KR20160132292A

KR20160132292A - Multi Peptide Conjugated Probe for Detecting of CD133

Info

Publication number: KR20160132292A
Application number: KR1020150064842A
Authority: KR
Inventors: 윤문영; 이상춘; 조준행
Original assignee: 한양대학교 산학협력단
Priority date: 2015-05-08
Filing date: 2015-05-08
Publication date: 2016-11-17
Also published as: KR101726212B1

Abstract

The present invention relates to a polypeptide probe for detecting CD133 functioning as a neuroglioma stem cell marker. The use of the polypeptide probe of the present invention enables the effective detection of neuroglioma stem cells. To this end, the polypeptide probe includes: (a) a polypeptide backbone made of an amino acid sequence represented by general formula 1, HxGC(GK)yC; and (b) a plurality of monopeptide probes coupled to the backbone and made of a first sequence on the sequence list.

Description

Multi-Peptide Conjugated Probe for Detection of CD133 < RTI ID = 0.0 >

The present invention relates to a multiple peptide probe for the detection of glioma stem cell marker CD133.

Although there have been significant technological advances in the diagnosis and treatment of cancer for the past decade, cancer mortality is still high. Currently, the biggest problem in chemotherapy is that cancer cells that are resistant to chemotherapy remain, and recurrence of cancer occurs.

Cancer stem cells associated with recurrence of these cancers are defined as specific cell populations within the tumor that have both self-renewal and differentiation into different types of cells. According to cancer stem cell theory, cancer stem cells can form new cancer masses, so if tumor cells that are not cancerous stem cells are completely removed by surgery and cancer stem cells remain, even if cancer chemotherapy is performed, the cancer recurs again.

More specifically, there are adult stem cells unique to each organ. These cells are essential for regenerating and maintaining organs when organs are damaged. However, there are cancer stem cells in cancer tissues that play a role in maintaining cancer tissues like normal organs, and it is known that cancer cells have a profound effect on recurrence or metastasis by participating in regenerating cancer cells that have been reduced after cancer treatment. Cancer stem cells are known to be present in the tissues of solid tumors such as breast cancer and brain cancer, as well as blood cancers such as leukemia. Therefore, attention should be paid to cancer treatment targeting cancer stem cells, which occupy only a small part of cancer tissues rather than conventional cancer treatments that have been used only for general cancer cells, which constitute the majority of cancer tissues, .

Based on carbon nanotubes developed by CH Wang et al. (Photothermolysis of glioblastoma stem-like cells targeted by carbon nanotubes conjugated with CD133 monoclonal antibody) There is one CD133 recognition / therapy technique. CD133 exists in the extracellular membrane and is a cluster of differentiation (CD) family of proteins well known as a protein whose expression is induced when cells grow. CD133 is the first protein identified by markers of these markers after it has been found that cells with similar characteristics to stem cells are found in glioma, a malignant brain tumor. CH Wang et al. Used GBM-CD133 ⁺ , a cell line with autologous differentiation, to grow the tumor in a mouse tissue, and then analyzed for the removal of the tumor when treated with carbon nanotubes and near-infrared radiation. As a result, the carbon nanotubes bound to the tumor tissue through the antibody effectively removed the surrounding GBM-CD133 + cells upon exposure to NIR. This demonstrates the role of CD133 as a cancer stem cell marker and is also effective in target diagnosis. However, this suggests that treatment with GBM-CD133 ⁺ in a mouse can provide early treatment for early detection of CD133- ^positive peptides.

In addition to the above-mentioned achievements of C. H. Wang et al., Diagnostic methods using antibodies as a general approach have been widely known, and various studies have been conducted from diagnosis and treatment systems using antibody-carbon nanotubes (CNTs). However, cancer stem cells are known to be present in trace amounts in common cancer cells scattered within cancer tissues. The technology that can effectively diagnose CD133 present in such a very small amount of cancer stem cells provides a possibility to selectively target only cancer stem cells in cancer cells. In addition, studies on the diagnosis of CD133 protein, a stem cell-specific marker derived from glioma glioma, provide a clue to identify the existence of cancer stem cells and to solve various problems encountered in the current chemotherapy.

Through these studies on cancer stem cells, it is possible to develop an effective therapeutic agent by early detection of cancer stem cells capable of differentiating into cancer cells as a side effect of early chemotherapy. In addition, based on this, it is possible to develop a new concept treatment and treatment method which can prevent recurrence of cancer, thereby contributing to diagnosis, prognosis, and prediction of cancer patient disease.

The combination of a conventional target-sensing probe and a new, versatile imaging technique with image technology and anticancer materials has provided an opportunity to improve treatment for early detection of cancer and to develop potential diagnostic methods. In order to develop a probe for the early detection of cancer, a probe having a high affinity and selectively attached to early cancer cells is to be used for early diagnosis of cancer. There have also been many studies in the field of genes. Based on the results of genetic analysis, genetic modification research is underway to prevent the function of cancer genes from being cut off or expressed.

According to the related art, there is a method of using PCR (Polymerase Chain Reaction) as the most common method for diagnosing a 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. In addition, this method uses the inherent nucleotide sequence of the gene to analyze the unique nucleotide sequence of the diagnostic object, and broadens the application range through securing a comprehensive gene information database through the recent Human Genome Project can do. In addition, the PCR method uses single-strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) methods to identify genetic mutations of the amplified genes using various probes And provides useful information in conjunction with them.

Cancer marker markers capable of detecting and early detection of cancer are under investigation for antibodies capable of identifying early development of tumors. Blood is the most representative body fluids and is a sort of 'in-vivo barometer' that changes sensitively during disease progression and progression. Plasma proteases are predicted to be more than about 50,000 constituents, and the concentration of each protein component is also very variable (1-10-12), suggesting an antigen-antibody reaction with a detection limit of about 10-6-10-9 Detection and quantitative analysis of biomarker proteins present in low / medium concentration is very difficult. However, diagnostic probes having properties such as high sensitivity, rapidity and high physicochemical stability, sensitivity, specificity, and applicability have not been proposed yet. Since antibodies have high specificity for cancer, many studies have been carried out for the discovery of cancer and drug delivery, but antibodies in vivo cause problems in generating immunoreactivity.

Also, in the above-mentioned prior art diagnostic method using PCR, there is an advantage that it has high sensitivity in obtaining a small amount of a unique base sequence possessed only by a diagnostic object, but there are problems such as difficulty in pretreatment of a sample and difficulty in field adaptability There are limits to the diagnostic system.

In this respect, the recent ELISA (enzyme-linked immunosorption assay) method determines the increase and the change of expression of a specific protein caused by disease. In order to recognize such a specific protein, an antibody that specifically binds to these specific proteins is used as a diagnostic probe to separate a diagnostic target from a biological sample such as blood or urine, and an enzyme-antibody reaction (ECL) is used for this purpose. However, the sensitivity of the nano-mol (10 ^-9 M) reaction sensitivity is lower than that of the PCR method, despite the advantage of being able to target in vivo proteins.

As part of related research that can overcome the above disadvantages, surface enhanced Raman spectroscopy (SERS) and bio-bar code technology incorporating nanotechnology (NT) based on current diagnosis method PCR and ELISA method have been developed , And femtomolar (10 ^-15 M) levels, respectively. Both of these methods are based on the signal properties (narrow Raman bands: low sensitivity to moisture, oxygen and dyes; and low optical reflectance) of Raman spectroscopy and high sensitivity corresponding to fluorescence analysis Amplification of the femtomole-level diagnostic object has become possible, and an approach to prostate specific antigen (PSA) using an easy sample such as blood has been proposed. However, these methods are based on a system using an antibody that recognizes a biomarker (a diagnostic target). All of these methods are limited not only in the number of binding due to the size limitation due to the physical and chemical structure of the antibody, And shows instability due to structural modification under reaction.

Therefore, it is necessary to develop new low molecular probe differentiated from diagnostic system using existing antibody.

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 sought to develop a novel probe targeting CD133, a glioma-specific stem cell-specific marker. As a result, when a plurality of single peptide probes that specifically bind to CD133 are bound to a polypeptide backbone having a predetermined amino acid sequence, it is possible to provide a multiple peptide probe having a greatly enhanced sensitivity compared to a single peptide probe, Thereby completing the invention.

Accordingly, it is an object of the present invention to provide a multiple peptide probe for CD133 targeting as a glioma stem cell-specific marker.

Another object of the present invention is to provide a glioma stem cell detection kit comprising the above-mentioned multiple peptide probe.

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 a method for detecting a polypeptide comprising the steps of: (a) a polypeptide backbone consisting of the amino acid sequence represented by the following general formula (1); and (b) a plurality of single peptide probes consisting of the sequence listing The present invention provides a multiple peptide probe for CD133 targeting as a glioma stem cell specific marker comprising:

1

HxGC (GK) yC;

In the general formula (1), x is an integer of 2-5 and y is an integer of 3-8.

The amino acid sequence of the single peptide probe consisting of the first sequence of the present invention is the M13 page peptide library (peptide library-PhD ^™ phage display peptide lirary kit, manufactured by New England Biolabs (NEB) , Which has about 2.7 × 10 ⁹ amino acid sequences) to find a sequence that binds to CD133, a glioma-specific stem cell-specific marker. The "single peptide probe" or "single peptide" of the present invention is a low molecular peptide having a small size, which is three-dimensionally stabilized and has an advantage of easily passing through the mucosa and recognizing a molecular target even in deep tissues. In addition, the low-molecular single peptide according to the present invention is relatively simple in mass production and has little toxicity. In addition, the low-molecular peptide according to the present invention has a high binding strength to a target substance and does not undergo denaturation even during heat / chemical treatment. Also, because of its small size, it can be used as a fusion protein by attaching it to other proteins.

The peptides of the present invention can be produced by chemical synthesis known in the art. Representative methods include, but are not necessarily limited to, liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry. The peptides of the present invention can also be produced by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, for example, using automated DNA synthesizers (such as those sold by Biosearch or Applied Biosystems). The constructed DNA sequence is operatively linked to the DNA sequence and contains one or more expression control sequences (e.g., promoters, enhancers, etc.) that regulate the expression of the DNA sequence , And the host cells are transformed with the recombinant expression vector formed therefrom. The resulting transformant is cultured under appropriate medium and conditions so that the DNA sequence is expressed, and the substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery can be performed using methods known in the art (e.g., chromatography). By "substantially pure peptide" herein is meant that the peptide according to the invention is substantially free of any other proteins derived from the host. Genetic engineering methods for peptide synthesis of the present invention can be found in the following references: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N. Y., Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif., 1991; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080, 1990. The peptides of the present invention are capable of known denaturation for eliminating the reactivity of the amino terminal of the N-terminus, for example, acetylation, no.

The biological functional equivalents that can be included in the scope of the multiple peptide probe of CD133, which is a glioma-specific stem cell-specific marker of the present invention, include a single peptide probe contained in the multiple peptide probe of the present invention and an amino acid sequence having an equivalent biological activity It will be apparent to those skilled in the art that the present invention will be limited to including variations.

Such amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are both positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing the mutation, the hydrophobic index of the amino acid can be considered. Each amino acid is assigned a hydrophobic index according to its hydrophobicity and charge: isoruicin (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in imparting the interactive biological function of peptides. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When the mutation is introduced with reference to the hydrophobic index, substitution is made between amino acids showing preferably a hydrophobic index difference of within 2, more preferably within 1, even more preferably within 0.5.

On the other hand, it is also well known that the substitution between amino acids with similar hydrophilicity values leads to peptides with homogeneous biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartate (+3.0 ㅁ 1); Glutamate (+3.0 ㅁ 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ㅁ 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoru Isin (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When the mutation is introduced with reference to the hydrophilicity value, the substitution is carried out between amino acids showing preferably a hydrophilic value difference of within 2, more preferably within 1, even more preferably within 0.5.

Amino acid exchange in peptides that do not globally alter the activity of the molecule is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

The single peptide contained in the CD133-labeled multiple peptide probe of the present invention, which is a glioma-specific stem cell-specific marker of the present invention, has a sequence substantially identical to the sequence described in the sequence listing And the like. The above substantial identity is determined by aligning the single peptide probe sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art, Or more homology, and more preferably 90% or more homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

The above-mentioned "single peptide probe" of the present invention is provided in a form bound to the "polypeptide backbone " The "polypeptide backbone" of the present invention corresponds to the sequence represented by the general formula 1 described above. In the sequence of the general formula 1, "Hx (x is an integer of 2-5)" corresponds to a sequence for adhering and fixing the backbone on the column. Specifically, for example, when using a Ni column, It can be effectively fixed. The "H" indicates histidine, and the Hx indicates that two to five histidine are continuously bonded. The two "C" s included in the above general formula are cysteines and serve to form a cyclic structure through the disulfide bond between -SH residues in the side chain. The above "K" represents lysine and serves to bind a single peptide probe using an amino group of a side chain. The "G" represents glycine, and when a single peptide probe is bound to the lysine, . This will be described in more detail below.

In the general formula 1, y is an integer of 3 to 8, and the number of lysine contained in the polypeptide backbone is determined according to the value of y, thereby determining the number of single peptide probes which can be bound to the maximum. The number of lysine contained in the polypeptide backbone of the present invention may be 3 to 8, preferably 4 to 6, depending on the y value.

The term "multiple peptide probe" of the present invention is defined to mean a complex in which two or more of the single peptide probes described above are bound.

In one embodiment of the present invention, the polypeptide backbone of the present invention forms a cyclic structure through the disulfide bond between two cysteines contained in the backbone. The present inventors have confirmed that when a cyclic structure is formed as compared with a linear peptide structure in which no disulfide bond is formed between two cysteines, the binding ability of a single peptide probe bound to the polypeptide backbone can be further increased.

In one embodiment of the present invention, the amino acid sequence represented by the general formula 1 of the present invention is composed of the first sequence of the sequence listing. The first sequence of the sequence listing is represented by HHHHGCGKGKGKGKGKC (N-terminal to C-terminal).

In order to easily identify, detect and quantify whether the multiple peptide probe of the present invention binds to glioma stem cells, a single peptide contained in the multiple peptide probe of the present invention may be provided in a labeled state. That is, they may be provided by linking (e.g., covalently binding or bridging) to a detectable label. The link may be formed directly between a single peptide of the present invention and a detectable label, but may also be formed through a linker. The linker can be any known in the art, and the sequence of a suitable peptide linker can be selected in consideration of the following factors: (a) the ability to be applied to a flexible extended conformation; (b) the ability to not create a secondary structure that interacts with the epitope; And (c) the absence of a hydrophobic moiety or moiety having charge capable of reacting with the epitope.

The detectable label may be a chromogenic enzyme such as a peroxidase, an alkaline phosphatase, a radioactive isotope such as 124I, 125I, 111In, 99mTc, 32P, 35S, a chromophore, , A luminescent material or a fluorescent material (e.g., FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots) .

Similarly, the detectable label may be an antibody epitope, a substrate, a cofactor, an inhibitor or an affinity ligand. Such labeling may be performed during the synthesis of the peptide of the present invention, or may be performed in addition to the peptide already synthesized.

If a fluorescent substance is used as a detectable marker, glioma cells can be detected by fluorescence tomography (FMT). For example, when a peptide of the present invention labeled with a fluorescent substance is administered into blood, fluorescence by peptide can be observed by fluorescence tomography. If fluorescence is observed, it is diagnosed as glioma.

The target of the peptide of the present invention, "CD133" is a CD (cluster of differentiation) family protein which is present in the extracellular membrane and is known as a protein whose expression is induced when the cell grows. It is known as a marker marker of cancer stem cells in glioma, a malignant brain tumor.

On the other hand, brain tumor is one of the cancer with the highest mortality rate. It is divided into 4 stages by histological characteristics and specific markers. The most frequent malignant brain tumor of the glioblastoma multiforme is resistant to radiotherapy and chemotherapy, and the median survival is only 14.6 months. In recent years, it has been reported that there are a few cells with similar characteristics to stem cells in gliomas (Identification of a cancer stem cell in human brain tumors, (2003), CANCER RESEARCH 63, 5821-5828).

In one embodiment of the present invention, the polypeptide backbone of the present invention forms a cyclic structure through the disulfide bond between two cysteines contained in the backbone. The present inventors have found that the binding of single peptide probes to a polypeptide backbone of a cyclic structure results in a further increase in sensitivity compared to binding to a chimeric polypeptide backbone.

In one embodiment of the present invention, y is 5 in the general formula 1 of the present invention. The polypeptide backbone when y is 5 can be described as a penta unit (PU) backbone. Multiple peptide probes in which 2-5, preferably 4-5 and most preferably 5 single peptide probes are attached to the pentameric backbone can be referred to as penta unit conjugated probes (PUCP).

In one embodiment of the present invention, the amino acid sequence represented by the general formula 1 of the present invention is composed of the second sequence of the sequence listing. The second sequence of the sequence listing represents an amino acid sequence of general formula 1 in the present specification, wherein x is 4 and y is 5. [

In one embodiment of the present invention, the multiple peptide probe of the present invention comprises a first functional group forming an amide bond with an amine group contained in the side chain of the lysine of the polypeptide backbone, and a second functional group forming an amide bond Further comprising both functional linkers comprising functional groups. As used herein, the term "first functional group" means a functional group capable of forming an amide bond with an amine group as described above, and is not particularly limited as long as it is a conventionally known functional group. As used herein, the term "second functional group" means a functional group capable of forming a bond with a single peptide probe of the present invention and capable of forming a disulfide bond or an amide bond according to a single peptide probe sequence.

As used herein, "bifunctional linker" means a linker comprising both the polypeptide backbone of the present invention and a respective functional group capable of forming a bond with a single peptide probe, and includes a linkage between a protein or a peptide Any linker can be used, and is not particularly limited. Specifically, for example, the linker of the present invention may be selected from the group consisting of N-succinimidyl iodoacetate, N-Hydroxysuccinimidyl Bromoacetate, m-maleimidobenzoyl Maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-maleimidobutyryloxysuccinamide ester, N-maleimidobutyryloxysulfosuccinamide ester, E-maleimidocaproic acid hydrazide 占 H HCl, , [N- (E-maleimidocaproyloxy) -succinamide]), [N-maleimidocaproyloxy) -sulfosuccinamide] ([N- maleimidocaproyloxy) -sulfosuccinamide]), maleimidop Maleimidopropionic acid N-hydroxysuccinimide ester, maleimidopropionic acid N-hydroxysulfosuccinimide ester, maleimidopropionic acid, maleimidopropionic acid, maleimidopropionic acid, maleimidopropionic acid, maleimidopropionic acid, N-succinimidyl-3- (2-pyridyldithio) propionate, N-succinimidyl-4- (iodo) Succinimidyl- (4-iodoacetyl) aminobenzoate, succinimidyl- (N-maleimidomethyl) cyclohexane-1-carboxylate, carboxylate, succinimidyl-4- (p-maleimidophenyl) butyrate, sulfosuccinimidyl- (4-iodoacetyl) aminobenzoate -iodoacetyl) aminobenzoate, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-car (P-maleimidophenyl) butyrate, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl- m-maleimidobenzoic acid hydrazide 占 Cl HCl, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid hydrazide 占 Cl H (4- (N-Maleimidomethyl) cyclohexane- (4-N-maleimidophenyl) butyric acid hydrazide.HCl), N-succinimidyl 3- (4-N-maleimidophenyl) (2-pyridyldithio) propionate, bis (sulfosuccinimidyl) suberate, 1,2-di [3 ' - (2'-pyridyldithio) propionamido] butane, 1,2-di [3 '- (2'pyridyldithio) propionamido] butane, Dissuccinimidyl Suberate, Dissuccinimidyl Tartarate (Disulfosuccinimdiyl tartarate), dithio-bis- (succinimidyl propionate), 3,3'-dithio-bis (thiophene) (3,3'-sulfosuccinimidyl-propionate), ethylene glycol bis (succinimidyl succinate) and ethylene glycol bis (sulfosuccinimidyl-propionate) (Ethylene Glycol bis (Sulfosuccinimidylsuccinate)). &Lt; / RTI >

In one embodiment of the present invention, the bond between the single peptide probe of the present invention and the second functional group of the linker of the present invention is a disulfide bond or an amide bond. When the bond between the single peptide probe of the present invention and the second functional group of the linker of the present invention is a disulfide bond, the single peptide probe of the present invention has a disulfide bond at one end of the "peptide sequence region for binding to the target" Linking oligopeptide moiety "that includes the amino acid cysteine (Cys) for < / RTI > The amino acid sequence of the linker-binding oligopeptide region is not particularly limited as long as it includes cysteine, and may be selected in consideration of the following factors: (a) ability to be applied to flexible extended conformation; (b) the ability to not create a secondary structure that interacts with the epitope; And (c) the absence of a hydrophobic moiety or moiety having charge capable of reacting with the epitope. Preferably glycine (Gly), asparagine (Asn) and / or serine (Ser) residues in addition to cysteine. Other neutral amino acids such as Thr and Ala may also be included in the "linker binding oligopeptide site" sequence. Suitable amino acid sequences for the linker-binding oligopeptide moieties described above may be comprised of 1-50 amino acid residues, see Maratea et al., Gene 40: 39-46 (1985); Murphy et al., Proc. Natl. Acad Sci. USA 83: 8258-8562 (1986); U.S. Patent Nos. 4,935,233, 4,751,180, and 5,990,275. Specifically, for example, an amino acid sequence such as -GCG used in one embodiment of the present invention can be used.

According to another aspect of the present invention, there is provided a glioma stem cell detection kit comprising the above-described multiple peptide probe. The glioma stem cell detection kit of the present invention uses the multi-peptide probe according to another embodiment of the present invention described above, and redundant contents are omitted in order to avoid the excessive complexity described in the present specification.

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

(a) The present invention provides a multiple peptide probe for CD133 targeting as a glioma-specific stem cell-specific marker.

(b) The present invention provides a glioma stem cell detection kit comprising the above-mentioned multiple peptide probe.

(C) Using the multiple peptide probe of the present invention, it is possible to effectively detect glioma stem cells.

Figure 1 shows the gene cloning results of the glioma glioma stem cell marker marker CD133 ECD. In FIG. 1 (a), P represents the CD133 ECD gene. In FIG. 1 (b), P1 and P2 are inserted into the expression vector pET-28a before the restriction enzyme treatment (P1) (P2) after treatment. It was confirmed that 873 bp of the gene encoding CD133 ECD was correctly inserted into the expression vector. M in Fig. 1 represents a molecular weight marker.
2 shows the protein expression ability and protein purification of the brain glioma stem cell marker marker CD133 ECD. In FIG. 2 (a), BI (before induction) represents the protein overexpressed at 18 ° C in O / N (overnight incubation) before adding IPTG. The ratio of soluble protein (Sol) to insoluble protein (InS) is shown in FIG. 2 (a). It was confirmed that the CD133 protein was expressed in an insoluble form. FIG. 2 (b) shows that CD133 protein was selected through affinity chromatography by performing a denaturing condition purification process to purify CD133 expressed in an insoluble protein form. F3-F11 represents the protein collected through affinity chromatography.
Figure 3 shows a plan view of M13 page screening for screening page peptides binding to CD133 protein. CD133 was immobilized on each well of the plate ((1) in FIG. 3), and a page library expressing peptides composed of 12 amino acids was added to the surface of M13 (FIG. 3 (2) And binding time are applied, the page peptide binding to the CD133 protein even under extreme conditions remains in the well ((3) in FIG. 3). Finally, when this is extracted, only the page peptide that specifically binds to the CD133 protein is finally obtained ((4) in FIG. 3). In FIG. 3, (1) to (4) are referred to as one round, and repeating the above process is called biopanning. The present inventors carried out three rounds of bio-panning.
Figure 4 shows each round of bio-panning conditions for detecting page peptides with high specificity and binding affinity for the CD133 catalytic domain protein. Increasing the ratio of NaCl to Tween-20 per round, while shortening the page peptide binding time, detected page peptide binding to the CD133 protein even under extreme conditions.
FIG. 5 shows the result of securing a total of 35 page plaques binding to CD133 protein. The 35 page plaques were classified into four different sequences of peptides.
Figure 6 shows a total of 35 page plaques binding to CD133 protein: 35 page plaques were classified into four different sequences of peptides. All four peptides were found to bind effectively to CD133 protein at the picomole level.
FIG. 7 shows the result of performing binding assay with CD133 protein, synthesizing a secondarily selected peptide sequence among the pages binding to CD133 protein, with a fluorescent probe. CD133-GCGK-FITC was found to have strong binding with the target substance at the nanomolar (nM) level.
Figure 8 shows a schematic diagram of multiple peptide probe synthesis for CD133 detection. 8 (a) shows PUCP in which the pyridyldithiol group is activated by using the reactivity of the succinimidyl group of SPDP, which is a both functional linker. 8 (b) shows a schematic diagram of the PUCP structure, which is an example of multiple peptide probes for the reaction of cysteine amino acid residues with -SH using the activity of the pyrimidyl group and the final synthesized CD133 detection. 8 (c) is a sequence diagram of the synthesized PUCP. The blue cysteine residue forms a cyclic PUCP with a cis-terminal cysteine disulfide bond, and Peptide * specifically binds to CD133 Is a peptide probe that functions to bind to a target substance.
Figure 9 shows the results of comparative analysis of the binding strength of a single peptide and PUCP binding to CD133. FIG. 9 (a) shows binding force with CD133 protein for a single peptide, (b) shows binding force with CD133 protein against PUCP, (c) shows binding force with CD133 protein against PDPP, d) shows a table comparing the binding strength between each probe and the CD133 protein.
Fig. 10 shows the binding efficiency analysis results of single peptide, PUCP, and PDPP with CD133 expressing cell line using cell immuno staining. (a) The binding to each probe (200 nM) was confirmed by a cell immunochemical method using CD133 protein in U87 neuronal glioma cells. It was confirmed that a single peptide and PUCP have similar fluorescence signals and bind to each other. In addition, each of the probes showed fluorescence signals similar to those possessed by the antibodies, and thus proved useful as diagnostic probes for PUCP and peptides. (b) Each of the probes (IgG-FITC, sc-peptide, antibody, single peptide, PDPP, PUCP) at the same concentration was treated with the target cells and the fluorescence signal was measured for signal analysis. PUCP emitted a weak signal compared to the PDPP formed of poly-D-lysine polymer, but it emitted a strong fluorescence signal compared to other probes.
Fig. 11 shows the results of cell stability analysis of PUCP through comparison of cytotoxicity. Figure 11 (a) shows the stability of RAW 264.7 cells regardless of their concentration, through analysis of the cell stability of the peptide binding to CD133. (b) shows that the cytostability analysis of PDPP bound to CD133 shows toxicity to RAW 264.7 cells even at low concentrations.
(b-1) shows that PDL-SPDP shows cytotoxicity from a low concentration as a precursor before PDPP synthesis, and cytotoxic effect on PDL itself also shows toxicity to RAW 264.7 cells. (c) shows the stability of RAW 264.7 cells regardless of their concentration through analysis of cell stability of PUCP binding to CD133.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Gene cloning of CD133 cell external domain (ECD)

First, the CD133 protein was expressed on E. coli and the CD133 gene was amplified by PCR as follows for the mass production and isolation: CD133 sense primer was 5'-ATAT GGATCC (BamH1) ATGGCTCTTGTCTTCAGTGCCCTGCTGTTACT-3 ', CD133 antisense primer 5'-ATAT GCGGCC (Not1) GCTCAGTATCGAGACGGGTCGTCGTATACATCCTCTGA-3 'was used. Denaturation at 95 DEG C for 1 minute, denaturation at 94 DEG C for 30 seconds, and annealing at 70 DEG C for 45 minutes in a reaction solution containing 5% DMSO containing the synthetic primer and template DNA. Sec, and extension at 72 ° C for 2 minutes and 45 sec. The template DNA obtained after 20 cycles of the PCR reaction was identified through the gene base sequence (see FIG. 1). The amplified gene was inserted into the bacterial expression vector pET-28a (+) by cutting with restriction enzymes (BamH1, Not1) and cloned according to the manufacturer's manual. The expression vector was then used for expression of E. coli BL21 DE3), and then the production of CD133 protein was induced for 12 hours under the conditions of 0.5 mM IPTG and 18 ° C. The above transformation method is briefly described as follows. First, 5 μl of DNA is added to 100 μl of competent cells, mixed well and placed on ice for 30 minutes. Thereafter, heat shock was applied at 42 DEG C for 90 seconds. In order to prevent contamination, 800 l of the LB medium was added to the alcohol lamp, followed by incubation at 37 DEG C for 45 minutes. Finally, it was applied to an LBA plate and incubated overnight in a 37 ° C incubator.

Example 2 Mass Production and Separation of CD133 Cell External Domain (ECD)

CD133 gene was amplified by PCR and inserted into bacterial expression vector pET-28a. Then, the cloned CD133 expression plasmid was transformed into the expression host BL21 (DE3) to induce CD133 production at 0.5 mM IPTG and 18 ° C Respectively. The bacterial cells were harvested by centrifugation to examine the solubility of the protein. As a result, it was confirmed that most proteins were insoluble (see FIG. 2). Therefore, proteins were isolated and purified under denaturing conditions to obtain such insoluble proteins.

The bacterial cells were obtained by centrifugation and the solubility of the protein was examined. As a result, it was confirmed that most proteins were insoluble. Therefore, for the isolation of CD133, only the intracellular insoluble protein was obtained, and it was dissolved in the denaturation condition (6M guanidium-HCl), then the CD133 protein was passed through the Ni-Sepharose column and denatured CD133 Refolding < / RTI > CD133 by slowly removing the denature through a linear gradient (reducing the concentration of Urea) while bound to the column to refold the CD133, lt; RTI ID = 0.0 > imidazole < / RTI > linear gradient. The resulting protein was dialyzed against a dialysate buffer (50 mM Tris, 2 mM β-mercaptoethanol, pH 7.4) to remove excess imidazole, and the purified protein was identified by SDS / PAGE See FIG. 2). Using the Bradford reagent, 0.6 mg / L culture was obtained.

Example 3: M13 phage peptide library screening-phage display

The phage display begins with fixing the protein obtained in Example 1 to a 96-well plate. The 96-well plate used here was a polystyrene plate (SPR) made of polystyrene on the surface, which stably fixed the protein using a hydrophobic interaction between the protein and the plate surface, Thereby increasing the display efficiency. Thus the protein in a fixed random peptide library (manufactured so as to have 27 million different amino acid sequence by the random array of a 12 amino acid peptide library ^{-Ph.D TM (phage display peptide library kit} , New England Biolabs (NEB) The peptide display method was designed to select only specific peptides having a good binding force with proteins by inducing interaction between proteins and peptides by introducing the peptide into the sample.

Specifically, in FIG. 3, CD133 protein is immobilized on a surface of a plate (SPL) to screen for a peptide binding to CD133 protein, and then the M13 phage peptide library (composed of 12 amino acids having about 2.7 billion different amino acid sequences And the peptide was fused to the M13 phage gp3 minor coat protein) to induce binding. Thereafter, peptide expression phages binding with high affinity were selected through various binding times and washing conditions. The screening plan and reaction conditions are shown in Fig. 3 and Fig. 4 (screening condition), respectively. Specifically, FIG. 3 is a plan view of M13 phage screening for peptide screening binding to CD133 protein, which comprises (1) immobilizing CD133 protein on the (SPL) surface of a 96-well plate, (2) After binding the phage library expressing the peptide library consisting of the amino acids to the CD133 protein, (3) washing it in various conditions, and (4) finally combining the phages were obtained by chemical elution or competitive elution. The thus obtained phage was infected with E. coli (ER2738) and amplified. The amplified phage was again bound to the CD133 protein, and then the amplified phage was repeated under the conditions such as the intensity of the washing condition and the short reaction time. Repeat the search process. This series of processes is called bio-panning (Bio-panning, Biopanning). FIG. 4 is a graph showing a total of three steps of bio-panning conditions for detecting a phage expressing a peptide having high specificity and binding ability against CD133 protein. The reaction conditions were varied with strong washing conditions and short reaction times for each step. Bio-panning was performed for a total of 3 times to obtain a phage having a peptide binding to CD133 protein. The thus obtained phage was infected with E. coli ER2738 cells, which were host cells, and amplified in LB medium. Then, about 45 phage plaques were selected and M13 phage genomic DNA (single stranded circular DNA) was isolated and purified, The amino acid sequence of the peptide fragment, which was expressed in the gp3 minor coat protein and bound to the CD133 protein by triplet code, was identified. The results are shown in Fig.

Example 4 Analysis of Binding Ability of Peptides Binding to CD133

In order to analyze the binding force of the screened peptides, the phage expressing each of the peptides was amplified, and then the concentration of the phage solution was determined by titering. Thereafter, the binding force of the peptide was deduced by measuring the amount of the phage bound to the CD133 protein-bound plate by adding the phage to the plate.

Specifically, the phage was added to the plate to which the CD133 protein was bound, and washed after a certain period of time. Subsequently, an antibody recognizing the phage surface protein (mouse anti-M13 monoclonal antibody, diluted 1: 5000 in TBST, Amersham Bioscience) was added and reacted for 1 hour. After washing 5 times with washing solution, secondary antibody (mouse IgG-HRP, 1: 4000 diluted with TBST, Santacruz), which binds to mouse anti-M13 monoclonal antibody, was reacted for 1 hour. After 5 washing steps, TMB substrate solution (3,3 ', 5'-tetramethylbenzidine (TMB) / H ₂ O ₂ , Chemicon) was added. After 15 minutes, 1 M sulfuric acid was added to terminate the reaction. This whole process is called ELISA (Enzyme-linked immunosorbant assay) using antibodies. The Kd value obtained as a result of this experiment is determined by measuring the ELISA signal intensity according to the corresponding concentration of the page peptide reacted with the fixed protein in each well at 450 nm As shown in FIG.

As a result, as shown in FIG. 6, the four peptides showed excellent binding force at the level of picomolarity, confirming the binding force corresponding to the binding force of the existing antibody-antigen reaction. In order to analyze the specificity of the CD133-1 peptide showing the best binding force with the target substance, a fluorescence probe was synthesized at the terminal of the peptide sequence.

Example 5 Analysis of Binding Capability of Synthetic Peptide Sequence with Fluorescence Probe Binding to CD133

CD133 protein on page peptide was synthesized by attaching a fluorescence substance (FITC; fluorescein isothiocyanate) to one sequence that specifically recognizes the target substance with high binding force, and the sequence is as follows.

CD133-GCGK-FITC: Ac-KMPKENPSSWLS GCGK-FITC

The indicated amino acid sequence represents the amino terminal. Acetylation was performed at the N-terminal to remove the reactivity of the amino terminal, and amino acid K (Lysine, lysine) was added at the C-terminal to attach FITC to the carboxy terminal. GCG was added near the carboxy terminus of each amino acid sequence for the process to maximize the sensitivity of the fluorescent peptide probe Ac-KMPKENPSSWLS GCGK-FITC. For analysis of the binding ability of CD133-GCGK-FITC to CD133 protein, each peptide was diluted by concentration on a plate on which CD133 protein was immobilized. The method of inferring the binding force of each peptide by measuring the amount of peptide bound to CD133 protein in each well is similar to that described in Example 4. [ However, in the case of FITC-synthesized peptides, there is no need for a marker to indicate a signal as in the case of a page peptide test, such as addition of a primary antibody, a secondary antibody, or a TMB substrate solution. Thus, CD133 protein and peptide are bound, The fluorescence intensity of each peptide concentration was measured at the maximum wavelength of FITC (Excitation @ 495 nm, Emission @ 520 nm) connected to the peptide by the intensity meter.

Peptide The FITC signal intensity at the corresponding concentration was plotted to determine the Kd value of the peptide (see Figure 6).

Example 6 Synthesis of PUCP with Multiple Recognition Probes

One example of a polypeptide backbone is the Penta-Unit (PU) backbone, which is a short peptide consisting of a total of 17 amino acids. The designed backbone was synthesized through Anisen Co., Ltd. The PU backbone was composed of amino acid residues with four functions in total. The 17 amino acids are HHHHGCGKGKGKGKGKC (SEQ ID No. 2), and the amino acid sequence is from N-terminal to C-terminal. The first four H (1-4) have the function of binding to Ni ^{2+ in} the column of the PU backbone, and the C located at 6 and 17 have a side chain of C -SH bonds in the side chain. In the case of lysine (K), when SPDP, a bi-functional linker for linking a target substance binding probe to a PU backbone, is used, it induces the formation of NH ₃ ⁺ binding of lysine. Since there are a total of five lysine residues in the PU backbone, five probes can be connected to form a total of five cyclic chain backbones. Finally, G (glycine) is the smallest amino acid and has no other functional groups in the side chain, so it is located between the lysines of the PU backbone and used to minimize interference with other probes when binding to other probes 1).

To connect multiple single peptide probes to the designed PU backbone, the PU backbone was first reacted with a SPDP (N-Succinimidyl 3- (2-pridyldithio) -propionate) linker. This is the principle that the succinimidyl group in SPDP acts as a good leaving group and reacts with NH ₃ ⁺ present in the side chain of lysine to bind SPDP to the PU backbone. In order to secure the SPDP-PU backbone after a Ni ² ⁺ - was reacted for 1 hour to agarose (Ni ² ⁺ -charged chelating sepharose) as the charged chelating Sepharose column secure the SPDP-PU backbone. Then, a probe (CD133-binding peptide, Ac- KMPKENPSSWLS GCGK (FITC) -NH ₂ ) to be bound was reacted for 1 hour to finally form a Penta-Unit conjugated probe (PUCP). The synthesized PUCP was isolated by treatment with imidazole to separate it from the Ni ² ⁺ - charged chelating sepharose column, and the separated PUCP was finally synthesized after desalting for 24 hours. The concentration of the synthesized PUCP was determined by measuring the fluorescence intensity for each peptide concentration at the maximum wavelength (excitation: 495 nm, emission: 520 nm) of the FITC connected to the ATR binding peptide using a fluorescence spectrometer.

Example 7 Analysis of Binding Capacity of Single Peptide and PUCP Binding to CD133

In order to compare the binding ability of CD133 - specific single peptides to multiple - bound PUCPs, fluorescence - based binding force was deduced. The binding force of the peptide was deduced by measuring the amount of the phage bound to the CD133 protein-bound plate by adding the phage to the plate.

Concretely, CD133 binding affinity analysis for PUCP and single peptide synthesized by the method of Example 6 was performed. The CD133 protein (2 μg) was immobilized on a polystyrene plate and reacted at room temperature for 2 hours, followed by washing. The washed plate was treated with 5% skim milk to minimize nonspecific reaction, and the reaction was allowed to proceed at room temperature for 1 hour. After the reaction, the residues were washed through 10 washing steps, and the reaction was induced by treating each probe (PUCP, single peptide) diluted by concentration immediately after the washing was completed. After the reaction was induced for 2 hours at room temperature, the binding strength was determined by measuring the fluorescence intensity for each peptide concentration at the maximum wavelength of FITC (excitation: 495 nm, emission: 520 nm) after washing. Fig. 2 shows the binding force of each of the probes and a table in which the probabilities are quantified.

Each peptide was diluted by concentration on a plate on which CD133 protein was immobilized. The method of inferring the binding force of each peptide by measuring the amount of the peptide bound to the CD133 protein in each well is similar to that described in Example 4. [ However, in the case of FITC-synthesized peptides, there is no need for a marker to indicate a signal as in the case of a page peptide test, such as addition of a primary antibody, a secondary antibody, or a TMB substrate solution. Thus, CD133 protein and peptide are bound, The fluorescence intensity of each peptide concentration was measured at the maximum wavelength of FITC (Excitation @ 495 nm, Emission @ 520 nm) connected to the peptide by a fluorometer. Peptide The FITC signal intensity at the corresponding concentration was plotted to determine the Kd value of the peptide.

As a result, strong binding force at nano-mol level was obtained (Kd of PUCP: 1.4 ㅁ 0.12 nM, Kd of single peptide: 8.0 ㅁ 0.7 nM). This confirms that PUCP has 8 times stronger binding force than a single peptide.

Example 8: Analysis of binding efficiency between PUCP and CD133 expressing cell line of single peptide using cell immuno-staining method

Immunocytochemistry was used to analyze the binding of the single peptide, PUCP, and PDVP (Polyvalent Directed Peptide Polymer) to the actual CD133 expressing cell line through fluorescence microscopy. CD133 antibody-PE with phycoerythrin as a control was used, and a peptide (scramble peptide, sc-peptide) with an arbitrary sequence was simultaneously treated for peptide specificity analysis. For this experiment, U87 cells were cultured in a DMEM medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin, 100 μg / ml streptomycin, And were mixed together and grown under the condition of 37 ° C, 5% CO ₂ . The grown cells were first reacted with blocking buffer (2% FBS) for 1 hour for binding with the peptide, followed by washing with washing buffer PBS. The cells were then reacted at 4 ° C for 4 hours using CD133 antibody-PE, sc-peptide, single peptide, PUCP, and PDPP. The sc-peptide refers to a control peptide having an arbitrary sequence as a scramble-peptide. The single peptide means a CD133 target peptide of Sequence Listing 1 sequence. The PDPP refers to a CD133 target peptide of Sequence Listing 1 sequence, D-lysine (150-300 kDa).

After washing, the cells are further stained with DAPI for 5 minutes. Finally, the binding specificity and specificity of each probe (antibody, sc-peptide, single peptide, PUCP, PDPP) binding to the plasma membrane protein CD133 was analyzed through a fluorescence microscope.

As a result, it was confirmed that a single peptide, PUCP and PDPP except for the sc-peptide (control group) all emitted fluorescence signals by binding to CD133 at the cell level. Numerically, PUCP is 2-3 times stronger than a single peptide Fluorescence signals were observed. These results indicate that PUCP is a novel probe that is available as a diagnostic probe and has stronger binding and cell-selective binding ability than a single peptide.

Example 9: Cell stability analysis of PUCP through comparison of cytotoxicity

To test the toxicity of anthrax toxin in cells for excised peptides, cytotoxicity tests were performed on Raw264.7 macrophages. First, 10 ⁵ cells were cultured in a 96-well cell culture plate under 5% CO ₂ for 24 hours under DMEM (10% FBS, 1% penicillin-streptomycin) medium. The cultured cells were reacted with each probe (single peptide, PDPP, PUCP) for 2 hours depending on the concentration. After that, all the reaction solutions were removed, 1 mg / ml MTT solution was treated for 4 hours, and finally DMSO was added to terminate the reaction. The intracellular anthrax toxin inhibitory effect on the peptide was measured by measuring the absorbance at 570 nm and measuring the cell signal (Fig. 4).

As shown in FIG. 4, PDPP exhibited stronger binding force and higher fluorescence signal than PUCP (see FIG. 3), but cell death was induced in proportion to the increase in concentration for cytotoxicity test. It is known that the positive charge of the lysine residue of PDPP adversely affects cells. On the other hand, PUCP, which has stronger binding force than single peptide and weaker binding force than PDPP, has no effect on the characteristics of the cell itself even when the concentration is increased. This means that PUCP with high sensitivity and cell stability can be used as a single peptide substitute, replacing PDPP with the highest sensitivity.

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.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Multi Peptide Conjugated Probe for Detection of CD133 <130> PN150122 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Single peptide probe for CD133 <400> 1 Lys Met Pro Lys Glu Asn Pro Ser Ser Trp Leu Ser 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Polypeptide backbone for multi-peptide probe <400> 2 His His His His Gly Cys Gly Lys Gly Lys Gly Lys Gly Lys Gly Lys 1 5 10 15 Cys

Claims

(a) a polypeptide backbone consisting of an amino acid sequence represented by the following general formula (1) and (b) a plurality of single peptide probes consisting of the first sequence listed in the sequence listing linked to said backbone, as glioma stem cell specific markers Multiple peptide probes for CD133 targeting:
1
HxGC (GK) yC;
In the general formula (1), x is an integer of 2-5 and y is an integer of 3-8.

2. The multiple peptide probe according to claim 1, wherein the polypeptide backbone forms a cyclic structure through a disulfide bond between two cysteines contained in the backbone.

2. The multiple peptide probe according to claim 1, wherein y in the general formula (1) is 5.

2. The multiple peptide probe according to claim 1, wherein the amino acid sequence represented by the general formula (1) is composed of the second sequence of the sequence listing.

The method of claim 1, wherein the multiple peptide probe comprises a first functional group that forms an amide bond with an amine group contained in the side chain of the lysine of the polypeptide backbone, and a second functional group that forms a bond with the single peptide probe Wherein the probe further comprises a functional linker.

6. The multiple peptide probe according to claim 5, wherein the bond between the single peptide probe and the second functional group of the bi-functional linker is a disulfide bond or an amide bond.

A glioma stem cell detection kit comprising the multiple peptide probe of any one of claims 1 to 6.