Dnase 1 inhibitor

Abstract

Disclosed is a DNase I inhibitor comprising a peptide sequence of GLSRPGLVKDV or a peptide sequence of CGGDVGVPDF. The peptide can specifically bind to the active site of DNase I to inhibit DNA hydrolysis activity. The inhibitor according to the present invention may be implemented in the form of a recombinant protein (Fv-antibody) or a synthetic peptide, and these inhibitors exhibit high binding affinity for DNase I and inhibitory activity against DNase I.

Description

DNASE I inhibitor

[0001] The present invention relates to a DNase I inhibitor. More specifically, the present invention relates to a peptide that inhibits DNase I, a composition for inhibiting DNase I containing the same, and a DNA degradation inhibitor.

[0002] Fv antibodies target the variable region (V) of immunoglobulin G (IgG). H It consists of three complementarity determining regions (CDRs) and four frame regions (FRs). In the present invention, the variable region (V) of the heavy chain IgG H An Fv-antibody library was generated through random mutation of the CDR3 amino acid sequence using ). As shown in Figures 1 and 2, the Fv-antibody library was prepared by site-specific mutagenesis of the CDR3 region consisting of 11 amino acid residues. Subsequently, the Fv-antibody library containing the randomized CDR3 amino acid sequence was expressed on the outer membrane of Escherichia coli (E. coli) using self-display technology. The Fv-antibody library thus produced was expressed at a rate of 10 per E. coli. 5 High surface density of more than 10 6It has been reported to possess high diversity in individual populations and libraries. Through these characteristics, target Fv-antibodies for specific antigens can be effectively screened without performing multiple biopannings. Screening for enzyme inhibitors using Fv-antibody libraries has been successfully performed for various enzymes. In a recent study, inhibitors of monoamine oxidase B (MAO-B) responsible for dopamine oxidation were screened using an Fv-antibody library. Anti-dopamine antibodies were used as targets for the screening of MAO-B inhibitors, and the screened inhibitors (Fv-antibodies and synthesized CDR3 peptides) were able to specifically bind to the anti-dopamine antibodies and the active site of MAO-B. Using the same screening method, inhibitors of monoamine oxidase A (MAO-A), which oxidizes serotonin, could also be screened using an Fv-antibody library targeting anti-serotonin antibodies. Enzyme inhibitors can also be screened using screening probes that target specific enzymes. Monocarboxylate carrier 1 and enzyme inhibitors of ribonuclease A were successfully screened, and the expressed Fv-antibody and the synthesized CDR3 peptide inhibitor showed high inhibitory activity.

[0003] Deoxyribonuclease I (DNase I), derived from bovine pancreas, is a glycoprotein (molecular weight 30 kDa) with endonuclease activity that hydrolyzes single and double-stranded DNA. Through its endonuclease activity, it hydrolyzes phosphodiester bonds to produce 3'-hydroxyl and 5'-phospho-oligonucleotides. DNase I possesses four ionic binding pockets, and for endonuclease activity, Ca 2+ and Mg 2+It requires. DNase I inhibitors have been developed based on proteins, nucleotides, and synthetic compounds. Among protein-based inhibitors, monomeric actin (G-actin) has been used for the specific inhibition of DNase I, while fibrous actin (F-actin) is known to have no inhibitory effect. Nogalamicin is a nucleotide-based DNase I inhibitor capable of forming stable complexes with DNA through interactions with adenine and thymine. 2-nitro-5-thiocyanobenzoic acid (NTCB) is Ca 2+ / Mg 2+ It is a well-known synthetic compound capable of inhibiting DNase I activity through thiol-specific modification at pH 7.5 where [substance] is present. Therefore, inhibition by NTCB is achieved by the reaction between the cysteine ​​residue of DNase I and the cyano group of NTCB, thereby irreversibly modifying DNase I. In the present invention, an amino acid sequence (CDR3) with DNase I inhibitory activity was selected using an Fv-antibody library expressed on the outer membrane of E. coli. The selected inhibitor was expressed in the form of a soluble recombinant protein (Fv-antibody) and a synthetic peptide. The binding affinity (K) of the Fv-antibody and the synthetic peptide for DNase I D ) was analyzed using a surface plasmon resonance (SPR) biosensor. The inhibitory activity (IC10) of the expressed Fv-antibody and synthetic peptide 50 ) was evaluated via agarose gel assay and TaqMan-like fluorescence assay. Finally, the interaction between the selected inhibitor and DNase I was analyzed using computer-based docking simulations.

[0004] The problem that the present invention aims to solve is to effectively suppress DNA degradation that may occur due to excessive activity of DNase I, thereby maintaining the stability of extracellular DNA and mitigating autoimmune responses. By regulating the activity of DNase I in various biological and medical situations, this can contribute to delaying tissue damage, inflammatory responses, or the progression of specific diseases. Furthermore, it has the potential to help improve therapeutic efficiency in fields such as gene therapy or cell-based therapy by preventing the degradation of foreign DNA.

[0005] In one aspect, the present invention provides a peptide having a function of specifically binding to DNase I and comprising the peptide sequence of SEQ ID NO. 1 or the peptide sequence of SEQ ID NO. 2.

[0006] [Sequence No. 1]

[0007] GLSRPGLVKDV

[0008] [Sequence No. 2]

[0009] CGGDVGVPPDF

[0010] In one embodiment, the peptide can specifically bind to the active site where DNase I degrades DNA and inhibit DNase I from degrading DNA.

[0011] In another aspect, the present invention provides a nucleic acid encoding the peptide.

[0012] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0013] In another aspect, the present invention provides a cell transformed with the recombinant expression vector.

[0014] In one embodiment, the cell may comprise one or more cells selected from the group comprising animal cells, plant cells, yeast, E. coli, and insect cells.

[0015] In one embodiment, the cell is monkey kidney cell 7 (COS7) cell, NSO cell, SP2 / 0 cell, Chinese hamster ovary (CHO) cell, W138, baby hamster kidney (BHK) cell, MDCK, myeloma cell line, HuT 78 cell and HEK293 cell, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp and It may include one or more cells selected from the group including Neurosporacrassa.

[0016] In another aspect, the present invention provides a composition for inhibiting DNase I, comprising an amino acid fragment having the peptide sequence of SEQ ID NO. 1 or the peptide sequence of SEQ ID NO. 2, having the function of specifically binding to DNase I.

[0017] [Sequence No. 1]

[0018] GLSRPGLVKDV

[0019] [Sequence No. 2]

[0020] CGGDVGVPPDF

[0021] In one embodiment, the peptide included in the DNase I inhibitory composition can specifically bind to the active site where DNase I degrades DNA, thereby inhibiting DNase I from degrading DNA.

[0022] Deoxyribonuclease (DNase) I inhibitors were screened using a library of Fv-antibodies expressed on the outer membrane of Escherichia coli. This Fv-antibody library was generated by including random sequences of the complementarity determining region 3 (CDR3). From the Fv-antibody library, two clones with binding affinities for DNase I were screened, and the amino acid sequences of their CDR3 were determined. The two screened Fv-antibodies were expressed as soluble recombinant proteins, and CDR3 was synthesized into a peptide. The binding affinities of the Fv-antibodies and peptides for DNase I were analyzed using a surface plasmon resonance (SPR) biosensor. The inhibitory activity (IC10) of the Fv-antibodies and peptides 50 IC was measured via agarose gel analysis and TaqMan-like fluorescence analysis. 50 The values ​​showed that both the Fv-antibody and the peptide from the screened clones exhibited inhibitory activity against DNase I, and indicated that the binding of the Fv-antibody occurred at the DNA substrate hydrolysis active site of DNase I. The interaction between the screened inhibitors and DNase I was analyzed through computer-based docking simulations, and it was confirmed that the binding of the Fv-antibody and the peptide occurred at the active site of DNase I.

[0023] The effect of the present invention is to prevent DNA degradation by selectively inhibiting the activity of DNase I, thereby contributing to reducing extracellular DNA damage occurring in autoimmune or inflammatory diseases. Additionally, it has the potential to be usefully applied to enhance the efficiency of biotechnologies, such as gene therapy, by increasing the stability of foreign DNA. These effects can help protect the cellular environment during the management and treatment of various diseases and ultimately contribute to improving the quality of life of patients.

[0024] Figure 1 illustrates the generation of position-specific mutations using a sequence of 33 nucleotides in the CDR3 region during the production of an Fv-antibody library.

[0025] Figure 2 shows an Fv-antibody library containing randomized CDR3 expressed on the outer membrane of E. coli via an auto-display plasmid.

[0026] Figure 3 shows the results of flow cytometry analysis after treating an Fv-antibody library with fluorescently labeled DNase I.

[0027] Figure 4 shows the results of flow cytometry analysis of normal E. coli.

[0028] Figure 5 shows the results of flow cytometry analysis of mutant E. coli strains containing only CDR1 and CDR2.

[0029] Figure 6 illustrates the screening of Fv antibodies using magnetic beads immobilized with DNase I.

[0030] Figure 7 shows the results of binding analysis and oligonucleotide sequence analysis of the CDR3 region performed on the screened clones.

[0031] Figure 8 shows the binding affinity of screened clone #1 determined by flow cytometry.

[0032] Figure 9 illustrates the quantitative analysis results according to the binding affinity of screened clone #1.

[0033] Figure 10 shows the binding affinity of screened clone #19 determined by flow cytometry.

[0034] Figure 11 illustrates the quantitative analysis results according to the binding affinity of the screened clone #19.

[0035] Figure 12 shows the expression results of the screened Fv-antibody expressed as a soluble protein, which was confirmed by SDS-PAGE with a molecular weight of approximately 40 kDa.

[0036] Figure 13 illustrates a schematic of a binding analysis using an SPR biosensor for an expressed Fv-antibody.

[0037] Figure 14 shows the binding affinity (K) of the Fv-antibody using the concentration response curve of screened clone #1. D Displays the analysis results.

[0038] Figure 15 shows the real-time binding curve of clone #1 screened using an SPR biosensor.

[0039] Figure 16 shows the binding affinity (K) of the Fv-antibody using the concentration response curve of screened clone #19. D Displays the analysis results.

[0040] Figure 17 shows the real-time binding curve of clone #19 screened using an SPR biosensor.

[0041] Figure 18 illustrates a schematic of the binding analysis for a peptide (screened CDR3 sequence) synthesized using an SPR biosensor.

[0042] Figure 19 shows the binding affinity (K) using the concentration response curve of screened peptide-1. D Displays the analysis results.

[0043] Figure 20 shows the real-time binding curve of peptide-1 synthesized using an SPR biosensor.

[0044] Figure 21 shows the binding affinity (K) using the concentration response curve of screened peptide-19. D Displays the analysis results.

[0045] Figure 22 shows the real-time binding curve of peptide-19 synthesized using an SPR biosensor.

[0046] Figure 23 shows the inhibitory activity analysis of Fv-1 via agarose gel electrophoresis.

[0047] Figure 24 shows the IC for the inhibitory activity of Fv-1. 50 This is a diagram of the value evaluation.

[0048] Figure 25 shows the analysis of the inhibitory activity of Fv-19 via agarose gel electrophoresis.

[0049] Figure 26 shows the IC for the inhibitory activity of Fv-19. 50 This is a diagram of the value evaluation.

[0050] Figure 27 shows the inhibitory activity analysis of Peptide-1 via agarose gel electrophoresis.

[0051] Figure 28 shows the IC for the inhibitory activity of Peptide-1. 50 This is a diagram of the value evaluation.

[0052] Figure 29 shows the inhibitory activity analysis of Peptide-19 via agarose gel electrophoresis.

[0053] Figure 30 shows the IC for the inhibitory activity of Peptide-19 50 This is a diagram of the value evaluation.

[0054] Figure 31 illustrates the procedure for analyzing inhibitory activity using a TaqMan-type fluorescence analysis kit.

[0055] Figure 32 shows the IC through the analysis of the inhibitory activity of Fv-1. 50 It is an evaluation.

[0056] Figure 33 shows the IC after correcting the results of the inhibitory activity analysis of Fv-1. 50 This is a graph showing the estimated values.

[0057] Figure 34 shows the IC through the analysis of the inhibitory activity of Fv-19. 50 It is an evaluation.

[0058] Figure 35 shows the IC after correcting the results of the inhibitory activity analysis of Fv-19. 50 This is a graph showing the estimated values.

[0059] Figure 36 shows the IC through the analysis of the inhibitory activity of Peptide-1. 50 It is an evaluation.

[0060] Figure 37 shows the IC after correcting the results of the inhibitory activity analysis of Peptide-1. 50 This is a graph showing the estimated values.

[0061] Figure 38 shows the IC through the analysis of the inhibitory activity of Peptide-19. 50 It is an evaluation.

[0062] Figure 39 shows the IC after correcting the results of the inhibitory activity analysis of Peptide-19. 50 This is a graph showing the estimated values.

[0063] Figure 40 illustrates the interaction site of DNase I using a computer-based docking simulation.

[0064] Figure 41 illustrates the analysis of the interaction between Fv-1 and DNase I using computer-based docking simulation.

[0065] Figure 42 illustrates the analysis of the interaction between Fv-19 and DNase I using computer-based docking simulation.

[0066] Figure 43 illustrates the analysis of the interaction between a commercial DNase I inhibitor and DNase I using computer-based docking simulation.

[0067] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. Since the present invention is susceptible to various modifications and may take various forms, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to the specific disclosed forms, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. Similar reference numerals have been used for similar components in the description of each drawing. In the attached drawings, the dimensions of the structures are shown enlarged compared to the actual dimensions for the clarity of the present invention.

[0068] The terms used in this application are used merely to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, terms such as “comprising” or “having” are intended to specify the presence of the features, numbers, steps, actions, components, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, or combinations thereof. In the context of this specification, terms such as “about” may mean about ± 1%, about ± 2%, about ± 3%, about ± 4%, about ± 5%, about ± 6%, about ± 7%, about ± 8%, about ± 9%, or about ± 10% of the figures described in the specification.

[0069] In addition, the description of one aspect of the present invention may be applied identically or similarly to the description of other aspects for identical or similar configurations or terms.

[0070] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0071] A peptide according to an embodiment of the present invention has the function of specifically binding to DNase I and may include the peptide sequence of SEQ ID NO. 1 or the peptide sequence of SEQ ID NO. 2.

[0072] [Sequence No. 1]

[0073] GLSRPGLVKDV

[0074] [Sequence No. 2]

[0075] CGGDVGVPPDF

[0076] In the context of this specification, the dictionary definition of DNase I is a DNA-degrading enzyme, meaning an endonuclease that has the function of hydrolyzing the double and single strands of DNA. DNase I plays a role in degrading DNA in the extracellular environment, which can induce autoimmune reactions or impair the stability of genetic material within cells. In the present invention, a peptide that inhibits DNase I may have the function of selectively inhibiting DNA hydrolysis by binding to the active site of such enzyme.

[0077] In the context of this specification, the dictionary definition of a peptide refers to a short chain of amino acids in which two or more amino acids are connected by peptide bonds. Peptides have physiological functions capable of interacting with specific proteins or enzymes and can perform various in vivo actions depending on their structure. The peptide presented in this invention includes a functional sequence capable of specifically binding to DNase I and inhibiting its activity.

[0078] In one embodiment, the peptide can specifically bind to the active site where DNase I degrades DNA, thereby inhibiting DNase I from degrading DNA. In this way, the peptide can play an important role in regulating enzyme activity in situations where excessive DNA degradation by DNase I is problematic. In particular, it can help alleviate unnecessary immune responses by inhibiting the degradation of extracellular DNA in pathological conditions such as autoimmune diseases or inflammatory reactions.

[0079] In this sense, the peptide according to the present invention can act as a microtop of DNA. In the context of this specification, a microtop refers to a fragment that mimics the three-dimensional structural features of a specific molecule and has a structure capable of binding to the active site of a specific protein or enzyme to regulate its function. The peptide according to the present invention has structural features that allow it to interact with the active site of DNase I and function similarly to the way DNA binds, thereby inhibiting the action of DNase I. Such a microtop may possess the characteristic of being able to selectively bind to a target enzyme.

[0080] The nucleic acid according to an embodiment of the present invention may encode the peptide. In the context of this specification, the dictionary definition of nucleic acid is a biochemical substance that stores and transmits genetic information, is an essential component of genes, exists in the form of DNA and RNA, and enables the expression of a desired peptide within a cell. Such nucleic acid sequences can help effectively produce DNase I inhibitors and have the potential to be utilized in biotechnological applications using gene delivery systems or recombinant expression vectors.

[0081] In the context of this specification, the literal meaning of a nucleic acid encoding a peptide is that a nucleic acid (DNA or RNA) carries genetic information that directs the synthesis of a peptide composed of a specific amino acid sequence. This information is converted into a peptide through the processes of transcription and translation. During transcription, the genetic information of DNA is copied into mRNA, and during translation, the code of mRNA is decoded into an amino acid sequence at the ribosome to synthesize a peptide. Through this process, the nucleic acid plays a role in directly determining the structure and function of the peptide.

[0082] As is known in the art, combinations of nucleic acids encoding amino acids contained in peptides can vary. Accordingly, the present invention includes not only the peptide described above, but also, as nucleic acids encoding the peptide described above, theoretically 5,308,416 nucleic acid sequences encoding SEQ ID NO. 1 and theoretically 262,144 nucleic acid sequences encoding SEQ ID NO. 2. These diverse nucleic acid sequences are attributed to the codon variability of each amino acid. A codon is a sequence of three bases in a nucleic acid that codes for a single amino acid; since multiple codons can code for the same amino acid, various nucleic acid sequences may exist to code for the same peptide sequence. This enhances the flexibility of the present invention and can help in selecting a nucleic acid sequence optimized for a specific biological system or application. For example, nucleic acid sequences can be optimized by considering the use of codons that are translated more efficiently in a specific biological species, which can contribute to improving the expression level and stability of the peptide.

[0083] Meanwhile, the recombinant expression vector according to an embodiment of the present invention may include the nucleic acid. In the context of this specification, the dictionary definition of a recombinant expression vector is a molecule used to introduce a gene into another cell to induce the expression of a protein or peptide. This vector may take the form of a plasmid, virus, artificial chromosome, etc., and includes essential elements for replication and gene expression, such as selection markers, promoters, reporter genes, etc. Such a vector enables high-efficiency expression of a target peptide within a specific cell type and can improve the stability and function of the peptide within the cell. For example, this vector can be utilized not only in laboratory research but also in biomanufacturing processes for mass production. Furthermore, a recombinant expression vector containing appropriate regulatory elements can finely regulate gene expression, thereby contributing to the optimization of the production of a desired protein or peptide.

[0084] Meanwhile, cells according to the embodiments of the present invention may be transformed with the recombinant expression vector. In the context of this specification, the dictionary definition of transformation is the process of changing the genetic composition of a cell by introducing DNA from an external source. Through this process, the cell acquires a new gene, which can be expressed within the cell to produce a specific protein. Such transformed cells can be used for various purposes in research and industrial applications. For example, when the mass production of a specific protein is required in drug development, the protein can be produced efficiently using transformed cells. Furthermore, these cells can be utilized for disease modeling or gene function research, serving as an important tool in life science research.

[0085] The continuous production of specific peptides or proteins can be enabled by transforming the above cells with the above recombinant expression vector. These transformed cells maintain stability during the production process and can produce target proteins with high yields. This allows researchers to rapidly obtain large quantities of protein, which can contribute to increasing the repeatability and reliability of experiments. Furthermore, these cells can play an important role in the development of protein-based therapeutics and can be usefully utilized in disease modeling and drug screening processes.

[0086] In one embodiment, the cell may comprise one or more cells selected from the group comprising animal cells, plant cells, yeast, E. coli, and insect cells. In one embodiment, the cell is monkey kidney cell 7 (COS7) cell, NSO cell, SP2 / 0 cell, Chinese hamster ovary (CHO) cell, W138, baby hamster kidney (BHK) cell, MDCK, myeloma cell line, HuT 78 cell and HEK293 cell, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp and It may include one or more cells selected from the group including Neurosporacrassa. These diverse cells have different physiological characteristics and culture conditions, allowing for optimal selection based on the production requirements of specific proteins. This enables efficient and economical production of target proteins in research and industrial applications.

[0087] Non-limiting examples of other possible cells in addition to those mentioned above include mouse fetal fibroblasts (L929), human lung epithelial cells (A549), porcine kidney epithelial cells (PK-15), deer naked mammary gland epithelial cells (BMGE), chicken embryonic fibroblasts (DF-1), dog kidney cells (MDCK), porcine liver cells (PLHC-1), insect cells (Sf9, Sf21, High Five), dwarf poplar embryo cells (PoET), mammalian liver cells (HepaRG), human colon cancer cells (Caco-2), human liver cells (HepG2), syringe nematode cells (N2), Hydra cells (Hym-248), quail muscle cells (QM7), cephalopod cells (Eflo-1), squid neurons (SfNC), zebrafish macrophages (DLF), amoeba cells (AX2), African clove embryo cells (A6), avian liver cells (CHL), neonatal human epidermal cells (NHEK), and Bacillus Bacillus licheniformis, Corynebacterium glutamicum, Clostridium acetobutylicum, Escherichia coli strain (BL21, DH5α), Lipid-degrading bacteria (Acinetobacter), Red algae culture cells (RHS), Yeast strain (S. cerevisiae CEN.PK, Y1000), Spore-forming bacteria (B. coagulans), Green algae cell line (Tetraselmis suecica), Marine bacterium strain (Maricaulis maris), Fungal strain (Trichoderma reesei), Coral cells (CoRS2), Termite culture cells (Psilocybe cyanescens), Protozoa cells (Tetrahymena thermophila), Fish kidney cells (EPC), Microbial fungus cells (Fusarium venenatum), Laboratory nematode gene cells (RMCE), Shellfish cells (Mytilus edulis), Shark cartilage cells (SC3), Fungal spores (Alternaria), pneumococcal cells (Streptococcus pneumoniae), pink algae (Rhodophyta sp.), malaria parasite (Plasmodium falciparum), psychrophilic bacterial strain (Pseudomonas syringae), cyanobacteria spirulina (Arthrospira platensis), Staphylococcus aureus, marine orchid cell (Zostera marina), seaweed cell (Porphyra umbilicalis), Antarctic seafloor microorganism (Pseudoalteromonas haloplanktis), genus Penicillium (Penicillium chrysogenum), symbiotic worm (Symbion pandora), bamboo plant cell (P. pubescens), snail worm (Gyraulus), mussel cell (Mytilus galloprovincialis), Actinomyces, cabbage peripherite cell (Peronospora parasitica), cotton root cell (Gossypium spp.), asexual plant cell (Chara sp.), methoxycoke (Glarea lozoyensis), meadow grass Cell (Festuca arundinacea), Copia Microcrococcus (Micrococcus luteus), Fungal heterotype (Aspergillus oryzae), Bacteriophage variant (T4 phage), Fungal pathogen (Candida albicans), Paprika cell (Capsicum annuum), Micronostictus texanus, Pineapple cell (Ananas comosus), Black mushroom cell (Auricularia auricula), Oak plant cell (Quercus sp.There may be ), sebaceous fungus (Exophiala dermatitidis), Bacillus subtilis natto, Brucella bacteria (Brucella melitensis), papaya cells (Carica papaya), germ cells, non-pathogenic microbial taxa (Parvibaculum lavamentivorans), Austroococcus tauri, marine Escherichia coli (Marina sp.), wild grape cells (Vitis vinifera), lily cells (Lilium spp.), rotifers (dog worms), Tulipa gesneriana, Chlorella cells (Chlorella vulgaris), roundworm cells (Ascaris lumbricoides), etc.

[0088] Meanwhile, a DNase I inhibitory composition according to an embodiment of the present invention may include an amino acid fragment having the peptide sequence of SEQ ID NO. 1 or the peptide sequence of SEQ ID NO. 2, which has the function of specifically binding to DNase I.

[0089] [Sequence No. 1]

[0090] GLSRPGLVKDV

[0091] [Sequence No. 2]

[0092] CGGDVGVPPDF

[0093] In one embodiment, the peptide included in the DNase I inhibitory composition can specifically bind to the active site where DNase I degrades DNA, thereby inhibiting DNase I from degrading DNA. This can help maintain DNA stability or prevent excessive DNA degradation in various physiological and pathological conditions where the regulation of DNase I activity is required. In particular, in situations where excessive degradation of extracellular DNA may occur and potentially induce inflammatory responses or autoimmune diseases, this composition can play a role in mitigating excessive activation of the immune response by selectively inhibiting the activity of DNase I. Furthermore, in fields such as gene therapy or cell therapy, it may contribute to increasing therapeutic efficiency by ensuring that foreign DNA remains stable.

[0094] The present invention does not exclude the addition of other substances necessary for the actual use of the DNase I inhibitory composition according to the embodiments of the present invention. Accordingly, the composition may include additional components, such as preservatives for improving stability, buffers for maintaining the activity of the peptide, or antioxidants for increasing biocompatibility, in addition to the peptide. Furthermore, to increase the delivery efficiency of the composition, it may be combined with a drug delivery system such as nanoparticles, lipid nanoparticles, or microemulsions, and these components may help the peptide effectively reach the target site and exert a sustained DNase I inhibitory effect.

[0095] The embodiments of the present invention are described below. However, the embodiments described below are merely partial embodiments of the present invention, and the scope of the present invention is not limited to the following embodiments.

[0096] [[Experimental Materials and Methods]]

[0097] [ingredient]

[0098] DNase I derived from bovine pancreas and actin derived from rabbit muscle were purchased from Sigma-Aldrich (Seoul, Korea). 90% purity synthetic peptides (peptide-1 and peptide-19) were ordered from Peptron Co. (Daejeon, Korea). FastDigest DpnI, Dynabeads™ M-280 tosyl-activated reagent, and other PCR reagents were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Luria-Bertani (LB) medium, high-salt medium, and agar were ordered from Duchefa (Haarlem, Netherlands). Primers used in the Fv-antibody library were custom synthesized by Bionics Co. (Seoul, Korea).

[0099] [Preparation of Fv-Antibody Library]

[0100] The Fv-antibody library was generated using a site-specific mutagenic method as previously reported. A single-stranded forward primer containing a 75 bp CDR3 sequence randomized was combined with a 22 bp backward primer, and the primer sequences applied to the Fv-antibody library are provided in Supplementary Table S1. To obtain double-stranded DNA primers containing randomized CDR3 sequences, 2 μL of synthetic single-stranded primer containing randomized CDR3 sequences, 2 μL of backward primer, and 4 μL of NEBuffer 2 were mixed. Finally, distilled water (DW) was added up to 40 μL. After hybridization (5 minutes at 95 °C, followed by cooling to 36 °C), this mixture was polymerized at 37 °C for 15 minutes. This mixture contained 8 μL of dNTPs, 3 μL of Klenow (exo-) enzyme, 16 μL of NEBuffer 2, and 133 μL of DW in a final volume of 200 μL. After inactivating the enzyme reaction mixture (at 75 °C for 20 min), double-stranded Fv-antibody library primers containing randomized CDR3 sequences were purified using a PCR purification kit. The PCR reaction to generate the Fv-antibody library was performed using 10 μL of HF buffer, template plasmid (pST009, 160 ng), 160 ng of purified double-stranded Fv-antibody library primers, 0.5 μL of Phusion high-precision DNA polymerase, 1 μL of dNTPs (10 mM), and a total of 50 μL of DW. The PCR cycle consisted of an initial denaturation at 98°C for 1 minute, followed by denaturation at 98°C for 30 seconds, conjugation at 68°C for 1 minute, and an extension step at 72°C for 5 minutes. The denaturation and extension steps were repeated 30 times, and the final termination step was carried out at 72°C for 10 minutes.After PCR was completed, the template plasmid was cleaved with DpnI enzyme at 37°C for 16 hours, and the Fv-antibody library plasmid obtained after cleaving was purified through a 100 K Amicon filter (Millipore, Billerica, Massachusetts, USA).

[0101] The prepared Fv-antibody library was expressed on the surface of E. coli via self-display technology as previously reported. Subsequently, plasmid modification (electroporation) was performed on E. coli cells. Transformed cells were cultured with LB medium and 50 mg / L kanamycin at 37°C for 16 hours. Then, 10 mM β-mercaptoethanol, 50 mg / L kanamycin, and 10 μM ethylenediaminetetraacetic acid (EDTA) were mixed in LB medium (10 mL), and 100 μL of cultured cells were incubated at 37°C until an OD600 nm value reached 0.5. Expression of the Fv-antibody library was induced by adding 1 mM IPTG, and cultured cells (OD600 nm = 0.5) were cultured at 30°C for 3 hours.

[0102] [Selection of Fv-antibodies Targeting DNase I]

[0103] DNase I inhibitors are selected using magnetic beads immobilized on DNase I with a CDR3 randomized Fv-antibody library. First, DNase I is conjugated to M-280 tosylation-activated magnetic beads. To prepare the mixture, 5 mg of M-280 tosylation-activated magnetic beads (165 μL), 100 μg of DNase I, 150 μL of 0.1 M borate buffer at pH 9.5, and 100 μL of 3 M ammonium sulfate are mixed and reacted at 37°C for 16 hours while shaking at 120 rpm. The magnetic beads immobilized on DNase I are collected using an external magnet and washed with phosphate-buffered saline (PBS). The screening process for Fv-antibodies that specifically bind to DNase I is carried out as follows: (1) An Fv-antibody library (100 μL, OD600 nm = 1.0) and DNase I (10 mg / mL, 10 μL) are immobilized on magnetic beads at room temperature and reacted while shaking at 15 rpm. (2) The beads are collected using magnetism and washed with PBS and 0.01% PBST to isolate Fv-antibody library clones that specifically bind to DNase I. (3) The isolated Fv-antibody library-DNase I-magnetic beads are suspended in 100 μL of PBS. (4) The resulting suspension is plated onto an agar medium to obtain only the Fv-antibody library that specifically binds to DNase I.

[0104] To select the final clone from the agar medium, a subset of candidate clones is randomly selected, and their binding affinity to fluorescently labeled DNase I is evaluated via Fluorescence Activated Cell Analysis (FACS). DNase I is labeled with FITC by mixing 0.5 mg of FITC and 1 mg of DNase I and reacting in 2 mL of PBS at 15 rpm in the dark at room temperature for 24 hours. The mixture is purified using a 10 K Amicon filter (Millipore, Billerica, MA, USA). The FACS screening process is as follows: (1) 20 MACS-screened clones (OD600 nm = 1.0, 100 μL) are reacted with FITC-labeled DNase I (500 nM) at 15 rpm at room temperature for 1 hour. (2) The samples are washed 6 times with PBS and 0.01% PBST. (3) λexcitation / λ using a FACSCalibur flow cytometer (Becton Dickinson, NJ, USA) emission = Perform FACS analysis at 488 nm / 525 nm. (4) Separate 50,000 E. coli cells using a FACSCalibur flow cytometer to isolate Fv-antibody clones that specifically bind to FITC-labeled DNase I. (5) Culture the isolated E. coli cells in LB medium and perform sequencing analysis.

[0105] [Preparation and Purification of Fv-Antibody]

[0106] Two selected Fv antibodies were obtained in the form of soluble recombinant proteins and expressed as fusion proteins containing green fluorescent protein (GFP) at the N-terminus and a His-tag at the C-terminus. Recombinant plasmids (pHE004, pHE005) were custom-made by Cosmogenetech (Seoul, Korea) and subsequently transformed into E. coli cells via electroporation. Transformed E. coli cells were cultured in LB medium containing 1 mM IPTG and 60 mg / L cabbageillin at 30°C for 16 hours. After culture, the cells were harvested by centrifugation at 3000 ×g for 3 minutes, and the resulting pellet was resuspended in 20 mL of binding buffer (5 mM Tris-HCl, 1 M NaCl, 0.5 mM EDTA, 3 M urea). Subsequently, the cells were lysed using a sonicator (Vibra Cell VCX-130). The lysate containing Fv-antibodies was recovered by centrifugation at 25,000 ×g for 10 minutes. Purification of GFP- and His-tag-bound Fv-antibodies was performed using a His-tag purification column, and a binding buffer containing 100 mM imidazole and 3 M urea was used as the elution buffer. After purification, dialyzation was performed at 100 rpm at 4 ℃ for 16 hours to remove urea and imidazole.

[0107] [Measurement of binding affinity using SPR biosensors]

[0108] Binding affinity between DNase I, expressed Fv-antibody, and synthesized peptide (K D ) is evaluated using an SPR biosensor provided by I-Cluebio (Seongnam, Korea). The gold chip for SPR measurement is 1 × 1 cm 2 A 2 nm thick titanium and a 48 nm thick gold layer are prepared by sputter coating a BK-7 glass of size. Then, DNase I is treated at a concentration of 100 μg / mL and attached to the gold surface of the coated SPR chip at 4°C for 16 hours.

[0109] K between DNase I, Fv-antibody, and synthesized peptide D To evaluate the values, Fv-antibodies (7.4 nM to 600 nM) and peptides (11.1 nM to 900 nM) were conjugated (15 min) and dissociated (8 min), respectively. Washing with PBS was performed, and SPR analysis was conducted with a flow rate set to 15 μL / min. SPR signal data were analyzed using Hill's equation.

[0110] y = [(ad) / {1+(x / c) b}]+d

[0111] Here, 'a' represents the maximum signal, 'd' the minimum signal, 'b' the Hill slope of the curve, and 'c' the protein concentration in the solution.

[0112] [Measurement of DNase I Inhibitory Activity]

[0113] DNase I inhibitory activity was evaluated using an agarose gel-based assay. The inhibitory activity of DNase I against Fv antibodies or synthetic peptides was analyzed as follows using DNA samples extracted from the pJB030 plasmid: (1) DNase I (40 ng) was reacted with Fv antibodies or synthetic peptides (concentration range: 16 nM to 10 μM) at room temperature for 2 hours (for synthetic peptides, the binding time was set to 4 hours). (2) 300 ng of pJB030 DNA was added to the mixture. (3) The mixture was loaded onto a 1% agarose gel and electrophoresis (90 V, 40 min) was performed. DNase I inhibitory activity was evaluated by densitometric analysis of DNA bands degraded on the agarose gel using a ChemiDoc XRS (Bio-Rad, CA, USA).

[0114] DNase I inhibitory activity was further evaluated using TaqMan-like fluorescence assay. To evaluate DNase I activity inhibition, the commercial resDetect from Acrobiosystems Inc. (1 Innovation Way, Newark, DE 19711, USA) was used. TM DNA fluorescence substrate-based analysis was also performed using a DNase I assay kit. The fluorescence of the DNA substrate was λexcitation / λ emission = Measured at 535 nm / 565 nm. First, DNase I (1.7 mU) was mixed with Fv-antibody or synthetic peptide (concentration range: 3.2 nM to 10 μM). Subsequently, fluorescence was evaluated using a Promega (Madison, WI, USA) Microplate Reader. The reaction was initiated by adding DNA labeled with a fluorescent dye to the sample, and fluorescence was measured repeatedly at 5-minute intervals for a total of 120 minutes at 37 °C.

[0115]

[0116] [[Results and Discussion]]

[0117] [Selection of DNase I Inhibitors from Fv-Antibody Library]

[0118] Fv antibodies are heavy chain IgG (V HIt corresponds to the highly variable region of ) and consists of three CDRs and FRs. The Fv-antibody library was generated through site-specific mutation induction, which randomized 11 amino acid residues in the CDR3 region. The template sequence of the Fv-antibody was derived from a monoclonal antibody against thyroid peroxidase (TPO). Subsequently, as shown in Figures 1 and 2, the Fv-antibody library was expressed on the outer membrane of Escherichia coli (E. coli). When the Fv-antibody library reacted with fluorescently labeled DNase I, target E. coli cells expressing Fv-antibodies with binding affinity for DNase I exhibited fluorescence high enough to be detected by flow cytometry. Flow cytometry results obtained after treating the Fv-antibody library containing the randomized CDR3 with DNase I (fluorescent label) showed E. coli cells with high fluorescence signals at the screening gate, as shown in Figures 3, 4, and 5. In the case of control E. coli or mutant E. coli having only CDR1 and CDR2 regions, no E. coli cells with a meaningful fluorescent signal appeared at the screening gate. These data indicate that the Fv-antibody library can target E. coli cells with binding affinity for DNase I, and that the CDR3 region contributes to the binding of E. coli cells to DNase I.

[0119] Clones with binding affinity for DNase I were screened from the Fv-antibody library. To isolate E. coli cells, DNase I was attached to tosyl-activated magnetic beads via a covalent bond between the tosyl and primary amine groups of DNase I, as shown in Fig. 6. Next, the magnetic beads immobilized with DNase I reacted with the Fv-antibody library, and the bound E. coli cells were isolated using an external magnet. These E. coli cells were cultured in agar medium and selected as candidate clones. Some of the selected clones were grown in agar medium and then reacted with DNase I labeled with a fluorescent substance. As shown in Fig. 7, three clones with high fluorescence signals (Nos. 1, 9, and 19) were selected, and two clones (Nos. 1 and 19) were determined as final clones through oligonucleotide sequencing of CDR3. The unselected clone (No. 9) was found to have the same sequence as the anti-TPO antibody template prior to site-specific mutation induction. When these two clones were treated with DNase I labeled with a fluorescent substance, it was estimated that the fluorescence signal of the clones increased quantitatively as the concentration of DNase I increased, as shown in Figures 8 and 9.

[0120] For control E. coli lacking the CDR3 region and mutant E. coli possessing only the CDR1 and CDR2 regions, no significant change in the fluorescence signal was observed upon treatment with the same concentration of DNase I. As shown in Figures 10 and 11, the KD values ​​of DNase I for the selected clones were evaluated as 485.3 nM for Clone #1 and 736.2 nM for Clone #19 through fluorescence signal analysis. Binding affinity (K) including the selected nucleotide and amino acid sequences D ) is summarized in Table 1.

[0121] Screened CDR3SequencesK D, FACS [nM](Clone)KD, SPR [nM](Fv)K D, SPR [nM](Peptide)Inhibitor-1 (Clone #1)Oligo-nucleotide5'- 1 GGCCT 6 GAGTC 11 GGCCC 16 GGTCT 21 TGTTA 26 AAGAT 31 GT 33 C-3'485.373.4279.2Amino acid sequences 1 GLSRP- 6 GLVKD- 11 VInhibitor-19 (Clone #19)Oligo-nucleotide5'- 1 TGCGG 6 GGGTG 11 ATGTC 16 GGGGT 21 TCCTC 26 CCGAT 31 TT 33 C-3'736.289.0243.2Amino acid sequences 1 CGGDV- 6 GVPPD- 11 F

[0122] Table 1. Screened nucleotide and amino acid sequences with specific binding affinities for DNase I. [Inhibitory activity of selected Fv-antibodies]

[0123] Fv-antibodies obtained from two selected clones were expressed as soluble recombinant proteins. These two Fv-antibodies, containing the selected CDR3 sequences, were expressed with GFP and a histidine tag for purification. As shown in Fig. 12, the nucleotide sequences of the Fv-antibodies were cloned into an expression vector (pJB030), and the molecular weight of the expressed protein was confirmed to be 40.3 kDa via SDS-PAGE. The binding affinities (K) of the two Fv-antibodies D) was evaluated using an SPR biosensor. DNase I was attached to a Ti / Au-coated SPR chip, and Fv-antibodies reacted with DNase I at a series of known concentrations (Fig. 13). The KD values ​​of the Fv-antibodies were analyzed using an isothermal model. Calculations confirmed that the KD values ​​were 73.4 nM for Fv-1 (Clone #1) and 89.0 nM for Fv-19 (Clone #19) (Figs. 14, 15, 16, and 17). Additionally, selected CDR3 sequences were chemically synthesized into peptides, and their binding affinity (K) for DNase I was D ) was measured using an SPR biosensor immobilized with DNase I (Fig. 18). The KD value of peptide-1 (clone #1) was evaluated to be 279.2 nM, and the KD value of peptide-19 (clone #19) was evaluated to be 243.2 nM (Figs. 19, 20, 21, and 22). For F-actin, a commercial DNase I inhibitor, the KD value was evaluated to be 100 μM. These data indicate that not only the selected Fv-antibodies but also the selected CDR3 sequence peptides possess binding affinity for DNase I. The amino acid sequences of the synthesized peptides and their KD values ​​are summarized in Table 1.

[0124] IC of the expressed Fv-antibody and the synthesized peptide 50To estimate the value, agarose gel analysis was performed using DNA plasmid samples before and after treatment with the DNase I inhibitor. For the agarose gel analysis, the amount of plasmid DNA (6,428 kb) was determined to be 300 ng after ethidium bromide staining. The amount of DNase I required for the complete hydrolysis of the plasmid DNA (300 ng) was determined to be 100 ng. The concentrations of plasmid DNA and DNase I used to evaluate the inhibitory activity of the expressed Fv-antibody were 300 ng and 40 ng, respectively. As shown in Figures 23 and 24, the plasmid DNA band was completely hydrolyzed in the absence of DNase I inhibitor treatment (lane 2). As hydrolysis progressed from the plasmid DNA band before DNase I treatment (lane 3), the intensity of the plasmid DNA band gradually decreased as the Fv-1 concentration increased. IC of Fv-1 through plasmid DNA band density measurement 50 It was calculated to be 550.0 nM. As shown in Figures 25 and 26, the same inhibition analysis was performed, and the IC of Fv-19 50 It was calculated to be 660.2 nM. When a synthetic peptide of the CDR3 sequence was used in the same inhibition assay, the IC50 of Peptide-1 50 It was calculated to be 864.5 nM (Figs. 27 and 28), and the IC of Peptide-19 50 It was calculated to be 974.6 nM (Figs. 29 and 30). The IC50 of G-actin, a commercial DNase I inhibitor, was calculated to be 974.6 nM. 50It was estimated to be 231.0 nM, which was a level comparable to the inhibitory activity of the Fv-antibody and the peptide. These results indicate that both the Fv-antibody and the peptide derived from the selected clones have inhibitory activity against DNase I, and that the Fv-antibody and the peptide bind to the active site of DNase I for the hydrolysis of DNA substrates, as shown in Figs. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22.

[0125] Agarose gel-based inhibition assays can be effectively used for a clear comparison of inhibitory activities rather than for a quantitative comparison of inhibitory activities. For this reason, the inhibitory activities of the expressed Fv-antibody and the synthesized peptide were also confirmed through TaqMan-like fluorescence assays. The fluorescence assay for DNase I activity is based on a DNA substrate to which a fluorophore and a quencher are bound to each DNA strand, as shown in Figure 31. Due to the interaction between the fluorophore and the quencher, the DNA substrate does not generate a fluorescent signal. When DNase I hydrolyzes the DNA strand, the fluorophore detaches from the DNA strand bound to the quencher, generating a fluorescent signal, which is quantitatively related to the activity of DNase I.

[0126] In this invention, the fluorescence signal in the absence of DNase I was set as a positive signal (100%), and it was confirmed that the fluorescence signal could be quantitatively reduced depending on the DNase I inhibitory activity of the expressed Fv-antibody and the synthesized peptide. As the Fv-1 concentration increased, the fluorescence signal decreased, and the IC of Fv-1 50 It was evaluated to be 124.7 nM, which is shown in Figs. 32 and 33. The IC of the Fv-19 50 It was evaluated to be 171.1 nM (Figs. 34 and 35), and the IC50 of Peptide-1 in the same inhibition assay. 50285.3 nM (Figs. 36 and 37), IC of Peptide-19 50 It was estimated to be 311.5 nM (Figs. 38 and 39). In the case of G-actin, a commercial DNase I inhibitor, IC 50 It was estimated to be 77.9 nM, which was very similar to the previously reported 64.8 nM. These results indicate that both the expressed Fv-antibody and peptide derived from the selected clones have inhibitory activity against DNase I, and that, as shown in Figure 3, the Fv-antibody and peptide bind to the active site of DNase I for the hydrolysis of DNA substrates.

[0127] [Analysis of Interactions Between DNase I and Selected Inhibitors]

[0128] The interaction between DNase I and selected inhibitors was analyzed using computer-based docking simulations with the open-source program Autodock Vina. The interaction was examined using PyMOL software (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC). As shown in Figure 40, DNase I (PDB number: 4AWN) was reported to bind to a local binding site on the DNA substrate consisting of seven interaction sites (Glu9, Arg41, Tyr76, Arg111, Asn170, Tyr175, Tyr211 amino acid sequences).

[0129] In the present invention, it was confirmed that the interaction between DNase I and the selected inhibitor occurs at the same binding site. As shown in Fig. 41, the interaction between Fv-1 and DNase I was analyzed using the CDR3 region. The interaction was calculated to occur at the same binding site corresponding to the binding sites of Arg41 and Tyr76. The amino acids participating in hydrogen bonding were Glu13, Thr14, and Arg41, while the amino acids participating in hydrophobic interactions were His44 and Tyr76. These results demonstrate that Fv-1 can exert inhibitory activity through interaction with some binding sites of DNase I.

[0130] As shown in Figure 42, the interaction between Fv-19 and DNase I was also evaluated using the CDR3 region. The interaction was calculated to occur at the binding sites of Gln9, Tyr76, Arg111, Tyr175, and Tyr211. The amino acids participating in hydrogen bonding were analyzed to be Gln9, Tyr76, Tyr175, and Tyr211, while the amino acid participating in hydrophobic interaction was Pro137. These results demonstrate that Fv-19 can exert inhibitory activity through interaction with the binding site of DNase I. Docking simulation results also suggest that the selected CDR3 sequence covers only a portion of the DNase I binding site, indicating that Fv-antibodies can block the DNase I binding site more effectively than synthetic peptides.

[0131] Figure 43 illustrates the interaction between a commercial DNase I inhibitor and DNase I. Analysis revealed that the commercial inhibitor forms hydrogen bonds and electrical interactions with Arg41, Tyr76, Arg111, etc., within the same binding site of DNase I. Additionally, amino acids such as Glu78, Ala136, and Pro137 were calculated to contribute further to the interaction. The binding affinity was found to be -6.3 kcal / mol, which indicates relatively low binding stability compared to Fv-antibodies (Fv-1, Fv-19). These results suggest that while the commercial DNase I inhibitor can partially block the binding site, the selected Fv-antibodies can inhibit the active site of DNase I more effectively.

[0132] [[conclusion]]

[0133] DNase I inhibitors were selected using a library of Fv-antibody expressed on the outer membrane of Escherichia coli (E. coli). This Fv-antibody library was designed to contain randomly modified CDR3. As a result of selecting clones from the Fv-antibody library based on binding affinity for DNase I, two clones were identified, and the amino acid sequences of CDR3 were determined. Subsequently, the two selected Fv-antibodies were expressed as soluble recombinant proteins, and the selected CDR3 was synthesized into a peptide. These two inhibitors were expressed and synthesized as a soluble protein and a peptide, respectively. The binding affinity (K) of the Fv-antibodies and the synthesized peptides for DNase I D ) was measured using an SPR biosensor. The binding affinities of Fv-1 (peptide-1) and Fv-19 (peptide-19) for DNase I were analyzed to be 73.4 nM (279.2 nM) and 89.0 nM (243.2 nM), respectively. In addition, IC50 was determined through the analysis of expressed Fv-antibodies, synthesized peptides, agarose gel analysis, and TaqMan-like fluorescence analysis. 50 The IC50 of DNase I was measured.50 The values ​​were analyzed to be 124.7 nM (285.3 nM) and 171.1 nM (311.5 nM) for Fv-1 (peptide-1) and Fv-19 (peptide-19), respectively. These results indicate that both the Fv-antibody and the peptide from the selected clones possess inhibitory activity against DNase I, and that the binding of the Fv-antibody and the peptide occurs at the active site of DNase I for the hydrolysis of DNA substrates. The analysis of the interaction between the selected inhibitor and DNase I was performed using computer-aided docking simulations. The results of the docking simulations suggest that the selected CDR3 sequence occupies only a portion of the active site of DNase I, and that the Fv-antibody can block the active site of DNase I more effectively than the synthetic peptide.

[0134] At least some of the sequences used in the present invention are as follows.

[0135] SequenceIDNumberSequenceNameMoleculeTypeOrganismSequence1Inhibitor-1AAsynthetic constuctGLSRPGLVKDV2Inhibitor-19AAsynthetic constuctCGGDVGVPPDF3Inhibitor-1-nucnucleotidesynthetic constuctGGCCTGAGTCGGCCCGGTCTTGTTAAAGATGTC4Inhibitor-19-nucnucleotidesynthetic constuctTGCGGGGGTGATGTCGGGGTTCCTCCCGATTTC

[0136] Although the present invention has been described above with reference to preferred embodiments, those skilled in the art will understand that various modifications and changes can be made to the invention without departing from the spirit and scope of the invention as set forth in the following claims.

Claims

1. Having the function of specifically binding to DNase I, A peptide sequence comprising the peptide sequence of SEQ ID NO. 1 or the peptide sequence of SEQ ID NO. 2, Peptide: [Sequence No. 1] GLSRPGLVKDV [Sequence No. 2] CGGDVGVPPDF.

2. In Paragraph 1, The above peptide specifically binds to the active site where DNase I degrades DNA, thereby inhibiting DNase I from degrading DNA. Peptide.

3. Coding for the peptide according to paragraph 1, Nucleic acid.

4. Comprising nucleic acids pursuant to paragraph 3, Recombinant expression vector.

5. Transformed with a recombinant expression vector according to paragraph 4, cell.

6. In Paragraph 5, The above cell comprises one or more cells selected from the group including animal cells, plant cells, yeast, E. coli, and insect cells, cell.

7. In Paragraph 6, The above cells are monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells and HEK293 cells, E. coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp, and Neurospora Comprising one or more cells selected from the group including Neurosporacrassa, cell.

8. Having the function of specifically binding to DNase I, Comprising an amino acid fragment having the peptide sequence of SEQ ID NO. 1 or the peptide sequence of SEQ ID NO. 2, Composition for inhibiting DNase I: [Sequence No. 1] GLSRPGLVKDV [Sequence No. 2] CGGDVGVPPDF.

9. In Paragraph 8, The peptide included in the above DNase I inhibitory composition specifically binds to the active site where DNase I degrades DNA, thereby inhibiting DNase I from degrading DNA. Composition for inhibiting DNase I.

Images