WO2016013828A1 - Extracellular domain 1 mutant of sulfated duffy chemokine receptor, and use thereof - Google Patents

Extracellular domain 1 mutant of sulfated duffy chemokine receptor, and use thereof Download PDF

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WO2016013828A1
WO2016013828A1 PCT/KR2015/007510 KR2015007510W WO2016013828A1 WO 2016013828 A1 WO2016013828 A1 WO 2016013828A1 KR 2015007510 W KR2015007510 W KR 2015007510W WO 2016013828 A1 WO2016013828 A1 WO 2016013828A1
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variant
disease
tyrosine
pharmaceutical composition
extracellular domain
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French (fr)
Korean (ko)
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이형근
이준행
변석호
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연세대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons

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  • the present invention relates to extracellular domain 1 variants of one or more tyrosine sulfated Duffy chemokine receptors and uses thereof.
  • Chemokines is a relatively small secretory protein with a size of 8 to 15 Kd and collectively refers to substances causing proliferation, migration and activation of blood cells such as lymphocytes and neutrophils and vascular endothelial cells. About 40 chemokines have been found and they are known to exhibit activity through their respective receptors. Chemokines are known to be involved in the development and progression of many diseases, such as autoimmune diseases (eg, RA, SLE, Sjogren), and autoinflammatory diseases (crohn's Ds). It is known to be related. In addition, fatal rejection in organ or tissue transplantation is also mediated by chemokines. Accordingly, research is being actively conducted to develop various kinds of inhibitors capable of inhibiting chemokine activity.
  • autoimmune diseases eg, RA, SLE, Sjogren
  • autoinflammatory diseases crohn's Ds
  • DARCs Duffy chemokine receptors
  • DARCs are also known as decoy receptors, which are mainly present in vascular endothelial cells and are involved in the regulation of inflammatory responses, angiogenesis, etc. (Blood. (2010) 115 (26): 5289-5299).
  • Angiogenesis is an exaggeration that must be involved in the wound repair process, which is essential for the rejuvenation of wounded tissue.
  • cells die and inflammatory reactions occur due to the destruction of blood vessels.
  • devascularization of blood components activation of platelets, blood coagulation, biological media such as kallikrein, thrombin, plasmin, etc. It goes through a series of processes called formation.
  • new tissue is formed at the site of the wound, and the synthesis and reconstitution of the substrate of the cell occurs, so that the wounded tissue is healed to function again.
  • a complex cascade reaction of endothelial cells, inflammatory cells, or immune-related granulocytes, mast cells, and platelets is performed, and a defense mechanism that inhibits foreign body penetration into the wound site is performed.
  • epidermal cells, macrophages, endothelial cells and fibroblasts secrete various growth factors to promote cell division for the formation of new tissues, and at the same time epidermal cells, endothelial cells,
  • the reorganization of the cell matrix occurs by the matrix proteins of various cells secreted by fibroblasts, thereby completing wound healing and forming new tissues at the wound site.
  • An important phenomenon that accompanies this complex wound healing process is the formation of new blood vessels.
  • Blood vessel formation plays a role in helping the tissues regenerate and function normally as new tissues are formed during wound healing, supplying appropriate oxygen and nutrients to these tissues.
  • Such agents that can promote angiogenesis play a very important role in the postoperative recovery process in various fields such as plastic surgery and cardiology, which are in charge of surgery in addition to wound healing. Development of a formulation for the situation is not active.
  • the inventors of the present invention have completed the present invention by using an Duffy chemokine receptor having high binding strength with chemokines, and trying to develop an accelerator capable of controlling the inflammatory response and simultaneously promoting blood vessel formation.
  • the present invention has been made to solve the above-mentioned problems in the prior art, to provide an extracellular domain 1 variant of the sulfated Duffy chemokine receptor that can regulate the inflammatory response and at the same time promote the formation of blood vessels It is done.
  • angiogenesis refers to both a series of processes in which new blood vessels are made and / or a series of processes in which existing blood vessels are extended, and migration of endothelial cells constituting blood vessels It may proceed through the process of invasion through the extracellular matrix (ECM), an intercellular barrier, proliferation, differentiation into blood vessels (tube production), and the like.
  • ECM extracellular matrix
  • endothelial cell refers to all cells that make up the endothelium aligned on the inner surface of blood vessels, which may include capillaries, coronary vessels, endocardium, veins, arterial vessels, etc. If it is a cell constituting is not limited thereto.
  • wound means any damage to which skin, tissue, organs, etc., are torn, penetrated, cut, cracked, or ruptured as a result of a disease, accident, surgery, or the like.
  • the present invention provides an extracellular domain 1 variant of the duffy chemokine receptor, wherein 1-3 tyrosine of the extracellular domain 1 of the Duffy chemokine receptor is sulfated.
  • the extracellular domain 1 of the Duffy chemokine receptor may include the amino acid sequence of SEQ ID NO: 1, but is not limited to the extracellular domain 1 of the Duffy chemokine receptor.
  • the tyrosine may be the 6th, 32nd, or 43rd amino acid, or may be a combination of two or more thereof, but is not limited thereto.
  • the present invention also provides a pharmaceutical composition comprising an extracellular domain 1 variant of the Duffy chemokine receptor.
  • the pharmaceutical composition may be used for promoting angiogenesis, for treating inflammatory diseases, for treating wounds, and the like.
  • the pharmaceutical composition for promoting angiogenesis is a non-union of bone. It can be used to treat a variety of wounds such as malunion, skin wound defects, and ischemic diseases.
  • the skin defect may be caused by diabetes, ischemic disease, Burger's disease, etc.
  • the ischemic disease may include, but is not limited to, myocardial infarction and stroke.
  • the pharmaceutical composition for treating inflammatory diseases may be used for various diseases such as asthma, atherosclerosis, glomerulonephritis, pancreatitis, restenosis, rheumatoid arthritis, diabetic nephropathy, pulmonary fibrosis, inflammatory bowel disease, Crohn's disease, transplant rejection, etc. If the disease can be treated through the regulation of angiogenesis and / or inflammatory response is not limited thereto.
  • the inflammatory diseases are ischemic lesions caused by embolisms such as chronic skin ulcer and pressure sore, cellulitis, atherosclerotic lesions (ischemic disease from atherosclerotic lesions), blood clots, etc.
  • ischemic disease from embolic lesions ischemic brain disease and lesions, including stroke, visceral ischemic syndrome, ocular ischemic syndrome in the eye or retina, Peripheral vascular disease (peripheral vascular disease, peripheral artery occlusive disease, peripheral obliterative arteriopathy), diabetic vasculopathy.
  • it can be used for adjuvant therapy for cellulitis, or for adjuvant and / or postoperative management for skin transplantation, for skin transplantation in deep burn patients.
  • the pharmaceutical composition may be characterized in that the capsule, tablets, granules, injections, ointments, powder or beverage form, the pharmaceutical composition may be characterized in that it is intended for humans.
  • Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal. Oral or parenteral release is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.
  • compositions of the present invention may be used in the form of oral dosage forms, such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.
  • the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc.
  • buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used.
  • the formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms. have. And others, solutions, suspensions, tablets, capsules, sustained release preparations and the like.
  • suitable carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.
  • the pharmaceutical compositions of the present invention vary depending on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed.
  • the dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, and 0.0001 to 50 mg / kg or It may be administered at 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • the pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.
  • the present invention also provides an angiogenesis treatment method, an inflammatory disease treatment method, a wound treatment method, etc. using the extracellular domain 1 variant of the Duffy chemokine receptor.
  • the extracellular domain 1 variant of one or more tyrosine sulfated Duffy chemokine receptors can regulate the inflammatory response and promote blood vessel formation, so plastic surgery, cardiology, etc. It is expected to be widely used in the field.
  • Figure 2 shows the results of electrophoresis confirming that the variant DNA of the extracellular domain 1 of the Duffy chemokine receptor is inserted into the pET41a vector normally according to an embodiment of the present invention.
  • Figure 3 shows the results confirmed by SDS-PAGE that the extracellular domain 1 of the Duffy chemokine receptor normally sulfated according to an embodiment of the present invention.
  • Figure 4 is a result of confirming the inflammatory response control capacity and blood vessel formation promoting ability of the extracellular domain 1 variant of Duffy chemokine receptor according to an embodiment of the present invention.
  • Extracellular domain 1 of the duffy chemokine receptor SEQ ID NO: 1
  • primers were synthesized to sulfate each of the three tyrosine groups present in MASSGYVLQAELSPSTENSSQLDFEDVWNSSYGVNDSFPDGDYDANLEAAAPCHSCLEAAAPCHSC). Primer sequences are shown in Table 1 below.
  • variant 1 (DaMT1) of the first position tyrosine (sixth amino acid) of the extracellular domain 1 of the Duffy chemokine receptor
  • 1.1F, 1.1R, 1.2F, 1.2R, 1.3F, 1.3R, 1.4F, 1.4R , 1.5F, and 1.5R primer sets were used and 1.1F, 1.1R, 1.2F, 1.2R, 1.3F, 1.3R were prepared to prepare variant 2 (DaMT2) of the first and second position tyrosine (32th amino acid).
  • Each primer was prepared by mixing at a concentration of 10 pmole, polymerase chain reaction was performed by mixing 5uL of dNTP, 5uL of 10X Taq buffer, 0.5uL of Ex Taq, and 35.5uL of tertiary distilled water to 4uL of the mixed primers.
  • the polymerase chain reaction was performed after the first DNA denaturation step (94 ° C., 5 minutes), 30 seconds at 94 ° C. (DNA denaturation step), 30 seconds at 52 ° C. (DNA annealing step), and 30 seconds at 72 ° C. (DNA extension). ) was repeated 55 times and reacted at 72 ° C. for 5 minutes.
  • Secondary polymerase chain reaction was performed using DNA amplified by the overlapping polymerase chain reaction as a template. Secondary polymerase chain reaction was performed by amplifying DNA 1.25uL, 1.1p primer 10pmol 0.5uL, 1.1R primer 10pmol 0.5uL, dNTP 5uL, 5X band 10uL, 10X taq buffer 5uL, Ex Taq 0.5uL, and tertiary distilled water 27.25uL Mix, and after the first DNA denaturation step (94 ° C., 5 minutes), 30 seconds at 94 ° C. (DNA denaturation step), 30 seconds at 50 ° C. (DNA annealing step), and 30 seconds at 72 ° C. (DNA extension) was repeated 23 times and reacted at 72 ° C. for 5 minutes.
  • first DNA denaturation step 94 ° C., 5 minutes
  • 30 seconds at 94 ° C. DNA denaturation step
  • 30 seconds at 50 ° C. DNA annealing step
  • Amplified DNA was checked for DNA size by 1.5% agarose gel electrophoresis (agarosegel electophoresis). After gel extraction (gel extraction) to confirm DNA sequence, it was inserted into pGEM T easy vector and transformed into Escherichia coli DH5a, and then the amplified vector was extracted to check whether the amplified DNA was inserted by electrophoresis. It was. The results are shown in FIG.
  • DNA sequencing was performed using a vector in which the amplified DNA was inserted. As a result of sequencing analysis, it was confirmed that a total of three variants in which the TAT sequence of each tyrosine position was converted to the TAG sequence was normally prepared.
  • Variant DNAs of the extracellular domain 1 of the three Duffy chemokine receptors prepared by the method of Example 1.2 were inserted into expression vectors (pET41a) containing the GST-His tag, respectively, and their size was confirmed by electrophoresis. The results are shown in FIG. The produced vector was finally transformed into E. coli BL21.
  • the pET41a vector into which each variant DNA was inserted was transformed into E. coli BL21, and the pSUPAR6-L3-3SY vector was further transformed into the transformed E. coli BL21.
  • the pSUPAR6-L3-3SY vector was available from Scripps Laboratories.
  • the transformed E. coli was inoculated in 5 mL of 2YT liquid medium, pre-incubated for 18 hours in a 37 ° C shaking incubator, inoculated to 1% in YT liquid medium containing 10 mM sulfotyrosine and shaken at 37 ° C. Main culture was incubator. When the OD600nm value reached 1.0, IPTG was added so as to have a final concentration of 1 mM and incubated at 30 ° C. for 20 hours.
  • the cultured cells were centrifuged (13,000 g, 1 minute) to remove the supernatant, and the cells from which the supernatant was removed were subjected to cell resuspensin buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.1% Triton X-100). 1 mL was added and mixed well. And sonication (breaking 30 seconds per 1ml buffer and 2 minutes rest twice) to break the cells, centrifugation (14,000g, 20 minutes) to recover only the supernatant containing protein. Proteins were separated from the supernatant recovered using His column. The isolated protein was quantified and confirmed by electrophoresis using 10% SDS-PAGE. As a control, extracellular domain 1 (DARCex1) of the Duffy chemokine receptor, which was not sulfated, was used. The results are shown in FIG.
  • Example 3 Duffy Chemokines Extracellular domain of receptor 1 Variant Inflammatory response Controllability And blood vessel formation promoting ability
  • Administration was at a concentration of 20 ug / mL.
  • the control group (FIG. 4A) administered only with GST produced little infiltration of inflammatory cells and formation of neovascularization
  • the experimental group administered with variants (FIG. 4B) infiltrated inflammatory cells at the lower right corner.
  • This increased brightly observed confirmed that the increased blood vessel formation at the top.
  • the experimental group (FIG. 4D) the formation of new blood vessels is increased, and the infiltration of inflammatory cells is increased, thereby increasing the cornea. It became cloudy and confirmed that it was dark overall.
  • Example 3.1 a separation cornea in a manner of using the easy-spinTM Total RNA extraction kit (iNtRON) to extract RNA, and, FastAQ TM One-Step SYBR Green qRT-PCR Kit (Excellgen) using a vascular endothelial growth factor Quantitative PCR (qPCR) of vascular endothelial growth factor A and vascular endothelial growth factor C was performed.
  • the results are shown in FIG.
  • the extracellular domain 1 variants of the sulfated Duffy chemokine receptor are easy to penetrate into vascular cells or inflammatory cells, and therefore, do not directly stimulate blood vessels, but after binding to chemokines to help the formation of blood vessels and / or blood vessels. Infiltrating into inflammatory cells increases the concentration of chemokines within the cells, thereby promoting the formation of new blood vessels, as well as controlling the inflammatory response. In addition, it was confirmed that the formation of new blood vessels was more actively promoted in the case of DaMT2 and DaMT3, which are complex gene variants than DaMT1, which is a single gene variant, through which the cell penetration and / or chemokine binding capacity of the complex gene was increased. I could confirm that.
  • the extracellular domain 1 variants of sulfated Duffy chemokine receptors produce blood vessels such as non-union of bone, malunion, skin wound defects and ischemic disease. It can be found that it can be widely used for various diseases that are needed, can be used for treating various wounds, and can be used to prevent or treat inflammatory diseases because it can control inflammatory diseases.
  • the extracellular domain 1 variant of one or more tyrosine sulfated Duffy chemokine receptors can regulate the inflammatory response and promote blood vessel formation, so plastic surgery, cardiology, etc. It is expected to be widely used in the field.
  • Extracellular domain 1 of duffy chemokine receptor Extracellular domain 1 of duffy chemokine receptor:
  • SEQ ID NO: 2 (1.1F): AAA GGA TCC GCC TCC TCT GGG TAG GTC CTC CAG GCG GAG
  • SEQ ID NO: 3 (1.2F): CTC TCC CCC TCA ACT GAG AAC TCA AGT CAG CTG GAC TTC
  • SEQ ID NO: 4 (1.3F): GAA GAT GTA TGG AAT TCT TCC TAT GGT GTG AAT GAT TCC
  • SEQ ID NO: 5 (1.4F): TTC CCA GAT GGA GAC TAT GGT GCC AAC CTG GAA GCA GCT
  • SEQ ID NO: 6 (1.5F): GCC CCC TGC CAC TCC TGT CTC GAG AAA AAA AAA AAA AAA AAA AAA AAA AAA;
  • SEQ ID NO: 7 (1.1R): TTT TTT CTC GAG ACA GGA GTG GCA GGG GGC AGC TGC TTC
  • SEQ ID NO: 8 (1.2R): CAG GTT GGC ACC ATA GTC TCC ATC TGG GAA GGA ATC ATT
  • SEQ ID NO: 9 (1.3R): CAC ACC ATA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAG
  • SEQ ID NO: 10 (1.4R): CTG ACT TGA GTT CTC AGT TGA GGG GGA GAG CTC CGC CTG
  • SEQ ID NO: 11 (1.5R): GAG GAC CTA CCC AGA GGA GGC GGA TCC TTT TTT TTT TTT TTT TTT
  • SEQ ID NO: 12 (2.1F): GAA GAT GTA TGG AAT TCT TCC TAG GGT GTG AAT GAT TCC
  • SEQ ID NO: 13 (2.1R): CAC ACC CTA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAG
  • SEQ ID NO: 14 (3.1F): TTC CCA GAT GGA GAC TAG GGT GCC AAC CTG GAA GCA GCT
  • SEQ ID NO: 15 (3.1R): CAG GTT GGC ACC CTA GTC TCC ATC TGG GAA GGA ATC ATT

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Abstract

The present invention relates to: an extracellular domain 1 mutant of a duffy chemokine receptor in which at least one tyrosine is sulfated; and a use thereof, has high cell penetration property and simultaneously has high binding property to chemokine, and thus can be widely used for effectively regulating inflammatory response and/or promoting angiogenesis.

Description

황산화된 더피 케모카인 수용체의 세포 외 도메인 1 변이체 및 이의 용도 Extracellular Domain 1 Variants of Sulfated Duffy Chemokine Receptors and Uses thereof
본 발명은 하나 이상의 타이로신이 황산화된 더피 케모카인 수용체의 세포 외 도메인 1 변이체 및 이의 용도에 관한 것이다.The present invention relates to extracellular domain 1 variants of one or more tyrosine sulfated Duffy chemokine receptors and uses thereof.
케모카인(Chemokine)은 크기가 8 내지 15 Kd의 비교적 작은 분비성 단백질로서 임파구, 중성구 등의 혈액세포와 혈관내피세포의 증식, 이동, 활성화를 일으키는 물질들을 통칭한다. 케모카인은 현재 약 40 여종이 발견되어 있으며 이들은 각각의 수용체를 통해 활성을 나타내는 것으로 알려져 있다. 케모카인은 여러 자가면역질환(e.g., RA, SLE, Sjogren), 자가 염증성질환(ulcerative colitis, Crohn's Ds)과 같은 많은 질병의 발생과 진행에 관여하는 것으로 알려져 있으며, 이 외에도 당뇨나 동맥경화의 발생에도 관련된 것으로 알려져 있다. 또한, 장기나 조직의 이식에 있어서 치명적인 거부반응도 케모카인에 의해 매개된다. 이에 따라 케모카인의 활성을 억제할 수 있는 다양한 종류의 억제제를 개발하기 위한 연구가 활발히 진행되고 있는 실정이다. Chemokines (Chemokine) is a relatively small secretory protein with a size of 8 to 15 Kd and collectively refers to substances causing proliferation, migration and activation of blood cells such as lymphocytes and neutrophils and vascular endothelial cells. About 40 chemokines have been found and they are known to exhibit activity through their respective receptors. Chemokines are known to be involved in the development and progression of many diseases, such as autoimmune diseases (eg, RA, SLE, Sjogren), and autoinflammatory diseases (crohn's Ds). It is known to be related. In addition, fatal rejection in organ or tissue transplantation is also mediated by chemokines. Accordingly, research is being actively conducted to develop various kinds of inhibitors capable of inhibiting chemokine activity.
한편, 우리의 몸은 케모카인의 과도한 활성을 조절하는 디코이 수용체(natural decoy receptor)들을 가지고 있다. 디코이 수용체는 고친화력을 가지고 있어 케모카인들과 강하게 결합하지만, 다른 케모카인 수용체들과 다르게 신호 전달의 기능이 없어 일반적으로 케모카인 고유의 활성을 중화(neutralization) 또는 억제(inhibition)하는 역할을 하는 것으로 알려져 있다. 더피 케모카인 수용체(duffy chemokine receptor, DARC) 역시 이러한 디코이 수용체의 일종으로 주로 혈관 내피 세포에 존재하며, 염증 반응의 조절, 혈관 형성 등에 관여하고 있는 것으로 알려져 있다(Blood. (2010) 115(26): 5289-5299).On the other hand, our bodies have natural decoy receptors that regulate the excess activity of chemokines. Decoy receptors have high affinity and bind strongly to chemokines, but unlike other chemokine receptors, decoy receptors are generally known to play a role in neutralizing or inhibiting chemokine-specific activity. . Duffy chemokine receptors (DARCs) are also known as decoy receptors, which are mainly present in vascular endothelial cells and are involved in the regulation of inflammatory responses, angiogenesis, etc. (Blood. (2010) 115 (26): 5289-5299).
한편, 혈관 형성은 상처받은 피부조직이 재생되기 위해서 필수적인 상처회복(wound repair) 과정에 반드시 수반되어야 하는 과장이다. 상처의 초기 단계에는 세포가 죽고 혈관의 파괴로 인한 염증반응이 일어나게 되며, 염증반응 이후에 혈액성분의 탈혈관 현상, 혈소판의 활성화, 혈액응고, 칼리크레인, 트롬빈, 플라스민 등과 같은 생물학적 매개물질의 형성이라는 일련의 과정을 거치게 된다. 결국 상처받은 부위에서 새로운 조직이 형성되고 세포의 기질의 합성과 재구성이 일어나 상처 입은 조직은 재기능을 할 수 있도록 치유되는 것이다. 염증의 과정을 살펴보면 내피세포, 염증세포, 또는 면역에 관여하는 과립구, 비만세포 및 혈소판 등의 복잡한 폭상반응(cascade reaction)을 일으키며 상처부위에의 이물질 침투를 억제하는 방어기작을 수행한다. 이러한 면역학적인 방어기작과 함께 표피세포, 대식세포, 내피세포, 섬유아세포 등이 여러 종류의 성장인자를 분비하여 새로운 조직의 형성을 위한 세포분열의 촉진이 일어나고, 그와 동시에 표피세포, 내피세포, 섬유아세포 등이 분비하는 여러 세포의 기질 단백질에 의한 세포 기질의 재구성이 일어나서 상처의 치유가 완성되고 새로운 조직이 상처부위에 형성된다. 이러한 복잡한 상처치유의 과정과 함께 수반되는 중요한 현상이 신생 혈관의 형성이다. 혈관 형성은 상처치유 과정 중에 새로운 조직이 형성되면서 이러한 조직에 적당한 산소와 영양분을 공급해 주어 그 조직이 정상적인 신체의 일부로서 재생이 되고, 정상적으로 기능을 수행할 수 있도록 도와주는 역할을 담당한다. 이와 같이 혈관 형성을 촉진할 수 있는 제제는 상처 치료 외에도 혈관 형성을 촉진하는 것을 외과 수술을 담당하고 있는 성형외과, 심장내과 등 다양한 분야에서 수술 후 회복 과정에 매우 중요한 역할을 담당하나, 혈관 형성 촉진용 제제의 개발은 활발히 이루어지고 있지 않은 실정이다.Angiogenesis, on the other hand, is an exaggeration that must be involved in the wound repair process, which is essential for the rejuvenation of wounded tissue. In the early stages of the wound, cells die and inflammatory reactions occur due to the destruction of blood vessels. After inflammatory reactions, devascularization of blood components, activation of platelets, blood coagulation, biological media such as kallikrein, thrombin, plasmin, etc. It goes through a series of processes called formation. Eventually, new tissue is formed at the site of the wound, and the synthesis and reconstitution of the substrate of the cell occurs, so that the wounded tissue is healed to function again. In the process of inflammation, a complex cascade reaction of endothelial cells, inflammatory cells, or immune-related granulocytes, mast cells, and platelets is performed, and a defense mechanism that inhibits foreign body penetration into the wound site is performed. With these immunological defense mechanisms, epidermal cells, macrophages, endothelial cells and fibroblasts secrete various growth factors to promote cell division for the formation of new tissues, and at the same time epidermal cells, endothelial cells, The reorganization of the cell matrix occurs by the matrix proteins of various cells secreted by fibroblasts, thereby completing wound healing and forming new tissues at the wound site. An important phenomenon that accompanies this complex wound healing process is the formation of new blood vessels. Blood vessel formation plays a role in helping the tissues regenerate and function normally as new tissues are formed during wound healing, supplying appropriate oxygen and nutrients to these tissues. Such agents that can promote angiogenesis play a very important role in the postoperative recovery process in various fields such as plastic surgery and cardiology, which are in charge of surgery in addition to wound healing. Development of a formulation for the situation is not active.
이에 본 발명자들은 케모카인과의 결합력이 높은 더피 케모카인 수용체를 이용하여, 염증 반응을 조절하며, 동시에 혈관 형성을 촉진시킬 수 있는 촉진제를 개발하고자 노력한 결과 본 발명을 완성하게 되었다.Therefore, the inventors of the present invention have completed the present invention by using an Duffy chemokine receptor having high binding strength with chemokines, and trying to develop an accelerator capable of controlling the inflammatory response and simultaneously promoting blood vessel formation.
본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위해 안출된 것으로, 염증 반응을 조절하며, 동시에 혈관 형성을 촉진시킬 수 있는 황산화된 더피 케모카인 수용체의 세포 외 도메인 1 변이체를 제공하는 것을 그 목적으로 한다.The present invention has been made to solve the above-mentioned problems in the prior art, to provide an extracellular domain 1 variant of the sulfated Duffy chemokine receptor that can regulate the inflammatory response and at the same time promote the formation of blood vessels It is done.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
이하, 본원에 기재된 다양한 구체예가 도면을 참조로 기재된다. 하기 설명에서, 본 발명의 완전한 이해를 위해서, 다양한 특이적 상세사항, 예컨대, 특이적 형태, 조성물, 및 공정 등이 기재되어 있다. 그러나, 특정의 구체예는 이들 특이적 상세 사항 중 하나 이상 없이, 또는 다른 공지된 방법 및 형태와 함께 실행될 수 있다. 다른 예에서, 공지된 공정 및 제조 기술은 본 발명을 불필요하게 모호하게 하지 않게 하기 위해서, 특정의 상세사항으로 기재되지 않는다. "한 가지 구체예" 또는 "구체예"에 대한 본 명세서 전체를 통한 참조는 구체예와 결부되어 기재된 특별한 특징, 형태, 조성 또는 특성이 본 발명의 하나 이상의 구체예에 포함됨을 의미한다. 따라서, 본 명세서 전체에 걸친 다양한 위치에서 표현 "한 가지 구체예에서" 또는 "구체예"의 상황은 반드시 본 발명의 동일한 구체예를 나타내지는 않는다. 추가로, 특별한 특징, 형태, 조성, 또는 특성은 하나 이상의 구체예에서 어떠한 적합한 방법으로 조합될 수 있다.Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the present invention, various specific details are described, such as specific forms, compositions, processes and the like. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the context of the expression “in one embodiment” or “embodiment” in various places throughout this specification does not necessarily represent the same embodiment of the invention. In addition, particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.
본 명세서에서 있어서, "혈관 형성(angiogenesis)"이란 새로운 혈관이 만들어지는 일련의 과정 및/또는 기존의 혈관이 연장되는 일련의 과정을 모두 의미하며, 혈관을 구성하고 있는 내피세포의 이동(migration), 세포간 장벽인 세포외기질(ECM)를 통과하는 침윤(invasion), 증식(proliferation), 혈관으로의 분화(튜브 생성)의 과정 등을 통하여 진행될 수 있다.In the present specification, "angiogenesis" refers to both a series of processes in which new blood vessels are made and / or a series of processes in which existing blood vessels are extended, and migration of endothelial cells constituting blood vessels It may proceed through the process of invasion through the extracellular matrix (ECM), an intercellular barrier, proliferation, differentiation into blood vessels (tube production), and the like.
본 명세서에서 있어서, "내피 세포"란 혈관의 내부 표면에 정렬된 내피를 구성하는 모든 세포를 의미하며, 여기에는 심장의 모세혈관, 관상혈관, 내막, 정맥, 동맥 혈관 등이 포함될 수 있으며, 내피를 구성하는 세포라면 이에 제한되지 않는다.As used herein, "endothelial cell" refers to all cells that make up the endothelium aligned on the inner surface of blood vessels, which may include capillaries, coronary vessels, endocardium, veins, arterial vessels, etc. If it is a cell constituting is not limited thereto.
본 명세서에 있어서, "상처"란 피부, 조직, 장기 등이 질병, 사고, 수술 등의 결과로서 찢어지거나, 관통되거나, 절단되거나, 갈라지거나 또는 파열되는 모든 손상을 의미한다.As used herein, "wound" means any damage to which skin, tissue, organs, etc., are torn, penetrated, cut, cracked, or ruptured as a result of a disease, accident, surgery, or the like.
본 발명은 더피 케모카인 수용체의 세포 외 도메인 1의 타이로신이 1 내지 3개가 황산화된, 더피 케모카인 수용체의 세포 외 도메인 1 변이체를 제공한다.The present invention provides an extracellular domain 1 variant of the duffy chemokine receptor, wherein 1-3 tyrosine of the extracellular domain 1 of the Duffy chemokine receptor is sulfated.
본 발명의 일 구체예에 있어서, 상기 더피 케모카인 수용체의 세포 외 도메인 1은 서열번호 1의 아미노산 서열을 포함할 수 있으나, 더피 케모카인 수용체의 세포 외 도메인 1이라면 이에 제한되지 않는다.In one embodiment of the present invention, the extracellular domain 1 of the Duffy chemokine receptor may include the amino acid sequence of SEQ ID NO: 1, but is not limited to the extracellular domain 1 of the Duffy chemokine receptor.
본 발명의 다른 구체예에 있어서, 상기 타이로신은 6번째, 32번째, 또는 43번째 아미노산일 수 있고, 또는 이들의 둘 이상의 조합일 수 있으나, 이에 제한되지 않는다.In another embodiment of the present invention, the tyrosine may be the 6th, 32nd, or 43rd amino acid, or may be a combination of two or more thereof, but is not limited thereto.
또한, 본 발명은 상기 더피 케모카인 수용체의 세포 외 도메인 1 변이체를 포함하는 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition comprising an extracellular domain 1 variant of the Duffy chemokine receptor.
본 발명의 일 구체예에 있어서, 상기 약학 조성물은 혈관 형성 촉진용, 염증성 질환 치료용, 상처 치료용 등으로 사용될 수 있으며, 상기 혈관 형성 촉진용 약학 조성물은 골의 위관절(Non-union of bone), 부전유합(Malunion), 피부결손상처(Skin wound defect), 허혈성 질환(ischemic disease) 등 다양한 상처 치료에 사용될 수 있다. 상기 피부결손상처는 당뇨병, 허혈성 질환, 버거씨 병 등으로 인해 유발될 수 있으며, 상기 허혈성 질환에는 심근경색, 뇌졸증 등이 포함될 수 있으나, 이에 제한되지 않는다. 상기 염증성 질환 치료용 약학 조성물은 천식, 죽상경화증, 사구체신염, 췌장염, 재협착, 류마티스성 관절염, 당뇨병성 신병증, 폐섬유증, 염증성 장질환, 크론씨 질환, 이식 거부반응 등 다양한 질환에 사용될 수 있으나, 혈관 형성 및/또는 염증 반응의 조절을 통하여 치료할 수 있는 질병이라면 이에 제한되지 않는다. 또한, 상기 염증성 질환은 만성 피부궤양(chronic skin ulcer and pressure sore), 봉와직염(cellulitis), 혈관경화성병변(atherosclerotic lesion)에 의한 허혈성질환(ischemic disease from atherosclerotic lesion), 혈전 등의 색전에 의한 허혈성병변(ischemic disease from embolic lesion), 뇌졸증을 포함하는 허혈성뇌질환 및 병변(ischemic brain disease and lesion, including stroke), 장허혈증후군(visceral ischemic syndrome), 안구나 망막에 생기는 허혈성질환(ocular ischemic syndrome), 말초혈관성질환(peripheral vascular disease, peripheral artery occlusive disease, peripheral obliterative arteriopathy), 당뇨성혈관질환(diabetic vasculopathy)이다. 이 외에도 봉와직염(cellulitis)의 보조치료(adjuvant therapy for cellulitis), 심부화상환자에서의 피부(skin)이식 후 혈관신생 촉진이 필요한 경우(adjuvant and/or postoperative management for skin transplantation) 등에도 사용가능하다.In one embodiment of the present invention, the pharmaceutical composition may be used for promoting angiogenesis, for treating inflammatory diseases, for treating wounds, and the like. The pharmaceutical composition for promoting angiogenesis is a non-union of bone. It can be used to treat a variety of wounds such as malunion, skin wound defects, and ischemic diseases. The skin defect may be caused by diabetes, ischemic disease, Burger's disease, etc. The ischemic disease may include, but is not limited to, myocardial infarction and stroke. The pharmaceutical composition for treating inflammatory diseases may be used for various diseases such as asthma, atherosclerosis, glomerulonephritis, pancreatitis, restenosis, rheumatoid arthritis, diabetic nephropathy, pulmonary fibrosis, inflammatory bowel disease, Crohn's disease, transplant rejection, etc. If the disease can be treated through the regulation of angiogenesis and / or inflammatory response is not limited thereto. In addition, the inflammatory diseases are ischemic lesions caused by embolisms such as chronic skin ulcer and pressure sore, cellulitis, atherosclerotic lesions (ischemic disease from atherosclerotic lesions), blood clots, etc. (ischemic disease from embolic lesions), ischemic brain disease and lesions, including stroke, visceral ischemic syndrome, ocular ischemic syndrome in the eye or retina, Peripheral vascular disease (peripheral vascular disease, peripheral artery occlusive disease, peripheral obliterative arteriopathy), diabetic vasculopathy. In addition, it can be used for adjuvant therapy for cellulitis, or for adjuvant and / or postoperative management for skin transplantation, for skin transplantation in deep burn patients.
본 발명에 있어서 상기 약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다.In the present invention, the pharmaceutical composition may be characterized in that the capsule, tablets, granules, injections, ointments, powder or beverage form, the pharmaceutical composition may be characterized in that it is intended for humans.
본 발명에 따른 약학 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. 경구 또는 비경구 투하가 바람직하다. 본원에 사용된 용어 "비경구"는 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골내 주사 또는 주입기술을 포함한다. 본 발명의 약학 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal. Oral or parenteral release is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.
본 발명의 약학 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명의 약학 조성물은 약제적으로 허용가능한 담체를 포함할 수 있다. 약제학적으로 허용되는 담체는 경구투여시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명의 약제학적 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구투여시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 기타, 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제형할 수 있다.The pharmaceutical compositions of the present invention may be used in the form of oral dosage forms, such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. have. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. In the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms. have. And others, solutions, suspensions, tablets, capsules, sustained release preparations and the like.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.Examples of suitable carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.
본 발명의 약학 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약무형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50mg/kg 또는 0.001 내지 50mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 의약 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형될 수 있다.The pharmaceutical compositions of the present invention vary depending on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed. The dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, and 0.0001 to 50 mg / kg or It may be administered at 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.
또한, 본 발명은 상기 더피 케모카인 수용체의 세포 외 도메인 1 변이체를 이용한 혈관 형성 치료 방법, 염증성 질환 치료 방법, 상처 치료 방법 등을 제공한다.The present invention also provides an angiogenesis treatment method, an inflammatory disease treatment method, a wound treatment method, etc. using the extracellular domain 1 variant of the Duffy chemokine receptor.
본 발명에 따른 하나 이상의 타이로신이 황산화된 더피 케모카인 수용체의 세포 외 도메인 1 변이체는 염증 반응을 조절하는 동시에 혈관 형성을 촉진시킬 수 있기 때문에 혈관 형성의 촉진이 필요한 분야인 성형외과, 심장내과 등의 분야에 폭넓게 사용될 수 있을 것으로 기대된다.The extracellular domain 1 variant of one or more tyrosine sulfated Duffy chemokine receptors according to the present invention can regulate the inflammatory response and promote blood vessel formation, so plastic surgery, cardiology, etc. It is expected to be widely used in the field.
도 1은 본 발명의 일 실시예에 따른 오버래핑 중합효소 연쇄반응에 의하여 증폭된 DNA가 벡터 안에 정상적으로 삽입된 것을 전기영동으로 확인한 결과이다.1 is a result of electrophoresis confirming that DNA amplified by an overlapping polymerase chain reaction according to an embodiment of the present invention is normally inserted into a vector.
도 2는 본 발명의 일 실시예에 따른 더피 케모카인 수용체의 세포 외 도메인 1의 변이체 DNA가 pET41a 벡터에 정상적으로 삽입된 것을 전기영동으로 확인한 결과이다.Figure 2 shows the results of electrophoresis confirming that the variant DNA of the extracellular domain 1 of the Duffy chemokine receptor is inserted into the pET41a vector normally according to an embodiment of the present invention.
도 3은 본 발명의 일 실시예에 따른 더피 케모카인 수용체의 세포 외 도메인 1이 정상적으로 황산화된 것을 SDS-PAGE로 확인한 결과이다.Figure 3 shows the results confirmed by SDS-PAGE that the extracellular domain 1 of the Duffy chemokine receptor normally sulfated according to an embodiment of the present invention.
도 4는 본 발명의 일 실시예에 따른 더피 케모카인 수용체의 세포 외 도메인 1 변이체의 염증 반응 조절능 및 혈관 형성 촉진능 확인한 결과이다.Figure 4 is a result of confirming the inflammatory response control capacity and blood vessel formation promoting ability of the extracellular domain 1 variant of Duffy chemokine receptor according to an embodiment of the present invention.
도 5는 본 발명의 일 실시예에 따른 더피 케모카인 수용체의 세포 외 도메인 1 변이체의 혈관 형성 촉진능을 qPCR로 확인한 결과이다.5 is a result confirming qPCR of the blood vessel formation promoting ability of the extracellular domain 1 variant of Duffy chemokine receptor according to an embodiment of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to 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. .
실시예Example
실시예 1: 더피 케모카인 수용체의 세포 외 도메인 1의 변이체 제조Example 1 Preparation of Variant of Extracellular Domain 1 of Duffy Chemokine Receptor
1.1. 프라이머 합성1.1. Primer synthesis
더피 케모카인 수용체의 세포 외 도메인 1(서열번호 1: Extracellular domain 1 of the duffy chemokine receptor (SEQ ID NO: 1)
MASSGYVLQAELSPSTENSSQLDFEDVWNSSYGVNDSFPDGDYDANLEAAAPCHSCLEAAAPCHSC)에 존재하는 세 개의 타이로신기를 각각 황산화시키기 위하여, 14개의 프라이머를 합성하였다. 프라이머 서열은 하기 표 1과 같다.Fourteen primers were synthesized to sulfate each of the three tyrosine groups present in MASSGYVLQAELSPSTENSSQLDFEDVWNSSYGVNDSFPDGDYDANLEAAAPCHSCLEAAAPCHSC). Primer sequences are shown in Table 1 below.
명칭designation 염기서열Sequence 서열번호SEQ ID NO:
1.1F1.1F AAA GGA TCC GCC TCC TCT GGG TAG GTC CTC CAG GCG GAGAAA GGA TCC GCC TCC TCT GGG TAG GTC CTC CAG GCG GAG 22
1.2F1.2F CTC TCC CCC TCA ACT GAG AAC TCA AGT CAG CTG GAC TTCCTC TCC CCC TCA ACT GAG AAC TCA AGT CAG CTG GAC TTC 33
1.3F1.3F GAA GAT GTA TGG AAT TCT TCC TAT GGT GTG AAT GAT TCCGAA GAT GTA TGG AAT TCT TCC TAT GGT GTG AAT GAT TCC 44
1.4F1.4F TTC CCA GAT GGA GAC TAT GGT GCC AAC CTG GAA GCA GCTTTC CCA GAT GGA GAC TAT GGT GCC AAC CTG GAA GCA GCT 55
1.5F1.5F GCC CCC TGC CAC TCC TGT CTC GAG AAA AAA AAA AAA AAAGCC CCC TGC CAC TCC TGT CTC GAG AAA AAA AAA AAA AAA 66
1.1R1.1R TTT TTT CTC GAG ACA GGA GTG GCA GGG GGC AGC TGC TTCTTT TTT CTC GAG ACA GGA GTG GCA GGG GGC AGC TGC TTC 77
1.2R1.2R CAG GTT GGC ACC ATA GTC TCC ATC TGG GAA GGA ATC ATTCAG GTT GGC ACC ATA GTC TCC ATC TGG GAA GGA ATC ATT 88
1.3R1.3R CAC ACC ATA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAGCAC ACC ATA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAG 99
1.4R1.4 R CTG ACT TGA GTT CTC AGT TGA GGG GGA GAG CTC CGC CTGCTG ACT TGA GTT CTC AGT TGA GGG GGA GAG CTC CGC CTG 1010
1.5R1.5R GAG GAC CTA CCC AGA GGA GGC GGA TCC TTT TTT TTT TTTGAG GAC CTA CCC AGA GGA GGC GGA TCC TTT TTT TTT TTT 1111
2.1F2.1F GAA GAT GTA TGG AAT TCT TCC TAG GGT GTG AAT GAT TCCGAA GAT GTA TGG AAT TCT TCC TAG GGT GTG AAT GAT TCC 1212
2.1R2.1R CAC ACC CTA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAGCAC ACC CTA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAG 1313
3.1F3.1F TTC CCA GAT GGA GAC TAG GGT GCC AAC CTG GAA GCA GCTTTC CCA GAT GGA GAC TAG GGT GCC AAC CTG GAA GCA GCT 1414
3.1R3.1R CAG GTT GGC ACC CTA GTC TCC ATC TGG GAA GGA ATC ATTCAG GTT GGC ACC CTA GTC TCC ATC TGG GAA GGA ATC ATT 1515
1.2. 더피 케모카인 수용체의 세포 외 도메인 1의 변이체 제조1.2. Preparation of Variant of Extracellular Domain 1 of Duffy Chemokine Receptor
더피 케모카인 수용체의 세포 외 도메인 1의 변이체를 제조하기 위하여 1차적으로 오버래핑 중합효소연쇄반응(overlapping polymerase chain reaction)을 수행하였다. 더피 케모카인 수용체의 세포 외 도메인 1의 첫번째 위치 타이로신(6번째 아미노산)의 변이체 1(DaMT1)을 제조하기 위해서는 1.1F, 1.1R, 1.2F, 1.2R, 1.3F, 1.3R, 1.4F, 1.4R, 1.5F, 및 1.5R 프라이머 세트를 사용하였고, 첫번째 및 두번째 위치 타이로신(32번째 아미노산)의 변이체 2(DaMT2)를 제조하기 위해서는 1.1F, 1.1R, 1.2F, 1.2R, 1.3F, 1.3R, 1.4F, 1.4R, 1.5F, 1.5R, 2.1F, 및 2.1R 프라이머 세트를 사용하였고, 첫번째, 두번째 및 세번째 위치 타이로신(43번째 아미노산)의 변이체 3(DaMT3)을 제조하기 위해서는 1.1F, 1.1R, 1.2F, 1.2R, 1.3F, 1.3R, 1.4F, 1.4R, 1.5F, 1.5R, 2.1F, 2.1R, 3.1F, 및 3.1R 프라이머 세트를 사용하였다. 각각의 프라이머를 10pmole의 농도로 혼합하여 준비하고, 혼합된 프라이머 4uL에 dNTP 5uL, 10X Taq buffer 5uL, Ex Taq 0.5uL, 3차 증류수 35.5uL를 혼합하여 중합효소연쇄반응을 수행하였다. 중합효소연쇄반응은 1차 DNA 변성 단계(94℃, 5분) 후, 94℃에서 30초(DNA 변성 단계), 52℃에서 30초(DNA 어닐링 단계), 및 72℃에서 30초(DNA 연장)를 55회 반복하고 72℃에서 5분 동안 반응시켜 수행하였다.In order to prepare a variant of the extracellular domain 1 of the Duffy chemokine receptor, an overlapping polymerase chain reaction was first performed. To prepare variant 1 (DaMT1) of the first position tyrosine (sixth amino acid) of the extracellular domain 1 of the Duffy chemokine receptor, 1.1F, 1.1R, 1.2F, 1.2R, 1.3F, 1.3R, 1.4F, 1.4R , 1.5F, and 1.5R primer sets were used and 1.1F, 1.1R, 1.2F, 1.2R, 1.3F, 1.3R were prepared to prepare variant 2 (DaMT2) of the first and second position tyrosine (32th amino acid). , 1.4F, 1.4R, 1.5F, 1.5R, 2.1F, and 2.1R primer sets were used, and 1.1F, to prepare variant 3 (DaMT3) of the first, second and third position tyrosine (43rd amino acid) 1.1R, 1.2F, 1.2R, 1.3F, 1.3R, 1.4F, 1.4R, 1.5F, 1.5R, 2.1F, 2.1R, 3.1F, and 3.1R primer sets were used. Each primer was prepared by mixing at a concentration of 10 pmole, polymerase chain reaction was performed by mixing 5uL of dNTP, 5uL of 10X Taq buffer, 0.5uL of Ex Taq, and 35.5uL of tertiary distilled water to 4uL of the mixed primers. The polymerase chain reaction was performed after the first DNA denaturation step (94 ° C., 5 minutes), 30 seconds at 94 ° C. (DNA denaturation step), 30 seconds at 52 ° C. (DNA annealing step), and 30 seconds at 72 ° C. (DNA extension). ) Was repeated 55 times and reacted at 72 ° C. for 5 minutes.
오버래핑 중합효소연쇄반응에 의하여 증폭된 DNA를 주형(template)으로 사용하여 2차 중합효소연쇄반응을 수행하였다. 2차 중합효소연쇄반응은 증폭된 DNA 1.25uL, 1.1F 프라이머 10pmol 0.5uL, 1.1R 프라이머 10pmol 0.5uL, dNTP 5uL, 5X band 10uL, 10X taq buffer 5uL, Ex Taq 0.5uL, 및 3차 증류수 27.25uL를 혼합하고, 1차 DNA 변성 단계(94℃, 5분) 후, 94℃에서 30초(DNA 변성 단계), 50℃에서 30초(DNA 어닐링 단계), 및 72℃에서 30초(DNA 연장)를 23회 반복하고 72℃에서 5분 동안 반응시켜 수행하였다.Secondary polymerase chain reaction was performed using DNA amplified by the overlapping polymerase chain reaction as a template. Secondary polymerase chain reaction was performed by amplifying DNA 1.25uL, 1.1p primer 10pmol 0.5uL, 1.1R primer 10pmol 0.5uL, dNTP 5uL, 5X band 10uL, 10X taq buffer 5uL, Ex Taq 0.5uL, and tertiary distilled water 27.25uL Mix, and after the first DNA denaturation step (94 ° C., 5 minutes), 30 seconds at 94 ° C. (DNA denaturation step), 30 seconds at 50 ° C. (DNA annealing step), and 30 seconds at 72 ° C. (DNA extension) Was repeated 23 times and reacted at 72 ° C. for 5 minutes.
증폭된 DNA는 1.5% 아가로스겔 전기영동(agarosegel electophoresis)을 이용하여 DNA 크기를 확인하였다. 그리고 DNA 서열을 확인하기 위하여 겔 추출(gel extraction)한 후 pGEM T easy vector에 삽입하고 대장균 DH5a에 형질전환시킨 후, 증폭된 벡터를 추출하여 전기영동을 이용하여 증폭된 DNA가 삽입되었는지 크기를 확인하였다. 그 결과는 도 1에 나타내었다.Amplified DNA was checked for DNA size by 1.5% agarose gel electrophoresis (agarosegel electophoresis). After gel extraction (gel extraction) to confirm DNA sequence, it was inserted into pGEM T easy vector and transformed into Escherichia coli DH5a, and then the amplified vector was extracted to check whether the amplified DNA was inserted by electrophoresis. It was. The results are shown in FIG.
도 1에 나타난 바와 같이, 벡터 안에 증폭된 DNA가 정상적으로 삽입된 것을 확인할 수 있었다.As shown in FIG. 1, it was confirmed that the amplified DNA was normally inserted into the vector.
증폭된 DNA가 삽입된 것이 확인된 벡터를 이용하여 염기서열 분석(DNA sequencing)을 수행하였다. 염기서열 분석 결과, 각각의 타이로신 위치의 TAT 서열이 TAG 서열로 변환된 총 3개의 변이체가 정상적으로 제조되었다는 것을 확인하였다.DNA sequencing was performed using a vector in which the amplified DNA was inserted. As a result of sequencing analysis, it was confirmed that a total of three variants in which the TAT sequence of each tyrosine position was converted to the TAG sequence was normally prepared.
실시예 2: 더피 케모카인 수용체의 세포 외 도메인 1 변이체의 황산화(sulfation)Example 2 Sulfation of Extracellular Domain 1 Variants of Duffy Chemokine Receptor
2.1. 단백질 발현용 균주 제작2.1. Production of protein expression strains
실시예 1.2의 방법으로 제조된 3개의 더피 케모카인 수용체의 세포 외 도메인 1의 변이체 DNA를 GST-His tag을 포함한 발현벡터(pET41a)에 각각 삽입하고, 전기영동을 통하여 크기를 확인하였다. 그 결과는 도 2에 나타내었다. 제조된 벡터는 최종적으로 대장균 BL21에 형질전환시켰다.Variant DNAs of the extracellular domain 1 of the three Duffy chemokine receptors prepared by the method of Example 1.2 were inserted into expression vectors (pET41a) containing the GST-His tag, respectively, and their size was confirmed by electrophoresis. The results are shown in FIG. The produced vector was finally transformed into E. coli BL21.
도 2에 나타난 바와 같이, 제조된 3개의 더피 케모카인 수용체의 세포 외 도메인 1의 변이체 DNA가 pET41a 벡터에 정상적으로 삽입된 것을 확인하였다.As shown in Figure 2, it was confirmed that the variant DNA of the extracellular domain 1 of the three Duffy chemokine receptor prepared was inserted into the pET41a vector normally.
그리고 타이로신기를 황산화시키기 위하여 각각의 변이체 DNA가 삽입된 pET41a 벡터를 각각 대장균 BL21에 형질전환시키고, pSUPAR6-L3-3SY 벡터를 형질전환된 대장균 BL21에 각각 추가 형질전환(co-transformation)시켰다. pSUPAR6-L3-3SY 벡터는 스크립스(Scripps) 연구소에서 분양받아 사용하였다.In order to sulfate the tyrosine group, the pET41a vector into which each variant DNA was inserted was transformed into E. coli BL21, and the pSUPAR6-L3-3SY vector was further transformed into the transformed E. coli BL21. The pSUPAR6-L3-3SY vector was available from Scripps Laboratories.
2.2. 황산화된 더피 케모카인 수용체의 세포 외 도메인 1 발현 및 분리2.2. Extracellular Domain 1 Expression and Isolation of Sulfated Duffy Chemokine Receptors
형질전환된 대장균은 5mL의 2YT 액체배지에 접종하여 37℃ 진탕배양기(shaking incubator)에서 18시간 동안 전배양한 후, 10mM의 sulfotyrosine이 첨가되어 있는 YT 액체배지에 1%가 되도록 접종하고 37℃ 진탕배양기에서 본배양하였다. 그리고 OD600nm값이 1.0에 도달했을 때 IPTG를 최종농도가 1mM이 되도록 첨가하고, 다시 30℃에서 20시간 동안 배양하였다. 배양된 세포는 원심분리(13,000g, 1분)하여 상층액을 제거하고, 상층액을 제거한 세포는 리서스펜션 버퍼(cell resuspensin buffer, 50mM Tris-HCl pH 8.0, 2mM EDTA, 0.1% Triton X-100) 1mL을 첨가하여 잘 혼합하였다. 그리고 초음파 처리(sonication, 1ml 버퍼 당 30초 깨고 2분 쉬고를 2회 실시)하여 세포를 깨고, 원심분리(14,000g, 20분)하여 단백질이 포함된 상층액만 회수하였다. His column을 이용하여 회수한 상층액으로부터 단백질을 분리하였다. 분리된 단백질은 정량 후, 10% SDS-PAGE를 이용하여 전기영동하여 확인하였다. 대조군으로는 황산화가 되지 않은 더피 케모카인 수용체의 세포 외 도메인 1(DARCex1)을 이용하였다. 그 결과는 도 3에 나타내었다.The transformed E. coli was inoculated in 5 mL of 2YT liquid medium, pre-incubated for 18 hours in a 37 ° C shaking incubator, inoculated to 1% in YT liquid medium containing 10 mM sulfotyrosine and shaken at 37 ° C. Main culture was incubator. When the OD600nm value reached 1.0, IPTG was added so as to have a final concentration of 1 mM and incubated at 30 ° C. for 20 hours. The cultured cells were centrifuged (13,000 g, 1 minute) to remove the supernatant, and the cells from which the supernatant was removed were subjected to cell resuspensin buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.1% Triton X-100). 1 mL was added and mixed well. And sonication (breaking 30 seconds per 1ml buffer and 2 minutes rest twice) to break the cells, centrifugation (14,000g, 20 minutes) to recover only the supernatant containing protein. Proteins were separated from the supernatant recovered using His column. The isolated protein was quantified and confirmed by electrophoresis using 10% SDS-PAGE. As a control, extracellular domain 1 (DARCex1) of the Duffy chemokine receptor, which was not sulfated, was used. The results are shown in FIG.
도 3에 나타난 바와 같이, 더피 케모카인 수용체의 세포 외 도메인 1 변이체들이 모두 정상적으로 황산화된 것을 확인하였다.As shown in FIG. 3, it was confirmed that all of the extracellular domain 1 variants of the Duffy chemokine receptor were normally sulfated.
실시예Example 3:  3: 더피Duffy 케모카인Chemokines 수용체의 세포 외 도메인 1  Extracellular domain of receptor 1 변이체의Variant 염증 반응  Inflammatory response 조절능Controllability 및 혈관 형성 촉진능 확인 And blood vessel formation promoting ability
3.1. 3.1. 더피Duffy 케모카인Chemokines 수용체의 세포 외 도메인 1  Extracellular domain of receptor 1 변이체의Variant 염증 반응  Inflammatory response 조절능Controllability 및 혈관 형성 촉진능 확인 And blood vessel formation promoting ability
실시예 2의 방법으로 제조된 3개의 황산화된 더피 케모카인 수용체의 세포 외 도메인 1의 변이체들이 염증 반응 조절능 및 혈관 형성 촉진능을 나타내는지 확인하기 위하여, 6 내지 8주된 BALB/C 마우스의 각막을 11-0 nylon으로 봉합하고 더피 케모카인 수용체의 세포 외 도메인 1의 변이체들을 1일 1회 20ug/mL의 농도로 각막에 투여하였으며, 대조군으로는 Glutathione-S-transferase(GST)를 1일 1회 20ug/mL의 농도로 투여하였다. 그리고 일주일 후에 마우스의 각막에서 혈관의 형성을 관찰하고, 쥐를 안락사시킨 후 눈을 적출하고 각막 윤부 둘레로 각막을 분리하여 혈관의 형성 및 염증세포의 침윤을 확인하였다. 신생 혈관 및 염증 세포의 침윤을 관찰하기 위하여 형광염색검사(Fluorescein dye)법을 이용하여 혈관을 염색한 후, 형광 현미경 하에서 관찰하였다. 그 결과는 도 4에 나타내었다.Corneals of 6-8 week old BALB / C mice to determine whether the variants of extracellular domain 1 of the three sulfated Duffy chemokine receptors prepared by the method of Example 2 exhibited inflammatory response regulation and angiogenesis promoting ability Was sutured with 11-0 nylon and the mutant of extracellular domain 1 of the Duffy chemokine receptor was administered to the cornea at a concentration of 20 ug / mL once a day, and Glutathione-S-transferase (GST) was used once a day as a control. Administration was at a concentration of 20 ug / mL. One week later, the formation of blood vessels was observed in the cornea of the mouse, the mice were euthanized, the eyes were extracted, and the cornea was separated around the limbal limbus to confirm the formation of blood vessels and infiltration of inflammatory cells. In order to observe the infiltration of neovascularization and inflammatory cells, blood vessels were stained by using a fluorescein dye method, and then observed under a fluorescence microscope. The results are shown in FIG.
도 4에 나타난 바와 같이, GST만 투여한 대조군(도 4A)에는 염증 세포의 침윤 및 신생 혈관의 형성이 거의 생성되지 않는 반면, 변이체들을 투여한 실험군(도 4B)에서는 오른쪽 하단부에 염증 세포의 침윤이 증가하여 밝게 관찰되며, 상단부에 혈관 형성이 증가된 것을 확인하였다. 또한, 대조군(도 4C)에서는 홍채의 혈관만 관찰되며 신생혈관이 관찰되지 않고 각막이 전반적으로 맑은 반면, 실험군(도 4D)에서는 신생 혈관의 형성이 증가하고, 염증 세포의 침윤이 증가하여 각막이 혼탁해져 전반적으로 어두운 것을 확인하였다. 상기 결과를 통하여, 더피 케모카인 수용체의 세포 외 도메인 1의 변이체들은 모두 혈관 형성을 촉진시키는 동시에 염증 반응을 조절할 수 있다는 것을 확인할 수 있었다.As shown in FIG. 4, the control group (FIG. 4A) administered only with GST produced little infiltration of inflammatory cells and formation of neovascularization, whereas the experimental group administered with variants (FIG. 4B) infiltrated inflammatory cells at the lower right corner. This increased brightly observed, confirmed that the increased blood vessel formation at the top. In addition, only the blood vessels of the iris are observed in the control group (FIG. 4C) and neovascularization is not observed, and the cornea is generally clear, whereas in the experimental group (FIG. 4D), the formation of new blood vessels is increased, and the infiltration of inflammatory cells is increased, thereby increasing the cornea. It became cloudy and confirmed that it was dark overall. Through the above results, it was confirmed that all the variants of the extracellular domain 1 of the Duffy chemokine receptor can promote vascular formation and regulate the inflammatory response.
3.2. 3.2. 더피Duffy 케모카인Chemokines 수용체의 세포 외 도메인 1  Extracellular domain of receptor 1 변이체의Variant 염증 반응  Inflammatory response 조절능Controllability 및 혈관 형성 촉진능 확인 And blood vessel formation promoting ability
실시예 3.1의 방법으로 분리한 각막에서 easy-spinTM Total RNA extraction kit(iNtRON)를 이용하여 RNA를 추출하고, FastAQTM One-Step SYBR Green qRT-PCR Kit(Excellgen)를 이용하여 혈관내피세포 성장인자 A(vascular endothelial growth factor A) 및 혈관내피세포 성장인자 C(vascular endothelial growth factor C)의 정량 PCR(qPCR)을 수행하였다. 그 결과는 도 5에 나타내었다.In Example 3.1, a separation cornea in a manner of using the easy-spinTM Total RNA extraction kit (iNtRON) to extract RNA, and, FastAQ TM One-Step SYBR Green qRT-PCR Kit (Excellgen) using a vascular endothelial growth factor Quantitative PCR (qPCR) of vascular endothelial growth factor A and vascular endothelial growth factor C was performed. The results are shown in FIG.
도 5에 나타난 바와 같이, 더피 케모카인 수용체의 세포 외 도메인 1의 변이체들을 투여한 군에서는 혈관내피세포 성장인자들의 발현이 촉진된 것을 확인하였다. 또한, 단일 유전자의 변이체와 비교하여, 복합 유전자 변이체의 경우에는 혈관내피세포 성장인자들의 발현이 효과적으로 발현된 것을 확인하였다.As shown in Figure 5, it was confirmed that the expression of vascular endothelial growth factor was promoted in the group administered with the variant of the extracellular domain 1 of the Duffy chemokine receptor. In addition, it was confirmed that the expression of vascular endothelial growth factors was effectively expressed in the case of the complex gene variant compared to the variant of the single gene.
상기 결과들을 통하여, 황산화된 더피 케모카인 수용체의 세포 외 도메인 1 변이체들은 혈관 세포 또는 염증 세포로의 침투가 용이하기 때문에 혈관을 직접 자극하는 것이 아니라 혈관 형성을 돕는 케모카인과 결합한 후 혈관 세포 및/또는 염증 세포로 침투하여 세포 내부의 케모카인 농도를 증가시켜, 신생 혈관의 형성을 촉진시킬 수 있을 뿐만 아니라, 염증 반응을 조절할 수 있다는 것을 확인할 수 있었다. 또한 단일 유전자의 변이체인 DaMT1 보다 복합 유전자의 변이체인 DaMT2 및 DaMT3의 경우 신생 혈관의 형성이 더욱 활발이 촉진되는 것을 확인하였는데, 이를 통하여 복합 유전자의 변이체의 경우 세포 침투력 및/또는 케모카인 결합능이 증가된 것을 확인할 수 있었다. 따라서 황산화된 더피 케모카인 수용체의 세포 외 도메인 1 변이체들은 골의 위관절(Non-union of bone), 부전유합(Malunion), 피부결손상처(Skin wound defect), 허혈성 질환(ischemic disease) 등 혈관 생성을 필요로 하는 다양한 질병들에 폭넓게 사용 가능하며, 다양한 상처 치료용으로도 사용 가능하며, 염증성 질환을 조절할 수 있으므로 염증성 질환을 예방 또는 치료하는 데에도 사용할 수 있다는 것을 확인할 수 있었다.Based on these results, the extracellular domain 1 variants of the sulfated Duffy chemokine receptor are easy to penetrate into vascular cells or inflammatory cells, and therefore, do not directly stimulate blood vessels, but after binding to chemokines to help the formation of blood vessels and / or blood vessels. Infiltrating into inflammatory cells increases the concentration of chemokines within the cells, thereby promoting the formation of new blood vessels, as well as controlling the inflammatory response. In addition, it was confirmed that the formation of new blood vessels was more actively promoted in the case of DaMT2 and DaMT3, which are complex gene variants than DaMT1, which is a single gene variant, through which the cell penetration and / or chemokine binding capacity of the complex gene was increased. I could confirm that. Thus, the extracellular domain 1 variants of sulfated Duffy chemokine receptors produce blood vessels such as non-union of bone, malunion, skin wound defects and ischemic disease. It can be found that it can be widely used for various diseases that are needed, can be used for treating various wounds, and can be used to prevent or treat inflammatory diseases because it can control inflammatory diseases.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
본 발명에 따른 하나 이상의 타이로신이 황산화된 더피 케모카인 수용체의 세포 외 도메인 1 변이체는 염증 반응을 조절하는 동시에 혈관 형성을 촉진시킬 수 있기 때문에 혈관 형성의 촉진이 필요한 분야인 성형외과, 심장내과 등의 분야에 폭넓게 사용될 수 있을 것으로 기대된다.The extracellular domain 1 variant of one or more tyrosine sulfated Duffy chemokine receptors according to the present invention can regulate the inflammatory response and promote blood vessel formation, so plastic surgery, cardiology, etc. It is expected to be widely used in the field.
서열번호 1(Extracellular domain 1 of duffy chemokine receptor): Extracellular domain 1 of duffy chemokine receptor:
MASSGYVLQAELSPSTENSSQLDFEDVWNSSYGVNDSFPDGDYDANLEAAAPCHSCLEAAAPCHSCMASSGYVLQAELSPSTENSSQLDFEDVWNSSYGVNDSFPDGDYDANLEAAAPCHSCLEAAAPCHSC
서열번호 2(1.1F): AAA GGA TCC GCC TCC TCT GGG TAG GTC CTC CAG GCG GAGSEQ ID NO: 2 (1.1F): AAA GGA TCC GCC TCC TCT GGG TAG GTC CTC CAG GCG GAG
서열번호 3(1.2F): CTC TCC CCC TCA ACT GAG AAC TCA AGT CAG CTG GAC TTCSEQ ID NO: 3 (1.2F): CTC TCC CCC TCA ACT GAG AAC TCA AGT CAG CTG GAC TTC
서열번호 4(1.3F): GAA GAT GTA TGG AAT TCT TCC TAT GGT GTG AAT GAT TCCSEQ ID NO: 4 (1.3F): GAA GAT GTA TGG AAT TCT TCC TAT GGT GTG AAT GAT TCC
서열번호 5(1.4F): TTC CCA GAT GGA GAC TAT GGT GCC AAC CTG GAA GCA GCTSEQ ID NO: 5 (1.4F): TTC CCA GAT GGA GAC TAT GGT GCC AAC CTG GAA GCA GCT
서열번호 6(1.5F): GCC CCC TGC CAC TCC TGT CTC GAG AAA AAA AAA AAA AAASEQ ID NO: 6 (1.5F): GCC CCC TGC CAC TCC TGT CTC GAG AAA AAA AAA AAA AAA
서열번호 7(1.1R): TTT TTT CTC GAG ACA GGA GTG GCA GGG GGC AGC TGC TTCSEQ ID NO: 7 (1.1R): TTT TTT CTC GAG ACA GGA GTG GCA GGG GGC AGC TGC TTC
서열번호 8(1.2R): CAG GTT GGC ACC ATA GTC TCC ATC TGG GAA GGA ATC ATTSEQ ID NO: 8 (1.2R): CAG GTT GGC ACC ATA GTC TCC ATC TGG GAA GGA ATC ATT
서열번호 9(1.3R): CAC ACC ATA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAGSEQ ID NO: 9 (1.3R): CAC ACC ATA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAG
서열번호 10(1.4R): CTG ACT TGA GTT CTC AGT TGA GGG GGA GAG CTC CGC CTGSEQ ID NO: 10 (1.4R): CTG ACT TGA GTT CTC AGT TGA GGG GGA GAG CTC CGC CTG
서열번호 11(1.5R): GAG GAC CTA CCC AGA GGA GGC GGA TCC TTT TTT TTT TTTSEQ ID NO: 11 (1.5R): GAG GAC CTA CCC AGA GGA GGC GGA TCC TTT TTT TTT TTT
서열번호 12(2.1F): GAA GAT GTA TGG AAT TCT TCC TAG GGT GTG AAT GAT TCCSEQ ID NO: 12 (2.1F): GAA GAT GTA TGG AAT TCT TCC TAG GGT GTG AAT GAT TCC
서열번호 13(2.1R): CAC ACC CTA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAGSEQ ID NO: 13 (2.1R): CAC ACC CTA GGA AGA ATT CCA TAC ATC TTC GAA GTC CAG
서열번호 14(3.1F): TTC CCA GAT GGA GAC TAG GGT GCC AAC CTG GAA GCA GCTSEQ ID NO: 14 (3.1F): TTC CCA GAT GGA GAC TAG GGT GCC AAC CTG GAA GCA GCT
서열번호 15(3.1R): CAG GTT GGC ACC CTA GTC TCC ATC TGG GAA GGA ATC ATTSEQ ID NO: 15 (3.1R): CAG GTT GGC ACC CTA GTC TCC ATC TGG GAA GGA ATC ATT

Claims (19)

  1. 더피 케모카인 수용체의 세포 외 도메인 1의 아미노산 서열 중 타이로신 1 내지 3개가 황산화된, 더피 케모카인 수용체의 세포 외 도메인 1 변이체.The extracellular domain 1 variant of the duffy chemokine receptor, wherein 1 to 3 tyrosine in the amino acid sequence of extracellular domain 1 of the Duffy chemokine receptor is sulfated.
  2. 제 1 항에 있어서,The method of claim 1,
    상기 더피 케모카인 수용체의 세포 외 도메인 1의 아미노산 서열은 서열번호 1의 서열을 포함하는 것을 특징으로 하는, 변이체.The amino acid sequence of the extracellular domain 1 of the Duffy chemokine receptor, characterized in that it comprises the sequence of SEQ ID NO: 1.
  3. 제 1 항에 있어서,The method of claim 1,
    상기 타이로신은 6번째 아미노산인 것을 특징으로 하는, 변이체.The tyrosine is characterized in that the sixth amino acid, variant.
  4. 제 1 항에 있어서,The method of claim 1,
    상기 타이로신은 32번째 아미노산인 것을 특징으로 하는, 변이체.The tyrosine is characterized in that the 32nd amino acid, variant.
  5. 제 1 항에 있어서,The method of claim 1,
    상기 타이로신은 43번째 아미노산인 것을 특징으로 하는, 변이체.The tyrosine is characterized in that the 43rd amino acid, variant.
  6. 제 1 항에 있어서,The method of claim 1,
    상기 타이로신은 6번째 및 32번째 아미노산인 것을 특징으로 하는, 변이체.The tyrosine is characterized in that the sixth and 32nd amino acids, variants.
  7. 제 1 항에 있어서,The method of claim 1,
    상기 타이로신은 6번째 및 43번째 아미노산인 것을 특징으로 하는, 변이체.The tyrosine is characterized in that the sixth and 43rd amino acids, variants.
  8. 제 1 항에 있어서,The method of claim 1,
    상기 타이로신은 32번째 및 43번째 아미노산인 것을 특징으로 하는, 변이체.The tyrosine is characterized in that the 32nd and 43rd amino acids, variants.
  9. 제 1 항에 있어서,The method of claim 1,
    상기 타이로신은 6번째, 32번째 및 43번째 아미노산인 것을 특징으로 하는, 변이체.The tyrosine is characterized in that the sixth, 32nd and 43rd amino acids, variants.
  10. 제 1 항 내지 제 9 항 중 어느 하나의 변이체를 포함하는, 혈관 형성 촉진용 약학 조성물.10. A pharmaceutical composition for promoting angiogenesis, comprising the variant of any one of claims 1 to 9.
  11. 제 10 항에 있어서,The method of claim 10,
    상기 약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 하는, 약학 조성물.The pharmaceutical composition is characterized in that the capsule, tablets, granules, injections, ointments, powders or drinks form.
  12. 제 1 항 내지 제 9 항 중 어느 하나의 변이체를 포함하는, 상처 치료용 약학 조성물.A pharmaceutical composition for treating wounds, comprising the variant of any one of claims 1 to 9.
  13. 제 12 항에 있어서,The method of claim 12,
    상기 약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 하는, 약학 조성물.The pharmaceutical composition is characterized in that the capsule, tablets, granules, injections, ointment, powder or beverage form.
  14. 제 1 항 내지 제 9 항 중 어느 하나의 변이체를 포함하는, 염증성 질환 치료용 약학 조성물.10. A pharmaceutical composition for treating an inflammatory disease, comprising the variant of any one of claims 1 to 9.
  15. 제 14 항에 있어서,The method of claim 14,
    상기 염증성 질환은 천식, 죽상경화증, 사구체신염, 췌장염, 재협착, 류마티스성 관절염, 당뇨병성 신병증, 폐섬유증, 염증성 장질환, 크론씨 질환, 및 이식 거부반응으로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 약학 조성물.The inflammatory disease is selected from the group consisting of asthma, atherosclerosis, glomerulonephritis, pancreatitis, restenosis, rheumatoid arthritis, diabetic nephropathy, pulmonary fibrosis, inflammatory bowel disease, Crohn's disease, and transplant rejection , Pharmaceutical composition.
  16. 제 14 항에 있어서,The method of claim 14,
    상기 염증성 질환은 만성 피부궤양(chronic skin ulcer and pressure sore), 봉와직염(cellulitis), 혈관경화성병변(atherosclerotic lesion)에 의한 허혈성질환(ischemic disease from atherosclerotic lesion), 혈전 등의 색전에 의한 허혈성병변(ischemic disease from embolic lesion), 뇌졸증을 포함하는 허혈성뇌질환 및 병변(ischemic brain disease and lesion, including stroke), 장허혈증후군(visceral ischemic syndrome), 안구나 망막에 생기는 허혈성질환(ocular ischemic syndrome), 말초혈관성질환(peripheral vascular disease, peripheral artery occlusive disease, peripheral obliterative arteriopathy), 및 당뇨성혈관질환(diabetic vasculopathy)으로 이루어진 군으로 선택되는 것을 특징으로 하는, 약학 조성물.The inflammatory disease is ischemic due to embolism such as chronic skin ulcer and pressure sore, cellulitis, atherosclerotic lesions (ischemic disease from atherosclerotic lesions, blood clots, etc.) disease from embolic lesions, ischemic brain disease and lesions, including stroke, visceral ischemic syndrome, ocular ischemic syndrome in the retina or the retina, peripheral vascular disease Pharmaceutical composition, characterized in that it is selected from the group consisting of (peripheral vascular disease, peripheral artery occlusive disease, peripheral obliterative arteriopathy), and diabetic vasculopathy.
  17. 제 14 항에 있어서,The method of claim 14,
    상기 약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 하는, 약학 조성물.The pharmaceutical composition is characterized in that the capsule, tablets, granules, injections, ointments, powders or drinks form.
  18. 제 1 항 내지 제 9 항 중 어느 하나의 변이체를 투여하는 단계를 포함하는, 상처 치료 방법.A method of treating a wound, comprising administering a variant of claim 1.
  19. 제 1 항 내지 제 9 항 중 어느 하나의 변이체를 투여하는 단계를 포함하는, 염증성 질환 치료 방법.A method of treating an inflammatory disease comprising administering a variant of any one of claims 1 to 9.
PCT/KR2015/007510 2014-07-21 2015-07-20 Extracellular domain 1 mutant of sulfated duffy chemokine receptor, and use thereof WO2016013828A1 (en)

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