KR102648130B1 - Pharmaceutical composition for the prevention and treatment of sepsis comprising LRR domain of NLRX1 protein - Google Patents

Abstract

The present invention relates to a composition for preventing or treating sepsis based on the LRR domain of the NLRX1 protein, which can effectively suppress and alleviate the disease severity of sepsis and restore serious inflammatory responses and abnormal physiological functions in the whole body, thereby treating sepsis. Alternatively, it can be usefully used for preventive purposes.

Description

A composition for preventing or treating sepsis based on the LRR domain of NLRX1 protein {Pharmaceutical composition for the prevention and treatment of sepsis comprising LRR domain of NLRX1 protein}

The present invention relates to a composition for preventing or treating sepsis based on the LRR domain of the NLRX1 protein.

Sepsis is a disease that accounts for the majority of deaths in critically ill patients. In the United States, 2,000 new patients occur every day and the mortality rate is high at 30-50%. Sepsis refers to cases in which patients with suspected or proven infection meet the diagnostic criteria for systemic inflammatory response syndrome, specifically body temperature >38˚C or <36˚C, heart rate >90 beats/min, and respiratory rate >20. It can be defined as meeting two or more of the following: beats/min or PaCO ₂ <32 mmHg, white blood cell count >12,000 cells/mm ³ or <4,000 cells/mm ³ or undifferentiated form >10%. Severe sepsis is defined as sepsis accompanied by hypotension, organ failure, and decreased tissue perfusion. Septic shock is defined as hypotension (systolic blood pressure less than 90 mmHg or mean arterial pressure less than 65 mmHg) despite appropriate fluid treatment. .

Sepsis is mainly caused by bacterial infection, and certain patterns such as fat, nucleic acid, and protein, which are components of all pathogens or microorganisms that invade the human body (PAMP; pathogen-associated molecular patterns, or MAMP; microorganism associated molecular pattern, or DAMP; danger- It begins by detecting associated molecular patterns (recognized by pattern recognition receptors) (PRR).

The immune system is divided into the innate and adaptive immune systems, which have different functions and roles. The innate immune system includes macrophages, neutrophils, and complement, and the adaptive immune system includes T and B lymphocyte populations with various types of antigen receptors.

Macrophages are a major immune system that expresses pattern recognition receptors and respond to PAMPs and DAMPs. Upon recognition of the ligand, the NF-κB and inflammasome signaling pathways are activated, leading to the activation of NF-κB and inflammasome signaling pathways, including TNFα, IL-1β, and IL-6. Produces pro-inflammatory cytokines. Excessive production of cytokines in the macrophages induces serious inflammatory responses and organ damage.

In this way, various immunomodulatory substances have been developed and studied to treat sepsis. For example, attempts have been made to treat sepsis using immunomodulators such as corticosteroids, anti-endotoxin antibodies, TNF antagonists, and IL-1 receptor antagonists, but there is still no way to improve sepsis. No results were obtained, and there is controversy about side effects in some studies.

Currently, antibiotics are the only bioapplicable treatment for sepsis. However, for conditions in which the immune system itself functions abnormally, the effectiveness of antibiotics is reduced to less than half, and they only provide symptom relief, making complete prevention or treatment impossible. There is a problem. Therefore, there is an urgent need to develop a new sepsis treatment that can be stably applied to clinical treatment and cell therapy with a direct therapeutic effect by targeting macrophages to suppress the excessive inflammatory response of macrophages within a short period of time after the invention of sepsis. This is the situation.

To date, most sepsis treatment strategies have targeted TNFα, IL-1R, and TLR4, which were not found to improve patient survival in large multicenter clinical trials. In cancer and rheumatic diseases, immunomodulatory substances are actively developed as treatments and are widely used clinically, but in sepsis, treatments that are clinically sufficient to improve the prognosis of sepsis have not yet been developed.

Accordingly, there is a need to identify new targets that regulate the complex pathophysiological phenomenon of sepsis, which can be used to develop new sepsis treatments.

The object of the present invention is a cell-penetrating peptide; and a peptide consisting of an LRR domain region derived from the NLRX1 protein represented by SEQ ID NO: 25. The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating sepsis, which contains a fusion protein as an active ingredient.

Additionally, the present invention provides a cell-penetrating peptide; and a peptide consisting of an LRR domain region derived from the NLRX1 protein represented by SEQ ID NO: 25. The purpose is to provide a pharmaceutical use for the treatment and prevention of sepsis using a fusion protein containing as an active ingredient.

In order to achieve the above object, the present invention provides a cell-penetrating peptide; and a peptide consisting of an LRR domain region derived from the NLRX1 protein represented by SEQ ID NO: 25. It provides a pharmaceutical composition for the prevention or treatment of sepsis containing a fusion protein as an active ingredient.

The LRR domain region may be composed of the amino acid sequence represented by SEQ ID NO: 3.

The cell-penetrating peptide may be any one selected from the amino acid sequences represented by SEQ ID NOs: 1 to 11.

The cell-penetrating peptide may be any one selected from the amino acid sequences represented by SEQ ID NOs: 1 to 7.

The cell-penetrating peptide may be the amino acid sequence represented by SEQ ID NO: 1.

The fusion protein may be composed of any one selected from the amino acid sequences represented by SEQ ID NO: 32, 36, and 39, and preferably, the fusion protein may be composed of the amino acid sequence represented by SEQ ID NO: 35.

The fusion protein may be delivered to 1.5 to 2 times as many macrophages as total immune cells (total dendritic cells).

The fusion protein may act specifically on macrophages compared to immune cells to inhibit the activation of the inflammation regulatory complex pathway of NF-κB signaling and inflammasome signaling, thereby suppressing the hyper-inflammatory response of macrophages.

The sepsis may be sepsis caused by gram-negative bacteria.

The sepsis may be caused by LPS, which constitutes the cell membrane of Gram-negative bacteria.

In addition, the present invention provides a cell-penetrating peptide for manufacturing a medicine for preventing or treating sepsis disease; and a peptide consisting of an LRR domain region derived from the NLRX1 protein represented by SEQ ID NO: 25. It provides a use of a pharmaceutical composition comprising a fusion protein including as an active ingredient.

In addition, the present invention provides a cell-penetrating peptide to patients with sepsis disease; and a peptide consisting of an LRR domain region derived from the NLRX1 protein represented by SEQ ID NO: 25. A method of treating sepsis disease is provided, comprising administering a pharmaceutical composition containing a fusion protein as an active ingredient.

The present invention relates to a fusion protein containing a cell-penetrating peptide and an LRR domain region derived from the NLRX1 protein, which can effectively suppress and alleviate the disease severity of sepsis and restore serious inflammatory reactions and abnormal physiological functions in the whole body, thereby preventing sepsis. It can be useful for therapeutic or preventive purposes.

Figures 1 and 2 are a diagram schematically showing the pharmacological mechanism by which C10-LRR acts on sepsis (Figure 1) and a diagram showing the mechanism (Figure 2).
Figure 3 is a graph analyzing NLRX1 expression in PBMC samples from healthy or septic patients.
Figure 4 schematically shows the pharmacokinetics of the recombinant proteins C10-LRR, C10-NBD, and C10-EGFP.
Figure 5 shows the DNA structures encoding recombinant proteins of C10-LRR, C10-NBD, and C10-EGFP, and the results of their analysis by 12% SDS-PAGE.
Figure 6 is a diagram schematically showing the experimental design of a sepsis animal model.
Figure 7 is a graph showing the results of measuring the survival rate of animal models divided into group 1 (PBS), group 2 (C10-EGFP), group 3 (C10-NBD), and group 4 (C10-LRR) for 7 days. .
Figure 8 is a graph showing the results of measuring the weight of animal models divided into group 1 (PBS), group 2 (C10-EGFP), group 3 (C10-NBD), and group 4 (C10-LRR) for 7 days. .
Figure 9 shows IL-6, TNF-α and This is a graph showing the results of measuring the expression level of IL-1β using ELISA.
Figure 10 shows IL-6, TNF-α and This is a graph showing the results of measuring the expression level of IL-1β using ELISA.
Figure 11 is a graph showing the results of measuring AST activity in animal models divided into group 1 (PBS), group 2 (C10-EGFP), group 3 (C10-NBD), and group 4 (C10-LRR) after 2 hours.
Figure 12 is a graph showing the survival rate of animal models divided into group 4 (C10-LRR) and group 5 (C10-LRR 0.2 mg/kg) measured for 6 days.
Figures 13 and 14 are ELISA graphs measuring the levels of L-6 and TNF-α after treating peritoneal macrophages with LPS and each recombinant protein.
Figure 15 is a graph analyzing cell survival rate after treating peritoneal macrophages with LPS and each recombinant protein.
Figure 16 shows the results of peritoneal macrophages treated with 1 μM C10-LRR and 1 mg/ml LPS, cultured for 15 and 30 minutes, and analyzed by Western blot.
Figures 17 and 18 are graphs showing the results of treating PMA-stimulated THP-1 cells with C10-LRR and LPS, and analyzing the levels of IL-6 and TNF-α by ELISA.
Figure 19 shows the results of treating THP-1 cells with 1 μM C10-LRR and 1 mg/ml LPS for 30 minutes, and measuring the levels of PlκBα Ser32, IκBα, p65 Ser536, and p65 expressed therefrom by Western blot.
Figure 20 is a graph showing the level of IL-1β measured by ELISA after culturing peritoneal macrophages with LPS and recombinant proteins (C10-LRR, C10-NBD, C10-EGFP) or nigericin.
Figure 21 is an experimental design to confirm the inhibitory effect of C10-LRR on inflammasome activation in peritoneal macrophages.
Figure 22 shows IL in the Prim+Stim group, Prim group, and Stim group targeting peritoneal macrophages administered various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-EGFP). This is a graph showing the results of analyzing -1β levels using ELISA.
Figure 23 shows the Prim+Stim group, Prim group, and Stim group for MEF (murine embryonic fibroblast) cells administered various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-EGFP). This is a graph showing the results of ELISA analysis of IL-1β levels in the group.
Figure 24 shows Pro-Casp1 after culturing peritoneal macrophages induced with inflammation with LPS and nigericin with various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-EGFP). , This is the result of measuring the expression level of Casp1 P20 by Western blot.
Figure 25 shows the expression of Pro-Casp1 and Casp1 P20 after adding recombinant proteins (C10-LRR, C10-NBD) to mitochondrial fractions and whole cell lysates induced by inflammation with LPS and nigericin and culturing them. This is the result of measuring the expression level by Western blot.
Figure 26 shows the results of primary peritoneal macrophages treated with C10-LRR, TAT-LRR, LRR, and PBS (control), cultured for 1 hour, and analyzed by flow cytometry.
Figure 27 shows the results of confirming the distribution within the cell using a confocal microscope when C10-LRR was treated in HeLA cells.
Figure 28a is a graph comparing the relative intracellular delivery efficiency in CD11b ^high macrophages compared to dendritic cells, Figure 28b is a graph comparing the relative intracellular delivery efficiency in CD11b ^med macrophages compared to dendritic cells, and Figure 28c is a graph comparing the relative intracellular delivery efficiency in CD11b med macrophages compared to dendritic cells. This is a graph comparing the relative intracellular delivery efficiency in neutrophil cells compared to cells.
Figures 29 and 30 show the results of treating Jurkat cells with the recombinant proteins of Examples 4 to 9, TAT-EGFP and EGFP (control group), culturing them for 1 hour, and analyzing them by flow cytometry.
Figures 31 and 32 show the results of Hela cells treated with the recombinant proteins of Examples 4 to 9, TAT-EGFP and EGFP (control group), cultured for 1 hour, and analyzed by flow cytometry.
Figure 33 schematically shows the experimental design to confirm the sepsis treatment effect of C10-LRR recombinant protein and αTNFαAb.
Figure 34 is a graph showing the results of measuring the survival rate of animal models divided into group 1 (PBS), group 2 (C10-LRR), group 3 (αTNFαAb), and group 4 (C10-LRR+αTNFαAb) for 7 days. .
Figure 35 is a graph showing the results of measuring the weight of animal models divided into group 1 (PBS), group 2 (C10-LRR), group 3 (αTNFαAb), and group 4 (C10-LRR+αTNFαAb) for 7 days. .
Figure 36 schematically shows the experimental design to confirm the sepsis treatment effect when the number of administrations of C10-LRR recombinant protein and αTNFαAb was varied.
Figure 37 is a graph showing the results of measuring the survival rate of animal models divided into group 1 (PBS), group 2 (C10-LRR), group 3 (αTNFαAb), and group 4 (C10-LRR+αTNFαAb) for 7 days. .
Figure 38 is a graph showing the results of measuring the weight of animal models divided into group 1 (PBS), group 2 (C10-LRR), group 3 (αTNFαAb), and group 4 (C10-LRR+αTNFαAb) for 7 days. .

The present inventors have tried to develop efficient materials that can overcome and replace conventional limitations in the prevention or treatment of sepsis. As a result, in order to identify the function of the domain present in the NLRX1 protein and ensure its efficient delivery into cells, a treatment strategy using a cell-penetrating peptide was designed, and treatment with a fusion protein using this peptide clearly showed a preventive or therapeutic effect. The present invention was completed by confirming that it exists.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating sepsis, which contains as an active ingredient a fusion protein containing a cell-penetrating peptide and a peptide consisting of an LRR domain region derived from the NLRX1 protein represented by SEQ ID NO: 25; or a cell-penetrating peptide and a peptide consisting of an LRR domain region derived from the NLRX1 protein represented by SEQ ID NO: 25; It provides a sepsis treatment method comprising applying to a human or a mammal other than a human a fusion protein.

In the present invention, the term 'peptide' refers to a polymer in the form of a chain formed by linking 4 to 1000 amino acid residues together through peptide bonds, and is interchangeable with 'polypeptide'.

In addition, in the present invention, the term 'polynucleotide' refers to a polymer compound in which nucleotides, which are chemical monomers composed of three elements: a base, sugar, and phosphoric acid, are connected in a chain form through multiple phosphate ester bonds.

In addition, in the present invention, the term 'cell penetrating peptide (CPP)' refers to a peptide that has the ability to deliver the cargo to be transported into cells in vitro or in vivo. It is interchangeable with ‘cell membrane penetrating domain’ or ‘cell membrane penetrating peptide’.

NLRX1 protein (NLR family member This is known as NOD-like receptor X1, NLR family X1, etc. It consists of an effector domain containing a mitochondrial localization signal at the N-terminus, a NACHT domain (NBD), and a C-terminal leucine-rich repeat (LRR) domain. The NLRX1 protein plays a very important role in the immune system and specifically interferes with the mitochondrial antiviral signaling protein (MAVS)/retinoic acid-inducible gene I (RIG-I) mitochondrial antiviral pathway, affecting innate immunity against viruses. It has been known to give . Additionally, NLRX1 is involved in host immunity during bacterial infections such as Chlamydia trachomatis and Helicobacter pylori by regulating bacterial burden and inflammation in mononuclear phagocytes. Although the NLRX1 protein has not yet been specifically identified, computational modeling predictions have confirmed that NLRX1 protein expression can be controlled by an initially induced negative feedback circuit after infection.

In the present invention, the function of each domain of NLRX1 was identified, and it was confirmed that a fusion protein obtained by fusing the LRR domain of the NLRX1 protein with a cell-penetrating peptide has not only a preventive effect on sepsis, but also a therapeutic effect.

Specifically, in one embodiment of the present invention, the LRR domain sequence was confirmed from the NLRX1 protein, a plasmid DNA in which a cephpermeable peptide was bound to the LRR domain was designed, and this was introduced into an expression vector and then transduced into the host strain, thereby producing cells. A fusion protein in which the penetrating peptide and the LRR domain region were linked was prepared.

The present invention relates to a fusion protein formed by combining a cell-penetrating peptide with an LRR domain derived from the NLRX1 protein, the function of which was previously unknown. Through the experimental examples described below, it was confirmed that the LRR domain derived from the NLRX1 protein represented by SEQ ID NO: 25 of the present invention binds to a cell-penetrating peptide and exhibits a prevention, treatment or improvement effect on sepsis, and the cell-penetrating peptide is It was confirmed that the effect was the best in the case of C10 peptide.

The NLRX1 protein or its genetic information can be obtained from known databases such as GenBank of the National Center for Biotechnology Information (NCBI). Specifically, the NLRX1 protein may be the NLRX1 protein represented by SEQ ID NO: 23. In addition, the LRR domain derived from the NLRX1 protein is a fragment of the NLRX1 protein represented by SEQ ID NO: 25, and may be the amino acid sequence represented by SEQ ID NO: 25. A more specific example may be the amino acid sequence shown in SEQ ID NO: 25, or one that has the activity and homology to the above sequence. In addition, the LRR domain derived from the NLRX1 protein is at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% of SEQ ID NO: 25, may have a sequence identity of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. However, in the examples described above, Not limited.

The LRR domain region may be the amino acid sequence represented by SEQ ID NO: 25.

The cell-penetrating peptide used in the present invention may be any one selected from the amino acid sequences shown in SEQ ID NOS: 1 to 11, and preferably may be any one selected from the amino acid sequences shown in SEQ ID NOS: 1 to 7, Most preferably, it may be the amino acid sequence represented by SEQ ID NO: 1. When the amino acid sequence represented by SEQ ID NO: 1 is used as the cell-permeable peptide, the fusion protein has a specific and remarkable delivery efficiency for macrophages and can exhibit significant prevention, treatment, and improvement effects on sepsis. Most preferably, the peptide is the amino acid sequence represented by SEQ ID NO: 1. Specifically, it is most preferable when the cell-penetrating peptide has an amino acid sequence represented by SEQ ID NO: 1 because it has the effect of being delivered to 1.5 to 2 times more macrophages than all immune cells (total dendritic cells).

The fusion protein may be composed of any one selected from the amino acid sequences represented by SEQ ID NO: 32, 36, and 39. Considering the above, most preferably, the fusion protein is composed of the amino acid sequence represented by SEQ ID NO: 32. You can.

The fusion protein according to the present invention not only allows it to pass through the cell membrane, which conventional biologically active substances could not easily pass through, but also acts directly inside the cell, including macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, Biologically active substances can be delivered to act within immune cells by targeting one or more immune cells selected from the group consisting of eosinophils, natural killer cells, basophils, and neutrophils. Therefore, the fusion protein can be a groundbreaking opportunity in the development of drug delivery systems.

The fusion protein according to the present invention preferably acts favorably on macrophages among immune cells, and thus can specifically and efficiently deliver biologically active substances into macrophages.

The fusion protein according to the present invention penetrates cells efficiently, especially into macrophages, significantly improving the survival of an animal model of sepsis induced by LPS-mediated lethal endotoxemia, and preventing IκB degradation and p65 phosphorylation. It was confirmed that not only did it efficiently suppress IL-6 production in peritoneal macrophages, but it also negatively regulated IL-1β production by preventing caspase-1 activation with sustained mitochondrial MAVS levels.

Therefore, the fusion protein of the present invention can be useful as a medicine that can prevent or treat not only sepsis but also septic shock.

The fusion protein may further include a nuclear localization signal (NLS) to localize the fusion protein in the nucleus.

The fusion protein may be linked to a tag that is advantageous for isolation and/or purification. For example, small peptide tags such as His tag, Flag tag, S tag, etc., GST (Glutathione S-transferase) tag, MBP (Maltose binding protein) tag, etc. may be linked depending on the purpose, but are not limited thereto.

The fusion protein may be extracted from nature, synthesized, or produced by genetic recombination methods based on DNA sequences.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers are those commonly used for presentation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly Includes, but is not limited to, vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

In the present invention, the term “prevention” refers to all actions that inhibit or delay sepsis by administering a composition containing the fusion protein of the present invention as an active ingredient.

In the present invention, the term “treatment” refers to any action in which sepsis is improved or beneficial by administration of a composition containing the fusion protein of the present invention as an active ingredient.

Additionally, in the present invention, 'sepsis' refers to a condition in which a serious inflammatory response occurs throughout the body due to infection with microorganisms. Symptoms of fever where body temperature rises above 38 degrees or hypothermia where body temperature drops below 36 degrees, respiratory rate increased above 24 times per minute (tachypnea), heart rate above 90 beats per minute (tachycardia), two of the following: increase or significant decrease in white blood cell count on blood test If more than one symptom is present, it is called systemic inflammatory response syndrome (SIRS). When this systemic inflammatory response syndrome is caused by microbial infection, it is called sepsis. Pathogens enter the bloodstream continuously or intermittently from an infection site in the body and settle in various organ tissues, creating lesions and causing severe systemic symptoms. Causative bacteria include staphylococci, streptococci, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Klebsiella pneumoniae, fungi, and anaerobic bacteria. Preferably, the sepsis may be sepsis caused by Gram-negative bacteria, and more preferably, the sepsis may be harmful due to LPS, which constitutes the cell membrane of Gram-negative bacteria.

Since management of infectious lesions is most important in the treatment of sepsis, drugs with antibiotics or anti-inflammatory functions are mainly used. However, because the distribution volume, metabolism, and excretion of the drug are different from those of normal people, it is difficult to predict the blood concentration and effect of the drug, and because the causes/causing bacteria of sepsis are diverse, it is difficult to use appropriate antibiotics, which limits treatment. Accordingly, various cytokine inhibitors have been developed, but none has yet been recognized as a method showing clear therapeutic efficacy, and no molecular target therapy has been developed.

Anti-TNFα, anti-TNFR antibodies, and TNFα inhibitors targeting TNFα were effective in animal models, but failed in phase 2 and 3 clinical studies. TNFα is a major factor in the early stages of sepsis, but since various other factors influence it, inhibitors that only inhibit TNFα are considered to be ineffective.

Accordingly, while the present inventors were conducting research to develop a sepsis prevention and treatment agent, the fusion protein of the present invention significantly improved the survival of an LPS-mediated endotoxemic sepsis animal model and prevented IκB degradation and p65 phosphorylation in peritoneal macrophages. IL-6 production was efficiently suppressed and IL-1β production was negatively regulated by preventing caspase-1 activation at the mitochondrial MAVS level, making the fusion protein of the present invention useful as an active ingredient in pharmaceutical compositions for preventing or treating sepsis. It was confirmed that it can be used.

That is, although the composition according to the present invention contains a fusion protein as a single active ingredient, it can effectively control NF-κB and inflammatory signal transduction at the same time, thereby efficiently treating systemic inflammation in sepsis.

In addition, since the composition according to the present invention has a complex anti-inflammatory mechanism, it can be seen that when administered in combination with a sepsis treatment, the effect of the drug in treating sepsis is significantly increased without interfering with the effect.

Additionally, the pharmaceutical composition for preventing and treating sepsis of the present invention may include a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration may also include water, suitable oils, saline solutions, aqueous glucose and glycols, and the like. Additionally, stabilizers and preservatives may be additionally included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As for other pharmaceutically acceptable carriers, those described in the following literature may be referred to (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally, and the parenteral administration method is not limited to this, but is intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, and intraperitoneal. , may be administered intranasally, enterally, topically, sublingually, or rectally.

The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration according to the administration route described above. When formulated, one or more buffers (e.g. saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g. EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g. aluminum hydroxide) side), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, and such solid preparations may contain at least one excipient, for example, in the pharmaceutical composition of the present invention. , starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose. , methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropylmethyl-cellulose or gelatin can be mixed. For example, tablets or sugar tablets can be obtained by combining the active ingredient with a solid excipient, grinding it, adding suitable auxiliaries and processing it into a granule mixture.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water or liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, or preservatives may be included. .

In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention can be formulated with a suitable parenteral carrier in the form of injections, transdermal administration, and nasal inhalation according to methods known in the art. The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administration, forms such as ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations are included. In the above, 'transdermal administration' means administering a pharmaceutical composition topically to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

For inhalation administration, the compounds used according to the invention may be packaged in pressurized packs or using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a text generally known in all pharmaceutical chemistry.

The pharmaceutical composition for preventing and treating sepsis of the present invention can provide desirable sepsis prevention and treatment effects when it contains the fusion protein in an effective amount. In this specification, the term 'effective amount' refers to an amount that shows a greater response than the negative control, and preferably refers to an amount sufficient to prevent or treat sepsis. The pharmaceutical composition of the present invention may contain 0.01 to 99.99% of the fusion protein, and the remaining amount may be comprised of a pharmaceutically acceptable carrier. The effective amount of the fusion protein included in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized.

The total effective amount of the pharmaceutical composition of the present invention can be administered to a patient in a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. When administered parenterally, the amount is preferably 0.01 to 50 mg, more preferably 0.1 to 30 mg per kg of body weight per day based on the fusion protein, and when administered orally, the amount is administered per day based on the fusion protein. It can be administered in one to several divided doses, preferably in an amount of 0.01 to 100 mg, more preferably 0.1 to 50 mg per kg of body weight. However, the dose of the fusion protein is determined by taking into account various factors such as the administration route and number of treatments of the pharmaceutical composition, as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate. Therefore, considering this, a person with ordinary knowledge in the art will be able to determine an appropriate effective dosage of the fusion protein according to a specific use for preventing and treating sepsis. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

The pharmaceutical composition for preventing and treating sepsis of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemical therapy, biological response regulators, or sepsis treatment agents.

In particular, the fusion protein according to the present invention is characterized in that it is administered in combination with a sepsis treatment agent, and the sepsis treatment agent alleviates the symptoms of sepsis, such as antibiotics, activated protein C, glucocorticoids, cytokine antagonists, fluids, and blood transfusions. There is no particular limitation as long as it is a treatment agent or method.

The antibiotic may be any one or more selected from the group consisting of aminoglycosides, tetracyclines, peptides, and rifamycins.

In the present invention, “antibiotics” are substances used for the treatment and prevention of bacterial infections and refer to substances that prevent the growth or life of microorganisms. The above antibiotics include natural antibiotics, which are chemical substances produced by microorganisms such as bacteria and molds, semisynthetic antibiotics, which are derivatives obtained through changes in the structural form of natural antibiotics, and synthetic antibiotics (antibacterial agents) that are chemically synthesized. .

Antibiotics of the present invention include aminoglycosides, penicillin, cephalosporin, tetracycline, macrolides, streptogramins, and glycosides. Glycopeptide, peptide, flavophospholipol, polyether, phenicol, lincosamide, rifamycin, polyene These include, but are not limited to, polynene, sulfonamide, benzylperimidine, quinolone, fluoroquinolone, and nitrofuran antibiotics. The aminoglycoside antibiotics include gentamicin, kanamycin, ribostamycin, tobramycin, sisomicin, astromycin, isepamycin, abecacin, dibecasin, spectinomycin, netilmycin, micronomycin, and streptomycin. , dihydrostreptomycin, apramycin, destomycin, hygromycin, amikacin, neomycin, etc., but are not limited thereto. The penicillin antibiotics include penicillin, benzylpenicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafucillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, etc., It is not limited to this. The above cephalosporin (cephem) antibiotics include cepharotin, cefazolin, cephaloridin, cephalexin, cefacetril, cephaloniom, cefoxazole, cefaphyrin, cefadroxil, cefamandole, cefoxitin, and cefu. Includes, but is not limited to, Roxime, cefoperazone, cephamethazole, cefotaxime, ceputiofur, etc. The tetracycline-based antibiotics include, but are not limited to, chlortetracycline, oxytetracycline, tetracycline, minocycline, and doxycycline. The macrolide antibiotics include, but are not limited to, erythromycin, kitasamicin, spiramycin, oleandomycin, josamycin, sedecamicin, tyrosin, and roxithromycin. The streptogramin-based antibiotics include, but are not limited to, virginiamycin and micamycin. The glycopeptide antibiotics include, but are not limited to, abopacin and vancomycin. The peptide-based antibiotics include polymyxins (e.g., colistin, polymyxin B, etc.), cyclic (e.g., bacitracin, etc.), and cyclic depsis (e.g., enramycin, thiopeptin, etc.) ), etc. are included, but are not limited thereto. The flavophospholipol-based antibiotics include, but are not limited to, bambermycin, macabomycin, quebemycin, and the like. The polyethyl antibiotics include, but are not limited to, monensin, salinomycin, lasaroside, naracin maduramycin, etc. The phenicol-based antibiotics include, but are not limited to, chloramphenicol, thiamphenicol, and florfenicol. The lincosamide antibiotics include, but are not limited to, lincomycin and clindamycin. The rifamycin-based antibiotics include, but are not limited to, limfampicin. The polyene antibiotics include, but are not limited to, nystatin, pimaricin, pentamycin, amphotericin B, trichomycin, candicidin, etc. The sulfonamide antibiotics include, but are not limited to, sulfapyridine, sulfadiazine, sulfadimidine, sulfafurazole, and sulfamonomethox. The benzylperimidine antibiotics include, but are not limited to, trimethoprim, ometoprim, and tetroxoprim. The quinolone antibiotics include, but are not limited to, naridisic acid, oxolinic acid, cinoxacin, and acrosoxacin. The fluoroquinolone antibiotics include, but are not limited to, flumequin, sipuroxacin, ennoxacin, furoxacin, and mabofloxacin. The nitrofuran-based antibiotics include, but are not limited to, furazolidone, furaltadone, nitrobin, nitrofurazone, etc.

In the present invention, a cytokine antagonist may be any substance or mixture of substances that reduces or inhibits at least one, preferably substantially all, biological activities of one or more cytokines in the patient's body. The antagonistic effect may occur directly by the antagonist or indirectly, for example by activating or inhibiting additional signaling pathways that also have an effect on the biological activity of the cytokine. Preferably the biological activity of the cytokine is inhibited by blocking its interaction with one or more receptors to which it can bind. This can be achieved, for example, by competitive binding of the antagonist to the receptor(s) in question or by binding of the antagonist to the cytokine itself.

The cytokines are members of the TNF-alpha superfamily (e.g., TNF-alpha, TNF-C, OX40L, CD154, FasL, LIGHT, TL1A, CD70, Siva, CD153, 4-1BB Ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF , CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand, EDA-A, EDA-A2), TGF-beta superfamily, IL-1 family (e.g., IL-1 and IL-8), IL One or more molecules may be selected from molecules belonging to the -2 family, IL-10 family, IL-17 family, and interferon family, such as TNF-alpha, TNF-alpha, IL-1, IL-8, IL-10, and IL17. , LIGHT, TLIA, Siva, TRAIL, RANKL, TWEAK, APRIL, NGF, BDNF, NT-3, and EDA-A2, TNF-C, OX40L, CD154, FasL, CD70, CD153, 4-1BB ligand, EDA-A , IL-2, IFN-alpha, IFN-beta, IFN-gamma, Flt3 ligand, GM-CSF, IL-15, IL-12, and fms-related tyrosine kinase 3 ligand (FLT3L).

The cytokine antagonist may be or include an antibody or antigen-binding fragment of an antibody, particularly an antibody or antibody fragment capable of binding to the corresponding cytokine or cytokine receptor. Examples of suitable cytokine antagonists include interleukin antagonists, especially IL-1 antagonists such as IL-1Ra, tumor necrosis factor (TNF) antagonists, especially TNF-α antagonists, such as anti-TNF-α antibodies, interferon antagonists and chemokines. It may be an antagonist, and most preferably it may be an anti-TNF-α antibody.

As seen above, conventional sepsis treatments do not show any effect on their own, but when administered in combination with the fusion protein of the present invention, it can be seen that they show a synergistic effect.

When used in combination with a sepsis treatment, the fusion protein according to the present invention can exert a synergistic effect compared to single administration, and can be administered simultaneously or independently of the sepsis treatment.

In the present invention, “synergistic” or “synergistic effect” means a therapeutic effect achieved by combining the fusion protein of the present invention with a sepsis treatment agent and/or achieved through the treatment method of the present invention; This means that it is greater than the sum of the effects generated by using the fusion protein and the sepsis treatment agent alone or separately. Advantageously, this synergy between therapeutic agents allows one or both of the two therapeutic agents to be used in lower doses, provides higher efficacy at the same dose, and/or reduces drug resistance. Prevent or delay its occurrence. The synergistic effect may be achieved by co-formulating the therapeutic substances contained in the pharmaceutical compositions described herein, or by administering the therapeutic substances simultaneously via unit dosage form, or by administering them simultaneously or sequentially. This can be achieved by administering them as separate dosage forms.

The pharmaceutical composition for preventing and treating sepsis of the present invention can also be provided in the form of an external preparation containing the fusion protein as an active ingredient.

When the pharmaceutical composition for preventing and treating sepsis of the present invention is used as an external skin agent, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, and foaming agents are added. , fragrance, surfactant, water, ionic emulsifier, non-ionic emulsifier, filler, sequestering agent, chelating agent, preservative, vitamin, blocking agent, wetting agent, essential oil, dye, pigment, hydrophilic activator, lipophilic activator or It may contain adjuvants commonly used in the field of dermatology, such as lipid vesicles or any other ingredients commonly used in topical skin preparations. Additionally, the ingredients may be introduced in amounts commonly used in the field of dermatology.

When the pharmaceutical composition for preventing and treating sepsis of the present invention is provided as an external skin preparation, it is not limited thereto, but may be in the form of an ointment, patch, gel, cream, or spray.

The composition of the present invention can be added to a feed additive or a feed composition containing the same for the purpose of preventing or improving sepsis.

In the present invention, the term "feed additive" refers to a substance added to feed for the purpose of various effects such as supplementing nutrients and preventing weight loss, improving digestibility of fiber in feed, improving milk quality, preventing reproductive disorders and improving conception rate, and preventing high temperature stress in the summer. Includes. The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act, and includes mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, mineral preparations such as trace minerals such as zinc, copper, cobalt, and selenium, and kerotene. , vitamins such as vitamins A, D, E, nicotinic acid, and vitamin B complex, protective amino acids such as methionine and lysine, protective fatty acids such as fatty acid calcium salts, probiotics (lactic acid bacteria), yeast cultures, and live bacteria such as mold fermentation products, Yeast agents, etc. may be additionally included.

In the present invention, the term "feed" refers to any natural or artificial diet, meal, etc., or an ingredient of the meal, for or suitable for eating, ingestion, and digestion by animals, and to prevent or improve muscle disease according to the present invention. Feed containing the composition as an active ingredient can be manufactured from various types of feed known in the art, and may preferably include concentrated feed, roughage and/or special feed, but is not limited thereto.

Concentrated feed includes seeds and fruits, including grains such as wheat, oats, and corn; bran, which includes rice bran, bran, and barley bran, which are by-products obtained from refining grains; soybeans; oil, sesame seeds, linseed, and coco oil; Fish soluble (fish solution) is a concentrated product of fresh liquid obtained from fish, fish meal, fish waste, and residual starch, which is the main component of the starch waste that is left after removing the starch from the by-products such as seed jelly, sweet potatoes, and potatoes. There are animal feeds such as meat meal, blood meal, feather meal, skim milk powder, dried whey, which is the residue from milk to cheese, and casein from skim milk, yeast, chlorella, and seaweed. It is not limited to this.

Forage includes raw grass feed such as wild grass, grass, and green cuttings, turnips for feed, beets for feed, root vegetables such as rutterbearger, a type of turnip, raw grass, green cuttings, and grains, etc., in silos. These include, but are not limited to, silage, which is stored feed that has been filled and fermented with lactic acid, field grass, hay made by cutting and drying grass, straw from breeding crops, and leaves from legumes. Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea and its derivative diureide isobutane, and mixed feeds to supplement ingredients that are likely to be lacking when mixing only natural feed ingredients, or to increase the storability of the feed. Substances added in trace amounts include feed additives and dietary supplements, but are not limited to these.

The feed additive for preventing or improving sepsis according to the present invention can be manufactured by adding the fusion protein in an appropriate effective concentration range according to various feed manufacturing methods known in the art.

The feed additive according to the present invention can be applied without limitation to any object aimed at preventing or improving sepsis. For example, it can be applied to any entity, including non-human animals such as cows, horses, pigs, goats, sheep, dogs, cats, rabbits, birds, and fish.

The present invention provides a method for preventing or treating sepsis, comprising administering the pharmaceutical composition.

In the method of the present invention, the animal is applicable to animals such as chicken, pig, monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, goat, etc. without limitation. Preferably, there is no particular limitation as long as it is an animal other than a human.

In the present invention, the administration may be administered by any administration means known in the art. For example, the administration can be administered directly to the subject intravenously, intramuscularly, intraperitoneally, orally, transdermal, mucosally, intranasally, intratracheally or subcutaneously. The administration may be administered systemically or locally.

In the method of the present invention, the composition of the present invention may be administered in a therapeutically or prophylactically effective amount. The “therapeutically or prophylactically effective amount” can be appropriately selected by those skilled in the art, taking into account the severity of symptoms, gender, age, and weight of the individual. The therapeutically or prophylactically effective amount may be, for example, 0.0001-100 mg of the fusion protein per 1 kg of the administered subject.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1. Production of C10-LRR recombinant protein

Construction of polynucleotide sequences encoding C10 and LRR recombinant proteins

To prepare C10-LRR recombinant protein, it was performed as follows. ① NheI restriction enzyme recognition site, ② polynucleotide sequence of SEQ ID NO: 12 encoding C10 polypeptide (SEQ ID NO: 1), ③ BamHI restriction enzyme recognition site, and ④ part of NRR domain (903 bp) with 2022 bp of NLRX1 protein N terminus deleted. A forward primer of SEQ ID NO. 29 containing the coding polynucleotide sequence sequentially in the 5' → 3' direction was produced (Bionics), and the polynucleotide sequence encoding ① HindIII restriction enzyme recognition site and ② part of the C-terminus of EGFP was 5'. → A reverse primer of SEQ ID NO: 30 sequentially included in the 3' direction was produced (Bionics). In the forward primer of SEQ ID NO: 29, ① the NheI restriction enzyme recognition site is for DNA cloning, and ③ the BamHI restriction enzyme recognition site includes the polynucleotide sequence of ② SEQ ID NO: 12 and ④ a polynucleotide sequence encoding part of the N-terminus of EGFP. It is for connection. In addition, the ①HindIII restriction enzyme recognition site of the reverse primer of SEQ ID NO: 30 is also for DNA cloning.

Using the pRSET-b vector containing the EGFP gene as a template, a PCR reaction was performed using the primer pair of SEQ ID NO: 29 and SEQ ID NO: 30, thereby encoding a fusion protein of C10 polypeptide derived from human IL-10 and LRR polypeptide. The polynucleotide sequence of SEQ ID NO: 31 was prepared. The PCR reaction includes an initial heat denaturation reaction at 95°C for 3 minutes, followed by heat denaturation of the template at 95°C for 20 seconds, primer and template binding at 50°C for 20 seconds, and extension reaction at 72°C for 30 seconds. One cycle was set to be performed, and 35 cycles were performed using a PCR reactor (Biorad).

Recombinant expression vector production

To express the C10-LRR fusion protein, the gene (DNA) fragment prepared above was cut into pRSETb, a protein expression vector, with restriction enzymes and then inserted into the vector using ligase. The amplified DNA fragment was subjected to an enzyme reaction using NheI and HindIII (NEB) to transform the 5'/3' ends of the DNA into sticky ends. Meanwhile, pRSETb was subjected to an enzymatic reaction using the same two restriction enzymes to create a linear pRSETb vector with NheI and HindIII insertion sites. After each enzyme reaction, it was separated using a PCR purification kit (Cosmogenetech).

The isolated C10-LRR fusion protein double-stranded DNA fragment and the pRSET-b vector were subjected to an enzyme reaction to be linked using T4 Ligase (NEB) for two hours at 25°C.

The circular pRSETb vector into which the ligated C10-LRR was inserted was transformed into a DH5α E. coli strain and cultured on LB plate medium containing 50 μg/ml of the antibiotic ampicillin to select transformed E. coli that formed colonies. The selected E. coli colonies were again inoculated into liquid LB medium containing 50 μg/ml of ampicillin and cultured, and then the plasmid vector was isolated using a plasmid Mini Preparation kit (Cosmogenetech).

Using the recombinant C10-LRR_pRSET-b vector constructed as above as a template, a PCR reaction was performed using the primer pair of SEQ ID NO: 29 and SEQ ID NO: 30 to produce a fusion protein of C10 polypeptide derived from human IL-10 and LRR. The coding polynucleotide sequence was obtained, and the polynucleotide sequence was submitted to Cosmogenetech for DNA sequence analysis to confirm whether the vector was accurately manufactured.

Recombinant protein isolation and purification

In order to express the 6-His tagged recombinant protein from E. coli obtained through the above steps, each colony was inoculated into LB liquid medium containing 50 μg/ml of ampicillin and cultured at 37°C for 10 hours, and then transferred to new LB liquid medium. After transferring to 500 ml, IPTG was added to a concentration of 0.2mM, the temperature was lowered to 20°C, and culture was continued at 150 rpm for an additional 14 hours. After the culture was completed, the culture medium was recovered.

To isolate and purify the 6-His tagged recombinant protein expressed in E. coli, Ni-NTA affinity chromatography was used. The E. coli culture was centrifuged to obtain a pellet, the pellet was treated with lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0), and the cell wall was destroyed by sonication. The cell lysate was centrifuged to recover the supernatant, and then cultured on Ni-NTA agarose (Qiagen) beads.

The beads to which the recombinant protein was bound were washed with wash buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH 8.0), and then washed with eluent buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH). 8.0), and the recovered solution was desalted using a PD-10 Sephadex G-25 column (GE Healthcare). To measure the degree of microbial endotoxin contamination in the purified recombinant protein, the desalted recombinant protein was incubated in 1% Triton , the above process was repeated four times, and then desalted using a PD-10 Sephadex G-25 column. After quantitatively measuring protein concentration through Bradford assay, it was placed in HBSS solution containing 10% glycerol until the experiment. Stored at 80°C.

Example 2. Production of TAT-LRR recombinant protein

Instead of the forward primer of SEQ ID NO: 29 and the reverse primer of SEQ ID NO: 30, the polynucleotide sequence (SEQ ID NO: 21) encoding the TAT cell-penetrating peptide (SEQ ID NO: 10) is included in the 5'-> 3' direction. A recombinant protein of TAT-LRR ( SEQ ID NO: 36) was obtained. The recombinant protein was confirmed through 12% SDS-PAGE.

Example 3. Production of dNP2-LRR recombinant protein

Instead of the forward primer of SEQ ID NO: 29 and the reverse primer of SEQ ID NO: 30, the polynucleotide sequence (SEQ ID NO: 22) encoding the dNP2 cell-penetrating peptide (SEQ ID NO: 11) is contained in the 5'-> 3' direction of SEQ ID NO: 37 A recombinant protein of dNP2-LRR ( SEQ ID NO: 40) was obtained. The recombinant protein was confirmed through 12% SDS-PAGE.

Comparative Example 1. Production of C10-NBD recombinant protein

Instead of the forward primer of SEQ ID NO: 29 and the reverse primer of SEQ ID NO: 30, a polynucleotide sequence (SEQ ID NO: 28) encoding the NRR domain (903 bp) (SEQ ID NO: 27), in which 2022 bp of the N-terminus of the NLRX1 protein is deleted, was used in the 5' -> The polynucleotide sequence of SEQ ID NO: 43 encoding the fusion protein of the C10 polypeptide derived from human IL-10 and NBD was produced through the forward primer of SEQ ID NO: 41 and the reverse primer of SEQ ID NO: 42 in the 3' direction. C10-NBD recombinant protein (SEQ ID NO: 44) was obtained in the same manner as in Example 1 except that. The recombinant protein was confirmed through 12% SDS-PAGE.

Comparative Example 2. Production of C10-EGFP recombinant protein

Instead of the forward primer of SEQ ID NO: 29 and the reverse primer of SEQ ID NO: 30, the forward primer of SEQ ID NO: 45 and the reverse primer of SEQ ID NO: 46, which contain a polynucleotide sequence encoding the EGFP polypeptide in the 5'-> 3' direction, are used to A recombinant protein of C10-EGFP was obtained in the same manner as in Example 1, except that the polynucleotide sequence of SEQ ID NO: 47 encoding the fusion protein of IL-10 derived C10 polypeptide and EGFP was prepared. The recombinant protein was confirmed through 12% SDS-PAGE.

Experimental Example 1. C10-LRR fusion protein design, production and purification

Public single-cell RNA sequencing (scRNA-seq)

Public ScRNA-seq data can be downloaded from the Broad Institute Single Cell Portal (https://singlecell.broadinstitute.org/single_cell) using the identifier SCP548 (subject PBMCs) [M. Reyes, M.R. Filbin, R.P. Bhattacharyya, K. Billman, T. Eisenhaure, D.T. Hung, B.D. Levy, R.M. Baron, P.C. Blainey, M.B. Goldberg, N. Hacohen, An immune-cell signature of bacterial sepsis, Nat Med 26(3) (2020) 333-340.]. PBMC samples were analyzed using three cohorts (healthy control, n=19; sepsis, n=4; sepsis-ICU, n=8).

Figure 3 is a graph analyzing NLRX1 expression in PBMC samples from healthy or septic patients, referring to public single-cell RNA sequencing (Reyes et al., 2020), Single Cell Portal (https://singlecell.broadinstitute.org) This is the result of analysis using /single_cell).

As shown in Figure 3, published single-cell RNA sequencing (scRNA-seq) data (PBMC samples were obtained from three cohorts (healthy control, n = 19; sepsis, n = 4; sepsis-ICU, n = 8)) Based on the analysis, it was confirmed that NLRX1 was decreased in sepsis patients compared to healthy controls. In other words, it can be confirmed that NLRX1 acts as a negative regulator of NF-κB signaling and inflammasome activation.

If NLRX1 can be effectively delivered into cells, it can be confirmed that NLRX1 will effectively control sepsis-related inflammatory responses by regulating NF-κB and inflammasome signals within cells. Accordingly, a fusion protein (recombinant protein) of the cell-penetrating peptide and NLRX1 according to the present invention was prepared to determine whether the fusion protein combining the cell-penetrating peptide of the present invention and each domain of NLRX1 is actually efficiently delivered into cells in vitro and in vivo. We wanted to confirm whether the delivered fusion protein showed actual effects. For this purpose, DNA encoding recombinant proteins (C10-LRR, C10-NBD, C10-EGFP) combining the two domains of NLRX1 (LRR-NBD) and C10 was designed (Figure 5). EGFP was used as a control. In addition, in order to confirm the binding and effect of conventional cell-penetrating peptides, recombinant proteins (dNP2-LRR, TAT-LRR) combining LRR polypeptide with TAT and dNP2 cell-penetrating peptides were designed and tested. Additionally, to confirm whether the C10 variant also has a cell delivery effect, a variant of the C10 cell-penetrating peptide was designed and tested.

Figure 5 shows the DNA structures encoding recombinant proteins of C10-LRR, C10-NBD, and C10-EGFP and the results of analyzing them by 12% SDS-PAGE. According to this, the recombinant proteins C10-LRR, C10-NBD, and C10-EGFP are It was confirmed that everything was manufactured properly.

The pharmacological mechanism of the fusion protein of C10 cell-penetrating peptide and NLRX1 protein of the present invention is schematically shown in Figure 4.

Experimental Example 2. Effect analysis in sepsis animal model

Fusion proteins (C10-LRR, C10-NBD) prepared by fusing the LRR domain or NBD domain of the NLRX1 protein with a cell-penetrating peptide were injected intraperitoneally into an animal model of sepsis to determine whether they had an effect on the disease. . PBS and C10-EGFP were used as controls.

laboratory animals

Male or female C57BL/6 mice aged 7 to 8 weeks raised at Hanyang University's SPF breeding facility (Specific pathogen free animal facility) were used, and the mice underwent an acclimatization process for 2 weeks before the experiment. Mice were raised on free diet in a breeding room maintained at a temperature of 22 ± 2 °C and humidity of 40-60% during the experiment, and the light and dark cycle was adjusted at 12-hour intervals. All animal experiments were conducted in compliance with the animal experiment operation regulations of the Institutional Animal Care and Use Committee of Hanyang University.

Administration and sampling of samples

For sample administration, mice were randomly divided into four groups of 24 each. For PBS, LPS (2 mg/kg) and PBS (1 mg/kg) were injected intraperitoneally (I.P.) at 0 hours, and LPS (5 mg/kg) and PBS (1 mg/kg) were injected intraperitoneally at 5 hours. It is an intraperitoneal injection negative control group, and the C10-EGFP administration group was intraperitoneally injected (I.P. injection) with LPS (2 mg/kg) and C10-EGFP recombinant protein (1 mg/kg) at 0 hours, and LPS (5 mg/kg) at 5 hours. mg/kg) and C10-EGFP recombinant protein (1 mg/kg) were injected intraperitoneally, and the C10-LRR administration group was injected with LPS (2 mg/kg) and C10-LRR recombinant protein (1 mg/kg) at 0 hours. was injected intraperitoneally (I.P. injection), and LPS (5 mg/kg) and C10-LRR recombinant protein (1 mg/kg) were injected intraperitoneally at 5 hours. The C10-NBD administration group received LPS (2 mg/kg) at 0 hours. mg/kg) and C10-NBD recombinant protein (1 mg/kg) were injected intraperitoneally (I.P. injection), and at 5 hours, LPS (5 mg/kg) and C10-NBD recombinant protein (1 mg/kg) were intraperitoneally injected. It was injected into my body. Additionally, a C10-LRR 0.2 mg/kg administration group was prepared in which the administration dose of C10-LRR recombinant protein was varied from 1 mg/kg to 0.2 mg/kg. The groups were measured for survival and weight changes every 24 hours for a total of 7 days. After sample injection at 5 hours, blood was harvested 2 or 24 hours later and analyzed for cytokine levels and AST activity. The Sham group is a normal group that has not undergone any treatment.

Animal models from 5 groups

Group 1 (PBS): LPS (2 mg/kg) and PBS (1 mg/kg) were administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and PBS (1 mg/kg) were administered intraperitoneally at 5 hours. administered intravenously

Group 2 (C10-EGFP): LPS (2 mg/kg) and C10-EGFP recombinant protein (1 mg/kg) were administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and C10-EGFP were administered at 5 hours. Recombinant protein (1 mg/kg) was administered intraperitoneally.

Group 3 (C10-NBD): LPS (2 mg/kg) and C10-NBD recombinant protein (1 mg/kg) were administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and C10-NBD were administered at 5 hours. Recombinant protein (1 mg/kg) was administered intraperitoneally.

Group 4 (C10-LRR): LPS (2 mg/kg) and C10-LRR recombinant protein (1 mg/kg) were administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and C10-LRR were administered at 5 hours. Recombinant protein (1 mg/kg) was administered intraperitoneally.

Group 5 (C10-LRR): LPS (2 mg/kg) and C10-LRR recombinant protein (0.2 mg/kg) were administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and C10-LRR recombinant protein were administered at 5 hours. Protein (0.2 mg/kg) administered intraperitoneally

ELISA

After 2 or 24 hours, blood was collected from the animal model through subconjunctival hemorrhage (eye bleeding). The recovered blood was centrifuged to separate serum and analyzed through ELISA. To measure the levels of IL-6, TNFα, and IL-1β in serum, an ELISA kit (BioLegend) was used according to the manufacturer's protocol.

statistics

For statistical analysis of the experiment, two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA were used to confirm the significant difference in mean values between groups. If the p value is lower than 0.05, *, lower than 0.01. If there is a p value, the significant difference is indicated by **, if the p value is lower than 0.001, it is indicated by ***, and if there is no significant difference, the significant difference is indicated by N.S. MFI represents median fluorescence intensity, and error bars represent S.D.

Figure 6 is an experimental design of a sepsis animal model. An LPS-induced lethal sepsis animal model was prepared, and C10-LRR was injected into the animal model in vivo to confirm the effect. Inflammasome signaling and pyroptosis are known to be very important in the early stages of sepsis-related mortality. Therefore, in order to produce a fatal sepsis animal model in the present invention, LPS was injected as a prime/challenge model to induce Caspase-11 and Caspase-1-mediated inflammation control complex (inflammasome signaling). A fatal sepsis animal model was prepared using . Specifically, 2 mg/kg LPS was first administered intraperitoneally, and 5 mg/kg was administered intraperitoneally 5 hours later for the second time (challenge). In this case, it was confirmed that the activity of macrophages increased compared to monocytes in the spleen, liver, kidneys, lungs, and peritoneum, and when external symptoms were combined, sepsis was induced. Recombinant proteins (1 mg/kg C10-LRR, C10-NBD, C10-EGFP) or PBS were administered at the same time as LPS, and the survival rate and weight of the animal model were monitored.

Figure 7 is a graph showing the results of measuring the survival rate of animal models divided into group 1 (PBS), group 2 (C10-EGFP), group 3 (C10-NBD), and group 4 (C10-LRR) for 7 days. , Figure 8 is a graph showing the results of measuring the weight of animal models divided into group 1 (PBS), group 2 (C10-EGFP), group 3 (C10-NBD), and group 4 (C10-LRR) for 7 days. am.

As shown in Figure 7, it was confirmed that in the 4 groups treated with C10-LRR, the survival rate was significantly improved compared to C10-EGFP and C10-NBD.

As shown in Figure 8, in the case of group 4 treated with C10-LRR, it was confirmed that the weight recovered significantly from 48 hours, unlike C10-EGFP and C10-NBD. It was confirmed that C10-LRR of the present invention restores the severe inflammatory response and abnormal physiological function of sepsis induced by LPS.

Figure 9 shows IL-6, TNF-α and A graph showing the results of measuring the expression level of IL-1β using ELISA, and Figure 10 shows the results of group 1 (PBS), group 2 (C10-EGFP), group 3 (C10-NBD), and group 4 (C10-LRR) after 24 hours. This is a graph showing the results of measuring the expression levels of IL-6, TNF-α, and IL-1β in animal models classified by ELISA.

As shown in Figure 9, 2 hours after injection of LPS 5 mg/kg (cahllenge), it was confirmed that the serum IL-1β level of group 4 treated with C10-LRR was significantly reduced compared to the other groups. .

As shown in Figure 10, 24 hours after injection of LPS 5 mg/kg (cahllenge), it was confirmed that the serum IL-6 level of group 4 treated with C10-LRR was significantly reduced compared to the other groups. . It was confirmed that the C10-LRR recombinant protein according to the present invention can achieve the effect of preventing or treating sepsis by reducing IL-1β and IL-6 levels in vivo.

Figure 11 is a graph showing the results of measuring AST activity in animal models divided into group 1 (PBS), group 2 (C10-EGFP), group 3 (C10-NBD), and group 4 (C10-LRR) after 2 hours.

As shown in Figure 11, the level of AST in the blood increases when 5 mg/kg of LPS is injected due to liver toxicity. However, it was confirmed that the CAST levels of group 4 treated with C10-LRR were maintained at a similar level to the Sham group (normal group). It can be seen that the AST level of the remaining groups significantly increased compared to the Sham (normal group). Therefore, it can be seen that C10-LRR shows a superior effect than other recombinant proteins.

Figure 12 is a graph showing the survival rate of animal models divided into group 4 (C10-LRR) and group 5 (C10-LRR 0.2 mg/kg) measured for 6 days.

As shown in Figure 12, as a result of treating C10-LRR at different doses, it was confirmed that the effect of C10-LRR increased in a dose-dependent manner in the LPS-induced sepsis animal model.

Experimental Example 3. C10-LRR inhibitory effect on NF-κB activation and IL-6 production in macrophages

Isolation of experimental animals and peritoneal macrophages

Male or female C57BL/6 mice aged 7 to 8 weeks raised at Hanyang University's SPF breeding facility (Specific pathogen free animal facility) were used, and the mice underwent an acclimatization process for 2 weeks before the experiment. Mice were raised on free diet in a breeding room maintained at a temperature of 22 ± 2 °C and humidity of 40-60% during the experiment, and the light and dark cycle was adjusted at 12-hour intervals. All animal experiments were conducted in compliance with the animal experiment operation regulations of the Institutional Animal Care and Use Committee of Hanyang University.

To isolate peritoneal macrophages from the mouse, 5 ml of PBS was administered into the mouse's abdominal cavity, and peritoneal fluid was obtained, distributed on a culture plate, and cultured in a 5% CO ₂ incubator at 37°C for 2 hours. did. After incubation was completed, the supernatant was removed, cells were washed with PBS, and non-adherent cells were measured. Adherent cells are primary peritoneal macrophages.

ELISA

The peritoneal macrophages were dispensed into a 96-well plate or a 12-well plate and cultured in a 5% CO ₂ incubator at 37°C until attachment. Floating cells were removed using PBS, and the dispensed macrophages were incubated with 1 mg/ml LPS for inflammasome activation in the attached macrophages for 4 hours. . At this time, various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-NBD, C10-EGFP) were treated and cultured together with LPS. After stimulation, the culture supernatant was recovered for ELISA. To measure the levels of IL-6, TNFα, and IL-1β in the culture supernatant, an ELISA kit (BioLegend) was used according to the manufacturer's protocol.

Cytotoxicity assay

1 × ¹⁰⁵ peritoneal macrophages were seeded on 96 well plates and incubated with various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant protein (C10-LRR) along with 1 mg/ml LPS (O55:B5, Sigma L2880). , C10-NBD, and C10-EGFP) were added and cultured for 24 hours. After incubation, 10 ㎕ of CCK-8 (cell counting kit-8) solvent (Dojindo) was added to each well, and absorbance at 450 nm was measured every 30 minutes for 4 hours using a microplate reader (iMark, Bio-Rad). ) was monitored using. Through this, the number of living cells was confirmed.

statistics

For statistical analysis of the experiment, two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA were used to confirm the significant difference in mean values between groups. If the p value is lower than 0.05, *, lower than 0.01. If there is a p value, the significant difference is indicated by **, if the p value is lower than 0.001, it is indicated by ***, and if there is no significant difference, the significant difference is indicated by N.S. MFI represents median fluorescence intensity, and error bars represent S.D.

Previously, when an LPS-induced sepsis animal model was treated with C10-LRR, it was confirmed that the level of IL-6 was reduced, and based on this, the function of macrophages was evaluated in vitro .

Peritoneal macrophages were isolated, LPS and each recombinant protein (C10-LRR, C10-NBD, C10-EGFP) were added together and cultured for 4 hours, and IL-6 and TNF-α were measured from the culture supernatant.

Figures 13 and 14 are ELISA graphs measuring the levels of L-6 and TNF-α after treating peritoneal macrophages with LPS and each recombinant protein. According to this, when treated with C10-LRR, the level of IL-6 was increased. It was confirmed that it was significantly reduced compared to when treated with other recombinant proteins.

No significant differences in TNF-α were observed between C10-LRR and other recombinant proteins.

Figure 15 is a graph analyzing the cell survival rate after treating peritoneal macrophages with LPS and each recombinant protein. It was confirmed that none of C10-LRR, C10-EGFP, and C10-NBD showed toxicity to cells.

Experimental Example 4. Effect of C10-LRR inhibition on NF-κB activation and IL-6 production in response to time and THP-1

Western Blot-1

Peritoneal macrophages used in this experiment were obtained and used in the same manner as in Experimental Example 3. Peritoneal macrophages were distributed in a 96-well plate or 12-well plate and cultured in a 5% CO ₂ incubator at 37°C until attachment. Floating cells were removed using PBS, and the dispensed macrophages were incubated with 1 mg/ml LPS for inflammasome activation in the attached macrophages for 4 hours. . At this time, various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-NBD, C10-EGFP) were treated and cultured together with LPS. After incubation (stimulation), cells were lysed with RIPA buffer (Cell Signaling Technology) for 30 minutes at 4°C, and the amount of protein in the lysate was analyzed using the Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's protocol. . After SDS-PAGE, the proteins were transferred to a PVDF membrane (Bio-Rad), and the membrane was treated with TBS buffer (Tris-buffered saline; TBS-T) containing 5% skim milk and 0.1% Tween-20. Then, the cells were incubated overnight at 4°C with the primary antibody: IκBα (Cell Signaling, 1:1000 diluted) antibody was used. The membrane was washed with TBS-T and incubated with secondary antibody for 1 hour at room temperature. Membranes were washed with TBS-T and treated with EZ-Western Lumi Pico or Femto reagent (DoGen). To measure the band intensity, it was analyzed using Fusion-Solo software (Vilber) according to the manufacturer's protocol.

Western Blot-2

THP-1 cells were purchased from ATCC and stored in DMEM or RPMI (Corning) medium. Culture medium containing 10% fetal bovine serum and 1% penicillin/streptomycin was used. Cells were cultured at 37°C in a 5% CO ₂ incubator.

The THP-1 cells were activated with 1 mg/ml LPS (O55:B5, Sigma L2880) for 15 minutes to 5 hours. At this time, various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-NBD, C10-EGFP) were treated and cultured together with LPS. After lysing the cells with RIPA buffer (Cell Signaling Technology) for 30 minutes at 4°C, the amount of protein in the lysate was analyzed using the Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's protocol. After SDS-PAGE, the proteins were transferred to a PVDF membrane (Bio-Rad), and the membrane was treated with TBS buffer (Tris-buffered saline; TBS-T) containing 5% skim milk and 0.1% Tween-20. And incubated overnight at 4°C with primary antibodies: IκBα (Cell Signaling, 1:1000 diluted), pIκBα Ser32 (Cell Signaling, 1:1000 diluted), p65 (Cell Signaling, 1:1000 diluted), p65 Ser536 ( Cell Signaling, 1:1000 diluted), PI3K p110β (Cell Signaling, 1:1000 diluted), pAKT Ser473, caspase (Adipogen, 1:1000 diluted), MAVS (Cell Signaling, 1:1000 diluted), NLRP3 (Adipogen, 1 :1000 diluted), or VDAC (Cell Signaling, 1:1000 diluted) antibody was used. The membrane was washed with TBS-T and incubated with secondary antibody for 1 hour at room temperature. Membranes were washed with TBS-T and treated with EZ-Western Lumi Pico or Femto reagent (DoGen). To measure the band intensity, it was analyzed using Fusion-Solo software (Vilber) according to the manufacturer's protocol.

ELISA

Peritoneal macrophages used in this experiment were obtained and used in the same manner as in Experimental Example 3. Peritoneal macrophages were distributed in a 96-well plate or 12-well plate and cultured in a 5% CO ₂ incubator at 37°C until attachment. Floating cells were removed using PBS, and the dispensed macrophages were incubated with 1 mg/ml LPS for inflammasome activation in the attached macrophages for 4 hours. . At this time, various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-NBD, C10-EGFP) were treated and cultured together with LPS. After stimulation, the culture supernatant was recovered for ELISA. To measure the levels of IL-6, TNFα, and IL-1β in the culture supernatant, an ELISA kit (BioLegend) was used according to the manufacturer's protocol.

statistics

For statistical analysis of the experiment, two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA were used to confirm the significant difference in mean values between groups. If the p value is lower than 0.05, *, lower than 0.01. If there is a p value, the significant difference is indicated by **, if the p value is lower than 0.001, it is indicated by ***, and if there is no significant difference, the significant difference is indicated by N.S. MFI represents median fluorescence intensity, and error bars represent S.D.

Figure 16 shows the results of peritoneal macrophages treated with 1 μM C10-LRR and 1 mg/ml LPS, cultured for 15 and 30 minutes, and analyzed by Western blot.

As shown in Figure 16, it was confirmed that treatment with C10-LRR significantly inhibited IκB degradation regardless of time. It can also be confirmed in human macrophages, that is, THP-1 cells stimulated with PMA (phorbol 12-myristate 13-acetate).

Figures 17 and 18 are graphs showing the results of treating PMA-stimulated THP-1 cells with C10-LRR and LPS, and analyzing the levels of IL-6 and TNF-α by ELISA.

As shown in Figures 17 and 18, it was confirmed that treatment with C10-LRR significantly inhibited IL-6 production. In contrast, no significant differences were observed in the production of TNF-α.

Figure 19 shows the results of treating THP-1 cells with 1 μM C10-LRR and 1 mg/ml LPS for 30 minutes, and measuring the levels of PlκBα Ser32, IκBα, p65 Ser536, and p65 expressed therefrom by Western blot.

As shown in Figure 19, it was confirmed that the activation of NF-κB signaling, including phosphorylation of lκBα Ser32, degradation of IκBα, and phosphorylation of p65 Ser536, was significantly reduced in THP-1 cells treated with C10-LRR.

Through the above-described experiment, it can be seen that C10-LRR suppresses IL-6 production in LPS-treated macrophages and plays a role in negatively regulating NF-κB signaling.

Experimental Example 5. C10-LRR inhibitory effect on NLRP3-inflammaseom in macrophages

Peritoneal macrophages used in this experiment were obtained and used in the same manner as in Experimental Example 3. Peritoneal macrophages were distributed in a 96-well plate or 12-well plate and cultured in a 5% CO ₂ incubator at 37°C until attachment. Floating cells were removed using PBS, and the attached cells were treated with various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-NBD, C10-EGFP), respectively. Cultured for 4 hours. After stimulation, the culture supernatant was recovered for ELISA, and the dispensed macrophages were primary cultured for 4 hours with 1 mg/ml LPS for inflammasome activation. After primary culture, cells were activated with 5 μM nigericin for 2 hours, and the culture supernatant was recovered for ELISA. To measure the levels of IL-6, TNFα, and IL-1β in the culture supernatant, an ELISA kit (BioLegend) was used according to the manufacturer's protocol.

For statistical analysis of the experiment, two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA were used to confirm the significant difference in mean values between groups. If the p value is lower than 0.05, *, lower than 0.01. If there was a p value, the difference was marked as **, if there was a p value lower than 0.001, it was marked as ***, and if there was no significant difference, the significant difference was marked as N.S. MFI represents median fluorescence intensity, and error bars represent S.D.

Figure 20 is a graph showing the level of IL-1β measured by ELISA after culturing peritoneal macrophages with LPS and recombinant proteins (C10-LRR, C10-NBD, C10-EGFP) or nigericin.

In order to confirm whether IL-1β, which was reduced in the sepsis animal model in the above experiment, is related to the regulation of inflammasome activation by C10-LRR, in the present invention, after priming with LPS and treating nigericin, macrophages were treated with IL-1β. We looked at the -1β change. As a result, as shown in Figure 20, when only LPS was treated, there was no significant difference in IL-1β change, but when treated with LPS and nigericin, IL-1β change was confirmed to significantly increase.

When treated with C10-LRR, it was confirmed that IL-1β was significantly suppressed.

Experimental Example 6. C10-LRR inhibitory effect on NLRP3-inflammaseom in macrophages-1

Administration of samples

To administer the samples, they were divided into nine groups. Peritoneal macrophages or MEFs (murine embryonic fibroblasts) were dispensed into 96-well plates or 12-well plates and cultured in a 5% CO ₂ incubator at 37°C until attachment.

The experiment of the present invention consists of a priming phase (LPS 1 mg/ml intraperitoneal administration, 4 hours) and a stimulation phase (5 μM nigericin intraperitoneal administration, 2 hours) (FIG. 21). Prim+Stim is a group that was cultured after treating the same concentration of recombinant protein (C10-LRR or C10-EGFP) in each of the priming and stimulation stages (the treatment concentration is indicated in the graph, and the prim and stim stages Each was administered once (total of 2 times - 0.2 μM, 0.5 μM, 1 μM).

Prim is a group cultured with recombinant proteins (C10-LRR, C10-EGFP) at corresponding concentrations (0.2 μM, 0.5 μM, 1 μM) only during the priming phase, and Stim is a group cultured with corresponding concentrations (0.2 μM, 0.5 μM) only at the stimulation phase. This group was cultured by treating with 1 μM) recombinant proteins (C10-LRR, C10-EGFP).

The presence or absence of treatment of each sample is indicated as - (untreated) and + (treated) in the graph.

ELISA

Culture supernatants from each sample administration group were recovered for ELISA. To measure the levels of IL-6, TNFα, and IL-1β in the culture supernatant, an ELISA kit (BioLegend) was used according to the manufacturer's protocol.

statistics

For statistical analysis of the experiment, two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA were used to confirm the significant difference in mean values between groups. If the p value is lower than 0.05, *, lower than 0.01. If there was a p value, the difference was marked as **, if there was a p value lower than 0.001, it was marked as ***, and if there was no significant difference, the significant difference was marked as N.S. MFI represents median fluorescence intensity, and error bars represent S.D.

Figure 21 is an experimental design to confirm the inhibitory effect of C10-LRR on inflammasome activation in peritoneal macrophages. The experiment consists of a priming phase (LPS 1 mg/ml intraperitoneally administered, 4 hours) and a stimulation phase (5 μM nigericin administered intraperitoneally, 2 hours). In the present invention, a total of three experiments were designed: Prim+Stim is a group treated with recombinant protein in both the priming and stimulation stages, Prim is a group treated with recombinant protein only in the priming stage, and Stim is a group treated with recombinant protein only in the stimulation stage. This is the group that processed proteins. At this time, when recombinant proteins (C10-LRR, C10-NBD, C10-EGFP) were treated, LPS was also treated. It is believed that the priming step involves NF-κB activation, and the stimulation step involves inflammasome activation.

Figure 22 shows IL in the Prim+Stim group, Prim group, and Stim group targeting peritoneal macrophages administered various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-EGFP). This is a graph showing the results of analyzing -1β levels by ELISA, and Figure 23 shows murine embryonic fibroblast (MEF) cells administered various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-EGFP). This is a graph showing the results of ELISA analysis of IL-1β levels in the Prim+Stim group, Prim group, and Stim group.

As shown in Figures 22 and 23, it was confirmed that the production of IL-1β was induced by treatment with LPS and nigericin. Additionally, when treated with C10-LRR, IL-1β production was confirmed to significantly decrease depending on the C10-LRR dose.

From the above results, it can be seen that C10-LRR has the effect of inhibiting both inflammasome signaling and NF-κB signaling. In contrast, when treated with C10-EGFP, no significant difference in IL-1β changes was observed.

Experimental Example 7. C10-LRR inhibitory effect on NLRP3-inflammaseom in macrophages-2

Isolation of mitochondrial fraction

Mitochondrial fraction was isolated using the Qproteome mitochondrial isolation kit (Qiagen) according to the manufacturer's protocol. Briefly, peritoneal macrophages were recovered from lysis buffer and centrifuged at 4°C and 1,000 g for 10 minutes. The supernatant was discarded, the pellet was crushed by passing it 10 times with a 23-gauge needle, and then centrifuged at 4°C and 1,000 g for 10 minutes. The supernatant was centrifuged at 4°C and 8,000 g for 15 minutes, the pellet was washed, and a mitochondrial fraction was obtained. For comparison, whole cell lysate was also collected and stored separately.

western blot

Peritoneal macrophages or mitochondrial fractions or whole cell lysates were dispensed into 96-well plates or 12-well plates and cultured in a 5% CO ₂ incubator at 37°C until adhesion. Floating cells were removed using PBS, and the dispensed macrophages were cultured with 1 mg/ml LPS for inflammasome activation on the attached cells for 4 hours. At this time, various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-NBD, C10-EGFP) were treated and cultured together with LPS. After culturing, the cells were treated with 5 μM nigericin and cultured for 2 hours. After lysing the cells with RIPA buffer (Cell Signaling Technology) for 30 minutes at 4°C, the amount of protein in the lysate was analyzed using the Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's protocol. After SDS-PAGE, the proteins were transferred to a PVDF membrane (Bio-Rad), and the membrane was treated with TBS buffer (Tris-buffered saline; TBS-T) containing 5% skim milk and 0.1% Tween-20. And incubated overnight at 4°C with primary antibodies: pro-Casp1 (Adipogen, 1:1000 diluted), casp1P20 (Adipogen, 1:1000 diluted), MAVS (Cell Signaling, 1:1000 diluted), NLRP3 (Adipogen, 1:1000 diluted), or VDAC (Cell Signaling, 1:1000 diluted) antibody was used. The membrane was washed with TBS-T and incubated with secondary antibody for 1 hour at room temperature. Membranes were washed with TBS-T and treated with EZ-Western Lumi Pico or Femto reagent (DoGen). To measure the band intensity, it was analyzed using Fusion-Solo software (Vilber) according to the manufacturer's protocol. Peritoneal macrophages used in this experiment were obtained and used in the same manner as in Experimental Example 3.

statistics

For statistical analysis of the experiment, two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA were used to confirm the significant difference in mean values between groups. If the p value is lower than 0.05, *, lower than 0.01. If there is a p value, the significant difference is indicated by **, if the p value is lower than 0.001, it is indicated by ***, and if there is no significant difference, the significant difference is indicated by N.S. MFI represents median fluorescence intensity, and error bars represent S.D.

Figure 24 shows Pro-Casp1 after culturing peritoneal macrophages induced with LPS and nigericin with various concentrations (0.2 μM, 0.5 μM, 1 μM) of recombinant proteins (C10-LRR, C10-EGFP). , This is the result of measuring the expression level of Casp1 P20 by Western blot.

It is known that caspase-1 p20 cleaves the N terminus of Pro-IL-1β, producing mature IL-1β in a secretable form. Therefore, in the present invention, we attempted to analyze the relationship between C10-LRR and caspase-1 p20.

As shown in Figure 24, when LPS and nigericin were treated, it was confirmed that the expression of caspase-1 p20 significantly increased compared to the normal group. In contrast, when treated with C10-LRR, the expression level of caspase-1 p20, which was increased by LPS and nigericin, was confirmed to be significantly reduced. In the case of C10-EGFP, the comparison group, no significant difference was observed compared to the group treated with LPS and nigericin.

Figure 25 shows the expression of Pro-Casp1 and Casp1 P20 after adding recombinant proteins (C10-LRR, C10-NBD) to mitochondrial fractions and whole cell lysates induced by inflammation with LPS and nigericin and culturing them. This is the result of measuring the expression level by Western blot. 'Mito' is a mitochondrial fraction, and WCL is a whole cell lysate.

MAVS is widely known to play a role in regulating the NLRP3-inflammasome in the outer membrane of mitochondria. Therefore, when treated with C10-LRR, we examined the levels of NLRP3 and MAVS in mitochondria and cytoplasm.

As shown in Figure 25, when stimulated with nigericin and LPS, the mitochondrial MAVS level increased significantly, and there was no difference in MCL.

When treated with C10-LRR, it was confirmed that MAVS expression was completely inhibited in mitochondria, and the levels of MAVS and NLRP3 were also significantly reduced in WCL. The above results show that C10-LRR negatively regulates inflammasome signaling and IL-1β production.

Experimental Example 8. Delivery efficiency of C10-LRR and TAT-LRR fusion proteins when administered to animals

laboratory animals

Male or female C57BL/6 mice aged 7 to 8 weeks raised at Hanyang University's SPF breeding facility (Specific pathogen free animal facility) were used, and the mice underwent an acclimatization process for 2 weeks before the experiment. Mice were raised on free diet in a breeding room maintained at a temperature of 22 ± 2 °C and humidity of 40-60% during the experiment, and the light and dark cycle was adjusted at 12-hour intervals. All animal experiments were conducted in compliance with the animal experiment operation regulations of the Institutional Animal Care and Use Committee of Hanyang University.

Administration and sampling of samples

To isolate peritoneal macrophages from the mouse, 5 ml of PBS was administered into the mouse's abdominal cavity, and peritoneal fluid was obtained, distributed on a culture plate, and cultured in a 5% CO ₂ incubator at 37°C for 2 hours. did. After incubation was completed, the supernatant was removed, cells were washed with PBS, and non-adherent cells were measured. Adherent cells are primary peritoneal macrophages.

Primary peritoneal macrophages obtained through the above process were distributed at a concentration of 2 × 10 ⁵ cells in 96-well plates or 12-well plates, and C10-LRR, TAT-LRR recombinant protein, and LRR were added to each well. Alternatively, 2 μM of PBS (control) was added and then cultured for 1 hour at 37°C in a 5% CO ₂ incubator. After incubation, cells were recovered through centrifugation and washed twice with PBS buffer. To remove proteins attached to the cell membrane, cells were treated with trypsin at 37°C for 10 minutes, and trypsin was neutralized using DMEM culture medium at 37°C containing 10% FBS. After washing the cells with PBS, flow cytometry (FACS Canto II, BD Bioscience) was performed. Data were analyzed using Flowjo software (Tree Star).

statistics

For statistical analysis of the experiment, two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA were used to confirm the significant difference in mean values between groups. If the p value is lower than 0.05, *, lower than 0.01. If there was a p value, the difference was marked as **, if there was a p value lower than 0.001, it was marked as ***, and if there was no significant difference, the significant difference was marked as N.S. MFI represents median fluorescence intensity, and error bars represent S.D.

Figure 26 shows the results of primary peritoneal macrophages treated with C10-LRR, TAT-LRR, LRR, and PBS (control), cultured for 1 hour, and analyzed by flow cytometry.

As shown in Figure 26, it was confirmed that LRR protein was effectively delivered by C10 and TAT cell-penetrating peptides in mouse peritoneal macrophages.

However, it was confirmed that the efficiency of LRR protein delivery to macrophages for the C10-LRR fusion protein using the TAT cell-penetrating peptide was lower than that of the C10 cell-penetrating peptide. Therefore, the effects of the present invention can be sufficiently achieved even if other cell-penetrating peptides are used in addition to the C10 cell-penetrating peptide. However, the C10 cell-penetrating peptide significantly performs macrophage-specific cell transfer, and is therefore most preferable for the treatment of sepsis. Able to know.

Experimental Example 10. Intracellular delivery efficiency of C10-LRR fusion protein

Cell lines and culture

HeLa cells were purchased from ATCC and stored in DMEM or RPMI (Corning) medium. Culture medium containing 10% fetal bovine serum and 1% penicillin/streptomycin was used. Cells were cultured at 37°C in a 5% CO ₂ incubator.

Fluorescence microscopy analysis

Hela cells were distributed in a 6-well plate at a cell concentration of 2 × 10 ⁵ , 2 μM of C10-LRR was added, and cultured in a 5% CO ₂ incubator at 37°C for 1 hour. After incubation, the cells were washed three times with PBS and fixed with 4% paraformaldehyde phosphate buffer solution (Wako) for 10 minutes. Nuclei were stained in PBS containing 0.01% Hoechst for 10 minutes, and the cells were washed twice with PBS. Fluorescent signals in the cytoplasm and nucleus were analyzed using a C2si confocal microscope (Nikon). The scale bar is 100 μm.

Figure 27 shows the results of confirming the distribution within the cell by confocal microscopy when C10-LRR was treated in HeLa cells. Red indicates Mitotracker, fluorescent signal in the mitochondria (cytoplasm), and blue indicates Hoechst. , shows a fluorescence signal in the nucleus, and green is anti 6His, indicating the fluorescence signal of C10-LRR.

As shown in Figure 27, C10-LRR can be seen to be distributed in both the cytoplasm and nucleus. It is believed that C10-LRR enters the cell through endocytosis and then spreads to exist. It was confirmed that the fusion protein of C10 cell-penetrating peptide and NRR protein can be effectively and selectively delivered into primary macrophages.

Experimental Example 11. Comparison of delivery efficiency of recombinant proteins in macrophages

Through previous experiments, it was confirmed that among the NLRX1 proteins, a fusion protein to which the LRR domain is bound has a prevention, treatment, or improvement effect on sepsis. Therefore, we sought to compare the delivery efficiency of C10 cell-penetrating peptide and other cell-penetrating peptides.

In order to compare the exact intracellular delivery efficiency of the C10 cell-penetrating peptide for dNP2 and TAT, the intracellular delivery efficiency in macrophages compared to dendritic cells was comparatively analyzed from the results of Experimental Example 3, and is shown in Figure 28.

At this time, dNP2-EGFP has a polynucleotide sequence (SEQ ID NO: 22) encoding dNP2 cell-penetrating peptide (SEQ ID NO: 11) instead of the forward primer of SEQ ID NO: 29 and the reverse primer of SEQ ID NO: 30 in the 5'->3' direction. A polynucleotide sequence of SEQ ID NO: 50 encoding a fusion protein of dNP2 polypeptide and EGFP was produced through the forward primer of SEQ ID NO: 48 and the reverse primer of SEQ ID NO: 49, which includes the polynucleotide sequence encoding the EGFP protein. The recombinant protein of dNP2-EGFP was obtained in the same manner as in Example 1 except that. The recombinant protein was confirmed through 12% SDS-PAGE.

For statistical analysis of the experiment, two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA were used to confirm the significant difference in mean values between groups. If the p value is lower than 0.05, *, lower than 0.01. If there was a p value, the difference was marked as **, if there was a p value lower than 0.001, it was marked as ***, and if there was no significant difference, the significant difference was marked as N.S. Error bars represent S.D.

Figure 28a is a graph comparing the relative intracellular delivery efficiency in CD11b ^high macrophages compared to dendritic cells, Figure 28b is a graph comparing the relative intracellular delivery efficiency in CD11b ^med macrophages compared to dendritic cells, and Figure 28c is a graph comparing the relative intracellular delivery efficiency in CD11b med macrophages compared to dendritic cells. This is a graph comparing the relative intracellular delivery efficiency in neutrophil cells compared to cells.

As shown in Figure 28, it was confirmed that the delivery efficiency of C10 cell-penetrating peptide was significantly superior to that of dNP2 and TAT in macrophages. However, it was confirmed that there was no significant difference in the delivery efficiency of dNP2, TAT, and C10 cell-penetrating peptide in neutrophils.

Through the above-mentioned results, the present invention confirmed that the C10 cell-penetrating peptide has a significantly higher affinity for macrophages, unlike conventional cell-penetrating peptides. Therefore, it can be seen that C10 cell-penetrating peptide is the most preferable cell-penetrating peptide fused with LRR for sepsis.

Experimental Example 12. Analysis of C10 variant

Through previous experiments, it was confirmed that among the NLRX1 proteins, a fusion protein to which the LRR domain is bound has a prevention, treatment, or improvement effect on sepsis. Accordingly, in order to confirm whether various variants in which the cell-penetrating peptide (C10) was substituted or deleted also maintained excellent delivery efficiency, the delivery efficiency of the variants to cells was first analyzed.

Preparation of fusion protein of C10 variant-EGFP

First, mutants represented by the amino acid sequences of SEQ ID NOs. 2 to 7 were created by substituting or deleting cysteine (C), leucine (L), and arginine (R) that constitute C10, and then EGFP, C10-EGFP, and TAT-EGFP were generated. Comparison was made using a control group, and the results can be seen in Figures 29 to 32.

The above mutants and the recombinant protein of EGFP were ② in Example 1. Instead, polynucleotide sequences of SEQ ID NOs: 13 to 18 encoding variants (SEQ ID NOs: 2 to 7) are used, and for this purpose, forward primers of SEQ ID NOs: 51, 54, 57, 60, 63, 66 and SEQ ID NOs: 52, 55 , Polynucleotide sequences of SEQ ID NOs: 53, 56, 59, 62, 65, and 68 were produced using reverse primers of 58, 61, 64, and 67, and each variant-EGFP was produced in the same manner as in Example 1. A recombinant protein was obtained. In order: C10(m)-EGFP, C10(c>A)-EGFP, C10(L del)-EGFP, C10(R del)-EGFP, C10(L>R)-EGFP, C10(L>K)- It was named EGFP. The recombinant protein was confirmed through 12% SDS-PAGE.

Cell lines and culture

HeLa and Jurkat cells were purchased from ATCC and stored in DMEM or RPMI (Corning) medium. Culture medium containing 10% fetal bovine serum and 1% penicillin/streptomycin was used. Cells were cultured at 37°C in a 5% CO ₂ incubator.

Flow cytometry

Jurkat and HeLa cells were distributed at a concentration of 2 × 10 ⁵ cells in 96-well plates or 12-well plates, and C10 variant-EGFP fusion protein (C10(m)-EGFP, C10(c>A)- was added to each well. 5 μM each of EGFP, C10(L del)-EGFP, C10(R del)-EGFP, C10(L>R)-EGFP, and C10(L>K)-EGFP) were added and incubated in a 5% CO ₂ incubator for 37 days. Cultured for 1 hour at ℃ conditions. After incubation, cells were recovered through centrifugation and washed twice with PBS buffer. After washing, the cells were treated with trypsin at 37°C for 10 minutes to remove proteins attached to the cell membrane. Next, DMEM culture medium containing 10% FBS was added to the cells to neutralize trypsin. After recovering the cells, they were washed with PBS and flow cytometry (FACS Canto II, BD Bioscience) was performed. Data were analyzed using Flowjo software (Tree Star).

statistics

For statistical analysis of the experiment, two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA were used to confirm the significant difference in mean values between groups. If the p value is lower than 0.05, *, lower than 0.01. If there was a p value, the difference was marked as **, if there was a p value lower than 0.001, it was marked as ***, and if there was no significant difference, the significant difference was marked as N.S. MFI represents median fluorescence intensity, and error bars represent S.D.

Figures 29 and 30 show C10 variant-EGFP fusion proteins (C10(m)-EGFP, C10(c>A)-EGFP, C10(L del)-EGFP, C10(R del)-EGFP, C10( The results were analyzed by flow cytometry after treatment with L>R)-EGFP, C10(L>K)-EGFP), TAT-EGFP and EGFP (control) and cultured for 1 hour. Figures 31 and 32 show Hela cells. C10 variant-EGFP fusion proteins (C10(m)-EGFP, C10(c>A)-EGFP, C10(L del)-EGFP, C10(R del)-EGFP, C10(L>R)-EGFP, C10 (L>K)-EGFP), TAT-EGFP, and EGFP (control) were treated and cultured for 1 hour, and then analyzed by flow cytometry.

As shown in Figures 29 to 32, in Jurkat cells, C10 variant-EGFP fusion proteins (C10(m)-EGFP, C10(c>A)-EGFP, C10(L del)-EGFP, C10(R del)-EGFP , C10(L>R)-EGFP, C10(L>K)-EGFP) were confirmed to have general cell penetration efficiency, and no significant differences in cell penetration efficiency were observed between each recombinant protein. Moreover, in Jurkat cells, C10 variant-EGFP fusion proteins (C10(m)-EGFP, C10(c>A)-EGFP, C10(L del)-EGFP, C10(R del)-EGFP, C10(L>R)- It was confirmed that the difference in cell penetration efficiency of EGFP, C10(L>K)-EGFP) and the conventional cell-penetrating peptide (TAT) was not significant. On the other hand, in Hela cells, C10 variant-EGFP fusion proteins (C10(m)-EGFP, C10(c>A)-EGFP, C10(L del)-EGFP, C10(R del)-EGFP, C10(L>R)- It was confirmed that EGFP, C10(L>K)-EGFP) exhibits significantly better cell penetration efficiency than TAT cell-penetrating peptide.

In particular, it can be seen that the cell penetration efficiency of the C10 cell-penetrating peptide represented by SEQ ID NO: 1 is significantly increased compared to its variants. Specifically, C10 variant-EGFP fusion proteins (C10(m)-EGFP, C10(c>A)-EGFP, C10(L del)-EGFP, C10(R del)-EGFP, C10(L>R)-EGFP, Looking at the cell penetration efficiency of C10 (L>K)-EGFP), when the first leucine residue is deleted, the efficiency decreases the most compared to the existing C10, indicating that the intracellular protein delivery of C10 is reduced. It was confirmed that leucine is preferable as the first amino acid residue for this role.

In addition, when the first amino acid residue is replaced with arginine (R) and lysine (K), which are mainly used in cell-penetrating peptides, the delivery efficiency is superior to that of the TAT cell-penetrating peptide, but is significantly lower than that of the C10 cell-penetrating peptide of SEQ ID NO: 1. It was confirmed that it had low cell penetration efficiency. This means that leucine plays a very important function and role in C10.

Next, when the arginine residue at the 10th position is deleted (SEQ ID NO: 5) and the cysteine residue at the 8th position is replaced with alanine (SEQ ID NO: 3), the TAT cell-penetrating peptide and SEQ ID NOS: 4, 6, and 7 It was confirmed that the cell penetration efficiency was superior to that of the variant, but that it had a significantly lower cell penetration efficiency than the C10 cell-penetrating peptide of SEQ ID NO: 1. This means that arginine at the 10th position and cysteine at the 8th position have the second most important functions and roles in C10.

Additionally, when the leucine residue at the third position was substituted with methionine (SEQ ID NO: 2), the cell penetration efficiency was superior to that of TAT cell-penetrating peptide and other variants of C10. However, since it has a significantly lower cell penetration efficiency than the C10 cell-penetrating peptide of SEQ ID NO: 1, it is less important than the first leucine in C10, but it plays an important function and role in cell transfer efficiency. .

Experimental Example 11. Analysis of the effect of improving sepsis on combined administration of conventional drugs and C10-LRR - 2 administrations

laboratory animals

Male or female C57BL/6 mice aged 7 to 8 weeks raised at Hanyang University's SPF breeding facility (Specific pathogen free animal facility) were used, and the mice underwent an acclimatization process for 2 weeks before the experiment. Mice were raised on free diet in a breeding room maintained at a temperature of 22 ± 2 °C and humidity of 40-60% during the experiment, and the light and dark cycle was adjusted at 12-hour intervals. All animal experiments were conducted in compliance with the animal experiment operation regulations of the Institutional Animal Care and Use Committee of Hanyang University.

Administration and sampling of samples

For sample administration, mice were randomly divided into four groups of five each. For PBS, LPS (2 mg/kg) and PBS (1 mg/kg) were injected intraperitoneally (I.P.) at 0 hours, and LPS (5 mg/kg) and PBS (1 mg/kg) were injected intraperitoneally at 5 hours. It is an intraperitoneal injection negative control group, and the C10-LRR administration group received intraperitoneal injection (I.P. injection) of LPS (2 mg/kg) and C10-LRR recombinant protein (1 mg/kg) at 0 hours, and LPS (5 mg/kg) at 5 hours. mg/kg) and C10-LRR recombinant protein (1 mg/kg) were injected intraperitoneally, and the αTNFαAb administration group was injected intraperitoneally (I.P.) with LPS (2 mg/kg) and αTNFαAb (1 mg/kg) at 0 hours. injection), LPS (5 mg/kg) and αTNFαAb (1 mg/kg) were injected intraperitoneally at 5 hours, and the C10-LRR+αTNFαAb administration group was administered LPS (2 mg/kg) and C10-LRR at 0 hours. Recombinant protein (1 mg/kg) and αTNFαAb (1 mg/kg) were injected intraperitoneally (I.P.), and at 5 hours, LPS (5 mg/kg), C10-LRR recombinant protein (1 mg/kg), and αTNFαAb were administered. (1 mg/kg) was injected intraperitoneally. The groups were measured for survival and weight changes every 24 hours for a total of 7 days. αTNFαAb refers to TNFα neutralizing antibody.

Animal models from 4 groups

Group 1 (PBS): LPS (2 mg/kg) and PBS (1 mg/kg) were administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and PBS (1 mg/kg) were administered intraperitoneally at 5 hours. administered intravenously

Group 2 (C10-LRR): LPS (2 mg/kg) and C10-LRR recombinant protein (1 mg/kg) were administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and C10-LRR were administered at 5 hours. Recombinant protein (1 mg/kg) was administered intraperitoneally.

Group 3 (αTNFαAb): LPS (2 mg/kg) and αTNFαAb (1 mg/kg) were administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and αTNFαAb (1 mg/kg) were administered intraperitoneally at 5 hours. administered intravenously

Group 4 (C10-LRR+αTNFαAb): LPS (2 mg/kg), C10-LRR recombinant protein (1 mg/kg), and αTNFαAb (1 mg/kg) were administered intraperitoneally at 0 hours, and LPS at 5 hours. (5 mg/kg) and C10-LRR recombinant protein (1 mg/kg) and αTNFαAb (1 mg/kg) were administered intraperitoneally.

Through previous experiments, it was confirmed that C10-LRR of the present invention exhibits a negative regulating effect on IL-6 and IL-1β. Furthermore, in the present invention, it was believed that a synergistic effect would be induced if C10-LRR was administered in combination with a conventional drug, and an experiment was conducted on this.

Figure 33 schematically shows the experimental design to confirm the sepsis treatment effect according to C10-LRR recombinant protein and αTNFαAb, and Figure 34 shows group 1 (PBS), group 2 (C10-LRR), and group 3 (αTNFαAb). and 4 groups (C10-LRR+αTNFαAb). It is a graph showing the results of measuring the survival rate of animal models for 7 days, and Figure 35 shows the results of group 1 (PBS), group 2 (C10-LRR), and group 3 (αTNFαAb). ) and 4 groups (C10-LRR+αTNFαAb). This is a graph showing the results of measuring the weight of animal models for 7 days.

As shown in Figures 34 and 35, it can be seen that the survival rate is greatly improved when treated with C10-LRR or αTNFαAb. In addition, it was confirmed that when treated with C10-LRR or αTNFαAb (72 hours), weight recovery was significantly faster than the group treated with PBS (120 hours). Furthermore, when C10-LRR and αTNFαAb were used together, the survival rate recovered to 100%, and body weight was also recovered in the fastest time (48 hours).

Therefore, it was confirmed that C10-LRR shows a significantly superior effect even when used alone, and that a significantly superior synergistic effect can be achieved when administered in combination with a conventional drug.

Experimental Example 14. Analysis of the effect of improving sepsis on combined administration of conventional drugs and C10-LRR - one-time administration

laboratory animals

Male or female C57BL/6 mice aged 7 to 8 weeks raised at Hanyang University's SPF breeding facility (Specific pathogen free animal facility) were used, and the mice underwent an acclimatization process for 2 weeks before the experiment. Mice were raised on free diet in a breeding room maintained at a temperature of 22 ± 2 °C and humidity of 40-60% during the experiment, and the light and dark cycle was adjusted at 12-hour intervals. All animal experiments were conducted in compliance with the animal experiment operation regulations of the Institutional Animal Care and Use Committee of Hanyang University.

Administration and sampling of samples

For sample administration, mice were randomly divided into four groups of five each. For PBS, LPS (2 mg/kg) was injected intraperitoneally (I.P.) at 0 hours, and LPS (5 mg/kg) and PBS (1 mg/kg) were injected intraperitoneally at 5 hours. This is a negative control group, and C10- In the LRR administration group, LPS (2 mg/kg) was injected intraperitoneally (I.P. injection) at 0 hours, and LPS (5 mg/kg) and C10-LRR recombinant protein (1 mg/kg) were injected intraperitoneally at 5 hours. In the αTNFαAb administration group, LPS (2 mg/kg) was injected intraperitoneally (I.P.) at 0 hours, and LPS (5 mg/kg) and αTNFαAb (1 mg/kg) were injected intraperitoneally at 5 hours. In the C10-LRR+αTNFαAb administration group, LPS (2 mg/kg) was injected intraperitoneally (I.P. injection) at 0 hours, and LPS (5 mg/kg) and C10-LRR recombinant protein (1 mg/kg) were administered at 5 hours. and αTNFαAb (1 mg/kg) were injected intraperitoneally. The groups were measured for survival and weight changes every 24 hours for a total of 7 days. αTNFαAb refers to TNFα neutralizing antibody.

Animal models from 4 groups

Group 1 (PBS): LPS (2 mg/kg) was administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and PBS (1 mg/kg) were administered intraperitoneally at 5 hours.

Group 2 (C10-LRR): LPS (2 mg/kg) was administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and C10-LRR recombinant protein (1 mg/kg) were administered intraperitoneally at 5 hours.

Group 3 (αTNFαAb): LPS (2 mg/kg) was administered intraperitoneally at 0 hours, and LPS (5 mg/kg) and αTNFαAb (1 mg/kg) were administered intraperitoneally at 5 hours.

Group 4 (C10-LRR+αTNFαAb): LPS (2 mg/kg) was administered intraperitoneally at 0 hours, and LPS (5 mg/kg), C10-LRR recombinant protein (1 mg/kg), and αTNFαAb were administered at 5 hours. (1 mg/kg) administered intraperitoneally

In the present invention, we attempted to analyze the effect of combined administration when the number of administrations was varied. Figure 36 schematically shows the experimental design to confirm the sepsis treatment effect when the number of administrations of C10-LRR recombinant protein and αTNFαAb was varied, and Figure 37 shows group 1 (PBS) and group 2 (C10-LRR). , It is a graph showing the results of measuring the survival rate of animal models divided into groups 3 (αTNFαAb) and 4 (C10-LRR+αTNFαAb) for 7 days, and Figure 38 shows the results of group 1 (PBS) and group 2 (C10-LRR). ), this is a graph showing the results of measuring the weight of animal models divided into groups 3 (αTNFαAb) and 4 (C10-LRR+αTNFαAb) for 7 days.

As shown in Figures 37 and 38, in the case of group 4 administered with C10-LRR and αTNFαAb, the survival rate was significantly increased compared to groups 2 and 3 administered alone, and weight was recovered significantly faster. confirmed. The above results indicate that combined administration of C10-LRR and a conventional drug (TNF-α antibody) is very effective in preventing or treating LPS-induced sepsis.

The C10 cell-penetrating peptide developed in the present invention has a selective delivery effect on macrophages, and it was confirmed that the fusion of C10 and LRR efficiently delivers LRR to macrophages. The C10-LRR acts selectively on macrophages to regulate NF-κB and inflammatory signaling, thereby inhibiting the production of IL-1β and IL-6, such as sepsis, a disease that continuously causes inflammatory reactions throughout the body. It was confirmed that the treatment or prevention effect was successful. Furthermore, it was confirmed that the C10-LRR of the present invention can induce a synergistic effect on the treatment of sepsis when administered in combination with a conventional drug (αTNFα Ab) to which C10 cell-penetrating peptide is not bound.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Pharmaceutical composition for the prevention and treatment of sepsis comprising LRR domain of NLRX1 protein <130> HPC9900 <160> 68 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> C10, h <400> 1 Leu Arg Leu Arg Leu Arg Arg Cys His Arg 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> C10, m <400> 2 Leu Arg Met Arg Leu Arg Arg Cys His Arg 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence < 220> <223> C10, C>A <400> 3 Leu Arg Leu Arg Leu Arg Arg Ala His Arg 1 5 10 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> C10, L del <400> 4 Arg Leu Arg Leu Arg Arg Cys His Arg 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> C10, R del <400> 5 Leu Arg Leu Arg Leu Arg Arg Cys His 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> C10, L>R <400> 6 Arg Arg Leu Arg Leu Arg Arg Cys His Arg 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> C10, L>K <400> 7 Lys Arg Leu Arg Leu Arg Arg Cys His Arg 1 5 10 <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> TP <400> 8 Lys Lys Arg Arg Lys Arg 1 5 <210> 9 <211> 9 <212> PRT < 213> Artificial Sequence <220> <223> TB <400> 9 Leu Arg Leu Leu Arg Leu Lys Leu Lys 1 5 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> TAT <400> 10 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 11 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> dNP2 <400> 11 Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys Gly Ser Lys Ile Lys 1 5 10 15 Lys Val Lys Lys Lys Gly Arg Lys 20 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> C10, h <400> 12 ctcaggctga ggctacggcg ctgtcatcga 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> C10, m <400> 13 ctcaggatgc ggctgaggcg ctgtcatcga 30 <210> 14 <211 > 30 <212> DNA <213> Artificial Sequence <220> <223> C10, C>A <400> 14 ctcaggctga ggctacggcg cgctcatcga 30 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> < 223> C10, L del <400> 15 aggctgaggc tacggcgctg tcatcga 27 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> C10, R del <400> 16 ctcaggctga ggctacggcg ctgtcat 27 < 210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> C10, L>R <400> 17 aagaggctga ggctacggcg ctgtcat 27 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> C10, L>K <400> 18 cggaggctga ggctacggcg ctgtcat 27 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TP <400> 19 aagaagagga gaaaaaggaa a 21 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> TB <400> 20 ctgcgtctgc tgaggctcaa gttaaaa 27 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TAT <400> 21 tatggacgca agaagcgccg ccagcgccgc cgc 33 <210> 22 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> dNP2 <400> 22 aaaattaaaa aagtcaagaa gaaaggaaga aaaggatcca aaattaaaaa agtcaagaag 60 aaaggaagaa aa 72 <210> 23 <211> 975 <212> PRT <213> Artificial Sequence <220> <223> NLRX1 <400> 23 Met Arg Trp Gly Cys His Leu Pro Arg Thr Ser Trp Gly Ser Gly Leu 1 5 10 15 Gly Arg Thr Pro Gln Leu Pro Asp Glu His Ile Ser Phe Leu Ile Gln 20 25 30 Trp Ser Trp Pro Phe Lys Gly Val His Pro Leu Arg Pro Pro Arg Ala 35 40 45 Phe Ile Arg Tyr His Gly Asn Ser Ala Asp Ser Ala Pro Pro Pro Gly 50 55 60 Arg His Gly Gln Leu Phe Arg Ser Ile Ser Ala Thr Glu Ala Ile Gln 65 70 75 80 Arg His Arg Arg Asn Leu Thr Glu Trp Phe Ser Arg Leu Pro Arg Glu 85 90 95 Glu Arg Gln Phe Gly Pro Thr Phe Ala Leu Asp Thr Val His Val Asp 100 105 110 Pro Val Ile Arg Glu Ser Thr Pro Asp Glu Leu Leu Arg Pro Ser Thr 115 120 125 Glu Leu Ala Thr Gly His Gln Gln Thr Gln Ala Gly Leu Pro Pro Leu 130 135 140 Ala Leu Ser Gln Leu Phe Asp Pro Asp Ser Cys Gly Arg Arg Val Gln 145 150 155 160 Thr Val Val Leu Tyr Gly Thr Val Gly Thr Gly Lys Ser Thr Leu Val 165 170 175 Arg Lys Met Val Leu Asp Trp Cys Tyr Gly Arg Leu Pro Ala Phe Glu 180 185 190 Leu Leu Ile Pro Phe Ser Cys Glu Asp Leu Ser Ser Leu Gly Ser Thr 195 200 205 Pro Ala Ser Leu Cys Gln Leu Val Thr Gln Arg Tyr Thr Pro Leu Lys 210 215 220 Glu Val Leu Pro Leu Met Thr Ala Ala Gly Ser Arg Leu Leu Phe Val 225 230 235 240 Leu His Gly Leu Glu Arg Leu Asn Leu Asp Phe Arg Leu Ala Gly Thr 245 250 255 Gly Leu Cys Ser Asp Pro Glu Glu Pro Gly Pro Pro Ala Ala Ile Ile 260 265 270 Val Asn Leu Leu Arg Lys Tyr Met Leu Pro Glu Ala Ser Ile Leu Val 275 280 285 Thr Thr Arg Pro Ser Thr Ile Ser Arg Ile Pro Ser Lys Tyr Val Gly 290 295 300 Arg Tyr Gly Glu Ile Cys Gly Phe Ser Asp Thr Asn Leu Gln Lys Leu 305 310 315 320 Tyr Phe Gln Leu Arg Leu Asn Gln Pro Asp Cys Gly Tyr Gly Ala Gly 325 330 335 Gly Ala Ser Val Ser Val Thr Pro Ala Gln Arg Asp Asn Leu Ile Gln 340 345 350 Met Leu Ser Arg Asn Leu Glu Gly His His Gln Ile Ala Ala Ala Cys 355 360 365 Phe Leu Pro Ser Tyr Cys Trp Leu Val Cys Ala Thr Leu His Phe Leu 370 375 380 His Ala Pro Thr Pro Ala Gly Gln Thr Leu Thr Ser Ile Tyr Thr Ser 385 390 395 400 Phe Leu Arg Leu Asn Phe Ser Gly Glu Thr Leu Asp Ser Thr His Thr 405 410 415 Ser Asn Leu Ser Leu Met Ser Tyr Ala Ala Arg Thr Met Gly Lys Leu 420 425 430 Ala Tyr Glu Gly Val Ser Ser Arg Lys Thr Tyr Phe Ser Glu Glu Asp 435 440 445 Val Arg Gly Cys Leu Glu Ala Gly Ile Lys Thr Glu Glu Glu Phe Gln 450 455 460 Leu Leu Gln Ile Phe Arg Arg Asp Ala Leu Arg Phe Phe Leu Ala Pro 465 470 475 480 Cys Val Glu Pro Gly His Leu Gly Thr Phe Val Phe Thr Val Pro Ala 485 490 495 Met Gln Glu Tyr Leu Ala Ala Leu Tyr Ile Val Leu Gly Leu Arg Lys 500 505 510 Thr Ala Leu Gln Arg Val Gly Lys Glu Val Val Glu Phe Val Gly Arg 515 520 525 Val Gly Glu Asp Val Ser Leu Val Leu Gly Ile Val Ala Lys Leu Leu 530 535 540 Pro Leu Arg Ile Leu Pro Leu Leu Phe Asn Leu Leu Lys Val Val Pro 545 550 555 560 Arg Val Phe Gly Arg Met Val Ser Lys Ser Arg Glu Ala Val Ala Gln 565 570 575 Ala Met Val Leu Glu Met Phe Arg Glu Glu Asp Tyr Tyr Asn Asp Asp 580 585 590 Val Leu Asp Gln Met Gly Ala Ser Ile Leu Gly Val Glu Gly Pro Arg 595 600 605 Arg His Pro Asp Glu Pro Ser Glu Asp Glu Val Phe Glu Leu Phe Pro 610 615 620 Met Phe Met Gly Gly Leu Leu Ser Ala His Asn Arg Ala Val Leu Ala 625 630 635 640 Gln Leu Gly Cys Pro Ile Lys Asn Leu Asp Ala Leu Glu Asn Ala Gln 645 650 655 Ala Ile Lys Lys Lys Leu Gly Lys Leu Gly Arg Gln Val Leu Pro Pro 660 665 670 Ser Glu Leu Leu Asp His Leu Phe Phe His Tyr Glu Phe Gln Asn Gln 675 680 685 Arg Phe Ser Ala Glu Val Leu Gly Ser Leu Arg Gln Leu Asn Leu Ala 690 695 700 Gly Val Arg Met Thr Pro Leu Lys Cys Thr Val Val Ala Ser Val Leu 705 710 715 720 Gly Ser Gly Arg His Pro Leu Asp Glu Val Asn Leu Ala Ser Cys Gln 725 730 735 Leu Asp Pro Ala Gly Leu His Thr Leu Met Pro Val Leu Leu Arg Ala 740 745 750 Arg Lys Leu Gly Leu Gln Leu Asn Asn Leu Gly Pro Glu Ala Cys Arg 755 760 765 Asp Leu Arg Asp Leu Leu Leu His Asp Gln Cys Gln Ile Thr Thr Leu 770 775 780 Arg Leu Ser Asn Asn Pro Leu Thr Ala Ala Gly Val Gly Leu Leu Met 785 790 795 800 Asp Gly Leu Ala Gly Asn Thr Ser Val Thr His Leu Ser Leu Leu His 805 810 815 Thr Asp Leu Gly Asp Glu Gly Leu Glu Leu Leu Ala Ala Gln Leu Asp 820 825 830 Arg Asn Lys Gln Leu Gln Glu Leu Asn Val Ala Tyr Asn Gly Ala Gly 835 840 845 Asp Thr Val Ala Leu Ala Leu Ala Lys Ala Ala Arg Glu His Pro Ser 850 855 860 Leu Glu Leu Leu His Leu Tyr Phe Asn Glu Leu Ser Ser Glu Gly Arg 865 870 875 880 Gln Val Leu Arg Asp Leu Gly Gly Ser Gly Glu Gly Gly Ala Arg Val 885 890 895 Val Ala Ser Leu Thr Glu Gly Thr Ala Val Ser Glu Tyr Trp Ser Val 900 905 910 Ile Leu Ser Glu Val Gln Arg Asn Val His Ser Trp Asp Pro Leu Arg 915 920 925 Val Gln Arg His Leu Lys Leu Leu Leu Arg Asp Leu Glu Asp Ser Arg 930 935 940 Gly Ala Thr Leu Asn Pro Trp Arg Lys Ala Gln Leu Leu Arg Val Glu 945 950 955 960 Gly Glu Val Lys Thr Leu Leu Glu Gln Leu Gly Gly Ser Gly His 965 970 975 <210> 24 <211> 2928 <212> DNA <213> Artificial Sequence <220> <223> NLRX1 <400> 24 atgaggtggg gctgccattt gcccaggacc tcttggggct ctggcctggg aagaacaccc 60 cagctaccag atgagcatat ctcc ttcttg atccagtgga gctggccctt taaaggggtg 120 catcccctga ggccccctag ggcctttatc cgttaccatg gaaactcggc agacagtgct 180 cccccaccag ggaggcatgg gcagctgttc aggagcatct ctgccacaga agctatccaa 240 aggcatcgcc ggaacctcac cgagtggttt agccgactgc ccagagagga gcgccagttt 300 ggaccaacct ttgctctaga cacagtt cat gttgaccccg tgatccgaga gagcacccca 360 gatgagctgc ttcgcccgtc cacggagctg gccacggggc atcagcaaac ccaggcaggg 420 ctccccccac tggccctgtc tcagcttttt gacccggatt cttgtgggcg ccgcgtgcag 480 accgtggtgt tg tatgggac cgtgggtact ggcaagagca cgttggtacg gaagatggtc 540 ttagactggt gttacgggag actgcctgcc tttgagcttc tcatcccctt ctcctgtgag 600 gacttgtcat ccctgggctc caccccagct tccctgtgcc aacttgtgac ccagcgttac 660 acacccctga aagaggtgtt gcccctgatg actgctgcgg gatcccgcct gctctttgtg 720 ctccatggct tggagcgcct caaccttg ac ttccggctgg caggcacagg gctttgcagt 780 gacccggagg aacccgggcc accagctgcc atcatagtca acctgctgcg caaatacatg 840 cttcccgagg ccagcattct ggtaaccacc cggccttcca ccattagccg aatccctagc 900 aagtatgtgg gccgctatgg tgagatctgt ggcttctctg ataccaacct gcagaagctc 960 tacttccagc tccgccttaa ccagcctgac tgtgggtacg gtgctggggg tgccagtgtc 1020 tcagtcacac cagctcagcg cgacaacctg attcaaatgc tctcccggaa cctggagggg 1080 caccaccaga ttgccgcagc ctgctttctg ccttcctatt gctggcttgt ctgtgctact 1140 ttgcacttcc tgcatgctcc cacacctgct ggt cagaccc tcacaagcat ctataccagc 1200 tttctacgcc tgaacttcag tggggaaaca ctggacagca cccacacgtc caatctatcc 1260 ctgatgtcct atgcagcccg gactatgggc aagctggcct acgagggcgt gtcatcccga 1320 aagacctact tctctgaaga ggatgtccg t ggctgcctgg aagctggcat caagacagag 1380 gaagagtttc aactgcttca gatcttccgc agggacgccc tgaggttttt cctggccccg 1440 tgtgtggaac cagggcacct gggtaccttc gtgttcaccg tgcccgccat gcaggagtat 1500 ctggctgccc tctacatcgt gcttggtttg cgcaagacag ccctgcagcg ggtgggcaaa 1560 gaagtggttg aatttgtggg ccgtgttggg gaagatgtca gcctggtatt gggcattgtg 1620 gccaagctgt tgcccctgcg gattctgcct ctgctcttca acttgctcaa ggtagttccg 1680 cgagtgtttg ggcgcatggt gagtaagagc cgggaggcag tggcccaggc catggtgctg 1740 gagatgttcc gggagga aga ctactacaat gacgatgttc tggatcagat gggtgccagc 1800 atcctgggtg tggagggccc ccggcgccac ccagatgaac cctctgagga tgaagtcttt 1860 gagctcttcc ccatgttcat gggcggactt ctctctgccc acaaccgggc ggtgctggct 1920 cagcttggct gtcccatcaa gaacctggat gccctggaga atgcccaggc catcaagaag 1980 aagctgggga agctgggtcg gcaggtgctg cccccctcgg agcttcttga ccatctcttc 20 40 ttccactatg agttccagaa ccagcgcttc tcagctgagg tgctgggctc cctacgccag 2100 ctcaatttag caggggtgcg catgacaccc ctcaagtgca cagtggtagc ctctgtactg 2160 ggaagtggaa ggcaccccct ggatgaggtg aacttggcct cctgccagct ggatccc gct 2220 gggctacaca ctctcatgcc tgtcctcctg cgtgcccgga aactggggtt gcaactcaac 2280 aatctgggcc ccgaggcctg cagagacctc cgagacctgc tcttacacga tcaatgccag 2340 atcaccactc ttaggctctc caacaaccca ctgacagcag ctggtgtggg cttactgatg 2400 gacgggctgg caggaaacac ttcggtgaca cacctgtctc tgctgcacac tgaccttgga 2460 gacgagggac tggaactgct ggctgcccag ctggaccgaa acaaacaact gcaggagctg 2520 aacgtggcct acaacggtgc tggtgacaca gtggctctgg ccttggctaa ggctgctcgg 2580 gagcaccctt ccctggagct gctgcacctc tacttcaatg agctgagttc agagggccgc 2640 caggtcctgc gggatttggg gggctctggt gaaggtggtg cccgggtcgt agcctcgctg 2700 acagaaggga cggcggtgtc tgagtactgg tcagtgatcc ttagtgaagt ccagcgcaac 2760 gtccacagct gggacccgct ccgggtccag aggcatctca agctgctgct ccgtgatctg 2820 gaggacagcc ggggcgccac ccttaatccc tggcgcaagg ctcagcttct gcgagtggag 2880 ggcgagg tca agactcttct ggagcagctg ggaggttctg gacactga 2928 <210> 25 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> LRR < 400> 25 Leu Leu Asp His Leu Phe Phe His Tyr Glu Phe Gln Asn Gln Arg Phe 1 5 10 15 Ser Ala Glu Val Leu Gly Ser Leu Arg Gln Leu Asn Leu Ala Gly Val 20 25 30 Arg Met Thr Pro Leu Lys Cys Thr Val Val Ala Ser Val Leu Gly Ser 35 40 45 Gly Arg His Pro Leu Asp Glu Val Asn Leu Ala Ser Cys Gln Leu Asp 50 55 60 Pro Ala Gly Leu His Thr Leu Met Pro Val Leu Leu Arg Ala Arg Lys 65 70 75 80 Leu Gly Leu Gln Leu Asn Asn Leu Gly Pro Glu Ala Cys Arg Asp Leu 85 90 95 Arg Asp Leu Leu Leu His Asp Gln Cys Gln Ile Thr Leu Arg Leu 100 105 110 Ser Asn Asn Pro Leu Thr Ala Ala Gly Val Gly Leu Leu Met Asp Gly 115 120 125 Leu Ala Gly Asn Thr Ser Val Thr His Leu Ser Leu Leu His Thr Asp 130 135 140 Leu Gly Asp Glu Gly Leu Glu Leu Leu Ala Ala Gln Leu Asp Arg Asn 145 150 155 160 Lys Gln Leu Gln Glu Leu Asn Val Ala Tyr Asn Gly Ala Gly Asp Thr 165 170 175 Val Ala Leu Ala Leu Ala Lys Ala Ala Arg Glu His Pro Ser Leu Glu 180 185 190 Leu Leu His Leu Tyr Phe Asn Glu Leu Ser Ser Glu Gly Arg Gln Val 195 200 205 Leu Arg Asp Leu Gly Gly Ser Gly Glu Gly Gly Ala Arg Val Val Ala 210 215 220 Ser Leu Thr Glu Gly Thr Ala Val Ser Glu Tyr Trp Ser Val Ile Leu 225 230 235 240 Ser Glu Val Gln Arg Asn Val His Ser Trp Asp Pro Leu Arg Val Gln 245 250 255 Arg His Leu Lys Leu Leu Leu Arg Asp Leu Glu Asp Ser Arg Gly Ala 260 265 270 Thr Leu Asn Pro Trp Arg Lys Ala Gln Leu Leu Arg Val Glu Gly Glu 275 280 285 Val Lys Thr Leu Leu Glu Gln Leu Gly Gly Ser Gly His 290 295 300 <210> 26 <211> 903 <212> DNA <213> Artificial Sequence <220> <223> LRR <400> 26 cttcttgacc atctcttctt ccactatgag ttccagaacc agcgcttctc agctga ggtg 60 ctgggctccc tacgccagct caatttagca ggggtgcgca tgacacccct caagtgcaca 120 gtggtagcct ctgtactggg aagtggaagg caccccctgg atgaggtgaa cttggcctcc 180 tgccagctgg atcccgctgg gctacacact ctcatgcctg tcctcctgcg tgcccggaaaa 24 0 ctggggttgc aactcaacaa tctgggcccc gaggcctgca gagacctccg agacctgctc 300 ttacacgatc aatgccagat caccactctt aggctctcca acaacccact gacagcagct 360 ggtgtgggct tactgatgga cgggctggca ggaaacactt cggtgacaca cctgtctctg 420 ctgcacactg accttggaga cgagggactg gaactgctgg ctgcccagct ggaccgaaac 480 aaacaactgc aggagctgaa cgtggcctac aacggtgctg gtgacacagt ggctctggcc 540 ttggctaagg ctgctcggga gcacccttcc ctggagctgc tgcacctcta cttcaatgag 600 ctgagttcag agggccgcca ggtcctgcgg gatttggggg gctctggtga aggtggtgcc 660 cgggtcgtag cctcgctgac agaagggacg gcggtgtctg agtactggtc agtgatcctt 720 agtgaagtcc agcgcaacgt ccacagctgg gacccgctcc gggtccagag gcatctcaag 780 ctgctgctcc gtgatctgga ggacagccgg ggcgccaccc ttaatccctg gcgcaaggct 840 cagcttctgc gagtggaggg cgaggtcaag actcttctgg agcagctggg aggttctgga 900 cac 903 <210> 27 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> NBD <400> 27 Ala Thr Glu Ala Ile Gln Arg His Arg Arg Asn Leu Thr Glu Trp Phe 1 5 10 15 Ser Arg Leu Pro Arg Glu Glu Arg Gln Phe Gly Pro Thr Phe Ala Leu 20 25 30 Asp Thr Val His Val Asp Pro Val Ile Arg Glu Ser Thr Pro Asp Glu 35 40 45 Leu Leu Arg Pro Ser Thr Glu Leu Ala Thr Gly His Gln Gln Thr Gln 50 55 60 Ala Gly Leu Pro Pro Leu Ala Leu Ser Gln Leu Phe Asp Pro Asp Ser 65 70 75 80 Cys Gly Arg Arg Val Gln Thr Val Val Leu Tyr Gly Thr Val Gly Thr 85 90 95 Gly Lys Ser Thr Leu Val Arg Lys Met Val Leu Asp Trp Cys Tyr Gly 100 105 110 Arg Leu Pro Ala Phe Glu Leu Leu Ile Pro Phe Ser Cys Glu Asp Leu 115 120 125 Ser Ser Leu Gly Ser Thr Pro Ala Ser Leu Cys Gln Leu Val Thr Gln 130 135 140 Arg Tyr Thr Pro Leu Lys Glu Val Leu Pro Leu Met Thr Ala Ala Gly 145 150 155 160 Ser Arg Leu Leu Phe Val Leu His Gly Leu Glu Arg Leu Asn Leu Asp 165 170 175 Phe Arg Leu Ala Gly Thr Gly Leu Cys Ser Asp Pro Glu Glu Pro Gly 180 185 190 Pro Pro Ala Ala Ile Ile Val Asn Leu Leu Arg Lys Tyr Met Leu Pro 195 200 205 Glu Ala Ser Ile Leu Val Thr Thr Arg Pro Ser Thr Ile Ser Arg Ile 210 215 220 Pro Ser Lys Tyr Val Gly Arg Tyr Gly Glu Ile Cys Gly Phe Ser Asp 225 230 235 240 Thr Asn Leu Gln Lys Leu Tyr Phe Gln Leu Arg Leu Asn Gln Pro Asp 245 250 255 Cys Gly Tyr Gly Ala Gly Gly Ala Ser Val Ser Val Thr Pro Ala Gln 260 265 270 Arg Asp Asn Leu Ile Gln Met Leu Ser Arg Asn Leu Glu Gly His His 275 280 285 Gln Ile Ala Ala Ala Cys Phe Leu Pro Ser Tyr Cys Trp Leu Val Cys 290 295 300 Ala Thr Leu His Phe Leu His Ala Pro Thr Pro Ala Gly Gln Thr Leu 305 310 315 320 Thr Ser Ile Tyr Thr Ser Phe Leu Arg Leu Asn Phe Ser Gly Glu Thr 325 330 335 Leu Asp Ser Thr His Thr Ser Asn Leu Ser Leu Met Ser Tyr Ala Ala 340 345 350 Arg Thr Met Gly Lys Leu Ala Tyr Glu Gly Val Pro Ser Arg Lys Thr 355 360 365 Tyr Phe Ser Glu Glu Asp Val Arg Gly Cys Leu Glu Ala Gly Ile Lys 370 375 380 Thr Glu Glu Glu Phe Gln Leu Leu Gln Ile Phe Arg Arg Asp Ala Leu 385 390 395 400 Arg Phe Phe Leu Ala Pro Cys Val Glu Pro Gly His Leu Gly Thr Phe 405 410 415 Val Phe Thr Val Pro Ala Met Gln Glu Tyr Leu Ala Ala Leu Tyr Ser 420 425 430 Val Leu Gly Leu Arg Lys Thr Ala Leu Gln Arg Val Gly Lys Glu Val 435 440 445 Val Glu Phe Val Gly Arg Val Gly Glu Asp Val Ser Leu Val Leu Gly 450 455 460 Ile Val Ala Lys Leu Leu Pro Leu Arg Ile Leu Pro Leu Leu Phe Asn 465 470 475 480 Leu Leu <210> 28 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> NBD <400> 28 gccacagaag ctatccaaag gcatcgccgg aacctcaccg agtggtttag ccgactgccc 60 agagaggagc gccagtttgg accaaccttt gctctagaca cagttcatgt tgaccccgtg 120 atccgagaga gcaccccaga tgagctgctt cgcccgtcca cgg agctggc cacggggcat 180 cagcaaaccc aggcagggct ccccccactg gccctgtctc agctttttga cccggattct 240 tgtgggcgcc gcgtgcagac cgtggtgttg tatgggaccg tgggtactgg caagagcacg 300 ttggtacgga agatggtctt agactggtgt tac gggagac tgcctgcctt tgagcttctc 360 atccccttct cctgtgagga cttgtcatcc ctgggctcca ccccagcttc cctgtgccaa 420 cttgtgaccc agcgttacac acccctgaaa gaggtgttgc ccctgatgac tgctgcggga 480 tcccgcctgc tctttgtgct ccatggcttg gagcgcctca acctt gactt ccggctggca 540 ggcacagggc tttgcagtga cccggaggaa cccgggccac cagctgccat catagtcaac 600 ctgctgcgca aatacatgct tcccgaggcc agcattctgg taaccacccg gccttccacc 660 attagccgaa tccctagcaa gtatgtgggc cgctatggtg agat ctgtgg cttctctgat 720 accaacctgc agaagctcta cttccagctc cgccttaacc agcctgactg tgggtacggt 780 gctgggggtg ccagtgtctc agtcacacca gctcagcgcg acaacctgat tcaaatgctc 840 tcccggaacc tggaggggca ccaccagatt gccgcagcct gctttctgcc ttcctattgc 900 tggcttgtct gtgctacttt gcacttcctg catgctccca cacctgctgg tcagaccctc 960 a caagcatct ataccagctt tctacgcctg aacttcagtg gggaaacact ggacagcacc 1020 cacacgtcca atctatccct gatgtcctat gcagcccgga ctatgggcaa gctggcctac 1080 gagggcgtgc catcccgaaa gacctacttc tctgaagagg atgtccgtgg ctgcctggaa 114 0 gctggcatca agacagagga agagtttcaa ctgcttcaga tcttccgcag ggacgccctg 1200 aggtttttcc tggccccgtg tgtggaacca gggcacctgg gtaccttcgt gttcaccgtg 1260 cccgccatgc aggagtatct ggctgccctc tacagcgtgc ttggtttgcg caagacagcc 1320 ctgcagcggg tgggcaaaga agtggttgaa tttgtgggcc gtgttgggga agatgtca gc 1380 ctggtattgg gcattgtggc caagctgttg cccctgcgga ttctgcctct gctcttcaac 1440 ttgctc 1446 <210> 29 <211> 60 <212> DNA <213> Artificial Sequence <220> <223 > forward primer of C10-LRR <400> 29 ctagctagcc tcaggctgag gctacggcgc tgtcatcgag tcgaccttct tgaccatctc 60 60 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of C10-LRR <400 > 30 aggttctgga cacgaattcc gg 22 <210> 31 <211> 1065 <212> DNA <213> Artificial Sequence <220> <223> C10-LRR <400> 31 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctag cc tcaggctgag gctacggcgc tgtcatcgag tcgaccttct tgaccatctc 120 ttcttccact atgagttcca gaaccagcgc ttctcagctg aggtgctggg ctccctacgc 180 cagctcaatt tagcaggggt gcgcatgaca cccctcaagt gcacagtggt agcctctgta 240 ctgggaagtg gaaggcaccc cctggatgag gtgaacttgg cctcctgcca gctggatccc 300 gctgggctac acactctcat gcctgtcctc ctgcgtgccc ggaaactggg gttgcaactc 360 aacaatctgg gccccgaggc ctgcagagac ctccgagacc tgctcttaca cgatcaatgc 420 cagatcacca ctcttaggct ctccaacaac ccactgacag cagctggtgt gggctt actg 480 atggacgggc tggcaggaaa cacttcggtg acacacctgt ctctgctgca cactgacctt 540 ggagacgagg gactggaact gctggctgcc cagctggacc gaaacaaaca actgcaggag 600 ctgaacgtgg cctacaacgg tgctggtgac acagtggctc tggccttggc taaggctgct 660 cgggagcacc cttccctgga gctgctgcac ctctacttca atgagctgag ttcagagggc 720 cgccagg tcc tgcgggattt ggggggctct ggtgaaggtg gtgcccgggt cgtagcctcg 780 ctgacagaag ggacggcggt gtctgagtac tggtcagtga tccttagtga agtccagcgc 840 aacgtccaca gctgggaccc gctccgggtc cagaggcatc tcaagctgct gctccgtgat 900 ctggaggaca gccggggcgc cacccttaat ccctggcgca aggctcagct tctgcgagtg 960 gagggcgagg tcaagactct tctggagcag ctgggaggtt ctggacacga attcgattac 1020 aaggatgacg atgacaagct cgagcaccac caccaccacc actga 1065 <210> 32 <211> 354 <212> PRT <213> Artificial Sequence <220> <223> C10-LRR <400> 32 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Leu Arg Leu Arg Leu Arg Arg Cys His 20 25 30 Arg Val Asp Leu Leu Asp His Leu Phe Phe His Tyr Glu Phe Gln Asn 35 40 45 Gln Arg Phe Ser Ala Glu Val Leu Gly Ser Leu Arg Gln Leu Asn Leu 50 55 60 Ala Gly Val Arg Met Thr Pro Leu Lys Cys Thr Val Val Ala Ser Val 65 70 75 80 Leu Gly Ser Gly Arg His Pro Leu Asp Glu Val Asn Leu Ala Ser Cys 85 90 95 Gln Leu Asp Pro Ala Gly Leu His Thr Leu Met Pro Val Leu Leu Arg 100 105 110 Ala Arg Lys Leu Gly Leu Gln Leu Asn Asn Leu Gly Pro Glu Ala Cys 115 120 125 Arg Asp Leu Arg Asp Leu Leu Leu His Asp Gln Cys Gln Ile Thr Thr 130 135 140 Leu Arg Leu Ser Asn Asn Pro Leu Thr Ala Ala Gly Val Gly Leu Leu 145 150 155 160 Met Asp Gly Leu Ala Gly Asn Thr Ser Val Thr His Leu Ser Leu Leu 165 170 175 His Thr Asp Leu Gly Asp Glu Gly Leu Glu Leu Leu Ala Ala Gln Leu 180 185 190 Asp Arg Asn Lys Gln Leu Gln Glu Leu Asn Val Ala Tyr Asn Gly Ala 195 200 205 Gly Asp Thr Val Ala Leu Ala Leu Ala Lys Ala Ala Arg Glu His Pro 210 215 220 Ser Leu Glu Leu Leu His Leu Tyr Phe Asn Glu Leu Ser Ser Glu Gly 225 230 235 240 Arg Gln Val Leu Arg Asp Leu Gly Gly Ser Gly Glu Gly Gly Ala Arg 245 250 255 Val Val Ala Ser Leu Thr Glu Gly Thr Ala Val Ser Glu Tyr Trp Ser 260 265 270 Val Ile Leu Ser Glu Val Gln Arg Asn Val His Ser Trp Asp Pro Leu 275 280 285 Arg Val Gln Arg His Leu Lys Leu Leu Leu Arg Asp Leu Glu Asp Ser 290 295 300 Arg Gly Ala Thr Leu Asn Pro Trp Arg Lys Ala Gln Leu Leu Arg Val 305 310 315 320 Glu Gly Glu Val Lys Thr Leu Leu Glu Gln Leu Gly Gly Ser Gly His 325 330 335 Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Leu Glu His His His His 340 345 350 His His <210> 33 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> forward primer of TAT-LRR <400> 33 ctagctagct atggacgcaa gaagcgccgc cagcgccgcc gcgtcgacct tcttgaccat 60 ctc 63 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of TAT-LRR <400> 34 aggttctgga cacgaattcc gg 22 <210 > 35 < 211> 1068 <212> DNA <213> Artificial Sequence <220> <223> TAT-LRR <400> 35 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagct atggacgcaa gaagcgccgc cagcgccgcc gcgtcgacct tcttgaccat 120 ctcttcttcc actatgagtt ccagaaccag cgcttctcag ctgaggtgct gggctcccta 180 cgccagctca atttagcagg ggtgcgcatg acacccctca agtgcacagt ggtagcctct 240 gtactgggaa gtggaaggca ccccctggat gaggtgaact tggcctcctg ccagctggat 300 cccgctgggc tacacactct catgcctgtc ctcctgcgtg cccggaaact ggggttgcaa 360 ctcaacaatc tgggccccga ggcctgcaga g acctccgag acctgctctt acacgatcaa 420 tgccagatca ccactcttag gctctccaac aacccactga cagcagctgg tgtgggctta 480 ctgatggacg ggctggcagg aaacacttcg gtgacacacc tgtctctgct gcacactgac 540 cttggagacg agggactgga actgctggct gcc cagctgg accgaaacaa acaactgcag 600 gagctgaacg tggcctacaa cggtgctggt gacacagtgg ctctggcctt ggctaaggct 660 gctcgggagc acccttccct ggagctgctg cacctctact tcaatgagct gagttcagag 720 ggccgccagg tcctgcggga tttggggggc tctggtgaag gtggtgcccg ggtcgtagcc 780 tcgctgacag aagggacggc ggtgtctgag tactggtcag tgatccttag tgaagtccag 840 cgcaacgtcc acagctggga cccgctccgg gtccagaggc atctcaagct gctgctccgt 900 gatctggagg acagccgggg cgccaccctt aatccctggc gcaaggctca gcttctgcga 960 gtggagggcg aggtcaagac tcttctggag cagctgggag g ttctggaca cgaattcgat 1020 tacaaggatg acgatgacaa gctcgagcac caccaaccacc accactga 1068 <210> 36 <211> 355 <212> PRT <213> Artificial Sequence <220> <223> TAT-LRR <400> 36 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg 20 25 30 Arg Arg Val Asp Leu Leu Asp His Leu Phe Phe His Tyr Glu Phe Gln 35 40 45 Asn Gln Arg Phe Ser Ala Glu Val Leu Gly Ser Leu Arg Gln Leu Asn 50 55 60 Leu Ala Gly Val Arg Met Thr Pro Leu Lys Cys Thr Val Val Ala Ser 65 70 75 80 Val Leu Gly Ser Gly Arg His Pro Leu Asp Glu Val Asn Leu Ala Ser 85 90 95 Cys Gln Leu Asp Pro Ala Gly Leu His Thr Leu Met Pro Val Leu Leu 100 105 110 Arg Ala Arg Lys Leu Gly Leu Gln Leu Asn Leu Gly Pro Glu Ala 115 120 125 Cys Arg Asp Leu Arg Asp Leu Leu Leu His Asp Gln Cys Gln Ile Thr 130 135 140 Thr Leu Arg Leu Ser Asn Asn Pro Leu Thr Ala Ala Gly Val Gly Leu 145 150 155 160 Leu Met Asp Gly Leu Ala Gly Asn Thr Ser Val Thr His Leu Ser Leu 165 170 175 Leu His Thr Asp Leu Gly Asp Glu Gly Leu Glu Leu Leu Ala Ala Gln 180 185 190 Leu Asp Arg Asn Lys Gln Leu Gln Glu Leu Asn Val Ala Tyr Asn Gly 195 200 205 Ala Gly Asp Thr Val Ala Leu Ala Leu Ala Lys Ala Ala Arg Glu His 210 215 220 Pro Ser Leu Glu Leu Leu His Leu Tyr Phe Asn Glu Leu Ser Ser Glu 225 230 235 240 Gly Arg Gln Val Leu Arg Asp Leu Gly Gly Ser Gly Glu Gly Gly Ala 245 250 255 Arg Val Val Ala Ser Leu Thr Glu Gly Thr Ala Val Ser Glu Tyr Trp 260 265 270 Ser Val Ile Leu Ser Glu Val Gln Arg Asn Val His Ser Trp Asp Pro 275 280 285 Leu Arg Val Gln Arg His Leu Lys Leu Leu Leu Arg Asp Leu Glu Asp 290 295 300 Ser Arg Gly Ala Thr Leu Asn Pro Trp Arg Lys Ala Gln Leu Leu Arg 305 310 315 320 Val Glu Gly Glu Val Lys Thr Leu Leu Glu Gln Leu Gly Gly Ser Gly 325 330 335 His Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Leu Glu His His 340 345 355 <210> 37 <210> 37 <211> 62 <212> DNA <213> Artificial sequence <220> <223> Forward Primer of DNP2-LRR <400> 37 CGGCTAAAAAAAAaaaaaaaaaaaaaaaaaaaaaaaaa of GA aaggaagaa AGTCGACCTT CTTGACCATC 60 TC 62 < 210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of dNP2-LRR <400> 38 ccggaattcg tgtccagaac ct 22 <210> 39 <211> 981 <212> DNA <213 > Artificial Sequence <220> <223> dNP2-LRR <400> 39 aagatcaaga aggttaaaaa aaagggtcgc aagggctcta aaattaaaaa agtcaagaag 60 aaaggaagaa aagtcgacct tcttgaccat ctcttcttcc actatgagtt ccagaaccag 120 cgcttctcag ctgaggtgct gggctcccta cgccagctca atttagcagg ggtgcgcatg 180 acacccctca agtgcacagt ggtagcctct gtactgggaa gtggaaggca ccccctggat 240 gaggtgaact tggcctcctg ccagctggat cccgctgggc tacacactct catgcctgtc 300 ctcctgcgtg cccggaaact ggggttgcaa ctcaacaatc tgggccccga ggcctgcaga 360 gacctccgag acctgctctt acacgatcaa tgccagatca ccactcttag gctctccaac 420 aacccactga cagcagctgg tgtgggctta ctgatggacg ggctggcagg aa acacttcg 480 gtgacacacc tgtctctgct gcacactgac cttggagacg agggactgga actgctggct 540 gcccagctgg accgaaacaa acaactgcag gagctgaacg tggcctacaa cggtgctggt 600 gacacagtgg ctctggcctt ggctaaggct gctcgggagc acccttccct ggag ctgctg 660 cacctctact tcaatgagct gagttcagag ggccgccagg tcctgcggga tttggggggc 720 tctggtgaag gtggtgcccg ggtcgtagcc tcgctgacag aagggacggc ggtgtctgag 780 tactggtcag tgatccttag tgaagtccag cgcaacgtcc acagctggga cccgctccgg 840 gtccagaggc atctcaagct gctgctccgt gatctggagg acagccgggg cgccaccctt 900 aatccctggc gcaaggctca gcttctgcga gtggagggcg aggtcaagac tcttctggag 960 cagctgggag gttctggaca c 981 <210> 40 <211> 368 <212> PRT <213> Artificial Sequence < 220> <223> dNP2-LRR <400> 40 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Lys Ile Lys Lys Val Lys Lys Lys Gly 20 25 30 Arg Lys Gly Ser Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys Val 35 40 45 Asp Leu Leu Asp His Leu Phe Phe His Tyr Glu Phe Gln Asn Gln Arg 50 55 60 Phe Ser Ala Glu Val Leu Gly Ser Leu Arg Gln Leu Asn Leu Ala Gly 65 70 75 80 Val Arg Met Thr Pro Leu Lys Cys Thr Val Val Ala Ser Val Leu Gly 85 90 95 Ser Gly Arg His Pro Leu Asp Glu Val Asn Leu Ala Ser Cys Gln Leu 100 105 110 Asp Pro Ala Gly Leu His Thr Leu Met Pro Val Leu Leu Arg Ala Arg 115 120 125 Lys Leu Gly Leu Gln Leu Asn Asn Leu Gly Pro Glu Ala Cys Arg Asp 130 135 140 Leu Arg Asp Leu Leu Leu His Asp Gln Cys Gln Ile Thr Thr Leu Arg 145 150 155 160 Leu Ser Asn Asn Pro Leu Thr Ala Ala Gly Val Gly Leu Leu Met Asp 165 170 175 Gly Leu Ala Gly Asn Thr Ser Val Thr His Leu Ser Leu Leu His Thr 180 185 190 Asp Leu Gly Asp Glu Gly Leu Glu Leu Leu Ala Ala Gln Leu Asp Arg 195 200 205 Asn Lys Gln Leu Gln Glu Leu Asn Val Ala Tyr Asn Gly Ala Gly Asp 210 215 220 Thr Val Ala Leu Ala Leu Ala Lys Ala Ala Arg Glu His Pro Ser Leu 225 230 235 240 Glu Leu Leu His Leu Tyr Phe Asn Glu Leu Ser Ser Glu Gly Arg Gln 245 250 255 Val Leu Arg Asp Leu Gly Gly Ser Gly Glu Gly Gly Ala Arg Val Val 260 265 270 Ala Ser Leu Thr Glu Gly Thr Ala Val Ser Glu Tyr Trp Ser Val Ile 275 280 285 Leu Ser Glu Val Gln Arg Asn Val His Ser Trp Asp Pro Leu Arg Val 290 295 300 Gln Arg His Leu Lys Leu Leu Leu Arg Asp Leu Glu Asp Ser Arg Gly 305 310 315 320 Ala Thr Leu Asn Pro Trp Arg Lys Ala Gln Leu Leu Arg Val Glu Gly 325 330 335 Glu Val Lys Thr Leu Leu Glu Gln Leu Gly Gly Ser Gly His Glu Phe 340 345 350 Asp Tyr Lys Asp Asp Asp Asp Lys Leu Glu His His His His His 355 360 365 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of C10-NBD <400> 41 ctagtcgacg ccacagaagc tatccaa 27 <210> 42 <211> 27 <212 > DNA <213> Artificial Sequence <220> <223> Forward primer of C10-NBD <400> 42 ccggaattcg agcaagttga agagcag 27 <210> 43 <211> 1608 <212> DNA <213> Artificial Sequence <220> <223> C10-NBD <400> 43 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagcc tcaggctgag gctacggcgc tgtcatcgag tcgacgccac agaagctatc 120 caaaggcatc gccggaacct caccgagtgg tttagccgac tgcccaga ga ggagcgccag 180 tttggaccaa cctttgctct agacacagtt catgttgacc ccgtgatccg agagagcacc 240 ccagatgagc tgcttcgccc gtccacggag ctggccacgg ggcatcagca aacccaggca 300 gggctccccc cactggccct gtctcagctt tttgacccgg at tcttgtgg gcgccgcgtg 360 cagaccgtgg tgttgtatgg gaccgtgggt actggcaaga gcacgttggt acggaagatg 420 gtcttagact ggtgttacgg gagactgcct gcctttgagc ttctcatccc cttctcctgt 480 gaggacttgt catccctggg ctccacccca gcttccctgt gccaacttgt gacccagcgt 540 tacacacccc tgaaagaggt gttgcccctg atgactgctg cgggatcccg cctgctcttt 600 gtgctccatg gcttggagcg cctcaacctt gacttccggc tggcaggcac agggctttgc 660 agtgacccgg aggaacccgg gccaccagct gccatcatag tcaacctgct gcgcaaatac 7 20 atgcttcccg aggccagcat tctggtaacc acccggcctt ccaccattag ccgaatccct 780 agcaagtatg tgggccgcta tggtgagatc tgtggcttct ctgataccaa cctgcagaag 840 ctctacttcc agctccgcct taaccagcct gactgtgggt acggtgctgg gggtgccagt 900 gtctcagtca caccagctca gcgcgacaac ctgattcaaa tgctctcccg gaacctggag 960 gggcaccacc agattg ccgc agcctgcttt ctgccttcct attgctggct tgtctgtgct 1020 actttgcact tcctgcatgc tcccacacct gctggtcaga ccctcacaag catctatacc 1080 agctttctac gcctgaactt cagtggggaa acactggaca gcacccacac gtccaatcta 1140 tccctga tgt cctatgcagc ccggactatg ggcaagctgg cctacgaggg cgtgccatcc 1200 cgaaagacct acttctctga agaggatgtc cgtggctgcc tggaagctgg catcaagaca 1260 gaggaagagt ttcaactgct tcagatcttc cgcagggacg ccctgaggtt tttcctggcc 1320 ccgtgtgtgg aaccagggca cctgggtacc ttcgtgttca ccgtgcccgc catgcaggag 1380 tatctggctg ccctct acag cgtgcttggt ttgcgcaaga cagccctgca gcgggtgggc 1440 aaagaagtgg ttgaatttgt gggccgtgtt ggggaagatg tcagcctggt attgggcatt 1500 gtggccaagc tgttgcccct gcggattctg cctctgctct tcaacttgct cgaattcgat 1560 tacaaggatg acgatgacaa gctcgagcac caccaccacc accactga 1608 <210> 44 < 211> 1028 <212> PRT <213> Artificial Sequence <220> <223> C10-NBD <400> 44 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Leu Arg Leu Arg Leu Arg Arg Cys His 20 25 30 Arg Val Asp Met Arg Trp Gly Cys His Leu Pro Arg Thr Ser Trp Gly 35 40 45 Ser Gly Leu Gly Arg Thr Pro Gln Leu Pro Asp Glu His Ile Ser Phe 50 55 60 Leu Ile Gln Trp Ser Trp Pro Phe Lys Gly Val His Pro Leu Arg Pro 65 70 75 80 Pro Arg Ala Phe Ile Arg Tyr His Gly Asn Ser Ala Asp Ser Ala Pro 85 90 95 Pro Pro Gly Arg His Gly Gln Leu Phe Arg Ser Ile Ser Ala Thr Glu 100 105 110 Ala Ile Gln Arg His Arg Arg Asn Leu Thr Glu Trp Phe Ser Arg Leu 115 120 125 Pro Arg Glu Glu Arg Gln Phe Gly Pro Thr Phe Ala Leu Asp Thr Val 130 135 140 His Val Asp Pro Val Ile Arg Glu Ser Thr Pro Asp Glu Leu Leu Arg 145 150 155 160 Pro Ser Thr Glu Leu Ala Thr Gly His Gln Gln Thr Gln Ala Gly Leu 165 170 175 Pro Pro Leu Ala Leu Ser Gln Leu Phe Asp Pro Asp Ser Cys Gly Arg 180 185 190 Arg Val Gln Thr Val Val Leu Tyr Gly Thr Val Gly Thr Gly Lys Ser 195 200 205 Thr Leu Val Arg Lys Met Val Leu Asp Trp Cys Tyr Gly Arg Leu Pro 210 215 220 Ala Phe Glu Leu Leu Ile Pro Phe Ser Cys Glu Asp Leu Ser Ser Leu 225 230 235 240 Gly Ser Thr Pro Ala Ser Leu Cys Gln Leu Val Thr Gln Arg Tyr Thr 245 250 255 Pro Leu Lys Glu Val Leu Pro Leu Met Thr Ala Ala Gly Ser Arg Leu 260 265 270 Leu Phe Val Leu His Gly Leu Glu Arg Leu Asn Leu Asp Phe Arg Leu 275 280 285 Ala Gly Thr Gly Leu Cys Ser Asp Pro Glu Glu Pro Gly Pro Pro Ala 290 295 300 Ala Ile Ile Val Asn Leu Leu Arg Lys Tyr Met Leu Pro Glu Ala Ser 305 310 315 320 Ile Leu Val Thr Thr Arg Pro Ser Thr Ile Ser Arg Ile Pro Ser Lys 325 330 335 Tyr Val Gly Arg Tyr Gly Glu Ile Cys Gly Phe Ser Asp Thr Asn Leu 340 345 350 Gln Lys Leu Tyr Phe Gln Leu Arg Leu Asn Gln Pro Asp Cys Gly Tyr 355 360 365 Gly Ala Gly Gly Ala Ser Val Ser Val Thr Pro Ala Gln Arg Asp Asn 370 375 380 Leu Ile Gln Met Leu Ser Arg Asn Leu Glu Gly His His Gln Ile Ala 385 390 395 400 Ala Ala Cys Phe Leu Pro Ser Tyr Cys Trp Leu Val Cys Ala Thr Leu 405 410 415 His Phe Leu His Ala Pro Thr Pro Ala Gly Gln Thr Leu Thr Ser Ile 420 425 430 Tyr Thr Ser Phe Leu Arg Leu Asn Phe Ser Gly Glu Thr Leu Asp Ser 435 440 445 Thr His Thr Ser Asn Leu Ser Leu Met Ser Tyr Ala Ala Arg Thr Met 450 455 460 Gly Lys Leu Ala Tyr Glu Gly Val Ser Ser Arg Lys Thr Tyr Phe Ser 465 470 475 480 Glu Glu Asp Val Arg Gly Cys Leu Glu Ala Gly Ile Lys Thr Glu Glu 485 490 495 Glu Phe Gln Leu Leu Gln Ile Phe Arg Arg Asp Ala Leu Arg Phe Phe 500 505 510 Leu Ala Pro Cys Val Glu Pro Gly His Leu Gly Thr Phe Val Phe Thr 515 520 525 Val Pro Ala Met Gln Glu Tyr Leu Ala Ala Leu Tyr Ile Val Leu Gly 530 535 540 Leu Arg Lys Thr Ala Leu Gln Arg Val Gly Lys Glu Val Val Glu Phe 545 550 555 560 Val Gly Arg Val Gly Glu Asp Val Ser Leu Val Leu Gly Ile Val Ala 565 570 575 Lys Leu Leu Pro Leu Arg Ile Leu Pro Leu Leu Phe Asn Leu Leu Lys 580 585 590 Val Val Pro Arg Val Phe Gly Arg Met Val Ser Lys Ser Arg Glu Ala 595 600 605 Val Ala Gln Ala Met Val Leu Glu Met Phe Arg Glu Glu Asp Tyr Tyr 610 615 620 Asn Asp Asp Val Leu Asp Gln Met Gly Ala Ser Ile Leu Gly Val Glu 625 630 635 640 Gly Pro Arg Arg His Pro Asp Glu Pro Ser Glu Asp Glu Val Phe Glu 645 650 655 Leu Phe Pro Met Phe Met Gly Gly Leu Leu Ser Ala His Asn Arg Ala 660 665 670 Val Leu Ala Gln Leu Gly Cys Pro Ile Lys Asn Leu Asp Ala Leu Glu 675 680 685 Asn Ala Gln Ala Ile Lys Lys Lys Leu Gly Lys Leu Gly Arg Gln Val 690 695 700 Leu Pro Pro Ser Glu Leu Leu Asp His Leu Phe Phe His Tyr Glu Phe 705 710 715 720 Gln Asn Gln Arg Phe Ser Ala Glu Val Leu Gly Ser Leu Arg Gln Leu 725 730 735 Asn Leu Ala Gly Val Arg Met Thr Pro Leu Lys Cys Thr Val Val Ala 740 745 750 Ser Val Leu Gly Ser Gly Arg His Pro Leu Asp Glu Val Asn Leu Ala 755 760 765 Ser Cys Gln Leu Asp Pro Ala Gly Leu His Thr Leu Met Pro Val Leu 770 775 780 Leu Arg Ala Arg Lys Leu Gly Leu Gln Leu Asn Asn Leu Gly Pro Glu 785 790 795 800 Ala Cys Arg Asp Leu Arg Asp Leu Leu Leu His Asp Gln Cys Gln Ile 805 810 815 Thr Thr Leu Arg Leu Ser Asn Asn Pro Leu Thr Ala Ala Gly Val Gly 820 825 830 Leu Leu Met Asp Gly Leu Ala Gly Asn Thr Ser Val Thr His Leu Ser 835 840 845 Leu Leu His Thr Asp Leu Gly Asp Glu Gly Leu Glu Leu Leu Ala Ala 850 855 860 Gln Leu Asp Arg Asn Lys Gln Leu Gln Glu Leu Asn Val Ala Tyr Asn 865 870 875 880 Gly Ala Gly Asp Thr Val Ala Leu Ala Leu Ala Lys Ala Ala Arg Glu 885 890 895 His Pro Ser Leu Glu Leu Leu His Leu Tyr Phe Asn Glu Leu Ser Ser 900 905 910 Glu Gly Arg Gln Val Leu Arg Asp Leu Gly Gly Ser Gly Glu Gly Gly 915 920 925 Ala Arg Val Val Ala Ser Leu Thr Glu Gly Thr Ala Val Ser Glu Tyr 930 935 940 Trp Ser Val Ile Leu Ser Glu Val Gln Arg Asn Val His Ser Trp Asp 945 950 955 960 Pro Leu Arg Val Gln Arg His Leu Lys Leu Leu Leu Arg Asp Leu Glu 965 970 975 Asp Ser Arg Gly Ala Thr Leu Asn Pro Trp Arg Lys Ala Gln Leu Leu 980 985 990 Arg Val Glu Gly Glu Val Lys Thr Leu Leu Glu Gln Leu Gly Gly Ser 995 1000 1005 Gly His Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Leu Glu His His 1010 1015 1020 His His His His 1025 <210> 45 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of C10-EGFP <400> 45 ctagctagcc tcaggctgag gctacggcgc tgtcatcgag gatccgtgag caagggcgag 60 60 <210> 46 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of C10-EGFP <400> 46 cccaagcttt tattacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac 60 gaactccag 69 <210> 47 <211 > 795 <212> DNA <213> Artificial Sequence <220> <223> C10-EGFP <400> 47 atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcctcaggct gaggctacgg 60 cgctgtcatc gaggatccgt gagcaaggc gaggagctgt tcaccggggt ggtgcccatc 120 ctggtc gagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 180 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 240 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 300 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 360 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagt tc 420 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 480 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 540 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 600 agcgtgcagc tcgccgacca ctaccagcag aacacccca tcggcgacgg ccccgtgctg 660 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 720 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 780 gagctgtaca agtaa 795 <210> 48 <211> 62 <212> DNA <213> Artificial Sequence <2 20> <223> Forward primer of dNP2-EGFP <400> 48 cggctagcaa aattaaaaaa gtcaagaaga aaggaagaaa agtcgacctt cttgaccatc 60 tc 62 <210> 49 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of dNP2-EGFP <400> 49 cccaagcttt tattacttgt acagctcgtc catgccga ga gtgatcccgg cggcggtcac 60 gaactccag 69 <210> 50 <211> 840 <212> DNA <213> Artificial Sequence <220> <223> dNP2-EGFP <400> 50 atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcaaaattaa aaaagtcaag 60 aagaaaggaa gaaaaggatc caaaattaaa aaagtcaaga agaaaggaag a aaagaattc 120 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 180 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 240 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 300 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct a ccccgacca catgaagcag 360 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 420 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 480 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gca acatcct ggggcacaag 540 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 600 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 660 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 720 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 7 80 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaataa 840 840 <210> 51 <211> 60 <212> DNA <213> Artificial Sequence <220> < 223> Forward primer of C10(m)-EGFP <400> 51 ctagctagcc tcaggatgcg gctgaggcgc tgtcatcgag gatccgtgag caagggcgag 60 60 <210> 52 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of C10 (m)-EGFP <400> 52 cccaagcttt tattacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac 60 gaactccag 69 <210> 53 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> C10(m)-EGFP <400> 53 atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcctcaggat gaggctacgg 60 cgctgtcatc gaggatccgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 120 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 180 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 240 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 300 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgt ccag 360 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 420 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 480 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 540 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 600 agcgt gcagc tcgccgacca ctaccagcag aacacccca tcggcgacgg ccccgtgctg 660 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 720 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 780 gag ctgtaca agtaa 795 <210> 54 < 211> 60 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of C10(C>A)-EGFP <400> 54 ctagctagcc tcaggctgag gctacggcgc gctcatcgag gatccgtgag caagggcgag 60 60 <210> 55 <211> 69 < 212> DNA <213> Artificial sequence <220> <223> reverse Primer of C10 (C> a) -EGFP <400> 55 CCCAAGCTTTGTCCGTC CATGCGAGA GTGATCGAGA 60 GaactCCAG 69 <210> 56 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> C10(C>A)-EGFP <400> 56 atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcctcaggct gaggctacgg 60 cgcgctcatc gaggatccgt gagcaaggc gaggagctgt tcaccggggt ggtgcccatc 120 ctggtcga gc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 180 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 240 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 300 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 360 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga gg tgaagttc 420 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 480 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 540 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 600 agcgtgcagc tcgccgacca ctaccagcag aacacccca tcggcgacgg ccccgtgctg 660 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 720 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 780 gagctgtaca agtaa 795 <210> 57 <211> 57 <212> DNA <213> Art ificial Sequence <220> <223> Forward primer of C10(L del)-EGFP <400> 57 ctagctagca ggctgaggct acggcgctgt catcgaggat ccgtgagcaa gggcgag 57 <210> 58 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of C10(L del)-EGFP <400> 58 cccaagcttt tattactt gt acagctcgtc catgccgaga gtgatcccgg cggcggtcac 60 gaactccag 69 <210> 59 <211> 792 <212> DNA <213> Artificial Sequence <220> <223> C10(L del)-EGFP <400> 59 atgcggggtt ctcatcatca tcatcatcat ggtatggcta g caggctgag gctacggcgc 60 tgtcatcgag gatccgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 120 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 180 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 240 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 300 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 360 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 420 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttca agga ggacggcaac 480 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 540 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 600 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 660 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 720 gatcacat gg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 780 ctgtacaagt aa 792 <210> 60 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of C10(R del)-EGFP <400> 60 ctagctagcc tcaggctgag gctacggcgc tgtcatggat ccgtgagcaa gggcgag 57 <210> 61 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of C10(R del)-EGFP <400> 61 cccaagcttt tattacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac 60 gaactccag 69 <210> 62 <211> 792 <212> DNA <213> Artificial Sequence <220> <223> C10(R del)-EGFP <400> 62 atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcctcaggct gaggctacgg 60 cgctgtcatg gatccgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 120 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 180 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 240 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 300 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 360 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 420 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 480 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 540 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc a caacatcga ggacggcagc 600 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 660 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 720 gatcacatgg tcctgctgga gttcgtgacc gccgccgg ga tcactctcgg catggacgag 780 ctgtacaagt aa 792 <210 > 63 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of C10(L>R)-EGFP <400> 63 ctagctagca agaggctgag gctacggcgc tgtcatggat ccgtgagcaa gggcgag 57 <210> 64 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of C10(L>R)-EGFP <400> 64 cccaagcttt tattacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac 60 gaactccag 69 <210> 65 <211> 795 <21 2 > DNA <213> Artificial Sequence <220> <223> C10(L>R)-EGFP <400> 65 atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcaagaggct gaggctacgg 60 cgctgtcatc gaggatccgt gagcaaggc gaggagctgt tcaccggggt ggtgcccatc 120 ctggtc gagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 180 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 240 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 300 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 360 gagcgcacca tcttcttcaa ggacgacggc aactac aaga cccgcgccga ggtgaagttc 420 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 480 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 540 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 600 agcgtgcagc tcgccgacca ctaccagcag aacacccca tcggcgacgg ccccgtgctg 660 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 720 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 780 gagctgtaca agtaa 795 <210> 66 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of C10(L>K )-EGFP <400> 66 ctagctagcc ggaggctgag gctacggcgc tgtcatggat ccgtgagcaa gggcgag 57 <210> 67 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of C10(L>K)-EGFP <400 > 67 cccaagcttt tattacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac 60 gaactccag 69 <210> 68 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> C10(L>K)-EGFP <400> 68 atgcggggt t ctcatcatca tcatcatcat ggtatggcta gccggaggct gaggctacgg 60 cgctgtcatc gaggatccgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 120 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 180 ggcgatgcca cctacggcaa gctgaccctg aagt tcatct gcaccaccgg caagctgccc 240 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 300 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 360 gagcgcacca tcttcttcaa ggacgacggc a actacaaga cccgcgccga ggtgaagttc 420 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 480 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 540 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 600 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg cccc gtgctg 660 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 720 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 780gagctgtaca agtaa 795

Claims (11)

A cell-penetrating peptide represented by any one selected from SEQ ID NOs: 1 to 7, 10, and 11; and a peptide consisting of the LRR domain region represented by SEQ ID NO: 25. A pharmaceutical composition for the prevention or treatment of sepsis containing a fusion protein as an active ingredient. delete delete According to paragraph 1,
A composition for preventing or treating sepsis, wherein the cell-penetrating peptide is any one selected from the amino acid sequences represented by SEQ ID NOs: 1 to 7. According to paragraph 1,
A composition for preventing or treating sepsis, wherein the cell-penetrating peptide has an amino acid sequence represented by SEQ ID NO: 1. According to paragraph 1,
A pharmaceutical composition for the prevention or treatment of sepsis, wherein the fusion protein consists of any one selected from the amino acid sequences represented by SEQ ID NOs: 32, 36, and 40. According to paragraph 1,
The fusion protein is a pharmaceutical composition for preventing or treating sepsis, characterized in that it consists of the amino acid sequence represented by SEQ ID NO: 32. According to paragraph 1,
A pharmaceutical composition for the prevention or treatment of sepsis, characterized in that the fusion protein is delivered to 1.5 to 2 times the number of macrophages compared to total immune cells (total dendritic cells). According to paragraph 1,
Sepsis, characterized in that the fusion protein acts specifically on macrophages compared to immune cells to inhibit the activation of the inflammation regulatory complex pathway of NF-κB signaling and inflammasome signaling, thereby suppressing the hyper-inflammatory response of macrophages. Pharmaceutical composition for prevention or treatment of. According to paragraph 1,
A pharmaceutical composition for preventing or treating sepsis, wherein the sepsis is sepsis caused by Gram-negative bacteria. According to paragraph 1,
A pharmaceutical composition for preventing or treating sepsis, characterized in that the sepsis is derived from LPS constituting the cell membrane of Gram-negative bacteria.