Peptides that specifically bind to vitamin-related biocomplex and use thereof

Abstract

The present invention relates to a peptide that specifically binds to a vitamin-related biocomplex and its use. Since the peptide that specifically binds to the vitamin D biocomplex of the present invention exhibits selective binding affinity to the vitamin D biocomplex, it can detect a protein in a form bound to vitamin D as a biomarker for vitamin D measurement and distinguish it from a vitamin D-binding protein in a simple protein form, thereby enabling more accurate measurement of the blood vitamin D concentration and making it useful for determining the vitamin D concentration in the body.

Description

Technology Field

[0001] The present invention relates to a peptide that specifically binds to a vitamin-related biocomplex and a biomarker composition for detecting vitamin D comprising said peptide. Background Technology

[0002] Vitamin D is known as a precursor to hormones important for bone growth and maintenance, as well as for maintaining mineral homeostasis. However, new light is being shed on the role of vitamin D as it has recently been discovered that not only the vitamin D receptor (VDR) but also 1-alpha-hydroxylase, which is necessary to convert vitamin D into its active form, is expressed in various tissues and cells within the human body. These findings suggest the possibility that vitamin D acts in tissues or organs other than bones, kidneys, and the small intestine, or is involved in biological processes unrelated to bone or mineral homeostasis, and results from laboratory and epidemiological studies support this.

[0003] Meanwhile, an analysis of data from the National Health and Nutrition Examination Survey revealed that the average vitamin D concentration of U.S. adults in 2001–2004 was 24 ng / mL, a decrease of 6 ng / mL compared to 1988–1994, with only 23% maintaining an adequate level of 30 ng / mL or higher. Additionally, various previous cohort studies have shown that 52–77% of subjects suffer from vitamin D deficiency. In Korea, a survey of menopausal women with osteoporosis found that 92% had levels below 30 ng / mL, and as many as 57% had levels below 12 ng / mL, indicating that vitamin D deficiency has now become a serious health issue globally, including in Korea.

[0004] Meanwhile, in general, the administration of an appropriate amount of vitamin D is required for vitamin D-related treatments. Prior to this, it is essential to check the vitamin D concentration in the body, and vitamin D concentration measurement is performed by measuring the concentration of 25-hydroxyvitamin D3 (25(OH)D3), which has a relatively long half-life. A blood 25(OH)D3 concentration of less than 20 ng / mL (50 nmol / L) is diagnosed as 'vitamin D deficiency,' 21-29 ng / mL as 'vitamin D insufficiency,' and 30 ng / mL (75 nmol / L) or higher as 'vitamin D sufficiency.'

[0005] Approximately 80% of vitamin D is synthesized by exposure to sunlight (UVB) and converted through the liver into 25(OH)D3, which is commonly found in the blood. When undergoing vitamin D screening, vitamin D levels are assessed by measuring the concentration of 25(OH)D3, which has a relatively long half-life. However, conventional detection methods have the disadvantage that 25(OH)D3 does not circulate independently but tends to exist in the form of a complex bound to vitamin D binding proteins, and that using a 150 kDa antibody to detect 25(OH)D3 fails to distinguish between the complex and the relatively small vitamin D binding protein (approx. 58 kDa). Additionally, there are issues such as difficulty in mass production and stability over time.

[0006] It is known that most vitamin D in the blood circulates in the form of a biocomplex (VDBP-Complex) bound to vitamin D binding protein (VDBP). Therefore, to determine vitamin D levels in the body, it is important to distinguish between vitamin D binding protein, which is a simple protein, and the vitamin-related biocomplex bound to it.

[0007] The inventors screened peptides characterized by specifically binding to vitamin-related biocomplexes using a phage display library, confirmed that the peptides recognize vitamin D biocomplexes and can be used as indicators for detecting vitamin D concentration, and completed the present invention. Prior art literature

[0008] Republic of Korea Registered Patent No. 10-2124352 The problem to be solved

[0009] The objective of the present invention is to provide a peptide that specifically binds to a vitamin D biocomplex.

[0010] In addition, another objective of the present invention is to provide a polynucleotide encoding a peptide that specifically binds to the vitamin D biocomplex.

[0011] In addition, another objective of the present invention is to provide a composition for detecting vitamin D comprising a peptide that specifically binds to the vitamin D biocomplex.

[0012] In addition, another objective of the present invention is to provide a vitamin D detection kit comprising the above-mentioned vitamin D detection composition.

[0013] In addition, another objective of the present invention is to provide a composition for diagnosing vitamin D deficiency or lack comprising the above peptide as an active ingredient.

[0014] In addition, another objective of the present invention is to provide a method for measuring blood vitamin D concentration. means of solving the problem

[0015] To achieve the above objectives, the present invention provides a peptide that specifically binds to a vitamin D biocomplex represented by the amino acid sequence of SEQ ID NO. 1 or 2, and a polynucleotide encoding the same.

[0016] Next, the present invention provides a composition for detecting vitamin D comprising a peptide that specifically binds to the vitamin D biocomplex.

[0017] Furthermore, the present invention provides a vitamin D detection kit comprising the above-mentioned vitamin D detection composition.

[0018] In addition, the present invention provides a composition for diagnosing vitamin D deficiency or lack, comprising the above-mentioned composition for detecting vitamin D.

[0019] Finally, the present invention provides a method for measuring blood vitamin D concentration, comprising the steps of: collecting a blood sample from a test subject; treating the blood sample with the vitamin D detection composition to detect a vitamin D biocomplex; and analyzing the amount of vitamin D biocomplex detected to measure the blood vitamin D concentration. Effects of the invention

[0020] Since the peptide that specifically binds to the vitamin D biocomplex of the present invention exhibits selective binding affinity to the vitamin D biocomplex, it can detect a protein in a form bound to vitamin D as a biomarker for vitamin D measurement and distinguish it from a vitamin D binding protein in a simple protein form, thereby enabling more accurate measurement of the blood vitamin D concentration and making it useful for determining the vitamin D concentration in the body. Brief explanation of the drawing

[0021] FIG. 1 is a simplified schematic diagram illustrating the peptide selection process using a phage display library in one embodiment of the present invention. FIG. 2 is a figure showing the results of sequence analysis of a peptide that specifically binds to a vitamin-related biocomplex (VDBP-Complex) in one embodiment of the present invention. Figure 3 is a figure showing the results of an affinity analysis of a peptide that specifically binds to a vitamin D biocomplex (VDBP-Complex) in one embodiment of the present invention. Specific details for implementing the invention

[0022] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalent scope interpreted therefrom.

[0023] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as “comprising” a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0024] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise noted.

[0025] The present invention will be described in more detail below.

[0027] The present invention provides a peptide that specifically binds to a vitamin D biocomplex represented by the amino acid sequence of SEQ ID NO. 1 or 2.

[0028] In the present invention, the term “vitamin D biocomplex” refers to a form in which 25-hydroxyvitamin D3 (25-hydroxyvitamin D3, 25(OH)D3) and vitamin D binding protein are combined.

[0029] In the present invention, the term "amino acid" includes not only the 22 standard amino acids that naturally incorporate into peptides, but also D-isomers and modified amino acids. Accordingly, the peptide may be a peptide containing D-amino acids. Meanwhile, amino acid sequences used in this specification are described by abbreviations according to the IUPAC-IUB nomenclature.

[0030] In the present invention, the term “peptide” means a polymer composed of two or more amino acids connected by an amide bond (or peptide bond). For the purposes of the present invention, it refers to a peptide that specifically binds to a vitamin D biocomplex.

[0031] Additionally, the peptide may be amino acid variants or fragments having different sequences due to post-translational modifications, such as deletion, insertion, or substitution of amino acid residues, within a range that does not affect function. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (e.g., acetylation, myristoylation, and palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-removal deimide, deamidation), and structural changes (e.g., formation of disulfide bridges). In addition, it includes changes in amino acids resulting from chemical reactions occurring during the bonding process with crosslinkers to form peptide conjugates, such as changes in amino groups, carboxyl groups, or side chains.

[0032] Accordingly, the present invention comprises a peptide substantially identical to the peptide comprising the amino acid sequence of SEQ ID NO. 1 or 2, a variant thereof, or an active fragment thereof.

[0033] The term "substantially identical peptide" means an amino acid sequence having at least 75%, preferably at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence homology with the amino acid sequence of SEQ ID NO. 1 or 2, but is not limited thereto; if it has at least 75% sequence homology and has the same activity, it is included within the scope of the present invention. Additionally, the peptide of the present invention may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability.

[0034] Additionally, the peptide may have a protecting group attached to the N- or C-terminus to obtain chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), modified specificity (e.g., broad spectrum of biological activity), and reduced antigenicity. In one embodiment, the N-terminus of the peptide may be attached to any one protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and polyethylene glycol (PEG); and / or the C-terminus of the peptide may be bonded to any one of the protecting groups selected from the group consisting of an amino group (-NH2), a tertiary alkyl group, and an azide (-NHNH2). Additionally, the peptide may optionally further include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability.

[0035] In this specification, the term "stability" may mean not only in vivo stability, which protects the peptide from attack by protein-cleaving enzymes in vivo, but also storage stability (e.g., room temperature storage stability).

[0036] The peptides of the present invention can be produced such that the purity of each peptide is 90% or higher through conventional peptide synthesis or manufacturing methods known to those skilled in the art, for example, by synthesizing them directly or by purchasing them after commissioning production from a peptide manufacturing company. The peptides can be produced and used in the form of a D-form, an L-form, a peptide in which only a portion of the sequence is in the D-form or L-form, or a racemic form thereof, all through conventional peptide synthesis or manufacturing methods known to those skilled in the art. In addition, other conventional modifications known in the art are possible to increase the stability of the peptides. In the present invention, the peptides were preferably synthesized using a solid-state peptide synthesis method, but as described above, the peptide synthesis method and conditions are not limited thereto.

[0038] In another aspect, the present invention provides a polynucleotide encoding a peptide represented by the amino acid sequence of SEQ ID NO. 1 or 2.

[0039] In one embodiment, the polynucleotide may be in the form of RNA or DNA, said DNA including cDNA and synthetic DNA. The DNA may be single-stranded or double-stranded. If it is single-stranded, it may be a coding strand or a non-coding (antisense) strand, and said coding sequences encode the same polypeptide as a result of degeneracy or redundancy of the genetic code.

[0040] The above polynucleotide may also include variants of the polynucleotide described herein, and the variants of the polynucleotide may be naturally occurring allelic variants of the polynucleotide or non-naturally occurring variants of the polynucleotide. An allelic variant is an alternate form of a polybase sequence that may have substitutions, deletions, or additions of one or more nucleotides that do not substantially alter the function of the polynucleotide being encoded. It is well known in the art that a single amino acid may be encoded by one or more nucleotide codons and that the polynucleotide can be easily modified to produce alternate polynucleotides encoding the same peptide.

[0042] Next, the present invention provides a composition for detecting vitamin D comprising a peptide that specifically binds to the vitamin D biocomplex.

[0043] Since the composition for detecting vitamin D of the present invention includes the peptide described above, the description of the content that overlaps with the peptide of the present invention described above is omitted to avoid excessive complexity in the present specification caused by the description of the overlapping content.

[0044] In addition, the above composition may detect a vitamin D biocomplex in a form in which vitamin D and a vitamin D binding protein are bound, but is not limited thereto.

[0045] In addition, the vitamin D may be 25-hydroxyvitamin D3 (25-hydroxyvitamin D3, 25(OH)D3), but is not limited thereto.

[0046] The vitamin D detection composition of the present invention is intended for detecting vitamin D in the blood, and given that vitamin D receptors are found in almost all tissues of the body, it is highly associated with various diseases. Therefore, the peptide of the present invention can be used in any form for the detection of vitamin D in the blood.

[0048] Furthermore, the present invention provides a vitamin D detection kit comprising the above-mentioned vitamin D detection composition.

[0049] The vitamin D detection kit of the present invention may take the form of a bottle, tub, sachet, envelope, tube, ampoule, etc., and may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may be equipped with a cap that is initially part of the container or may be attached to the container by mechanical, adhesive, or other means and may be fully or partially detachable. The container may also be equipped with a stopper that allows access to the contents by a needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0051] In addition, the present invention provides a composition for diagnosing vitamin D deficiency or lack, comprising a peptide that specifically binds to the vitamin D biocomplex as an active ingredient.

[0052] Since the diagnostic composition of the present invention includes the peptide described above, the description of the content that overlaps with the peptide of the present invention described above is omitted to avoid excessive complexity in the present specification caused by the description of the overlapping content.

[0053] In addition, the diagnostic composition of the present invention includes a peptide that specifically binds to the vitamin D biocomplex, which can measure the concentration of vitamin D present in a blood sample, and can diagnose vitamin D deficiency or lack based on the result of measuring the vitamin D concentration. For example, if the blood vitamin D concentration is less than 20 ng / mL (50 nmol / L), it can be diagnosed as vitamin D deficiency, and if it is between 21 and 29 ng / mL, it can be diagnosed as vitamin D deficiency.

[0055] Finally, the present invention provides a method for measuring blood vitamin D concentration, comprising the steps of: collecting a blood sample from a test subject; treating the blood sample with the vitamin D detection composition to detect a vitamin D biocomplex; and analyzing the amount of vitamin D biocomplex detected to quantify the vitamin D content in the blood.

[0056] Furthermore, the aforementioned test subject refers to a subject for which the concentration of vitamin D in the blood is to be measured, and may be used without restriction on humans, dogs, monkeys, cats, and rodents (e.g., mice, genetically modified mice, etc.). More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0057] In addition, the blood specimen may be one or more selected from the group consisting of whole blood, serum, and plasma, but is not limited thereto.

[0058] In one embodiment according to the method of the present invention, the concentration of vitamin D is confirmed / measured / detected in an immunoassay procedure. Such immunoassays are well known to those skilled in the art. Methods of performing such assays, as well as practical applications and procedures, are summarized in relevant textbooks. Examples of relevant textbooks include Tijssen, P., Preparation of enzyme-antibody or other enzyme-macromolecule conjugates, In: Practice and theory of enzyme immunoassays, pp. 221-278; Burdon, RH and v. Knippenberg, PH (eds.), Elsevier, Amsterdam (1990); and various volumes of Methods in Enzymology, Colowick, SP, and Caplan, NO (eds.), Academic Press, particularly volumes 70, 73, 74, 84, 92, and 121, which cover immunological detection methods.

[0059] In one embodiment, a method for determining the concentration of vitamin D in the blood is selected from the group consisting of enzyme-linked immunoassay (ELISA), electrochemiluminescent immunoassay (ECLIA), radioimmunoassay (RIA), and chemiluminescent immunoassay (CLIA). In a preferred embodiment, vitamin D is detected by an enzyme-linked immunoassay (ELISA). In a further preferred embodiment, vitamin D is detected by an (electro-)chemiluminescent immunoassay (ECLIA). In a further embodiment, vitamin D is detected by a radioimmunoassay (RIA). Also, a preferred embodiment is a chemiluminescent immunoassay (CLIA) for determining vitamin D. Additionally, preferably, the assay is a sandwich fluorescent immunoassay (FIA), a microparticle capture enzyme immunoassay (MEIA), a solid-phase fluorescent immunoassay (SPFIA), a particle concentration fluorescent immunoassay (PCFIA), and turbidity and non-turbidity assays with and without latex particle enhancement (LPIA). Furthermore, in one embodiment, the assay may be in the form of a test strip.

[0061] Hereinafter, embodiments of the present invention will be described in more detail with reference to the attached drawings. However, the following embodiments are intended only to embody the content of the present invention and are not to limit the present invention.

[0063] <Example 1> Immobilization of Vitamin-Related Biomaterials in a Petri Dish

[0064] Vitamin D binding protein (VDBP) and a vitamin D-related biocomplex (VDBP-Complex) in which vitamin D and vitamin D binding protein are bound were coated onto Petri dishes, and the coating method is as follows. A petri dish (SPL, Korea, Cat#10060) was used to coat the two types of vitamin-related biomaterials onto the Petri dishes. The vitamin-related biomaterials were diluted to a concentration of 20 μg / ml using 0.1 M NaHCO3 (pH 8.6), and 4 ml of the diluted solution was added to cover the surface of the Petri dish. The mixture was incubated in a humidified container at 4°C for at least 8 hours using an agitator. After incubation, the coating solution was poured out of each Petri dish to remove it, and any residual solution was removed by patting the coated area with a clean paper towel. 1 ml of TBST (TBS+0.1%[v / v] Tween-20) was added for washing, and the supernatant was removed again. This washing process was repeated a total of 5 times.

[0065] Vitamin-related biomaterials were immobilized on the surface of a Petri dish using the method described above, and a phage display experiment was performed using this.

[0067] <Example 2> Specific to vitamin-related biocomplexes using phage display

[0068] Selection of peptides that bind

[0069] Phage display technology was used to select peptides that specifically bind to the vitamin D biocomplex (see Fig. 1). Specifically, a phage library was prepared with a total reaction volume of 1.5 ml at a final concentration of 10^9 PFU / ml (Plaque forming units / ml). The phage library was first applied to a control group (Petri dish) and a negative control group (Petri dish immobilized with BSA). 1.1 ml of the phage library was incubated in a polystyrene Petri dish at room temperature for 45 minutes. Subsequently, only the supernatant was collected and incubated in a Petri dish immobilized with BSA at room temperature for 45 minutes. This series of processes, specifically the step of first applying the material to be used as a control, was termed "pre-binding." The supernatant phage library obtained after pre-binding was applied to a Petri dish immobilized with vitamin-related biomaterials and incubated at room temperature for 45 minutes. To select peptides that specifically bind to each vitamin-related biomaterial, weakly bound or unbound peptides were removed by washing a total of five times with 1 ml of TBST. 1 ml of elution buffer (0.2 M Glycine-HCl, pH 2.2) was added to each plate and incubated at room temperature for 15 minutes. The reaction mixture containing the phage library including the isolated specific peptides was recovered, and 150 µl of 1 M Tris-HCl (pH 9.1) buffer was added to neutralize it. The neutralized reaction mixture was placed in a Petri dish and incubated at room temperature for 30 minutes. This process, specifically the step of eluting phages bound to the target substance, treating them with a substance to be used as a control, and collecting only the supernatant to construct a new phage library, was named "after-binding." 500 µl of the isolated after-binding library phages was recombined and constructed for phage infection in the Kit. E. coliThe mixture was inoculated into ER2738, mixed with 20 ml of LB culture medium, and cultured at 37°C for 4.5 hours to amplify the reaction. The reaction mixture was centrifuged at 12,000 g at 4°C to recover 80% of the supernatant, mixed with 20% PEG / 2.5 M NaCl, and reacted at 4°C for 15 hours to precipitate library phages containing specific peptides. Subsequently, the mixture was centrifuged at 12,000 g at 4°C to precipitate and recover only the library phages containing specific peptides. The series of phage display library binding processes was carried out a total of 7 times. As the phage display library rounds progressed, the reaction conditions were tightened to obtain peptides that bind more specifically to the proteins of the target substances, which are vitamin-related biomaterials.

[0071] <Example 3> Extraction of total DNA from selected library phages

[0072] To select peptides specific to the vitamin D biocomplex, 40 library phages corresponding to each of rounds 4, 5, 6, and 7 were recovered, and the method for extracting phage DNA from the library phages containing the recovered specific peptides is as follows.

[0073] E. coliAfter culturing ER2738 overnight, it was inoculated into 1 ml of LB culture medium at a ratio of 1:100 and incubated at 37°C for 5 hours. After incubation, the host E. coli was removed by centrifugation at 14,000 rpm for 1 minute. Subsequently, 500 µl of the supernatant containing phage was mixed with 200 µl of 20% PEG / 2.5 M NaCl buffer and reacted at room temperature for 20 minutes. The reaction mixture was centrifuged at 14,000 rpm at 4°C for 10 minutes to remove the supernatant and isolate only the phage. The phage obtained through centrifugation was washed with ethanol to remove outer membrane proteins and obtain pure phage DNA. 100 µl of iodide buffer was added to the obtained DNA and mixed, followed by the addition of 200 µl of ethanol, and the mixture was reacted at room temperature for 15 minutes. The reaction mixture was centrifuged at 14,000 rpm at 4°C for 10 minutes to remove the supernatant, and the peptide was washed with 500 µl of 70% ethanol. Afterward, the mixture was centrifuged once more to completely remove the supernatant, and then dried at 37°C for 5 minutes to completely evaporate the ethanol. Subsequently, 30 µl of TE buffer was mixed with the purified DNA.

[0075] <Example 4> Amplification of gene DNA constituting peptides of selected library phages and identification of peptides specifically binding to vitamin-related biocomplexes

[0076] In order to amplify the DNA of a library phage that specifically binds to the vitamin D biocomplex obtained in Example 3 and to analyze the peptide portion constituting the phage terminus, a pair of primers was synthesized from Bioneer (Korea) (Table 1), [reverse primer 5'-CCCTCATAGTTAGCGTAACG-3' (Sequence No. 5)]. DNA oligonucleotides were amplified through a PCR reaction using the above primers, and the reaction composition consisted of 1 µl of template DNA, 5 µl of 10 X PCR buffer, 4 µl of dNTP mixture, 1 µl of 10 pM forward primer, 1 µl of 10 pM reverse primer, 0.2 µl of Ex Taq polymerase (Takara, Japan) (1 unit / µl), and 37.8 µl of distilled water. The reaction conditions for the amplification of template DNA involved denaturation at 95°C for 5 minutes, followed by 34 cycles of reaction at 95°C for 30 seconds, 53.5°C for 30 seconds, and 72°C for 1 minute and 30 seconds, and then an additional extension at 72°C for 7 minutes. After the PCR reaction, 4 µl was taken, the amplified product was verified by 0.7% agarose gel electrophoresis, purified using a PCR purification kit (GeneAll, Korea), and recovered in 30 µl of distilled water.

[0078] Primer sequence (5' -> 3') Phage-F CCG ATT CCT TTA GTG GTA CCT TTC TAT Phage-R CCC TCA TAG TTA GCG TAA CG

[0080] <Example 5> Analysis of specifically binding peptide sequences

[0081] In order to find a peptide sequence that specifically binds to a vitamin D biocomplex that binds to a vitamin D-binding protein in the blood, passes through the cell membrane, and moves into the cell, vitamin-related biomaterials were immobilized on a Petri dish in Example 1, and DNA of a phage having a peptide showing specific binding affinity obtained through Examples 2 and 3 was selected and extracted, and sequence analysis (Bioneer, Korea) was performed using the DNA amplified and recovered in Example 4.

[0082] Figure 2 shows the sequence analysis of peptides that specifically bind to the vitamin D biocomplex (VDBP-Complex).

[0083] As a result, as shown in Fig. 2, a peptide characterized by specifically binding to the vitamin D biocomplex was identified through sequence analysis, and the sequences that are repeated between rounds were indicated separately by color (see Fig. 2).

[0085] <Example 6> Confirmation of binding affinity of a peptide specifically binding to a selected vitamin-related biocomplex

[0086] A control group (Petri dish)-specific phage candidate peptide (SEQ No. 3) selected through 7 rounds of biopanning and 7 vitamin D biocomplex-specific phage candidate peptides (see Fig. 2) were selected and amplified for use in ELISA (enzyme-linked immunosorbent assay) experiments. Each phage stock was pre-cultured E. coliER2738 was inoculated, mixed with 20 ml of LB culture medium, and cultured at 37°C for 7 hours to amplify the cells. The reaction mixture was centrifuged at 12,000 g at 4°C to recover 80% of the supernatant, mixed with 20% PEG / 2.5 M NaCl, and reacted at 4°C for 15 hours to precipitate phage candidates containing specific peptides. Subsequently, the mixture was centrifuged at 12,000 g at 4°C to precipitate and recover only the phage candidates containing specific peptides. The precipitate was dissolved in TBS, and phage titration was performed to confirm that the concentration (PFU / ml) of each phage was at least 10^13 PFU / ml. Vitamin D-related biomaterials were diluted in the same manner as in Example 1, dispensed at a dose of 200 μl into a 96-well plate (Thermo Fisher Scientific, Cat#167008, USA), and fixed using the same method as in Example 1. Vitamin D was diluted to 200 μM using 100% EtOH, dispensed at a dose of 200 μl, and fixed by recrystallization. ELISA was performed to compare the binding affinities of each of the eight phages with the vitamin-related biomaterials. For the ELISA analysis, OD values ​​were measured and compared at 410 nm using the Anti-M13 antibody [B62-FE2] (HRP) and ABTS.

[0087] As a result, it was confirmed that the peptide SFTKTSTFTWRD (SEQ No. 1) and the peptide SLFTKQYDYFDT (SEQ No. 2) have high binding affinities for vitamin-related biocomplexes, and among them, the phage having the SFTKTSTFTWRD sequence was observed to show relatively high binding affinity (see Fig. 3).

[0089] The selection of significant biomarkers determines the reliability of detection and diagnostic results. A significant biomarker implies that the obtained results are accurate, indicating high validity, and that repeated data demonstrates a consistent trend, implying high reliability.

[0090] The peptide that specifically binds to the vitamin D biocomplex of the present invention includes a process of pre-removing phages that have strong selectivity for a substance used as a control in a phage library, and a process of additionally removing phages that have selectivity for the target substance and simultaneously selectivity for the control among the phages that have selectivity for the target substance. Therefore, since the peptide of the present invention specifically binds only to the vitamin D biocomplex, it can be seen that it is a biomarker with excellent validity and reliability.

[0092] As described above, specific embodiments of the present invention have been described in detail; however, those skilled in the art who understand the spirit of the present invention will be able to easily propose other inventions that are inferior or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the same spirit. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 A composition for detecting vitamin D comprising a peptide represented by the amino acid sequence of SEQ ID NO. 1 or 2, wherein the vitamin D is 25-hydroxyvitamin D3 (25-hydroxyvitamin D3, 25(OH)D3). Claim 5 delete Claim 6 delete Claim 7 A vitamin D detection kit comprising the vitamin D detection composition of claim 4. Claim 8 A composition for diagnosing vitamin D deficiency or insufficiency comprising, as an active ingredient, a peptide represented by the amino acid sequence of SEQ ID NO. 1 or 2, wherein the vitamin D is 25-hydroxyvitamin D3 (25-hydroxyvitamin D3, 25(OH)D3). Claim 9 A method for measuring blood vitamin D concentration, comprising: a step of detecting vitamin D by treating a blood sample separated from an individual with a composition for detecting vitamin D according to claim 4; and a step of quantifying the blood vitamin D content by analyzing the amount of vitamin D detected, wherein the vitamin D is 25-hydroxyvitamin D3 (25-hydroxyvitamin D3, 25(OH)D3). Claim 10 A method for measuring blood vitamin D concentration according to claim 9, characterized in that the blood sample is one or more selected from the group consisting of whole blood, serum, and plasma.

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