KR101826249B1 - Fusion protein and composition for controlling cell cycle comprising the fusion protein - Google Patents

Abstract

The present invention provides a method for producing a recombinant vector comprising the steps of: (a) selecting a group consisting of a central target catalase fusion protein, a polynucleotide encoding the fusion protein, a recombinant vector comprising the polynucleotide, a cell into which the recombinant vector has been introduced and a fusion protein, a polynucleotide and a recombinant vector And more particularly to a pharmaceutical composition for preventing and / or treating cancer comprising one or more active ingredients.

Description

[0001] The present invention relates to a fusion protein and a cell cycle regulator comprising the fusion protein,

A polynucleotide encoding the fusion protein, a recombinant vector comprising the polynucleotide, a cell into which the recombinant vector is introduced, and a recombinant vector selected from the group consisting of the fusion protein, the polynucleotide, and the recombinant vector Or more as an active ingredient. The present invention also relates to a pharmaceutical composition for preventing and / or treating cancer.

The division, proliferation and differentiation of the cells constituting the living body is the basic mechanism for maintaining the life phenomenon. Cell proliferation and growth are regulated by a sophisticated intracellular signaling pathway, which is a process in which a cell receives signals from the outside in response to various proteins (PLC, PKC, Shc, Grb2, Raf, MAPK, MEK, etc.) (GTP, cAMP, etc.) to the cell clock in the nucleus. If any part of this process occurs, it will develop into a disease in most cases. In particular, the cell cycle occurring in intracellular nuclei is one of the important processes controlling the maintenance of life of the cells.

The cell cycle consists of Gap1 (G1), DNA synthesis step (S), Gap2 (G2) and splitter (M). In addition to the cell cycle, if the cells are present at a high density or are left in the environment of a low concentration of growth factors for a long period of time, the cells will enter the growth state Go, called the dormant phase.

If several factors interfere with the regulation mechanism of the cell cycle, it increases the instability of the genetic material, leading to uncontrolled cell growth and inducing unstimulated cell growth such as cancer.

Therefore, by controlling the cell cycle, it is predicted that it will be possible to treat diseases such as cancer caused by unsteady cell growth, and development of the related technology is required.

Korean Patent Publication No. 2010-0124625

An example of the present invention provides a centrosome targeting catalase fusion protein. The fusion protein may be characterized in that it has cell cycle control activity.

Another example provides a polynucleotide encoding said fusion protein.

Another example provides a recombinant vector comprising the polynucleotide.

Another example provides a recombinant cell into which said recombinant vector has been introduced.

Another example provides a pharmaceutical composition for preventing and / or treating cancer comprising, as an active ingredient, at least one member selected from the group consisting of the fusion protein, the polynucleotide, the recombinant vector, and the recombinant cell.

Another example is a method for preventing and / or preventing cancer, comprising contacting at least one member selected from the group consisting of the fusion protein, the polynucleotide, the recombinant vector, and the recombinant cell to a subject in need of prevention and / Provide a treatment method.

Another example provides a cell cycle regulating composition comprising at least one member selected from the group consisting of the fusion protein, the polynucleotide, the recombinant vector, and the recombinant cell as an active ingredient.

Another example provides a cell cycle control method comprising contacting at least one member selected from the group consisting of the fusion protein, the polynucleotide, the recombinant vector, and the recombinant cell to a subject in need of cell cycle control.

And the beginning of mitosis controlled by the density of the core body periphery of the H ₂ O ₂ in the command, when the concentration of H ₂ O ₂ is lowered around the core body was confirmed that this inhibiting mitosis. This suggests that catalase, which catalyzes the decomposition of H ₂ O ₂ to produce water and oxygen, can be artificially inhibited from progressing to the cell division when expressed as a centrally expressed protein. This can be useful as an anticancer agent in cancer cells whose active oxygen is excessively increased compared to normal cells.

Hereinafter, the present invention will be described in more detail.

One example of the present invention,

(1) a catalase or catalase fragment; And

(2) centroid target domain

Lt; RTI ID = 0.0 > catalase < / RTI > fusion protein. The fusion protein has cell cycle regulatory activity.

The catalase is an enzyme that decomposes hydrogen peroxide into water. It is widely found in mammals, and plays a role in inhibiting the accumulation of peroxides continuously produced in various metabolic reactions in vivo and protecting cells or tissues from damage caused by peroxides. The catalase may be derived from mammals including humans.

In one example, the catalase may be of human origin and may be derived from, for example, the amino acid sequence of the NCBI accession number NP_001743.1 (SEQ ID NO: 1) or the coding sequence of the NCBI accession number NM_001752.2 (cDNA) (CDS: NM_001752.2 84-1667 site: SEQ ID NO: 5; 527 aa), but is not limited thereto.

The catalase fragment may be at least all of the polypeptides comprising a site essential for catalase activity, for example, 523 to 527 consecutive or 523 to 523 consecutive amino acids comprising the amino acids from position 1 to 523 of the amino acid sequence of SEQ ID NO: But is not limited to, a polypeptide consisting of 526 amino acids.

In the present specification, unless otherwise stated, the catalase may mean one or more selected from the full-length catalase and the catalase fragment.

The central target domain is a polypeptide targeting a centrosome at the time of cell division, and serves to target the catalase or catalase fragment to a centrosome. In one example, the centric target domain can be, for example, a pericentrin-AKAP450 centrosomal targeting (PACT) domain or a PACT domain fragment. The PACT domain is a centromere targeting domain conserved in mammals, and may be derived from mammals, including humans. In one example, the PACT domain comprises 168 amino acids (3644N-3811T) from 3644th (N) to 3811th (T) of Human AKAP450 protein (NCBI Accession No. CAB40713.1) (T) of 169 amino acids (3643A-3811T). For example, the PACT domain comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 6, or an amino acid sequence further comprising Ala at the N-terminus of the amino acid sequence of SEQ ID NO: 2 . The PACT domain fragment refers to a part of the PACT domain that includes an essential region for targeting a centrally located PACT domain. For example, the essential region for targeting the PACT domain may include the following two motifs: a region from the 62nd (K) to the 105th (P) of the amino acid sequence of SEQ ID NO: 2 (motif 1; SEQ ID NO: 16) and / or the 122nd (R) to 143th (W) region (motif 2; Therefore, the PACT domain fragment is composed of 44 to 168 or 44 to 167 amino acids including the 62nd (K) to 105th (P) region (SEQ ID NO: 16) in the amino acid sequence of SEQ ID NO: A polypeptide consisting of consecutive 22 to 168 or 22 to 167 amino acids including the 122nd (R) to 143th (W) region (SEQ ID NO: 17), or a 62nd (K (SEQ ID NO: 16) and the 122nd (R) to 143rd (W) region (SEQ ID NO: 18) ≪ / RTI > amino acids. For example, the PACT domain fragment has an amino acid sequence from the 62nd amino acid sequence (K: 3705th amino acid based on the CAB40713.1 amino acid sequence) to the 143th amino acid (W: 3786th amino acid based on the CAB40713.1 amino acid sequence) of the amino acid sequence of SEQ ID NO: (SEQ. ID. NO. 18). ≪ / RTI >

In this specification, unless otherwise stated, the PACT domain may refer to both the full-length PACT domain and the PACT domain fragment.

An example of the amino acid sequence is summarized in Table 1 below.

SEQ ID NO: denomination The amino acid sequence (5 '-> 3') Explanation One Cat MADSRDPASDQMQHWKEQRAAQKADVLTTGAGNPVGDKLNVITVGPRGPLLVQDVVFTDEMAHFDRERIPERVVHAKGAGAFGYFEVTHDITKYSKAKVFEHIGKKTPIAVRFSTVAGESGSADTVRDPRGFAVKFYTEDGNWDLVGNNTPIFFIRDPILFPSFIHSQKRNPQTHLKDPDMVWDFWSLRPESLHQVSFLFSDRGIPDGHRHMNGYGSHTFKLVNANGEAVYCKFHYKTDQGIKNLSVEDAARLSQEDPDYGIRDLFNAIATGKYPSWTFYIQVMTFNQAETFPFNPFDLTKVWPHKDYPLIPVGKLVLNRNPVNYFAEVEQIAFDPSNMPPGIEASPDKMLQGRLFAYPDTHRHRLGPNYLHIPVNCPYRARVANYQRDGPMCMQDNQGGAPNYYPNSFGAPEQQPSALEHSIQYSGEVRRFNTANDDNVTQVRAFYVNVLNEEQRKRLCENIAGHLKDAQIFIQKKAVKNFTEVHPDYGSHIQALLDKYNAEKPKNAIHTFVQSGSHLAAREKANL Catalase whole sequence 2 PACT NIEAIIASEKEVWNREKLTLQKSLKRAEAEVYKLKAELRNDSLLQTLSPDSEHVTLKRIYG KYLRAESFRKALIYQKKYLLLLGGFQECEDATLALLARMGGQP AFTDLEVITNRPKGFT RFRSAVRVSIAISRMKFLVRRW HRVTGSVSININRDGFGLNQGAEKT PACT sequence (Bold: Essential area for central targeting) 3 Cat-PACT QASNSAVDA NIEAIIASEKEVWNREKLTLQKSLKRAEAEVYKLKAELRNDSLRQTLSPDSEHVTLKRIYG KYLRAESFRKALIYQKKYLLLLGGFQECEDATLALLARMGGQP AFTDLEVITNRPKGFT RFRSAVRVSIAISRMKFLVRRW HRVTGSVSININRDGFGLNQGAEKT GSTGSR Fusion protein sequence
("QASNSAVDA" (underlined): Linker, of which the last residue A may be deleted;
C-terminal peptide "GSTGSR" (underlined) inserted in the cloning step and not included in the fusion protein) 4 Cat-delPACT QASNSAVDA NIEAIIASEKEVWNREKLTLQKSLKRAEAEVYKLKAELRNDSLLQTLSPDSEHVTLKRIYGHRVTGSVSININRDGFGLNQGAEKT GSTGSR The fusion protein sequence in which the important PACT site (shown in italic in SEQ ID NO: 3: SEQ ID NO: 18) 16 Motif 1 KYLRAESFRKALIYQKKYLLLLLGGFQECEDATLALLARMGGQP Motifs essential for central targeting among PACT sequences 17 Motif 2 RFRSAVRVSIAISRMKFLVRRW 18 KYLRAESFRKALIYQKKYLLLLGGFQECEDATLALLARMGGQPAFTDLEVITNRPKGFTRFRSAVRVSIAISRMKFLVRRW Motif 1 + 2 area

In the fusion protein, the position of the catalase or catalase fragment and the PACT domain or the PACT domain fragment is not particularly limited. For example, the catalase or catalase fragment is located at the N-terminal side (N-terminal portion in the fusion protein; Terminal region of the fusion protein (i.e., linked to the C-terminus of the catalase or catalase fragment) at the C-terminus side (linked to the N-terminus of the PACT domain or PACT domain fragment) .

The catalase or catalase fragment and the PACT domain or PACT domain fragment may be directly linked without a linker or linked via a linker (e.g., a peptide linker). The "linkage" means that the linkage is linked by a covalent bond such as a peptide bond. The peptide linker has no particular limitation on the type and length of the amino acid contained therein, but it is preferable that the peptide linker does not affect the structure of the fusion protein. Thus, the peptide linker has about 20 or less, about 18 or less, about 15 or less, about 12 or less 1 to 20, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 2 to 20, 2 to 18, 2 to 15, 12, 2, 10, 5, 20, 5 to 18, 5 to 15, 5 to 12, or 1 to 10 amino acids.

The fusion protein may contain up to about 20, up to about 18, up to about 15, up to about 12, up to about 10 amino acids, such as from 1 to 20 amino acids, for example, at the C-terminus, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 2 to 20, 2 to 18, 2 to 15, 2 to 12, 2 to 10, 5 to 20, 5 to 18, 5 to 15, 5 to 12, or 1 to 10 amino acids. The kind of the further included amino acid is not limited. In one example, the further included amino acid is inserted in the cloning step or is derived from a human AKAP450 protein (NCBI Accession No. CAB40713.1) containing the PACT domain (e. 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 2 to 20, 2 to 18, 2 to 15, 2 to 12, 2 to 10 , 5 to 20, 5 to 18, 5 to 15, 5 to 12, or 1 to 10 amino acids).

In one example, the centro-targeted Catalase (Cat-PACT)

A catalase or catalase fragment comprising consecutive 523 to 527 or 523 to 526 amino acids comprising the amino acids from position 1 to 523 of the amino acid sequence of SEQ ID NO: 1 at the N-terminal side; And

(44 to 168 or 44 to 167 amino acids including the 62nd to 105th amino acids in the amino acid sequence of SEQ ID NO: 2 (SEQ ID NO: 16), the 122nd to 143th amino acids (SEQ ID NO: 18) comprising consecutive 22 to 168 or 22 to 167 amino acids, or 62 to 143 amino acids (SEQ ID NO: 18) Lt; RTI ID = 0.0 > PACT < / RTI >

For example, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 3.

Another example of the present invention provides a polynucleotide encoding the centrally targeted Cat-PACT fusion protein.

The polynucleotide includes a polynucleotide encoding the catalase or catalase fragment as described above, and a polynucleotide encoding the PACT domain or PACT domain fragment.

The polynucleotide encoding the catalase may comprise the nucleotide sequence of SEQ ID NO: 5, and the polynucleotide encoding the catalase fragment may comprise the nucleotide sequence of SEQ ID NO: 5 with consecutive 1,569 Lt; RTI ID = 0.0 > nucleotides. ≪ / RTI >

The polynucleotide encoding the PACT domain may comprise the nucleotide sequence of SEQ ID NO: 6.

In one example, the polynucleotide encoding the centro-targeted Catalase (Cat-PACT) fusion protein may comprise the nucleotide sequence of SEQ ID NO: 7.

Another example provides a recombinant vector comprising a polynucleotide encoding the centrally-targeted Cat-PACT fusion protein.

In one example, the recombinant vector may have, but is not limited to, the cleavage map set forth in FIG.

In addition, the recombinant vector may be a viral or non-viral vector.

The viral vector may be, but is not limited to, adenovirus, adeno-associated virus, helper-dependent adenovirus, retroviral vector, and the like.

The recombinant vector may be constructed by a variety of methods known in the art.

The term "vector" means means for expressing a gene of interest in a host cell. The vector includes elements for expression of a target gene and may include a replication origin, a promoter, an operator, a transcription termination terminator, and the like into the genome of the host cell. Ribosome binding sites (RBS) for translation into selectable markers and / or proteins to confirm successful introduction into the host cell and / or appropriate enzyme sites for introduction of the IRES (e.g., IRES (Internal Ribosome Entry Site), and the like. The vector may be engineered in a conventional genetic engineering manner to have the fusion polynucleotide (fusion promoter) described above as a promoter. The vector may further comprise a transcription control sequence other than the promoter (e.g., an enhancer, etc.).

In another embodiment, the present invention provides a recombinant cell into which the recombinant vector is introduced.

The cell may be a mammalian cell and the mammalian cell may be a mammal such as a human (e.g. Hela, HEK-293, retinal-derived PER-C6, cells derived from diploid fibroblasts, myeloma cells, HepG2 etc.) But it is not so limited.

The transfer (introduction) of the recombinant vector into a cell can be carried out using a carrier method well known in the art. For example, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, gene bombardment, and the like can be used as the delivery method. For example, liposome-mediated transfection (Lipofector reagent). < / RTI >

The centrally-targeted catalase (Cat-PACT) fusion protein can control the cell cycle by removing H ₂ O ₂ around the central body (degrading with water) and lowering the concentration of H ₂ O ₂ . By such action, it is possible to treat diseases related to uncontrolled cell proliferation such as cancer.

Yet another example is a recombinant vector comprising a core-target catalase (Cat-PACT) fusion protein, a polynucleotide encoding the fusion protein, a recombinant vector comprising the polynucleotide, and a recombinant cell comprising the recombinant vector As an active ingredient, a pharmaceutical composition for preventing and / or treating cancer.

The fusion protein, the polynucleotide encoding the fusion protein, and the recombinant vector are as described above.

The cancer may be, but is not limited to, colon, bladder, breast, colon, kidney, liver, lung, ovary, testis, prostate, pancreas, stomach, cervix, thyroid, blood, bone, brain or skin .

The pharmaceutical composition may be formulated and provided in a suitable form. Each of the above-mentioned formulations may be formulated into oral formulations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups and aerosols, or parenteral formulations such as transdermal preparations, suppositories, And can be used as formulations.

In addition, the above-mentioned preparation may be one which additionally contains pharmaceutically suitable and physiologically acceptable carriers, excipients and diluents. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used can be used.

More specifically, the agent may include a carrier for addition to the pharmaceutical composition (active ingredient) for formulation thereof. The carrier may be a binder, a lubricant, a suspending agent, a solubilizer, a buffer, a preservative, a lubricant, an isotonic agent, an excipient, a stabilizer, a dispersant, a suspending agent,

In embodiments where the agent is to be administered to humans, the agent may be administered alone, but may be administered in admixture with a pharmaceutical carrier selected, generally taking into account the mode of administration and standard phamaceutical practice .

For example, when the preparation is provided for parenteral use, a topical preparation such as a liquid preparation, a gel preparation, a cleaning composition, a tablet for insertion, a suppository form, cream, ointment, dressing solution, spray, Liquid formulations such as solutions, suspensions, emulsions and the like and may be in the form of sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, creams, ointments, jellies, A skin external preparation such as a liquid preparation, a gel preparation, a cleaning composition, and a tablet for insertion may be included. The formulation can be prepared, for example, by adding a solubilizer, an emulsifying agent, a buffering agent for pH control, etc. to the sterilized water. Examples of the non-aqueous solvent or suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

In addition, when the preparation is provided for oral use, it may be in the form of tablets containing, for example, starch or lactose, alone or in capsules containing excipients, or as elixirs containing chemicals which taste or color Or in the form of a suspension, orally, buccally or sublingually.

The dose of the preparation may be varied depending on the patient's age, weight, sex, dosage form, health condition and disease severity, and may be administered once or several times a day at a predetermined time interval according to the judgment of a doctor or pharmacist have. For example, a daily dose of 0.001 to 10000 mg / kg, 0.01 to 10000 mg / kg, 0.1 to 10000 mg / kg, 0.5 to 10000 mg / kg, 0.001 to 1000 mg / kg, 0.01 to 1000 mg / Kg, 0.1 to 1000 mg / kg, 0.5 to 1000 mg / kg, 0.001 to 500 mg / kg, 0.01 to 500 mg / kg, 0.1 to 500 mg / kg, 0.5 to 500 mg / kg, / kg, 0.01 to 300 mg / kg, 0.1 to 300 mg / kg, or 0.5 to 300 mg / kg. The above-mentioned dosage is an average case, and the dose may be high or low depending on individual differences.

If the daily dose of the preparation is less than the above dose, a significant effect can not be obtained. If the daily dose exceeds the above-mentioned dose, it may not be economical and may cause undesirable side effects due to deviation from the commercial dose. Range.

The subject to be administered with the agent may be a mammal such as a human, a cell isolated from a mammal, a tissue, a body fluid, or a culture thereof.

Another example provides a cell cycle regulating composition comprising at least one member selected from the group consisting of the fusion protein, the polynucleotide, the recombinant vector, and the recombinant cell as an active ingredient. Another example provides a cell cycle control method comprising contacting at least one member selected from the group consisting of the fusion protein, the polynucleotide, the recombinant vector, and the recombinant cell to a subject in need of cell cycle control.

The subject may be a mammal other than a human, a cell, tissue, body fluid, or culture thereof isolated from a mammal including a human.

The cell cycle control may mean normalizing the cell cycle (inducing cell cycle arrest) to control the cell cycle or abnormal cell division or apoptosis.

The present invention provides more effective prevention and / or treatment of cancer by providing a core-target Catalase (Cat-PACT) fusion protein having cell cycle control activity.

1 is a cleavage map of a Cat-PACT-containing plasmid (pCat-PACT).
Fig. 2 is a schematic diagram of protein sequences of Cat-PACT and Cat-delPACT.
Figure 3 is a confocal microscope photograph showing the targeting of Cat-PACT as a central body.
FIG. 4 is a graph showing the proportion of cells entering the cell divider based on cell rounding after expressing Cat-PACT or Cat-delPACT using retrovirus and progressing G1 / S cells to mitosis.
FIG. 5 is a graph showing the proportion of cells entering a cell divider based on the condensation of DNA chromosomes after Cat-PACT or Cat-delPACT is expressed using retrovirus and G1 / S cells are transformed into mitosis.
FIG. 6 shows immunoblot results of inhibitory phosphorylation of Cdk1 (pY ¹⁵ -Cdk1), phosphorylation of Histone H3 (pHH3), Cdk1 and Actin in cells expressing Cat-PACT and Cat-delPACT.
FIG. 7 shows immunoblot intensities of inhibitory phosphorylation (pY ¹⁵ -Cdk1), Histone H3 phosphorylation (pHH3), Cdk1 and Actin of Cdk1 in cells expressing Cat-PACT and Cat-delPACT of Example, respectively This is a quantified graph.
FIG. 8 is a graph quantifying cell cycle entry time by changes in the expression pattern of H2B-GFP with time in cells expressing the Cat-PACT fusion protein or Cat-delPACT fusion protein of the example of the present invention.
FIG. 9 is a graph (right) of a confocal microscope photograph (left) and Cyclin B intensity quantification (right) observed using Cyclin B antibody in cells expressing Cat-PACT fusion protein or Cat-delPACT fusion protein.
FIG. 10 is a confocal microscope photograph (left) and a graph (right) quantifying the intensity of Plk1 in the central body observed using Plk1 antibody in cells expressing Cat-PACT fusion protein or Cat-delPACT fusion protein.
Fig. 11 is a graph (right) of a confocal microscope photograph (left) observed using Aurora A antibody in cells expressing Cat-PACT fusion protein or Cat-delPACT fusion protein and the intensity of Aurora A in the central body.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1. Preparation of plasmid

In order to construct a plasmid for expressing the pCat-PACT fusion protein, PACT was linked to the C-terminal of the dsRed gene in the pdsRed-C1 plasmid (provided by DS Hwang, Seoul National University, Seoul, ) Was used as a parental vector and the dsRed gene was transfected with the human catalase fragment coding sequence in which the C-terminal amino acid 'KANL' coding region was deleted from the human catalase coding gene (SEQ ID NO: 5).

(SEQ ID NOS: 10 and 11; see Table 2) in a plasmid containing the human catalase fragment coding sequence (SW Kang, Ewha Woman's University, Seoul, South Korea) Removal of C-terminal 'KANL' amino acid sequence region) The coding sequence was amplified by PCR and inserted into pdsRed-PACT using AgeI / HindIII enzyme to construct a plasmid for expression of pCat-PACT (see Fig. The plasmid for expressing pCat-PACT contains the nucleotide sequence of SEQ ID NO: 7 encoding the pCat-PACT fusion protein (SEQ ID NO: 3).

(motif 1: K62-P105 (KYLRAESFRKALIYQKKYLLLLLGGFQECEDATLALLARGGQP; SEQ ID NO: 16) in the PACT domain (coding gene: SEQ ID NO: 6) of SEQ ID NO: 2 to produce the pCat- delPACT plasmid. A PACT fragment (PACT fragment from which the amino acid sequence from K62 to W143 (SEQ ID NO: 18) was removed) from which all of the WACT3 (RFRSAVRVSIAISRMKFLVRRW; SEQ ID NO: 17) was removed was amplified from dsRed-PACT. (SEQ ID NOS: 12 and 15 and SEQ ID NOS: 13 and 14; see Table 2) for the amplification of the PACT fragment coding sequence (hereinafter referred to as delPACT) in which the two motif-containing portions were removed The product obtained by the second PCR amplification using the first PCR product as a template and the primer pair (SEQ ID NOS: 12 and 13; see Table 2) was inserted into the plasmid for expressing pCat-PACT using SaI / HpaI enzyme , a plasmid for expressing pCat-delPACT was constructed. The plasmid contains the nucleotide sequence of SEQ ID NO: 8, which encodes the pCat-delPACT fusion protein containing the PACT fragment removed from K62 to W143. Through the removal of the middle part, it is proved that the removed part is an essential part for targeting the core body.

The nucleotide sequences of the dsRed-PACT, PACT cDNA, and primer used are shown in Table 2 below, and a schematic diagram of the fusion protein expressed by the plasmid is shown in FIG.

SEQ ID NO: denomination The base sequence (5 '-> 3') 5 Cat
(Catalase coding sequence) Atggctgacagccgggatcccgccagcgaccagatgcagcactggaaggagcagcgggcc
gcgcagaaagctgatgtcctgaccactggagctggtaacccagtaggagacaaacttaat
gttattacagtagggccccgtgggccccttcttgttcaggatgtggttttcactgatgaa
atggctcattttgaccgagagagaattcctgagagagttgtgcatgctaaaggagcaggg
gcctttggctactttgaggtcacacatgacattaccaaatactccaaggcaaaggtattt
gagcatattggaaagaagactcccatcgcagttcggttctccactgttgctggagaatcg
ggttcagctgacacagttcgggaccctcgtgggtttgcagtgaaattttacacagaagat
ggtaactgggatctcgttggaaataacacccccattttcttcatcagggatcccatattg
tttccatcttttatccacagccaaaagagaaatcctcagacacatctgaaggatccggac
atggtctgggacttctggagcctacgtcctgagtctctgcatcaggtttctttcttgttc
agtgatcgggggattccagatggacatcgccacatgaatggatatggatcacatactttc
aagctggttaatgcaaatggggaggcagtttattgcaaattccattataagactgaccag
ggcatcaaaaacctttctgttgaagatgcggcgagactttcccaggaagatcctgactat
ggcatccgggatctttttaacgccattgccacaggaaagtacccctcctggactttttac
atccaggtcatgacatttaatcaggcagaaacttttccatttaatccattcgatctcacc
aaggtttggcctcacaaggactaccctctcatcccagttggtaaactggtcttaaaccgg
aatccagttaattactttgctgaggttgaacagatagccttcgacccaagcaacatgcca
cctggcattgaggccagtcctgacaaaatgcttcagggccgcctttttgcctatcctgac
actcaccgccatcgcctgggacccaattatcttcatatacctgtgaactgtccctaccgt
gctcgagtggccaactaccagcgtgacggcccgatgtgcatgcaggacaatcagggtggt
gctccaaattactaccccaacagctttggtgctccggaacaacagccttctgccctggag
cacagcatccaatattctggagaagtgcggagattcaacactgccaatgatgataacgtt
actcaggtgcgggcattctatgtgaacgtgctgaatgaggaacagaggaaacgtctgtgt
gagaacattgccggccacctgaaggatgcacaaattttcatccagaagaaagcggtcaag
aacttcactgaggtccaccctgactacgggagccacatccaggctcttctggacaagtac
aatgctgagaagcctaagaatgcgattcacacctttgtgcagtccggatctcacttggcg
gcaagggagaaggcaaatctgtga 6 PACT
(PACT Encoding Sequence) aacattgaagccatcattgcctctgaaaaagaagtatggaacagagaaaaattgactctccagaaatctttgaaaagggcagaggctgaagtatacaaactgaaagctgaactaagaaatgactctttacttcaaactctgagccctgattctgaacatgtcactttaaagagaatttatggtaaatacttgagggcagaaagttttcgaaaggctctcatttaccagaagaaatacctgctgctgttactgggtgggttccaggaatgtgaagatgccaccttggccctgcttgcccggatgggggggcagccagctttcacggatctagaggtgatcaccaatcgcccaaagggcttcaccaggtttcggtcggccgtcagagtatccattgcaatttccagaatgaaatttttggttcgacggtggcatcgagtcacaggttctgtttccatcaatattaacagagatggctttggactgaatcaaggtgcagaaaagact 7 Cat-PACT
(Fusion Protein Encoding Sequence) tcaatattaacagagatggctttggactgaatcaaggtgcagaaaagactggatccaccggatctagataa 8 Cat-delPACT
(A fusion protein coding sequence in which the important PACT region has been removed) cattgaggccagtcctgacaaaatgcttcagggccgcctttttgcctatcctgacactcaccgccatcgcctgggacccaattatcttcatatacctgtgaactgtccctaccgtgctcgagtggccaactaccagcgtgacggcccgatgtgcatgcaggacaatcagggtggtgctccaaattactaccccaacagctttggtgctccggaacaacagccttctgccctggagcacagcatccaatattctggagaagtgcggagattcaacactgccaatgatgataacgttactcaggtgcgggcattctatgtgaacgtgctgaatgaggaacagaggaaacgtctgtgtgagaacattgccggccacctgaaggatgcacaaattttcatccagaagaaagcggtcaagaacttcactgaggtccaccctgactacgggagccacatccaggctcttctggacaagtacaatgctgagaagcctaagaatgcgattcacacctttgtgcagtccggatctcacttggcggcaagggagcaagcttcgaattctgcagtcgacgccaacattgaagccatcattgcctctgaaaaagaagtatggaacagagaaaaattgactctccagaaatctttgaaaagggcagaggctgaagtatacaaactgaaagctgaactaagaaatgactctttacttcaaactctgagccctgattctgaacatgtcactttaaagagaatttatggtcatcgagtcacaggttctgtttccatcaatagagatggctttggactgaatcaaggtgcagaaaagactggatccaccggatctagataa 9 dsRed-PACT gggggggcagccagcttcacggatctagaggtgatcaccaatcgcccaaagggcttcaccaggtttcggtcggccgtcaggtatccattgcaatttccgaatgaaatttttggttcgacggtggcatcgagtcacaggttctgtttccatcaatattaacagagatggctttggactgaatcaaggtgcagaaaagactggatccaccggatctagataa 10 Cat Primer_F CTA CCGGTA TGG CTG ACA GCC 11 Cat Primer_R CGA AGC TTG CTC CCT TGC CGC CAA GTGA 12 PACT Primer_F GAG CAA GCT TCG AAT TCT GC 13 PACT Primer_R ACA AGT TAA CAA CAA CAA TTGCA 14 delPACT Primer_F AAG AGA ATT TAT GGT CAT CGA GTC ACA GGT TCT GT 15 delPACT Primer_R ACC ATA AAT TCT CTT TAA AGT GAC

Example  2. Expression of Cat-PACT fusion protein in centrosomes

In order to introduce the plasmid prepared in Example 1, each of the plasmids prepared above was introduced into HeLa cells (American Type Culture Collection [ATCC], USA) by liposome infusion. Then, 10% fetal bovine serum FBS (Gibco, USA)) and penicillin-streptomycin (Hyclone, USA) at 37 ° C for 24 hours.

Next, confocal immunofluorescence microscopy (A1R; Nikon, Japan) was used to confirm whether Cat-PACT fusion protein was expressed around the central body, and the results are shown in FIG. It was confirmed that the Cat-PACT fusion protein was targeted as a centrosome while the Cat-delPACT fusion protein was not targeted as a centrosome.

Example  3. Inhibitory effect of Cat-PACT fusion protein on cell cycle activity

3-1. Measure cell rounding and chromosome condensation

Cat-PACT plasmids, Cat-delPACT plasmids and empty vectors as control were used in the same manner as in Example 2 to determine whether the Cat-PACT fusion protein exhibited inhibitory effects on cell cycle activity. GFP-H2B stable HeLa cells (JH Lee, Ajou University, Suwon, South Korea, pEGFP-N1 vector was inserted with human Histone H2B gene) and cultured at 37 ° C for 10 hours.

Then, in order to equally adjust the cell cycle to G1-S, the cells were cultured in a medium containing 2 mM thymidine for 18 hours, left in an S-phase for 3 hours in a medium in which thymidine was removed, And treated for 10 hours in a medium containing 100 ng / ml nocodazole (Sigma-Aldrich, USA).

Cell rounding and chromosome condensation were then measured. The cell divider cell looks like a circular ball (cell rounding) compared to the interphase cell, and the chromosome condenses to show a strong GFP signal. The confocal microscope was used to obtain bright and GFP images, and the proportion of cells showing cell rounding and cells showing chromosome condensation in all cells was determined. The results are shown in FIG. 4 (cell rounding) and 5 (chromosome condensation), and in Tables 3 and 4.

Cell Rounding Measurement Results Cell Rounding (%) Empty 61.8 Cat-PACT 36.2 Cat-delPACT 54.5

Results of chromosome condensation measurement Condensed chromosome (%) Empty 62.5 Cat-PACT 35.3 Cat-delPACT 55.8

The results shown in Tables 3 and 4 and FIGS. 4 and 5 show that cells with reduced H ₂ O ₂ expression in the central body expressing Cat-PACT inhibited cell division in the G1 phase compared with control cells and cells expressing Cat-delPACT Indicating that cell cycle progression is inhibited. This result implies that the central H ₂ O ₂ is required for progression to the cell division, and artificially decreasing H ₂ O ₂ in the central body may interfere with cell cycle progression.

3-2. Phosphorylated Cdk1  Confirmation by measurement

Cat-PACT and control Cat-delPACT were cultured in the same conditions as in 3-1, and then the cell lysates were centrifuged at 15,000 × g for 30 minutes. . Cell lysates were separated by SDS-PAGE and then reacted with anti-catalase antibody, anti-pY ¹⁵ -Cdk1 antibody, anti-Cdk1 antibody (Santa Cruz), anti-pHistone H3 (pHH3) antibody ) And anti-Actin antibody (Sigma). The immunoblot results are shown in Figure 6, quantified and shown in Table 5 and Figure 7 below.

Relavitivi band intensity Cdk1 Empty 1.03 Cat-PACT 1.13 Cat-delPACT 1.13 pY15-Cdk1 Empty 1.00 Cat-PACT 3.28 Cat-delPACT 1.68 pHH3 Empty 1.00 Cat-PACT 0.49 Cat-delPACT 0.82

As shown in Table 5 and FIGS. 6 and 7, the cells expressing Cat-PACT showed an increase in the amount of pY ¹⁵ -Cdk1, which indicates the inactivation of Cdk1, compared with cells expressing the control empty vector and Cat-delPACT, pHH3 is decreased. These results indicate that a Cdk1 activity decreases when reducing the biochemical method the core body of the H ₂ O ₂ is a group of cell division proceeds important and artificially central body H ₂ O ₂ on the activity assays in Cdk1 through.

3-3. real time Cell image  Analysis through analysis

Cat-PACT and control Cat-delPACT were transfected with GFP-H2B stable HeLa cells in a small amount using retrovirus and cultured under conditions similar to those in Example 3-1, and confocal microscopy with live cell incubator in thymidine- The progression to the cell divider was analyzed by image acquisition at 30-minute intervals for 20 hours. 90 cells expressing Cat-PACT and 70 cells expressing Cat-delPACT were examined, and progression to cell division was analyzed by chromosome condensation and cell rounding. The percentage of cells entering the cell shredder obtained as a result of the test is shown in Table 6 and FIG.

Percentage of cells entering the cell divider Time Cat-PACT (%) Cat-delPACT (%) 06:00 - 07:59 3.19 1.43 08:00 - 09:59 0 6.27 10:00 - 10:59 4.12 20.74 11:00 - 11:59 6.34 27.37 12:00 - 12:59 14.89 15.39 13:00 - 13:59 15.45 8.06 14:00 - 14:59 21.69 7.70 15:00 - 15:59 12.82 4.47 16:00 - 16:59 12.40 1.43 17:00 - 17:59 7.36 2.86

As shown in Table 6 and FIG. 8, it can be confirmed that the Cat-PACT expressing cells are inhibited from progressing to the mitotic phase by about 2-4 hours as compared with the Cat-delPACT expressing cells. This indicates that an artificial decrease of the central body H ₂ O ₂ together with the results of Examples 3-1 and 3-2 suppresses cell cycle progression and Cdk1 activity.

Example  4. Cat-PACT fusion protein Cdk1  Stability control of the proteins of the higher regulator

Activation of Cdk1, an important phosphorylase for cell division, is initiated in the centrioles. The Cdk1 protein was found to be involved in the activation of Cdk1 by Cyclin B, Plk1, and Aurora A, and the amount of these proteins was determined by Cat-delPACT in cells expressing Cat-PACT in the central body, immunostaining using confocal microscopy And the amount of protein was quantitatively compared in the central body.

Specifically, samples were prepared in the same manner as in Example 3-1 using Cat-PACT, Cat-delPACT, and HeLa expressing the control vector. Each sample was fixed with 4% formaldehyde or 100% methanol and analyzed by immuno staining using each antibody (anti-CyclinB antibody (Santa Cruz), anti-Plk1 antibody (Invitrogen), anti-Aurora A antibody (Cell Signaling) .

The obtained results are shown in Tables 7 to 9 and Figs. 9 to 11.

CyclinB / pericentrin Empty 1.00 Cat-PACT 0.42 Cat-delPACT 0.94

Plk1 / pericentrin Empty 1.00 Cat-PACT 0.56 Cat-delPACT 0.80

Aurora A / γ-tubulin Empty 1.00 Cat-PACT 0.52 Cat-delPACT 0.85

FIG. 9 is a confocal microscope photograph (left) measuring the level of Cyclin B in the central body and a graph showing the relative fluorescence intensity (right). Figure 10 is a confocal microscope photograph (left) measuring the level of Plk1 and a graph showing the relative fluorescence intensity (right). Figure 11 is a confocal microscope photograph (left) measuring the level of Aurora A and a graph showing relative fluorescence intensity (right).

As can be seen in Tables 7 to 9 and Figs. 9 to 11, the expression levels of Cyclin B, Plk1, and Aurora A in the central body by expression of Cat-PACT fusion protein were significantly lower than those of Comparative Example in which Cat-delPACT fusion protein was expressed than it was found that 55, 30 decrease, and 40%, respectively, it was found that the core body is not exposed to H ₂ O ₂ needed in the core body preventing the Cyclin B, Plk1, Aurora a decomposition.

In conclusion, Cat-PACT fusion protein, a catalase targeted to the central body, was confirmed by biochemical and cytopathological methods to inhibit the progression of Cdk1 to the cell division in cancer cells and inhibit Cdk1 activation. It is expected that Cat-PACT fusion protein can induce apoptosis through inhibition of cellular progression and inhibition of a series of processes in many cancer cells with increased reactive oxygen species and can be applied to various types of cancer treatment.

<110> Ewha University - Industry Collaboration Foundation <120> Fusion protein and composition for controlling cell cycle          comprising the fusion protein <130> DPP20154296 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 527 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of catalase <400> 1 Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys   1 5 10 15 Glu Gln Arg Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly              20 25 30 Asn Pro Val Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly          35 40 45 Pro Leu Leu Val Gln Asp Val Val Phe Thr Asp Glu Met Ala His Phe      50 55 60 Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly  65 70 75 80 Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys                  85 90 95 Ala Lys Val Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg             100 105 110 Phe Ser Thr Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp         115 120 125 Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp     130 135 140 Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu 145 150 155 160 Phe Pro Ser Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu                 165 170 175 Lys Asp Pro Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser             180 185 190 Leu His Gln Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly         195 200 205 His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn     210 215 220 Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln 225 230 235 240 Gly Ile Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu                 245 250 255 Asp Pro Asp Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly             260 265 270 Lys Tyr Pro Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln         275 280 285 Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro     290 295 300 His Lys Asp Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg 305 310 315 320 Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro                 325 330 335 Ser Asn Met Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln             340 345 350 Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg Arg His Arg Leu Gly Pro         355 360 365 Asn Tyr Leu His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala     370 375 380 Asn Tyr Gln Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly 385 390 395 400 Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro                 405 410 415 Ser Ala Leu Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe             420 425 430 Asn Thr Ala Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val         435 440 445 Asn Val Leu Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala     450 455 460 Gly His Leu Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys 465 470 475 480 Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu                 485 490 495 Leu Asp Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe             500 505 510 Val Gln Ser Gly Ser His Leu Ala Ala Arg Glu Lys Ala Asn Leu         515 520 525 <210> 2 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of PACT domain <400> 2 Asn Ile Glu Ala Ile Ile Ala Ser Glu Lys Glu Val Trp Asn Arg Glu   1 5 10 15 Lys Leu Thr Leu Gln Lys Ser Leu Lys Arg Ala Glu Ala Glu Val Tyr              20 25 30 Lys Leu Lys Ala Glu Leu Arg Asn Asp Ser Leu Leu Gln Thr Leu Ser          35 40 45 Pro Asp Ser Glu His Val Thr Leu Lys Arg Ile Tyr Gly Lys Tyr Leu      50 55 60 Arg Ala Glu Ser Phe Arg Lys Ala Leu Ile Tyr Gln Lys Lys Tyr Leu  65 70 75 80 Leu Leu Leu Leu Gly Gly Phe Gln Glu Cys Glu Asp Ala Thr Leu Ala                  85 90 95 Leu Leu Ala Arg Met Gly Gly Gln Pro Ala Phe Thr Asp Leu Glu Val             100 105 110 Ile Thr Asn Arg Pro Lys Gly Phe Thr Arg Phe Arg Ser Ala Val Arg         115 120 125 Val Ser Ile Ala Ile Ser Arg Met Lys Phe Leu Val Arg Arg Trp His     130 135 140 Arg Val Thr Gly Ser Val Ser Ile Asn Ile Asn Arg Asp Gly Phe Gly 145 150 155 160 Leu Asn Gln Gly Ala Glu Lys Thr                 165 <210> 3 <211> 706 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Cat-PACT fusion protein <400> 3 Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys   1 5 10 15 Glu Gln Arg Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly              20 25 30 Asn Pro Val Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly          35 40 45 Pro Leu Leu Val Gln Asp Val Val Phe Thr Asp Glu Met Ala His Phe      50 55 60 Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly  65 70 75 80 Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys                  85 90 95 Ala Lys Val Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg             100 105 110 Phe Ser Thr Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp         115 120 125 Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp     130 135 140 Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu 145 150 155 160 Phe Pro Ser Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu                 165 170 175 Lys Asp Pro Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser             180 185 190 Leu His Gln Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly         195 200 205 His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn     210 215 220 Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln 225 230 235 240 Gly Ile Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu                 245 250 255 Asp Pro Asp Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly             260 265 270 Lys Tyr Pro Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln         275 280 285 Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro     290 295 300 His Lys Asp Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg 305 310 315 320 Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro                 325 330 335 Ser Asn Met Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln             340 345 350 Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg Arg His Arg Leu Gly Pro         355 360 365 Asn Tyr Leu His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala     370 375 380 Asn Tyr Gln Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly 385 390 395 400 Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro                 405 410 415 Ser Ala Leu Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe             420 425 430 Asn Thr Ala Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val         435 440 445 Asn Val Leu Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala     450 455 460 Gly His Leu Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys 465 470 475 480 Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu                 485 490 495 Leu Asp Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe             500 505 510 Val Glu Ser Gly Ser His Leu Ala Ala Arg Glu Gln Ala Ser Asn Ser         515 520 525 Ala Val Asp Ala Asn Ile Glu Ala Ile Ile Ala Ser Glu Lys Glu Val     530 535 540 Trp Asn Arg Glu Lys Leu Thr Leu Gln Lys Ser Leu Lys Arg Ala Glu 545 550 555 560 Ala Glu Val Tyr Lys Leu Lys Ala Glu Leu Arg Asn Ser Ser Leu Leu                 565 570 575 Gln Thr Leu Ser Pro Asp Ser Glu His Val Thr Leu Lys Arg Ile Tyr             580 585 590 Gly Lys Tyr Leu Arg Ala Glu Ser Phe Arg Lys Ala Leu Ile Tyr Gln         595 600 605 Lys Lys Tyr Leu Leu Leu Leu Leu Gly Gly Phe Gln Glu Cys Glu Asp     610 615 620 Ala Thr Leu Ala Leu Leu Ala Arg Met Gly Gly Gln Pro Ala Phe Thr 625 630 635 640 Asp Leu Glu Val Ile Thr Asn Arg Pro Lys Gly Phe Thr Arg Phe Arg                 645 650 655 Ser Ala Val Arg Ser Ser Ale Ile Ser Arg Met Lys Phe Leu Val             660 665 670 Arg Arg Trp His Arg Val Thr Gly Ser Val Ser Ile Asn Ile Asn Arg         675 680 685 Asp Gly Phe Gly Leu Asn Gln Gly Ala Glu Lys Thr Gly Ser Thr Gly     690 695 700 Ser Arg 705 <210> 4 <211> 624 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Cat-delPACT fusion protein <400> 4 Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys   1 5 10 15 Glu Gln Arg Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly              20 25 30 Asn Pro Val Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly          35 40 45 Pro Leu Leu Val Gln Asp Val Val Phe Thr Asp Glu Met Ala His Phe      50 55 60 Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly  65 70 75 80 Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys                  85 90 95 Ala Lys Val Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg             100 105 110 Phe Ser Thr Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp         115 120 125 Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp     130 135 140 Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu 145 150 155 160 Phe Pro Ser Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu                 165 170 175 Lys Asp Pro Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser             180 185 190 Leu His Gln Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly         195 200 205 His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn     210 215 220 Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln 225 230 235 240 Gly Ile Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu                 245 250 255 Asp Pro Asp Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly             260 265 270 Lys Tyr Pro Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln         275 280 285 Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro     290 295 300 His Lys Asp Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg 305 310 315 320 Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro                 325 330 335 Ser Asn Met Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln             340 345 350 Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg Arg His Arg Leu Gly Pro         355 360 365 Asn Tyr Leu His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala     370 375 380 Asn Tyr Gln Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly 385 390 395 400 Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro                 405 410 415 Ser Ala Leu Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe             420 425 430 Asn Thr Ala Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val         435 440 445 Asn Val Leu Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala     450 455 460 Gly His Leu Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys 465 470 475 480 Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu                 485 490 495 Leu Asp Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe             500 505 510 Val Glu Ser Gly Ser His Leu Ala Ala Arg Glu Gln Ala Ser Asn Ser         515 520 525 Ala Val Asp Ala Asn Ile Glu Ala Ile Ile Ala Ser Glu Lys Glu Val     530 535 540 Trp Asn Arg Glu Lys Leu Thr Leu Gln Lys Ser Leu Lys Arg Ala Glu 545 550 555 560 Ala Glu Val Tyr Lys Leu Lys Ala Glu Leu Arg Asn Ser Ser Leu Leu                 565 570 575 Gln Thr Leu Ser Pro Asp Ser Glu His Val Thr Leu Lys Arg Ile Tyr             580 585 590 Gly His Arg Val Thr Gly Ser Val Ser Ile Asn Ile Asn Arg Asp Gly         595 600 605 Phe Gly Leu Asn Gln Gly Ala Glu Lys Thr Gly Ser Thr Gly Ser Arg     610 615 620 <210> 5 <211> 1584 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding catalase <400> 5 atggctgaca gccgggatcc cgccagcgac cagatgcagc actggaagga gcagcgggcc 60 gcgcagaaag ctgatgtcct gaccactgga gctggtaacc cagtaggaga caaacttaat 120 gttattacag tagggccccg tgggcccctt cttgttcagg atgtggtttt cactgatgaa 180 atggctcatt ttgaccgaga gagaattcct gagagagttg tgcatgctaa aggagcaggg 240 gcctttggct actttgaggt cacacatgac attaccaaat actccaaggc aaaggtattt 300 gagcatattg gaaagaagac tcccatcgca gttcggttct ccactgttgc tggagaatcg 360 ggttcagctg acacagttcg ggaccctcgt gggtttgcag tgaaatttta cacagaagat 420 ggtaactggg atctcgttgg aaataacacc cccattttct tcatcaggga tcccatattg 480 tttccatctt ttatccacag ccaaaagaga aatcctcaga cacatctgaa ggatccggac 540 atggtctggg acttctggag cctacgtcct gagtctctgc atcaggtttc tttcttgttc 600 agtgatcggg ggattccaga tggacatcgc cacatgaatg gatatggatc acatactttc 660 aagctggtta atgcaaatgg ggaggcagtt tattgcaaat tccattataa gactgaccag 720 ggcatcaaaa acctttctgt tgaagatgcg gcgagacttt cccaggaaga tcctgactat 780 ggcatccggg atctttttaa cgccattgcc acaggaaagt acccctcctg gactttttac 840 atccaggtca tgacatttaa tcaggcagaa acttttccat ttaatccatt cgatctcacc 900 aaggtttggc ctcacaagga ctaccctctc atcccagttg gtaaactggt cttaaaccgg 960 aatccagtta attactttgc tgaggttgaa cagatagcct tcgacccaag caacatgcca 1020 cctggcattg aggccagtcc tgacaaaatg cttcagggcc gcctttttgc ctatcctgac 1080 actcaccgcc atcgcctggg acccaattat cttcatatac ctgtgaactg tccctaccgt 1140 gctcgagtgg ccaactacca gcgtgacggc ccgatgtgca tgcaggacaa tcagggtggt 1200 gctccaaatt actaccccaa cagctttggt gctccggaac aacagccttc tgccctggag 1260 cacagcatcc aatattctgg agaagtgcgg agattcaaca ctgccaatga tgataacgtt 1320 actcaggtgc gggcattcta tgtgaacgtg ctgaatgagg aacagaggaa acgtctgtgt 1380 gagaacattg ccggccacct gaaggatgca caaattttca tccagaagaa agcggtcaag 1440 aacttcactg aggtccaccc tgactacggg agccacatcc aggctcttct ggacaagtac 1500 aatgctgaga agcctaagaa tgcgattcac acctttgtgc agtccggatc tcacttggcg 1560 gcaagggaga aggcaaatct gtga 1584 <210> 6 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding PACT domain <400> 6 aacattgaag ccatcattgc ctctgaaaaa gaagtatgga acagagaaaa attgactctc 60 cagaaatctt tgaaaagggc agaggctgaa gtatacaaac tgaaagctga actaagaaat 120 gactctttac ttcaaactct gagccctgat tctgaacatg tcactttaaa gagaatttat 180 ggtaaatact tgagggcaga aagttttcga aaggctctca tttaccagaa gaaatacctg 240 ctgctgttac tgggtgggtt ccaggaatgt gaagatgcca ccttggccct gcttgcccgg 300 atgggggggc agccagcttt cacggatcta gaggtgatca ccaatcgccc aaagggcttc 360 accaggtttc ggtcggccgt cagagtatcc attgcaattt ccagaatgaa atttttggtt 420 cgacggtggc atcgagtcac aggttctgtt tccatcaata ttaacagaga tggctttgga 480 ctgaatcaag gtgcagaaaa gact 504 <210> 7 <211> 2121 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding Cat-PACT fusion protein <400> 7 atggctgaca gccgggatcc cgccagcgac cagatgcagc actggaagga gcagcgggcc 60 gcgcagaaag ctgatgtcct gaccactgga gctggtaacc cagtaggaga caaacttaat 120 gttattacag tagggccccg tgggcccctt cttgttcagg atgtggtttt cactgatgaa 180 atggctcatt ttgaccgaga gagaattcct gagagagttg tgcatgctaa aggagcaggg 240 gcctttggct actttgaggt cacacatgac attaccaaat actccaaggc aaaggtattt 300 gagcatattg gaaagaagac tcccatcgca gttcggttct ccactgttgc tggagaatcg 360 ggttcagctg acacagttcg ggaccctcgt gggtttgcag tgaaatttta cacagaagat 420 ggtaactggg atctcgttgg aaataacacc cccattttct tcatcaggga tcccatattg 480 tttccatctt ttatccacag ccaaaagaga aatcctcaga cacatctgaa ggatccggac 540 atggtctggg acttctggag cctacgtcct gagtctctgc atcaggtttc tttcttgttc 600 agtgatcggg ggattccaga tggacatcgc cacatgaatg gatatggatc acatactttc 660 aagctggtta atgcaaatgg ggaggcagtt tattgcaaat tccattataa gactgaccag 720 ggcatcaaaa acctttctgt tgaagatgcg gcgagacttt cccaggaaga tcctgactat 780 ggcatccggg atctttttaa cgccattgcc acaggaaagt acccctcctg gactttttac 840 atccaggtca tgacatttaa tcaggcagaa acttttccat ttaatccatt cgatctcacc 900 aaggtttggc ctcacaagga ctaccctctc atcccagttg gtaaactggt cttaaaccgg 960 aatccagtta attactttgc tgaggttgaa cagatagcct tcgacccaag caacatgcca 1020 cctggcattg aggccagtcc tgacaaaatg cttcagggcc gcctttttgc ctatcctgac 1080 actcaccgcc atcgcctggg acccaattat cttcatatac ctgtgaactg tccctaccgt 1140 gctcgagtgg ccaactacca gcgtgacggc ccgatgtgca tgcaggacaa tcagggtggt 1200 gctccaaatt actaccccaa cagctttggt gctccggaac aacagccttc tgccctggag 1260 cacagcatcc aatattctgg agaagtgcgg agattcaaca ctgccaatga tgataacgtt 1320 actcaggtgc gggcattcta tgtgaacgtg ctgaatgagg aacagaggaa acgtctgtgt 1380 gagaacattg ccggccacct gaaggatgca caaattttca tccagaagaa agcggtcaag 1440 aacttcactg aggtccaccc tgactacggg agccacatcc aggctcttct ggacaagtac 1500 aatgctgaga agcctaagaa tgcgattcac acctttgtgc agtccggatc tcacttggcg 1560 gcaagggagc aagcttcgaa ttctgcagtc gacgccaaca ttgaagccat cattgcctct 1620 gaaaaagaag tatggaacag agaaaaattg actctccaga aatctttgaa aagggagag 1680 gctgaagtat acaaactgaa agctgaacta agaaatgact ctttacttca aactctgagc 1740 cctgattctg aacatgtcac tttaaagaga atttatggta aatacttgag ggcagaaagt 1800 tttcgaaagg ctctcattta ccagaagaaa tacctgctgc tgttactggg tgggttccag 1860 gaatgtgaag atgccacctt ggccctgctt gcccggatgg gggggcagcc agctttcacg 1920 gatctagagg tgatcaccaa tcgcccaaag ggcttcacca ggtttcggtc ggccgtcaga 1980 gtatccattg caatttccag aatgaaattt ttggttcgac ggtggcatcg agtcacaggt 2040 tctgtttcca tcaatattaa cagagatggc tttggactga atcaaggtgc agaaaagact 2100 ggatccaccg gatctagata a 2121 <210> 8 <211> 1869 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding Cat-delPACT fusion protein <400> 8 atggctgaca gccgggatcc cgccagcgac cagatgcagc actggaagga gcagcgggcc 60 gcgcagaaag ctgatgtcct gaccactgga gctggtaacc cagtaggaga caaacttaat 120 gttattacag tagggccccg tgggcccctt cttgttcagg atgtggtttt cactgatgaa 180 atggctcatt ttgaccgaga gagaattcct gagagagttg tgcatgctaa aggagcaggg 240 gcctttggct actttgaggt cacacatgac attaccaaat actccaaggc aaaggtattt 300 gagcatattg gaaagaagac tcccatcgca gttcggttct ccactgttgc tggagaatcg 360 ggttcagctg acacagttcg ggaccctcgt gggtttgcag tgaaatttta cacagaagat 420 ggtaactggg atctcgttgg aaataacacc cccattttct tcatcaggga tcccatattg 480 tttccatctt ttatccacag ccaaaagaga aatcctcaga cacatctgaa ggatccggac 540 atggtctggg acttctggag cctacgtcct gagtctctgc atcaggtttc tttcttgttc 600 agtgatcggg ggattccaga tggacatcgc cacatgaatg gatatggatc acatactttc 660 aagctggtta atgcaaatgg ggaggcagtt tattgcaaat tccattataa gactgaccag 720 ggcatcaaaa acctttctgt tgaagatgcg gcgagacttt cccaggaaga tcctgactat 780 ggcatccggg atctttttaa cgccattgcc acaggaaagt acccctcctg gactttttac 840 atccaggtca tgacatttaa tcaggcagaa acttttccat ttaatccatt cgatctcacc 900 aaggtttggc ctcacaagga ctaccctctc atcccagttg gtaaactggt cttaaaccgg 960 aatccagtta attactttgc tgaggttgaa cagatagcct tcgacccaag caacatgcca 1020 cctggcattg aggccagtcc tgacaaaatg cttcagggcc gcctttttgc ctatcctgac 1080 actcaccgcc atcgcctggg acccaattat cttcatatac ctgtgaactg tccctaccgt 1140 gctcgagtgg ccaactacca gcgtgacggc ccgatgtgca tgcaggacaa tcagggtggt 1200 gctccaaatt actaccccaa cagctttggt gctccggaac aacagccttc tgccctggag 1260 cacagcatcc aatattctgg agaagtgcgg agattcaaca ctgccaatga tgataacgtt 1320 actcaggtgc gggcattcta tgtgaacgtg ctgaatgagg aacagaggaa acgtctgtgt 1380 gagaacattg ccggccacct gaaggatgca caaattttca tccagaagaa agcggtcaag 1440 aacttcactg aggtccaccc tgactacggg agccacatcc aggctcttct ggacaagtac 1500 aatgctgaga agcctaagaa tgcgattcac acctttgtgc agtccggatc tcacttggcg 1560 gcaagggagc aagcttcgaa ttctgcagtc gacgccaaca ttgaagccat cattgcctct 1620 gaaaaagaag tatggaacag agaaaaattg actctccaga aatctttgaa aagggagag 1680 gctgaagtat acaaactgaa agctgaacta agaaatgact ctttacttca aactctgagc 1740 cctgattctg aacatgtcac tttaaagaga atttatggtc atcgagtcac aggttctgtt 1800 tccatcaata gagatggctt tggactgaat caaggtgcag aaaagactgg atccaccgga 1860 tctagataa 1869 <210> 9 <211> 1248 <212> DNA <213> Artificial Sequence <220> <223> The nucleotide sequence of dsRed-PACT <400> 9 atggacaaca ccgaggacgt catcaaggag ttcatgcagt tcaaggtgcg catggagggc 60 tccgtgaacg gccactactt cgagatcgag ggcgagggcg agggcaagcc ctacgagggc 120 acccagaccg ccaagctgca ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc 180 ctgtcccccc agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc 240 gactacatga agctgtcctt ccccgagggc ttcacctggg agcgctccat gaacttcgag 300 gcggcggcg tggtggaggt gcagcaggac tcctccctgc aggacggcac cttcatctac 360 aaggtgaagt tcaagggcgt gaacttcccc gccgacggcc ccgtaatgca gaagaagact 420 gccggctggg agccctccac cgagaagctg tacccccagg acggcgtgct gaagggcgag 480 atctcccacg ccctgaagct gaaggacggc ggccactaca cctgcgactt caagaccgtg 540 tacaaggcca agaagcccgt gcagctgccc ggcaaccact acgtggactc caagctggac 600 atcaccaacc acaacgagga ctacaccgtg gtggagcagt acgagcacgc cgaggcccgc 660 cctccggct cccagtccgg actcagatct cgagctcaag cttcgaattc tgcagtcgac 720 gccaacattg aagccatcat tgcctctgaa aaagaagtat ggaacagaga aaaattgact 780 ctccagaaat ctttgaaaag ggcagaggct gaagtataca aactgaaagc tgaactaaga 840 aatgactctt tacttcaaac tctgagccct gattctgaac atgtcacttt aaagagaatt 900 tatggtaaat acttgagggc agaaagtttt cgaaaggctc tcatttacca gaagaaatac 960 ctgctgctgt tactgggtgg gttccaggaa tgtgaagatg ccaccttggc cctgcttgcc 1020 cggatggggg ggcagccagc tttcacggat ctagaggtga tcaccaatcg cccaaagggc 1080 ttcaccaggt ttcggtcggc cgtcagagta tccattgcaa tttccagaat gaaatttttg 1140 gttcgacggt ggcatcgagt cacaggttct gtttccatca atattaacag agatggcttt 1200 ggactgaatc aaggtgcaga aaagactgga tccaccggat ctagataa 1248 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cat Primer_F <400> 10 ctaccggtat ggctgacagc c 21 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Cat Primer_R <400> 11 cgaagcttgc tcccttgccg ccaagtga 28 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PACT Primer_F <400> 12 gagcaagctt cgaattctgc 20 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PACT Primer_R <400> 13 acaagttaac aacaacaatt gca 23 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> delPACT Primer_F <400> 14 aagagaattt atggtcatcg agtcacaggt tctgt 35 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> delPACT Primer_R <400> 15 accataaatt ctctttaaag tgac 24 <210> 16 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Motif 1 (aa 62-105) of PACT domain <400> 16 Lys Tyr Leu Arg Ala Glu Ser Phe Arg Lys Ala Leu Ile Tyr Gln Lys   1 5 10 15 Lys Tyr Leu Leu Leu Leu Leu Gly Gly Phe Gln Glu Cys Glu Asp Ala              20 25 30 Thr Leu Ala Leu Leu Ala Arg Met Gly Gly Gln Pro          35 40 <210> 17 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Motif 1 (aa 122-143) of PACT domain <400> 17 Arg Phe Arg Ser Ala Val Arg Val Ser Ile Ala Ile Ser Arg Met Lys   1 5 10 15 Phe Leu Val Arg Arg Trp              20 <210> 18 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Motif 1 (aa 62-143) of PACT domain <400> 18 Lys Tyr Leu Arg Ala Glu Ser Phe Arg Lys Ala Leu Ile Tyr Gln Lys   1 5 10 15 Lys Tyr Leu Leu Leu Leu Leu Gly Gly Phe Gln Glu Cys Glu Asp Ala              20 25 30 Thr Leu Ala Leu Leu Ala Arg Met Gly Gly Gln Pro Ala Phe Thr Asp          35 40 45 Leu Glu Val Ile Thr Asn Arg Pro Lys Gly Phe Thr Arg Phe Arg Ser      50 55 60 Ala Val Arg Val Ser Ile Ala Ile Ser Arg Met Lys Phe Leu Val Arg  65 70 75 80 Arg Trp        

Claims (13)

A fusion protein comprising the amino acid sequence of SEQ ID NO: 3. delete A polynucleotide encoding the fusion protein of claim 1. 4. The polynucleotide of claim 3, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7. A recombinant vector comprising the polynucleotide of claim 3. 6. The recombinant vector of claim 5, wherein the recombinant vector comprises an SV40 origin of replication, a CMV enhancer, a CMV promoter, a restriction enzyme recognition site, and a Polypeptide signal sequence. A recombinant cell comprising the recombinant vector of claim 5. 8. The recombinant cell of claim 7, wherein said cell is a mammalian cell. A pharmaceutical composition comprising the fusion protein of claim 1, a polynucleotide encoding the fusion protein, a recombinant vector comprising the polynucleotide, and a recombinant cell comprising the recombinant vector as an active ingredient. &Lt; / RTI &gt; 10. The pharmaceutical composition of claim 9, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 3. 10. The pharmaceutical composition according to claim 9, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7. 10. The method of claim 9, wherein the cancer is cancer of the colon, bladder, breast, colon, kidney, liver, lung, ovary, testis, prostate, pancreas, stomach, cervix, thyroid, blood, bone, Composition. A recombinant vector comprising the fusion protein of claim 1, a polynucleotide encoding the fusion protein, a recombinant vector comprising the polynucleotide, and a recombinant vector comprising the recombinant vector, wherein (i) , Or (ii) cells, tissues, body fluids, or cultures thereof isolated from mammals, including humans.

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