KR20210019319A - Fusion protein and bio-imaging composition comprising the same - Google Patents

Abstract

The present invention relates to a fusion protein comprising a binding part capable of binding to a target and a carrier protein, and a composition comprising the same. The fusion protein of the present invention comprises: the binding part capable of binding to at least one of osteoblasts and hydroxyapatite; and the carrier protein. By using the fusion protein according to the present invention, it is possible to exhibit an effect capable of bone imaging.

Description

Fusion protein and bio-imaging composition comprising the same {Fusion protein and bio-imaging composition comprising the same}

The present invention relates to a fusion protein comprising a binding moiety capable of binding to a target and a carrier protein, and a composition comprising the same.

Bone is a solid organ that makes up the body and plays an important role in protecting, forming and supporting various organs. Bone maintains a certain bone density by balancing the degree of activation of osteoblasts that form bone and osteoclasts that degrade bone. When the activation balance of each cell is broken, bone density increases or decreases, leading to related diseases such as osteosclerosis or osteoporosis. Therefore, in the diagnosis of bone-related diseases and the development of therapeutic agents, hydroxyapatite, an inorganic substance constituting osteoblasts and bone, is an important target material.

In order to diagnose or treat bone-related diseases in the body, a method of inducing the activity of osteoblasts or inhibiting the activity of osteoclasts using chemical substances or pharmaceuticals is typically used. However, these chemicals do not have target specificity and may cause unexpected phenomena in organs and cells other than bone. Therefore, it is very necessary to develop a mediator material that can be selectively delivered to targets related to bone disease.

Various types of nanoparticles such as inorganic substances, liposomes, polymers, and proteins are being developed as mediators that can be selectively delivered to targets in the body. Chemically synthesized nanoparticles usually have low biocompatibility and cytotoxicity, which has a fatal disadvantage for in vivo application. In addition, a ligand must be attached to the above nanoparticles through an additional process to give target specificity. On the other hand, nanoparticles composed of proteins have the advantage that they can be easily produced without complicated modification processes by fusion with a peptide or a part of a protein that can specifically bind to a target through genetic modification. Therefore, it is possible to develop new delivery mediators having the function of selectively binding to bone-related disease markers through genetic modification.

Under the above background, the present invention is to provide a new fusion protein that selectively binds to osteoblasts and/or hydroxyapatite.

In addition, it is intended to provide a composition for bio-imaging and a method of manufacturing the same by selective binding of the above fusion protein to osteoblasts and/or hydroxyapatite.

In order to achieve the above object, the present invention is a coupling portion that can be coupled to at least any one of osteoblasts and hydroxyapatite; It provides a fusion protein comprising; and a carrier protein.

In addition, the present invention provides a composition for bio-imaging comprising the fusion protein according to the present invention.

In addition, the present invention provides a method for producing a fusion protein comprising a step of amplifying the polynucleotide sequence encoding the fusion protein according to the present invention by PCR.

Since the fusion protein of the present invention has the specificity of selectively binding to osteoblasts and/or hydroxyapatite, it can be used for bone imaging to further increase imaging efficiency.

In addition, due to the specific binding as described above, there is an advantage of minimizing the problem caused by non-specific binding of chemical substances used in conventional bone imaging. In addition, based on these characteristics, it may be used to develop a delivery system that can be used for diagnosis or treatment of various diseases related to bone.

1 is a schematic diagram of a vector configuration for preparing a ferritin fusion protein capable of (a) a subunit structure of a ferritin protein, (b) an entire structure of a ferritin protein, and (c) an osteoblast or hydroxyapatite binding.
FIG. 2 is a photograph of electrophoresis results of osteoblast-binding fusion protein 1 (Ob-Fn) and hydroxyapatite-binding fusion protein 2 (HA-Fn).
3 is a photograph of (a) a TEM image of fusion protein 1 (Ob-Fn), (b) DLS measurement result, and (c) native gel electrophoresis result.
Figure 4 is a result of confirming the binding ability to (a) HeLa of fusion protein 1 (Ob-Fn) and wild-type ferritin protein (WT-Fn), (b) the result of confirming the binding ability to undifferentiated osteoblasts, and (c) 21 day differentiation This is a picture of the result of confirming the binding ability to posterior osteoblasts.
5 is a graph showing the results of evaluating the binding ability of fusion protein 2 (HA-Fn) and wild-type ferritin protein (WT) to hydroxyapatite.

Hereinafter, the present invention will be described in detail with reference to the drawings. The terms or words used in this specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventor may appropriately define the concept of terms in order to describe his own invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Fusion protein

The fusion protein of the present invention includes a binding site and a carrier.

The coupling portion may be coupled to at least one of osteoblasts and hydroxyapatite. Specifically, the binding portion may include at least one amino acid sequence represented by SEQ ID NO: 1 or 2. More specifically, the binding portion may include at least one amino acid sequence represented by SEQ ID NO: 3 or 4 in terms of improving the accuracy of binding to the carrier protein and stability of binding.

SEQ ID NO: 1 SDSSD SEQ ID NO: 2 DDDDDD SEQ ID NO: 3 C SDSSD HHHHHH GSSGSSGSSGSS SEQ ID NO: 4 C DDDDDD HHHHHH GSSGSSGSSGSS

The carrier serves to transport a biological tissue or a binding portion capable of being bonded to a biological material into a living body. The carrier may be a polymer or protein, such as a polymer such as a metal nanoparticle or a protein composed only of amino acids, such as polyethylene glycol, polyallylamine, and polyacrylamide, but may be a protein. In specific embodiments of the present invention, a fusion protein of the present invention was constructed using a ferritin protein having an amino acid sequence of SEQ ID NO: 5 as a representative example of the carrier protein, and its effect was confirmed.

In particular, the ferritin protein is an intracellular protein commonly produced in most organisms including humans. Ferritin has a spherical structure with an empty inside, and is known to use this space to control the concentration of iron ions in the body. In particular, the human-derived ferritin protein is characterized by low immunogenicity and a globular protein having a size of 10 to 15 nm, for example, 12 nm, which is suitable for application as a medium for delivering the binding portion into the body.

In addition, since the ferritin protein forms one structure by self-assembly of the same 24 subunits, a fusion protein in which the ferritin protein is used as a carrier protein also has a plurality of subunits to form a structure. You will be able to. In one structure formed by a plurality of fusion proteins as described above, a plurality of binding portions can also be present in an exposed state, and thus, there is an advantage that the binding force to osteoblasts or hydroxyapatite can be further doubled. In addition, when the ferritin protein is used, not only can the fusion protein of the present invention be mass-produced in E. coli, but also the fusion protein produced through it can achieve excellent structural stability and pH stability. It is very suitable as a carrier protein constituting a fusion protein.

Meanwhile, the fusion protein including the binding portion and the carrier may include at least one amino acid sequence represented by SEQ ID NO: 6 or 7.

SEQ ID NO: 5 TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELKMGDGDSLFDNLR SEQ ID NO: 6 C SDSSD HHHHHH GSSGSSGSSGSS TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNECALHLEKNVNQSLLELHKMGLATDKNDLCDNPHDKLATDKLDNPH SEQ ID NO: 7 C DDDDDD HHHHHH GSSGSSGSSGSS TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLVLIKEDKGLATDDNPHLCDKGLATDKDL

The fusion protein may further include at least one of a fluorescent protein and a fluorescent dye to be fluorescently labeled.

The fluorescent protein refers to a substance that generates light by changing physical conditions or chemical treatment. The fluorescent protein is a fluorescent protein or photoprotein such as a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), etc. Alternatively, it may be luciferase, but the present invention is not limited thereto, and all fluorescent materials used in the art may be included.

The fluorescent dye is not particularly limited as long as it can be a label that can be essentially detectable by the emission of radiation (radioactivity) or by interaction, for example, fluorescein, rhodamine, coumarin, cyanide Min, etc., in one aspect, organic fluorescent dyes Atto 488, Alexa Flour 488, Dy505, Rhodamine 123, Atto 520, Dy 530, ATTO 532, Alexa Fluor 532, Fluorescein, FITC, Cy2, Cy3B, Alexa Flour 568, TAMRA , Cy3, Cy3.5, SNAP-Cell TMR-Star, Atto 565, Atto 590, Alexa Fluor 647, Cy5, Atto 647, Atto 647N, Dyomics 654, Atto 655, TMP-ATTO 655, Atto 680, Cy5.5, Atto 680, Alexa Fluor 680, Atto 700, Alexa Fluor 700, DyLight 750, Cy7, Alexa Flour 750, Atto 740, Alexa Flour 790 or IRDye 800 CW may be selected, but is not particularly limited thereto.

In addition, at least one of the fluorescent protein and the fluorescent dye may be bound to the C-terminus, N-terminus, or C-terminus and N-terminus of the carrier protein, specifically, ferritin protein. In one example, a fusion protein was prepared using a fluorescently labeled vector using cysteine (Cys) at the N-terminus of the subunit structure of the ferritin protein.

The size of the fusion protein is not particularly limited, but an average size of 450 to 600 kDa, for example, 562 to 567 kDa, may be used in terms of improving mobility and imaging accuracy for use in bio-imaging.

In specific embodiments and experimental examples of the present invention, a fusion protein capable of specific binding to osteoblasts and hydroxyapatite by fusion of the peptides of SEQ ID NOs: 1 and 2, which specifically bind to osteoblasts and hydroxyapatite, with ferritin protein. Was prepared, and a fluorescent dye was attached to the fusion protein to confirm specific binding of each fusion protein to the target. As a result, it was confirmed that the fusion protein according to the present invention can form selective and specific binding to osteoblasts and/or hydroxyapatite.

Method for producing a fusion protein

The manufacturing method of the fusion protein includes a step of amplifying the polynucleotide sequence encoding the fusion protein according to the present invention by PCR.

The inventors of the present invention perform PCR by using at least one forward primer of SEQ ID NO: 8 or 9 and reverse primer of SEQ ID NO: 10 when amplifying a recombinant vector comprising a polynucleotide sequence encoding a fusion protein according to the present invention. It was confirmed that the efficiency can be improved.

SEQ ID NO: 8 ACGTGCCATATG TGT TCTGATTCATCTGAT CATCACCATCAC SEQ ID NO: 9 ACGTGCCATATG TGT GATGACGATGACGATGAT CATCACCATCAC SEQ ID NO: 10 TCGTATCTCGAG TTAGCTTTCATTATCACTGTCTCCCAG

The fusion protein of the present invention can be prepared by a conventional method of obtaining a protein using the recombinant vector after introducing the polynucleotide sequence encoding the fusion protein according to the present invention into a recombinant vector. For example, it may be prepared by introducing the recombinant vector into a microorganism to obtain a recombinant microorganism, culturing the recombinant microorganism to express the fusion protein, and then recovering the expressed fusion protein.

In the culture of the microorganism, conditions such as a medium component, a culture temperature, and a culture time can be appropriately adjusted, and specifically, the culture medium contains all nutrients essential for the growth and survival of the microorganism such as a carbon source, a nitrogen source, and a trace element component. can do. In addition, the pH of the medium can be appropriately adjusted and components such as antibiotics can be included. In addition, the inducer can be treated to induce the expression of 　 protein, and the type of the inducer to be treated can be determined according to the 　 vector 　 system, and conditions such as administration time and dosage of the inducer can be appropriately adjusted.

Composition for bio-imaging

The composition for bio-imaging includes the fusion protein according to the present invention.

The fusion protein can selectively or specifically bind to at least one of osteoblasts and hydroxyapatite, so when a composition containing the same is administered into a subject's body, tissues and organs in which osteoblasts and/or hydroxyapatite are present in the body The fusion protein is bound to the back. In particular, since the osteoblasts and hydroxyapatite are present in the bone, the surface of the bone, and around the bone, the composition including the fusion protein can be used for bone imaging.

The composition for bio-imaging may further include a pharmaceutically acceptable carrier in addition to the fusion protein. The pharmaceutically acceptable carrier is commonly used in formulation, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline Sex cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto.

In addition, the composition for bio-imaging may be administered orally or parenterally to a subject, and the method of administration thereof is not particularly limited. For example, for the oral administration, the bio-imaging composition may be formulated as a tablet, pill, powder, granule, capsule, etc., but is not limited thereto. In addition, the parenteral administration is intravenous injection, intramuscular injection, intra-articular injection, intra-synovial injection, intrameningal injection, intrahepatic injection, intralesional injection Alternatively, it may be administered by intracranial injection or the like, but is not limited thereto.

In addition, the'subject' to which the composition for bio-imaging is administered may be an animal, including humans, and is particularly preferably an animal having bones in a living body structure, but is not particularly limited thereto.

Hereinafter, in order to aid the understanding of the present invention, it will be described in detail with reference to the drawings and examples. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely describe the present invention to those with average knowledge in the field to which the present invention belongs.

Example 1. Vector construction for production of osteoblasts or hydroxyapatite-binding fusion protein

FIG. 1 shows a schematic diagram of a vector configuration for preparing a ferritin fusion protein capable of (a) a subunit structure of a ferritin protein, (b) an entire structure of a ferritin protein, and (c) an osteoblast or hydroxyapatite binding.

First, in the subunit structure of the ferritin protein, cysteine (Cys) for attaching a fluorescent dye at the N-terminal position, a peptide that specifically binds to osteoblasts or hydroxyapatite, His-tag for purification of fusion proteins, and structural And linkers for functional stability were sequentially inserted and fused.

Thereafter, PCR was performed using a human-derived ferritin gene in which a His-tag and a linker are sequentially inserted at the N-terminal position as a template, and a forward primer of SEQ ID NO: 8 or 9 and a reverse primer of SEQ ID NO: 10, respectively. The gene was amplified. The amplified gene was cloned into a pET-21a vector using restriction enzymes Nde 1 and Xho 1, and then the base sequence encoding the osteoblast or hydroxyapatite-binding ferritin protein was confirmed. As a control, the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 10 were used in the same manner as described above to obtain a wild-type ferritin protein without osteoblasts or hydroxyapatite-binding peptide sequence.

The amino acid sequence of the identified osteoblast-binding fusion protein 1 (Ob-Fn) was represented by SEQ ID NO: 6, and the amino acid sequence of the hydroxyapatite-binding fusion protein 2 (HA-Fn) was represented by SEQ ID NO: 7.

Example 2. Purification of Fusion Protein 1 (Ob-Fn) and Fusion Protein 2 (HA-Fn)

Vectors encoding the fusion proteins 1 and 2 prepared above were introduced into BL21 (DE3) E. coli to obtain a transformant, and then the obtained transformant was cultured at 37°C until an OD value of 0.5 to 0.8 was reached. Then, 0.5 mM IPTG (Isopropyl-bD-thio-galactoside) was added to the culture solution to induce protein expression.

Thereafter, after further incubation at 18° C. for 20 hours, the E. coli culture solution was centrifuged at 4,000 rpm to separate the E. coli and the culture solution, and the culture solution was removed, and then the separated E. , 10 mM imidazole, pH 8.0), and then E. coli was dissolved using an ultrasonic disruptor.

After the E. coli lysate was centrifuged at 16,000 rpm to obtain only an aqueous supernatant, the ferritin protein with His-tag was finally purified by affinity chromatography using Ni-NTA resin. The size of the protein subunit was confirmed through 15% SDS-PAGE analysis of the final purified ferritin protein.

As can be seen in FIG. 2, it was confirmed that a 24 kDa osteoblast or a subunit of a hydroxyapatite-binding fusion protein was expressed.

Experimental Example 1. Characterization of Fusion Protein 1 and Fusion Protein 2

First, fusion protein 1 was negatively stained with 2% phosphotungstric acid (ph 7.5), and then the TEM image was confirmed. As a result of checking in FIG. 3 (a), it was confirmed that 24 subunits of fusion protein 1 were self-assembled to form nanoparticles having a hollow spherical shape in the center.

In addition, an experiment was performed to measure the actual size of fusion protein 1 using DLS. The wild-type ferritin protein used as a control is known to have a size of 11 to 12 nm, and fusion protein 1 is an additional binding peptide inserted at the N-terminus of the human-derived ferritin protein, and the inserted peptide is exposed to the outside. Therefore, it was expected that the size was larger than that of the wild-type ferritin protein, and as a result of actual DLS measurement, the size of the fusion protein 1 was 14 nm, which was confirmed to conform to the above prediction (FIG. 3 (b)).

Finally, using fusion protein 1 and wild-type protein, an experiment to check the size and charge of the protein according to the presence or absence of fusion protein 1 through electrophoresis using a 6% native gel without SDS (sodium dodecyl sulfate). Was performed. As a result, there was only a difference in the number of 5 amino acid sequences per subunit, and thus staining bands were observed at similar positions (Fig. 3 (c)).

Example 3. Preparation of fluorescently labeled fusion protein

In a buffer (20 mM Tris, 50 mM NaCl, pH 7.5), each of the fusion proteins 1 and 2 purified in the experiment of Example 2 and Cy5-maleiminde were mixed and reacted at room temperature for 4 hours at a ratio of 1:30 equivalents. , After the reaction was over, unreacted Cy5-maleimide was removed using a PD-10 desalting column, and then concentrated using a centrifuge, and a Cy5 fluorescent dye was labeled on the cysteine located at the far end of the N-terminus of the fusion protein.

As a result of analysis using a spectrometer, it was confirmed that an average of four Cy5 fluorescent dyes were labeled on one finally prepared fusion protein.

Experimental Example 2. Evaluation of osteoblast binding ability of fusion protein 1

In order to confirm the selective binding of the fluorescently labeled fusion protein 1 obtained in Experimental Example 4 to osteoblasts, an experiment was performed using MC3T3-E1, an undifferentiated osteoblast derived from a mouse, and the results are shown in FIG. .

Specifically, for MC3T3-E1 differentiation, 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate, which are differentiation inducing agents, were added to α-MEM medium containing 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin. It was changed every 3 days and cultured for 3 weeks.

Fusion protein 1 was reacted in α-MEM medium without FBS at a concentration of 10 μg/ml for 1 hour, and then washed twice with DPBS solution. Cells were immobilized for 15 minutes with 4% paraformaldehyde, washed twice, and then nuclear stained with DAPI (2-(4-amidinophenyl)-1H-indole-6-carboxamidine) and observed under a fluorescence microscope.

The control cervical cancer cells HeLa and undifferentiated MC3T3-E1 were cultured for 2 days and then performed in the same manner as described above. As a result, it was confirmed that a strong fluorescent signal was detected in MC3T3-E1 cells differentiated for 3 weeks, and no fluorescent signal was detected in undifferentiated MC3T3-E1 cells and HeLa cells.

As a method for additionally confirming the selective binding of ferritin, it was performed in the same manner as the above method using a wild type ferritin protein. As a result, it was confirmed that the fluorescence signal was not detected not only in undifferentiated MC3T3-E1 cells, HeLa cells, but also in MC3T3-E1 cells differentiated for 3 weeks. Therefore, it was confirmed that osteoblast-bound ferritin nanoparticles selectively bind to osteoblasts.

Experimental Example 3. Evaluation of hydroxyapatite binding ability of fusion protein 2

In order to confirm the selective binding of the fluorescently labeled fusion protein 2 obtained in Example 3 to hydroxyapatite, an experiment was performed using hydroxyapatite having a particle size smaller than 200 nm, and the results are shown in FIG. I did.

6.25, 12.5, 25 μg/ml of fusion protein 2 and 2.5 mg/ml of hydroxyapatite particles were reacted for 1 hour in a 300 μl solution containing 0.05% Tween-20, washed twice, and then 600 with a plate fluorescent reader. nm excitation was performed to measure a 670 nm emission signal.

As a result, hydroxyapatite treated with 12.5 μg/ml ferritin nanoparticles showed the best results with a binding rate of 72%. On the other hand, the wild type ferritin nanoparticles as a control group had a binding rate of 10%.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Fusion protein and bio-imaging composition comprising the same <130> 2019-DPA-3177 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> osteablast and/or hydroxyapatite binding amino acid <400> 1 Ser Asp Ser Ser Asp 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> osteablast and/or hydroxyapatite binding amino acid <400> 2 Asp Asp Asp Asp Asp Asp 1 5 <210> 3 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> osteablast and/or hydroxyapatite binding amino acid <400> 3 Cys Ser Asp Ser Ser Asp His His His His His His Gly Ser Ser Gly 1 5 10 15 Ser Ser Gly Ser Ser Gly Ser Ser 20 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> osteablast and/or hydroxyapatite binding amino acid <400> 4 Cys Asp Asp Asp Asp Asp Asp His His His His His His Gly Ser Ser 1 5 10 15 Gly Ser Ser Gly Ser Ser Gly Ser Ser 20 25 <210> 5 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> ferritin protein <400> 5 Cys Asp Asp Asp Asp Asp Asp His His His His His His Gly Ser Ser 1 5 10 15 Gly Ser Ser Gly Ser Ser Gly Ser Ser 20 25 <210> 6 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> osteoblast and/or hydroxyapatite binding amino acid <400> 6 Cys Ser Asp Ser Ser Asp His His His His His His Gly Ser Ser Gly 1 5 10 15 Ser Ser Gly Ser Ser Gly Ser Ser Thr Thr Ala Ser Thr Ser Gln Val 20 25 30 Arg Gln Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln Ile 35 40 45 Asn Leu Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser Tyr Tyr 50 55 60 Phe Asp Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr Phe Leu 65 70 75 80 His Gln Ser His Glu Glu Arg Glu His Ala Glu Lys Leu Met Lys Leu 85 90 95 Gln Asn Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys Lys Pro 100 105 110 Asp Cys Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys Ala Leu 115 120 125 His Leu Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu His Lys Leu 130 135 140 Ala Thr Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr His 145 150 155 160 Tyr Leu Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp His Val 165 170 175 Thr Asn Leu Arg Lys Met Gly Ala Pro Glu Ser Gly Leu Ala Glu Tyr 180 185 190 Leu Phe Asp Lys His Thr Leu Gly Asp Ser Asp Asn Glu Ser 195 200 205 <210> 7 <211> 207 <212> PRT <213> Artificial Sequence <220> <223> osteoblast and/or hydroxyapatite binding amino acid <400> 7 Cys Asp Asp Asp Asp Asp Asp His His His His His His Gly Ser Ser 1 5 10 15 Gly Ser Ser Gly Ser Ser Gly Ser Ser Thr Thr Ala Ser Thr Ser Gln 20 25 30 Val Arg Gln Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln 35 40 45 Ile Asn Leu Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser Tyr 50 55 60 Tyr Phe Asp Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr Phe 65 70 75 80 Leu His Gln Ser His Glu Glu Arg Glu His Ala Glu Lys Leu Met Lys 85 90 95 Leu Gln Asn Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys Lys 100 105 110 Pro Asp Cys Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys Ala 115 120 125 Leu His Leu Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu His Lys 130 135 140 Leu Ala Thr Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr 145 150 155 160 His Tyr Leu Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp His 165 170 175 Val Thr Asn Leu Arg Lys Met Gly Ala Pro Glu Ser Gly Leu Ala Glu 180 185 190 Tyr Leu Phe Asp Lys His Thr Leu Gly Asp Ser Asp Asn Glu Ser 195 200 205 <210> 8 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> primer <400> 8 Ala Cys Gly Thr Gly Cys Cys Ala Thr Ala Thr Gly Thr Gly Thr Thr 1 5 10 15 Cys Thr Gly Ala Thr Thr Cys Ala Thr Cys Thr Gly Ala Thr Cys Ala 20 25 30 Thr Cys Ala Cys Cys Ala Thr Cys Ala Cys 35 40 <210> 9 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> primer <400> 9 Ala Cys Gly Thr Gly Cys Cys Ala Thr Ala Thr Gly Thr Gly Thr Gly 1 5 10 15 Ala Thr Gly Ala Cys Gly Ala Thr Gly Ala Cys Gly Ala Thr Gly Ala 20 25 30 Thr Cys Ala Thr Cys Ala Cys Cys Ala Thr Cys Ala Cys 35 40 45 <210> 10 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> primer <400> 10 Thr Cys Gly Thr Ala Thr Cys Thr Cys Gly Ala Gly Thr Thr Ala Gly 1 5 10 15 Cys Thr Thr Thr Cys Ala Thr Thr Ala Thr Cys Ala Cys Thr Gly Thr 20 25 30 Cys Thr Cys Cys Cys Ala Gly 35 <210> 11 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> primer <400> 11 Ala Cys Gly Thr Gly Cys Cys Ala Thr Ala Thr Gly Thr Gly Thr Cys 1 5 10 15 Ala Thr Cys Ala Cys Cys Ala Thr Cys Ala Cys Cys Ala Thr Cys Ala 20 25 30 Cys

Claims (13)

A coupling portion capable of binding to at least one of osteoblasts and hydroxyapatite; And a carrier protein.
The method according to claim 1,
The binding portion will comprise at least one amino acid sequence represented by SEQ ID NO: 1 or 2, the fusion protein.
The method according to claim 2,
The binding portion will include at least one amino acid sequence represented by SEQ ID NO: 3 or 4, fusion protein.
The method according to claim 1,
The carrier protein is ferritin (Ferritin) protein that will, fusion protein.
The method of claim 4,
The ferritin protein is a fusion protein comprising the amino acid sequence represented by SEQ ID NO: 5.
The method according to claim 1,
A fusion protein comprising at least one amino acid sequence represented by SEQ ID NO: 6 or 7.
The method according to claim 1,
The fusion protein further comprising at least one of a fluorescent protein and a fluorescent dye.
The method of claim 7,
At least one of the fluorescent protein and the fluorescent dye is bound to the C-terminus, N-terminus or C-terminus and N-terminus of the carrier protein.
The method according to claim 1,
The fusion protein will have an average size of 450 to 600 kDa, fusion protein.
Amplifying the polynucleotide sequence encoding the fusion protein according to any one of claims 1 to 9 by PCR; containing, a method for producing a fusion protein.
The method of claim 10,
The PCR is a method for producing a fusion protein to be performed using at least one forward primer of SEQ ID NO: 8 or 9 and a reverse primer of SEQ ID NO: 10.
A composition for bio-imaging comprising the fusion protein according to any one of claims 1 to 9.
The method of claim 12,
The bio-imaging composition for bio-imaging is bone imaging.

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