe Asn Ser Tyr Glu Leu Gly Ser   1 5 10 15 Leu Gln Ala Glu Asp Glu Ala Asn Gln Pro Phe Cys Ala Val Lys Met              20 25 30 Lys Glu Ala Leu Ser Thr Glu Arg Gly Lys Thr Leu Val Gln Lys Lys          35 40 45 Pro Thr Met Tyr Pro Glu Trp Lys Ser Thr Phe Asp Ala His Ile Tyr      50 55 60 Glu Gly Arg Val Ile Gln Ile Val Leu Met Arg Ala Ala Glu Glu Pro  65 70 75 80 Val Ser Glu Val Thr Val Gly Val Ser Val Leu Ala Glu Arg Cys Lys                  85 90 95 Lys Asn Asn Gly Lys Ala Glu Phe Trp Leu Asp Leu Gln Pro Gln Ala             100 105 110 Lys Val Leu Met Ser Val Gln Tyr Phe Leu Glu Asp Val Asp Cys Lys         115 120 125 Gln Ser Met Arg Ser Glu Asp Glu Ala Lys Phe Pro Thr Met Asn Arg     130 135 140 Arg Gly Ala Ile Lys Gln Ala Lys Ile His Tyr Ile Lys Asn His Glu 145 150 155 160 Phe Ile Ala Thr Phe Phe Gly Gln Pro Thr Phe Cys Ser Val Cys Lys                 165 170 175 Asp Phe Val Trp Gly Leu Asn Lys Gln Gly Tyr Lys Cys Arg Gln Cys             180 185 190 Asn Ala Ala Ile His Lys Lys Cys Ile Asp Lys Ile Ile Gly Arg Cys         195 200 205 Thr Gly Thr Ala Asp Ser Arg Asp Thr Ile Phe Gln Lys Glu Arg     210 215 220 Phe Asn Ile Asp Met Pro His Arg Phe Lys Val His Asn Tyr Met Ser 225 230 235 240 Pro Thr Phe Cys Asp His Cys Gly Ser Leu Leu Trp Gly Leu Val Lys                 245 250 255 Gln Gly Leu Lys Cys Glu Asp Cys Gly Met Asn Val His His Lys Cys             260 265 270 Arg Glu Lys Val Ala Asn Leu Cys Gly Ile Asn Gln Lys Leu Leu Ala         275 280 285 Glu Ala Leu Asn Gln Val Thr Gln Arg Ala Ser Arg Arg Ser Serp Ser     290 295 300 Ala Ser Ser Glu Pro Val Gly Ile Tyr Gln Gly Phe Glu Lys Lys Thr 305 310 315 320 Gly Val Ala Gly Glu Asp Met Gln Asp Asn Ser Gly Thr Tyr Gly Lys                 325 330 335 Ile Trp Glu Gly Ser Ser Lys Cys Asn Ile Asn Asn Phe Ile Phe His             340 345 350 Lys Val Leu Gly Lys Gly Ser Phe Gly Lys Val Leu Leu Gly Glu Leu         355 360 365 Lys Gly Arg Gly Glu Tyr Phe Ala Ile Lys Ala Leu Lys Lys Asp Val     370 375 380 Val Leu Ile Asp Asp Asp Val Glu Cys Thr Met Val Glu Lys Arg Val 385 390 395 400 Leu Thr Leu Ala Ala Glu Asn Pro Phe Leu Thr His Leu Ile Cys Thr                 405 410 415 Phe Gln Thr Lys Asp His Leu Phe Phe Val Met Glu Phe Leu Asn Gly             420 425 430 Gly Asp Leu Met Tyr His Ile Gln Asp Lys Gly Arg Phe Glu Leu Tyr         435 440 445 Arg Ala Thr Phe Tyr Ala Glu Ile Met Cys Gly Leu Gln Phe Leu     450 455 460 His Ser Lys Gly Ile Ile Tyr Arg Asp Leu Lys Leu Asp Asn Val Leu 465 470 475 480 Leu Asp Arg Asp Gly His Ile Lys Ile Ala Asp Phe Gly Met Cys Lys                 485 490 495 Glu Asn Ile Phe Gly Glu Ser Arg Ala Ser Thr Phe Cys Gly Thr Pro             500 505 510 Asp Tyr Ile Ala Pro Glu Ile Leu Gln Gly Leu Lys Tyr Thr Phe Ser         515 520 525 Val Asp Trp Trp Ser Phe Gly Val Leu Leu Tyr Glu Met Leu Ile Gly     530 535 540 Gln Ser Pro Phe His Gly Asp Asp Glu Asp Glu Leu Phe Glu Ser Ile 545 550 555 560 Arg Val Asp Thr Pro His Tyr Pro Arg Trp Ile Thr Lys Glu Ser Lys                 565 570 575 Asp Ile Leu Glu Lys Leu Phe Glu Arg Glu Pro Thr Lys Arg Leu Gly             580 585 590 Val Thr Gly Asn Ile Lys Ile His Pro Phe Phe Lys Thr Ile Asn Trp         595 600 605 Thr Leu Leu Glu Lys Arg Arg Leu Glu Pro Pro Phe Arg Pro Lys Val     610 615 620 Lys Ser Pro Arg Asp Tyr Ser Asn Phe Asp Gln Glu Phe Leu Asn Glu 625 630 635 640 Lys Ala Arg Leu Ser Tyr Ser Asp Lys Asn Leu Ile Asp Ser Met Asp                 645 650 655 Gln Ser Ala Phe Ala Gly Phe Ser Phe Val Asn Pro Lys Phe Glu His             660 665 670 Leu Leu Glu Asp         675 <210> 4 <211> 344 <212> PRT <213> Homo sapiens <400> 4 Thr Tyr Gly Lys Ile Trp Glu Gly Ser Ser Lys Cys Asn Ile Asn Asn   1 5 10 15 Phe Ile Phe His Lys Val Leu Gly Lys Gly Ser Phe Gly Lys Val Leu              20 25 30 Leu Gly Glu Leu Lys Gly Arg Gly Glu Tyr Phe Ala Ile Lys Ala Leu          35 40 45 Lys Lys Asp Val Val Leu Ile Asp Asp Asp Val Glu Cys Thr Met Val      50 55 60 Glu Lys Arg Val Leu Thr Leu Ala Ala Glu Asn Pro Phe Leu Thr His  65 70 75 80 Leu Ile Cys Thr Phe Gln Thr Lys Asp His Leu Phe Phe Val Met Glu                  85 90 95 Phe Leu Asn Gly Gly Asp Leu Met Tyr His Ile Gln Asp Lys Gly Arg             100 105 110 Phe Glu Leu Tyr Arg Ala Thr Phe Tyr Ala Glu Ile Met Cys Gly         115 120 125 Leu Gln Phe Leu His Ser Lys Gly Ile Ile Tyr Arg Asp Leu Lys Leu     130 135 140 Asp Asn Val Leu Leu Asp Arg Asp Gly His Ile Lys Ile Ala Asp Phe 145 150 155 160 Gly Met Cys Lys Glu Asn Ile Phe Gly Glu Ser Arg Ala Ser Thr Phe                 165 170 175 Cys Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile Leu Gln Gly Leu Lys             180 185 190 Tyr Thr Phe Ser Val Asp Trp Trp Ser Phe Gly Val Leu Leu Tyr Glu         195 200 205 Met Leu Ile Gly Gln Ser Pro Phe His Gly Asp Asp Glu Asp Glu Leu     210 215 220 Phe Glu Ser Ile Arg Val Asp Thr Pro His Tyr Pro Arg Trp Ile Thr 225 230 235 240 Lys Glu Ser Lys Asp Ile Leu Glu Lys Leu Phe Glu Arg Glu Pro Thr                 245 250 255 Lys Arg Leu Gly Val Thr Gly Asn Ile Lys Ile His Pro Phe Phe Lys             260 265 270 Thr Ile Asn Trp Thr Leu Leu Glu Lys Arg Arg Leu Glu Pro Pro Phe         275 280 285 Arg Pro Lys Val Lys Ser Pro Arg Asp Tyr Ser Asn Phe Asp Gln Glu     290 295 300 Phe Leu Asn Glu Lys Ala Arg Leu Ser Tyr Ser Asp Lys Asn Leu Ile 305 310 315 320 Asp Ser Met Asp Gln Ser Ala Phe Ala Gly Phe Ser Phe Val Asn Pro                 325 330 335 Lys Phe Glu His Leu Leu Glu Asp             340 <210> 5 <211> 1031 <212> DNA <213> Homo sapiens <400> 5 acctacggca agatctggga gggcagcagc aagtgcaaca tcaacaactt catcttccac 60 aaggtcctgg gcaaaggcag cttcgggaag gtgctgcttg gagagctgaa gggcagagga 120 gagtactttg ccatcaaggc cctcaagaag gatgtggtcc tgatcgacga cgacgtggag 180 tgcaccatgg ttgagaagcg ggtctgacac ttgccgcaga gaatcccttt ctcacccacc 240 tcatctgcac cttccagacc aaggaccacc tgttctttgt gatggagttc ctcaacgggg 300 gggacctgat gtaccacatc caggacaaag gccgctttga actctaccgt gccacgtttt 360 atgccgctga gataatgtgt ggactgcagt ttctacacag caagggcatc atttacaggg 420 acctcaaact ggacaatgtg ctgttggacc gggatggcca catcaagatt gccgactttg 480 ggatgtgcaa agagaacata ttcggggaga gccgggccag caccttctgc ggcacccctg 540 actatatcgc ccctgagatc ctacagggcc tgaagtacac attctctgtg gactggtggt 600 ctttcggggt ccttctgtac gagatgctca ttggccagtc ccccttccat ggtgatgatg 660 aggatgaact cttcgagtcc atccgtgtgg acacgccaca ttatccccgc tggatcacca 720 aggagtccaa ggacatcctg gagaagctct ttgaaaggga accaaccaag aggctgggag 780 tgacgggaaa catcaaaatc caccccttct tcaagaccat aaactggact ctgctggaaa 840 agcggaggtt ggagccaccc ttcaggccca aagtgaagtc acccagagac tacagtaact 900 ttgaccagga gttcctgaac gagaaggcgc gcctctccta cagcgacaag aacctcatcg 960 actccatgga ccagtctgca ttcgctggct tctcctttgt gaaccccaaa ttcgagcacc 1020 tcctggaaga t 1031 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide # 1 <400> 6 Leu Met Asn Pro Asn Asn His Pro Arg Thr Pro Arg   1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide # 2 <400> 7 His Met Ser Pro Asn Gly His Pro Thr Thr Glu Pro   1 5 10

Claims (12)

A polypeptide for detecting PKC-δ (Protein Kinase C-delta) comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7. The polypeptide according to claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 6. The polypeptide 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 according to claim 1, wherein the PKC-delta is derived from human. 5. The polypeptide according to claim 4, wherein the PKC-delta comprises the amino acid sequence of SEQ ID NO: 3. The polypeptide of claim 1, wherein said polypeptide binds to a catalytic domain having the amino acid sequence of SEQ ID NO: 4. A cancer diagnostic composition comprising the polypeptide of claim 1. 8. The composition for diagnosing cancer according to claim 7, wherein the composition comprises the amino acid sequence of SEQ ID NO: 6. The cancer diagnostic composition according to claim 7, wherein the cancer is any one or more selected from the group consisting of brain tumor, lung cancer, pancreatic cancer, liver cancer, prostate cancer, colon cancer, breast cancer, gastric cancer and rectal cancer. A cancer diagnostic kit comprising the polypeptide of claim 1. The cancer diagnostic kit according to claim 10, wherein the kit comprises the amino acid sequence of SEQ ID NO: 6. [Claim 11] The cancer diagnostic kit according to claim 10, wherein the cancer is any one or more 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.

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