KR102092782B1 - Novel hemostatic agent using mussel adhesive protein and method for preparing the same - Google Patents

Novel hemostatic agent using mussel adhesive protein and method for preparing the same Download PDF

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KR102092782B1
KR102092782B1 KR1020170153269A KR20170153269A KR102092782B1 KR 102092782 B1 KR102092782 B1 KR 102092782B1 KR 1020170153269 A KR1020170153269 A KR 1020170153269A KR 20170153269 A KR20170153269 A KR 20170153269A KR 102092782 B1 KR102092782 B1 KR 102092782B1
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차형준
김효정
이재윤
전은영
박태윤
최근호
최봉혁
주계일
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Abstract

본 발명은 제1 성분으로서 홍합 접착 단백질; 및 제2 성분으로서 홍합 접착 단백질 이외의 생체 유래 고분자;를 포함하는 지혈용 조성물, 이를 포함하는 지혈제 및 그 제조 방법에 관한 것으로서, 우수한 생체적합성, 생분해성, 수중 환경에서의 지혈 효능 및 조직 환경에 적합한 기계적 물성을 가지는 것을 특징으로 한다.The present invention is a mussel adhesive protein as a first component; And as a second component, a bio-derived polymer other than mussel adhesive protein; A composition for hemostasis comprising, a hemostatic agent comprising the same and a method for manufacturing the same, excellent biocompatibility, biodegradability, hemostatic efficacy in aquatic environment and tissue environment It is characterized by having suitable mechanical properties.

Description

홍합 접착 단백질을 포함하는 신규한 지혈제 및 이의 제조 방법 {Novel hemostatic agent using mussel adhesive protein and method for preparing the same}Novel hemostatic agent using mussel adhesive protein and preparation method thereof {Novel hemostatic agent using mussel adhesive protein and method for preparing the same}

본 발명은 홍합 접착 단백질을 포함하는 신규한 지혈제 및 이의 제조 방법에 관한 것이다.The present invention relates to a novel hemostatic agent comprising a mussel adhesive protein and a method for manufacturing the same.

일상 생활 및 작업 현장에서 상해가 발생할 경우 신속하고 안전한 응급 지혈이 과다 출혈과 이에 수반되는 생명의 위협을 막는 데에 중요한 역할을 한다. 외과 수술에서 환부의 효율적인 지혈은 환자의 출혈량을 줄이고 수혈량을 절약할 수 있으며 수술 의사의 시야를 확보함으로써 수술에 소요되는 시간과 비용을 절감할 수 있다. 지혈을 위해서는 붕대, 거즈, 수술용 봉합사, 스테이플러 등 물리적 압박 또는 전기, 초음파, 레이저 등 기기를 이용하는 방법이 사용되기도 하나 출혈 부위와 손상과 감염 등을 유발할 수 있다. 지혈의약품은 출혈 부위에 도입 시 제품에 따라 물리적, 화학적, 생리활성 등의 성질을 이용해 출혈을 차단하며 상처 조직의 손상과 감염을 방지할 수 있어 안전성 및 효율성 측면에서 비교우위에 있다.In the event of an injury in daily life and on the job site, rapid and safe emergency hemostasis plays an important role in preventing excessive bleeding and accompanying life threats. Efficient hemostasis of the affected area in surgical operation can reduce the patient's bleeding volume, save blood transfusion volume, and reduce the time and cost of surgery by securing the field of view of the surgeon. For hemostasis, physical compression such as bandage, gauze, surgical suture, and stapler, or a method using devices such as electricity, ultrasound, and laser may be used, but may cause bleeding, damage and infection. Hemostatic drugs have a comparative advantage in terms of safety and efficiency because they can prevent bleeding and prevent damage and infection of wound tissue by using physical, chemical, and physiological properties depending on the product when introduced to the bleeding site.

해양생물인 홍합은 족사에서 접착 단백질을 분비하여 수중의 표면에 부착함으로써 해류와 부력에 휩쓸리지 않고 위치를 유지할 수 있다. 이러한 홍합 접착 단백질은 수중에서 금속, 유리, 플라스틱 및 생체물질에 강하게 접착할 수 있고 생체 내 면역반응을 유발하지 않는 것으로 알려져, 접착 능력을 보유한 생체 소재로 활용 가능하다. 표면 코팅, 하이드로젤, 나노섬유, 나노입자 등의 제형화를 통한 조직공학 및 의료 분야로 적용 가능성이 확인된 바 있으며 생체적합성, 생분해성 및 생물학적 안전성 등이 검증된 바 있다.Mussels, a marine organism, can maintain their position without being swept away by currents and buoyancy by secreting adhesive proteins from the foot and attaching them to the surface of the water. These mussel adhesion proteins are known to strongly adhere to metals, glass, plastics, and biomaterials in water and are not known to induce an immune response in vivo, and thus can be used as a biomaterial having adhesion. Applicability to tissue engineering and medical fields through formulation of surface coatings, hydrogels, nanofibers, and nanoparticles has been confirmed, and biocompatibility, biodegradability, and biological safety have been verified.

말미잘은 해양생물 중 강장류에 해당하며 특유한 자포 기관을 이용하여 먹이와 외적 등을 쏘아 독을 주입한다. 스타렛 말미잘의 자포에 분포하는 구조 단백질들 중 누에 실크, 콜라겐 등과 유전자 반복 서열과 기계적 물성에서 유사성을 가지는 단백질이 존재하며 이 단백질을 하이드로젤, 나노섬유, 마이크로섬유 등으로 제형화할 수 있다는 것이 밝혀졌다. 이 말미잘 실크 단백질은 생체적합성, 생분해성을 보유하고 있으며 세포 포집 및 동물 실험 등을 통하여 생물학적 안전성이 확인되었다.Sea anemone is a tonic among marine organisms, and uses a unique spontaneous organ to inject poison by shooting food and external products. Among the structural proteins distributed in the astrocytes of Starlet anemone, there is a protein having similarity in mechanical properties with silk repeat, collagen, etc., and it can be formulated into hydrogel, nanofiber, microfiber, etc. lost. This anemone silk protein has biocompatibility and biodegradability, and biological safety has been confirmed through cell capture and animal experiments.

가재, 게, 새우 등의 해양생물을 비롯한 갑각류 껍데기의 주성분인 키틴을 탈아세틸화 처리하면 글루코사민 중합체인 키토산을 얻을 수 있다. 키토산 소재는 혈장 흡수, 적혈구 응집, 혈소판 부착 및 활성화 등 지혈 효과를 보유하고 있으며, 생체적합성, 생분해성, 항감염성 등이 우수하기 때문에 지혈 소재로 활용되고 있다.Chitinic acid, a glucosamine polymer, can be obtained by deacetylating chitin, the main component of crustacean shells, including marine life such as crawfish, crab, and shrimp. The chitosan material has hemostatic effects such as plasma absorption, red blood cell aggregation, platelet adhesion and activation, and is used as a hemostatic material because of its excellent biocompatibility, biodegradability, and anti-infective properties.

하이드로젤은 물 또는 체액 내에서 가교된 격자 안으로 많은 양의 물 또는 체액을 흡수하여 팽윤되며 삼차원 구조를 유지하는 재료를 의미한다. 팽윤된 이후에도 열역학적으로 안정하게 존재하여 액체와 고체의 중간 형태에 해당하는 기계적 및 물리화학적 특성을 가진다. 이러한 하이드로젤은 대게 생체 적합성, 높은 다공성 및 산소 투과도를 보이며, 생체 연조직과의 비슷한 물리적 특성을 나타낼 수 있다.Hydrogel refers to a material that absorbs and swells a large amount of water or body fluid into a cross-linked lattice in water or body fluid and maintains a three-dimensional structure. It remains thermodynamically stable even after swelling and has mechanical and physicochemical properties corresponding to the intermediate form of liquid and solid. These hydrogels usually show biocompatibility, high porosity, and oxygen permeability, and may exhibit similar physical properties to biosoft tissues.

전기방사는 고분자 물질을 포함한 용액을 포함한 주사기 등에 높은 전기장을 가하여 순간적으로 섬유 형태로 방사하는 방법이다. 휘발성이 높은 용매를 포함하는 낮은 점도 상태의 용액이 전기장 내에서 낙하 시, 고분자에 가해지는 정전기력이 표면장력을 극복하고 용매가 증발함에 따라 고분자가 직경 수십~수백 나노미터의 섬유를 형성하게 된다. 이러한 나노섬유는 그 형태가 세포외기질과 유사하고 다공성의 박막 형태로 제조할 수 있어 생체 소재로 활용이 가능하다.Electrospinning is a method of instantaneously spinning in the form of fibers by applying a high electric field to a syringe containing a solution containing a polymer material. When a low-viscosity solution containing a highly volatile solvent falls in an electric field, the electrostatic force applied to the polymer overcomes the surface tension, and as the solvent evaporates, the polymer forms fibers of tens to hundreds of nanometers in diameter. These nanofibers have a shape similar to the extracellular matrix and can be manufactured in a porous thin film form, and thus can be used as a biomaterial.

코아세르베이트는 음이온성 고분자 전해질과 양이온성 고분자 전해질이 특정 조건에서 혼합되었을 때 형성되는 콜로이드 물질의 일종으로, 코아세르베이트가 형성되는 경우 용액의 흡광도가 증가하며, 용액 상에서 동그란 구 형태로 외부 용액과 분리되어 존재한다. 코아세르베이트 형성 시, 참여 전해질은 용액에서 분리되어 응축되고 여전히 액상을 띠게 되며, 이때 표면장력이 줄어들고 점성이 늘어나는 등, 물성도 변화한다. 코아세르베이트는 단백질과 그 반대 성질을 띠는 고분자 전해질과의 혼합을 통해서도 일어날 수 있다. 코아세르베이트의 낮은 표면장력에 기인해 약물, 효소, 세포, 식품첨가물 등의 기능성 물질을 미세캡슐 안에 고정화 하는데 쓰이는 기술도 보고되어 있다.Coacervate is a type of colloidal material that is formed when anionic polymer electrolyte and cationic polymer electrolyte are mixed under specific conditions. When coacervate is formed, absorbance of the solution increases, and the solution exists as a spherical sphere separated from the external solution. do. When forming the coacervate, the participating electrolyte is separated from the solution, condensed, and still has a liquid phase. At this time, the physical properties also change, such as decreasing surface tension and increasing viscosity. Coacervates can also occur through the mixing of proteins with polymer electrolytes of opposite properties. A technique used to immobilize functional substances such as drugs, enzymes, cells, and food additives in microcapsules due to the low surface tension of coacervate has also been reported.

생물학적 지혈작용을 촉진하는 피브린 글루 등의 제품군은 상용화 지혈제의 주를 이루고 있으나 기계적 물성이 높지 않은 문제점이 있다. 물리적 압박을 이용하는 제품군은 대부분 비생체 고분자 소재를 기반으로 하고 있는데, 이러한 제품들은 공통적으로 단순 지혈효과만을 가지며 출혈 부위에 붙는 접착력을 충분히 보유하지 못하고 있다.A family of fibrin glues, etc., which promote biological hemostasis, constitutes a major hemostatic agent, but has a problem that mechanical properties are not high. Most of the products using physical compression are based on non-biopolymer materials, and these products have only a simple hemostatic effect and do not have sufficient adhesion to the bleeding site.

본 발명의 목적은 생체 적합성과 물성이 우수한 해양 생체 유래 소재를 이용하여 상처 부위의 지혈을 도와줄 수 있는 신규한 복합 기능성 지혈제를 제공하는 것이다. An object of the present invention is to provide a novel complex functional hemostatic agent that can help hemostasis of a wound site using a marine-derived material having excellent biocompatibility and physical properties.

또한, 본 발명의 목적은 제1 성분으로서 홍합 접착 단백질 및 제2 성분으로서 홍합 접착 단백질 이외의 생체 유래 고분자를 포함하는 지혈용 조성물 및 이를 포함하는 지혈제를 제공하는 것이다.In addition, an object of the present invention is to provide a composition for hemostasis including a mussel adhesive protein as a first component and a bio-derived polymer other than a mussel adhesive protein as a second component and a hemostatic agent comprising the same.

본 발명자들은 생체적합성과 물성이 우수한 해양생체 유래 소재를 이용하여 기능성 지혈제를 개발할 수 있다는 점에 착안하여, 홍합 접착 단백질을 포함하는 지혈용 조성물 및 이를 포함하는 지혈제를 제안하였다.The present inventors proposed a composition for hemostasis including mussel adhesive protein and a hemostatic agent comprising the same, with the view that a functional hemostatic agent can be developed using a material derived from a marine organism having excellent biocompatibility and physical properties.

따라서, 본 발명은 제1 성분으로서 홍합 접착 단백질 및 제2 성분으로서 홍합 접착 단백질 이외의 생체 유래 고분자를 포함하는 지혈용 조성물에 관한 것이다.Accordingly, the present invention relates to a composition for hemostasis comprising a mussel adhesive protein as the first component and a bio-derived polymer other than the mussel adhesive protein as the second component.

또한, 본 발명은 본 발명에 따른 지혈용 조성물을 포함하는 지혈제에 관한 것이다.In addition, the present invention relates to a hemostatic agent comprising the composition for hemostasis according to the present invention.

본 발명에 따라 해양생물 유래 구조단백질 원천소재를 포함하는 지혈제가 제공되며, 이는 기존 지혈제에서 부족했던 수중 상처부위 지혈 효능을 가지며, 우수한 생체적합성, 생분해성을 가진다.In accordance with the present invention, a hemostatic agent comprising a source of structural protein derived from marine life is provided, which has a hemostatic effect in the wounded area of the sea that was lacking in the existing hemostatic agent, and has excellent biocompatibility and biodegradability.

본 발명에 따른 지혈제는 수중 환경에서 강한 조직 접착력을 보유하는 홍합 접착 단백질을 함유하여, 출혈량이 많아 지혈이 어려운 부위에서 우수한 지혈 효능이 있다.The hemostatic agent according to the present invention contains a mussel adhesive protein that retains strong tissue adhesion in an aquatic environment, and has excellent hemostatic effect in areas where hemorrhage is difficult due to a large amount of bleeding.

본 발명에 따른 지혈제는 말미잘 실크 단백질을 함유하여, 팽창과 수축이 많은 조직 환경에서도 우수한 기계적 물성을 확보할 수 있다.The hemostatic agent according to the present invention contains anemone silk protein, and thus can secure excellent mechanical properties even in a tissue environment having a large amount of expansion and contraction.

본 발명에 따른 지혈제는 합성 고분자가 아닌 천연 해양 구조단백질 소재를 이용하여 높은 생체 안정성을 보유하며 미생물 시스템을 통한 대량생산이 가능하다.The hemostatic agent according to the present invention has a high bio-stability by using a natural marine structural protein material rather than a synthetic polymer, and mass production through a microbial system is possible.

본 발명에 따른 지혈제는 적용 부위의 특성에 따라 다양한 형태로의 제형이 가능하다.The hemostatic agent according to the present invention can be formulated in various forms depending on the characteristics of the application site.

도 1은 본 발명에 따른 지혈제 제조방법에 관한 개념도이다.
도 2는 실시예 1에 따라 제조한 홍합 접착 단백질을 포함하는 하이드로젤 소재 및 홍합 접착 단백질/말미잘 실크 단백질 광가교 하이드로젤 소재의 최대 인장 응력을 나타낸 그래프이다. 막대그래프 가운데에 표시된 선분은 표준편차를 의미한다.
도 3은 실시예 1에 따라 제조한 홍합 접착 단백질을 포함하는 하이드로젤 소재 및 홍합 접착 단백질/말미잘 실크 단백질 광가교 하이드로젤 소재의 영 계수를 나타낸 그래프이다. 막대그래프 가운데에 표시된 선분은 표준편차를 의미한다.
도 4는 실시예 1에 따라 제조한 하이드로젤을 이용하는 경우의 혈액 응고 정도를 540nm 파장 흡광도를 이용하여 측정한 결과를 나타낸 그래프이다. 막대그래프 가운데에 표시된 선분은 표준편차를 의미한다.
도 5는 실시예 1에 따라 제조한 하이드로젤이 신호전달물질 PF4 (Platelet Factor 4) 분비에 미치는 영향을 나타낸 그래프이다. 막대그래프 가운데에 표시된 선분은 표준편차를 의미한다.
도 6은 실시예 1에 따라 제조한 하이드로젤을 이용하는 경우 마우스에서 출혈량이 감소되는 것을 확인한 사진이다.
도 7은 실시예 2에 따라 제조한 콜로이드 용액이 혈소판 활성을 시키는 것을 나타내는 전자현미경 사진이다.
도 8은 실시예 2에 따라 제조한 콜로이드 용액의 상대적인 혈액응고 능력을 나타낸 그래프이다. 막대그래프 가운데에 표시된 선분은 표준편차를 의미한다.
도 9는 실시예 2에 따라 제조한 콜로이드 용액의 상대적인 PF4 분비량을 나타낸 그래프이다. 막대그래프 가운데에 표시된 선분은 표준편차를 의미한다.
도 10은 실시예 2에 따라 제조한 콜로이드 용액을 사용하는 경우 혈액 흡수 및 응고가 일어난 형태를 나타낸 전자현미경 사진이다.
도 11은 실시예 3에 따라 제조한 나노섬유에서 혈액이 통과하지 않고 표면에서 응고되는 것을 나타낸 사진이다.1 is a conceptual diagram of a method for manufacturing a hemostatic agent according to the present invention.
2 is a graph showing the maximum tensile stress of a hydrogel material and mussel adhesive protein / anemone silk protein photocrosslinked hydrogel material including mussel adhesive protein prepared according to Example 1. The line segment in the middle of the bar graph indicates the standard deviation.
Figure 3 is a graph showing the Young's modulus of the hydrogel material and mussel adhesive protein / anemone silk protein light crosslinked hydrogel material containing the mussel adhesive protein prepared according to Example 1. The line segment in the middle of the bar graph indicates the standard deviation.
Figure 4 is a graph showing the results of measuring the blood coagulation degree when using a hydrogel prepared according to Example 1 using a wavelength absorbance of 540nm. The line segment in the middle of the bar graph indicates the standard deviation.
5 is a graph showing the effect of the hydrogel prepared according to Example 1 on the secretion of the signal transducer PF4 (Platelet Factor 4). The line segment in the middle of the bar graph indicates the standard deviation.
6 is a photograph confirming that the amount of bleeding is reduced in mice when using the hydrogel prepared according to Example 1.
7 is an electron micrograph showing that the colloidal solution prepared according to Example 2 activates platelet activity.
8 is a graph showing the relative blood clotting ability of the colloidal solution prepared according to Example 2. The line segment in the middle of the bar graph indicates the standard deviation.
9 is a graph showing the relative amount of PF4 secretion of the colloidal solution prepared according to Example 2. The line segment in the middle of the bar graph indicates the standard deviation.
10 is an electron microscope photograph showing a form in which blood absorption and coagulation occurred when using the colloidal solution prepared according to Example 2.
11 is a photograph showing that the nanofibers prepared according to Example 3 did not pass blood and solidified on the surface.

본 발명은 제1 성분으로서 홍합 접착 단백질; 및 제2 성분으로서 홍합 접착 단백질 이외의 생체 유래 고분자;를 포함하는 지혈용 조성물에 관한 것이다. The present invention is a mussel adhesive protein as a first component; And a second component, a bio-derived polymer other than mussel adhesive protein.

본 발명의 조성물은 제1 성분으로서 홍합 접착 단백질을 포함한다. 상기 홍합 접착 단백질은 홍합에서 유래한 접착 단백질로, 미틸러스 에둘리스(Mytilus edulis), 미틸러스 갈로프로빈시얼리스(Mytilus galloprovincialis) 및 미틸러스 코루스커스(Mytilus coruscus)로 구성된 군에서 선택되는 홍합과 패류에서 유래된 것일 수 있으나, 이에 제한되지는 않는다. The composition of the present invention contains mussel adhesive protein as a first component. The mussel adhesive protein is an adhesive protein derived from mussel, and is composed of Mytilus edulis, Mytilus galloprovincialis, and Mytilus coruscus. It may be derived from mussels and shellfish selected from, but is not limited thereto.

본 발명에 따른 지혈용 조성물에서 상기 홍합 접착 단백질은 홍합에서 유래한 Mefp(Mytilus edulis foot protein)-1, Mgfp(Mytilus galloprovincialis foot protein)-1, Mcfp(Mytilus coruscus foot protein)-1, Mefp-2, Mefp-3, Mgfp-3 및 Mgfp-5로 이루어진 군에서 1 종 이상 선택되는 것일 수 있고, 바람직하게는 fp(foot protein)-1(서열번호 1), fp-2(서열번호 2), fp-3(서열번호 3), fp-4(서열번호 4), fp-5(서열번호 5), 및 fp-6(서열번호 6)로 이루어진 군에서 1 종 이상 선택되는 것일 수 있다. 또한, 본 발명의 홍합 접착 단백질은 WO2006/107183A1 또는 WO2005/092920A1에 기재된 모든 홍합 접착 단백질 중에서 1종 이상 선택되는 것일 수 있다. 바람직하게는, 상기 홍합 접착 단백질은 fp-151(서열번호 7), fp-131(서열번호 8), fp-353(서열번호 9), fp-153(서열번호 10) 및 fp-351(서열번호 11)으로 이루어진 군에서 1종 이상 선택되는 것일 수 있다. 홍합 접착 단백질은 접착 효과 뿐 아니라, 혈소판을 활성화시키며 지혈 효과가 있는 것으로 알려져 있다.In the composition for hemostasis according to the present invention, the mussel adhesion protein is Mytilus edulis foot protein (Mefp) -1, Mgtil (Mytilus galloprovincialis foot protein) -1, Mcfp (Mytilus coruscus foot protein) -1, Mefp-2 derived from mussels , Mefp-3, Mgfp-3 and Mgfp-5 may be one or more selected from the group consisting of, preferably fp (foot protein) -1 (SEQ ID NO: 1), fp-2 (SEQ ID NO: 2), It may be one or more selected from the group consisting of fp-3 (SEQ ID NO: 3), fp-4 (SEQ ID NO: 4), fp-5 (SEQ ID NO: 5), and fp-6 (SEQ ID NO: 6). Further, the mussel adhesion protein of the present invention may be one or more selected from all mussel adhesion proteins described in WO2006 / 107183A1 or WO2005 / 092920A1. Preferably, the mussel adhesion protein is fp-151 (SEQ ID NO: 7), fp-131 (SEQ ID NO: 8), fp-353 (SEQ ID NO: 9), fp-153 (SEQ ID NO: 10) and fp-351 (SEQ ID NO: Number 11) may be selected from one or more groups. It is known that mussel adhesion protein not only has an adhesion effect, but also activates platelets and has a hemostatic effect.

이때 본 발명에 따른 지혈용 조성물은 홍합 접착 단백질 20 중량% 이상, 바람직하게는 30 중량% 이상, 더욱 바람직하게는 50 중량% 이상을 포함할 수 있다. 홍합 접착 단백질이 20 중량%보다 적게 포함되는 경우에는 지혈 효과가 현저히 떨어지는 문제점이 있다.At this time, the composition for hemostasis according to the present invention may include at least 20% by weight of mussel adhesive protein, preferably at least 30% by weight, more preferably at least 50% by weight. When the mussel adhesive protein is contained less than 20% by weight, there is a problem in that the hemostatic effect is significantly reduced.

또한, 본 발명의 조성물은 제2 성분으로서 생체 유래 고분자를 포함한다. 상기 생체 유래 고분자는 홍합 접착 단백질 이외의 생체 유래 고분자로서, 말미잘 실크 단백질, 키토산, 히알루론산, 콜라젠(collagen), 젤라틴(gelatin), 알부민(albumin), 알긴산(alginate), 셀룰로오스(cellulose), 트롬빈(thrombin) 및 피브린(fibrin)으로 구성된 군에서 1종 이상 선택될 수 있다. 지혈용 조성물은 출혈 부위에서 혈액에 흘러내리지 않고, 조직의 신축에 따라 형태를 유지할 수 있어야 한다. 이를 위해 본 발명에서는 조성물 내에 생체 구조 소재로서 상기와 같은 생체 유래 고분자를 더 포함시켜 기계적 물성을 개선한다. 말미잘 실크 단백질은 아네로인(aneroin)이라고도 불리며, 제형화했을 때 강한 기계적 물성을 나타내고, 특히 인체 내 장기에 필요한 탄성을 가지고 있다. 말미잘 실크 단백질은 서열번호 12 내지 14로 구성된 군에서 1종 이상 선택될 수 있다. 키토산의 경우 지혈 효과를 가지고 있으며 생체 재료로 적합하다. 히알루론산은 N-아세틸-D-글루코사민과 D-글루쿠론산이 교대로 사슬 형태로 결합된 생체 유래 고분자 물질로서, 피부, 탯줄 등 동물의 조직에 많이 존재하며, 생체 재료로 적합하고, 물성 조절이 용이하다. 콜라젠은 피부, 모발, 연골, 뼈 등을 구성하는 단백질로 혈액이 콜라젠에 노출될 경우 혈액 응고가 촉진된다. 젤라틴은 콜라젠을 고온으로 처리하여 얻을 수 있으며 콜라젠과 마찬가지로 혈액 응고를 촉진시킬 수 있다. 알부민은 혈액 및 조직액에 포함되는 주 단백질로 삼투압 조절 등 역할을 한다. 알긴산은 갈조류에 함유되는 다당류로 칼슘 이온에 노출되면 겔화하는 성질이 있어 혈액 응고에 사용할 수 있다. 셀룰로오스는 식물을 주로 구성하는 다당류로 지혈 소재로 이용되어 혈액 응고를 촉진할 수 있다. 트롬빈은 혈장에 존재하는 분해효소로 피브리노겐을 피브린으로 분해하여 혈전을 형성한다. 피브린은 혈전의 주요 성분으로 혈구 및 혈장 단백질과 함께 응집하여 지혈 마개를 형성한다.In addition, the composition of the present invention contains a bio-derived polymer as a second component. The bio-derived polymer is a bio-derived polymer other than mussel adhesion protein, anemone silk protein, chitosan, hyaluronic acid, collagen, gelatin, albumin, albumin, alginate, cellulose, and thrombin (thrombin) and fibrin (fibrin) may be selected from one or more. The composition for hemostasis should not flow into the blood at the bleeding site, but must be able to maintain its shape according to the elasticity of the tissue. To this end, the present invention further improves mechanical properties by further including a bio-derived polymer as described above as a bio-structured material in the composition. Anemone silk protein, also called aneroin, exhibits strong mechanical properties when formulated, and has the necessary elasticity, especially for organs in the human body. Anemone silk protein may be selected from one or more from the group consisting of SEQ ID NOs: 12 to 14. Chitosan has a hemostatic effect and is suitable as a biomaterial. Hyaluronic acid is a bio-derived polymer substance in which N-acetyl-D-glucosamine and D-glucuronic acid are alternately bound in a chain form. This is easy. Collagen is a protein that makes up skin, hair, cartilage, bone, etc. When blood is exposed to collagen, blood clotting is promoted. Gelatin can be obtained by treating collagen at a high temperature and, like collagen, can promote blood clotting. Albumin is the main protein contained in blood and tissue fluids, and serves to regulate osmotic pressure. Alginic acid is a polysaccharide contained in brown algae, which has a gelling property when exposed to calcium ions and can be used for blood clotting. Cellulose is a polysaccharide mainly composed of plants and is used as a hemostatic material to promote blood clotting. Thrombin is a degrading enzyme in plasma that breaks down fibrinogen into fibrin to form blood clots. Fibrin is a major component of thrombus and aggregates with blood cells and plasma proteins to form a hemostatic stopper.

본 발명의 지혈용 조성물은 제1 성분인 홍합 접착 단백질과 제2 성분인 홍합 접착 단백질 이외의 생체 유래 고분자를 2:8~8:2의 중량비로 포함할 수 있다. 2 성분이 상기 중량비보다 낮은 범위로 포함되는 경우에는 지혈제로 사용하기 위한 기계적 물성을 확보할 수 없고, 상기 중량비보다 높은 범위로 포함되는 경우에는 지혈 및 접착 효과가 떨어지는 문제가 있다.The composition for hemostasis of the present invention may include a bio-derived polymer other than the first component mussel adhesive protein and the second component mussel adhesive protein in a weight ratio of 2: 8 to 8: 2. When the two components are included in a range lower than the weight ratio, mechanical properties for use as a hemostatic agent cannot be secured, and when included in a range higher than the weight ratio, there is a problem in that hemostasis and adhesion effects are deteriorated.

본 발명의 지혈용 조성물은 염화칼슘(CaCl2), 아데노신이인산(ADP), 비타민 K, 트롬보플라스틴, 포도당, 탈크, 탄산나트륨, 스테아린산마그네슘 및 폴리비닐알코올로 이루어진 군에서 1종 이상 선택되는 첨가제를 더 포함할 수 있으나, 이에 한정되지는 않으며, 본 발명의 지혈용 조성물의 형태, 용도, 투여 부위 및 경로, 사용량에 따라 필요한 약학적 성분들을 추가로 포함할 수 있다. 여기서, 염화칼슘을 염 또는 수용액의 형태로 지혈용 조성물 리터당 약 5~25 mM을 첨가하여 지혈 경로 활성에 필요한 칼슘 이온을 제공할 수 있다. 아데노신이인산을 염 또는 수용액의 형태로 지혈용 조성물 리터당 약 0.5~5 μM을 첨가하여 지혈 경로를 활성화할 수 있다. The composition for hemostasis of the present invention is one or more additives selected from the group consisting of calcium chloride (CaCl 2 ), adenosine diphosphate (ADP), vitamin K, thromboplastin, glucose, talc, sodium carbonate, magnesium stearate and polyvinyl alcohol. It may further include, but is not limited to, the form, use, administration site and route of the composition for hemostasis of the present invention may further include necessary pharmaceutical ingredients according to the amount used. Here, calcium chloride may be added in the form of a salt or an aqueous solution to about 5 to 25 mM per liter of the composition for hemostasis to provide calcium ions necessary for hemostatic pathway activity. Adenosine diphosphate can be activated in the form of a salt or an aqueous solution by adding about 0.5 to 5 μM per liter of the composition for hemostasis.

본 발명에 따른 지혈용 조성물은 하이드로젤, 콜로이드 용액, 나노섬유, 용액, 분말 또는 스프레이 형태로 제공될 수 있다.The composition for hemostasis according to the present invention may be provided in the form of a hydrogel, colloidal solution, nanofiber, solution, powder or spray.

본 발명의 조성물이 하이드로젤 형태로 제공되는 경우, 상기 하이드로젤은 제1 성분, 제2 성분 및 트리스비피리딘루테늄디클로라이드(Ru(II)bpy3 2+2Cl-) 또는 과황산나트륨(SPS)의 혼합물을 젤화하여 제조되는 것일 수 있다. 하이드로젤은 제1 성분 및 제2 성분의 혼합물이 광가교되어 형성된 것일 수 있다. 제1 성분 및 제2 성분을 광가교하여 젤화시킨 소재는 높은 기계적 물성과 다공성 구조 및 접착 능력을 지니게 된다. 이 하이드로젤은 세척 후 습윤 상태의 지혈 패치 또는 동결 건조한 스펀지 형태로 이용될 수 있다.When the composition of the present invention is provided in the form of a hydrogel, the hydrogel comprises a first component, a second component and trisbipyridine ruthenium dichloride (Ru (II) bpy 3 2+ 2Cl − ) or sodium persulfate (SPS). It may be prepared by gelling the mixture. In the hydrogel, a mixture of the first component and the second component may be formed by photocrosslinking. The material obtained by crosslinking the first component and the second component and gelling has high mechanical properties, a porous structure, and adhesion. This hydrogel can be used in the form of a wet hemostatic patch or freeze-dried sponge after washing.

본 발명의 조성물이 콜로이드 용액의 형태로 제공되는 경우, 상기 콜로이드 용액은 제1 성분 및 제2 성분으로 형성된 코아세르베이트를 포함할 수 있다. 이 경우 제2 성분은 음이온성 고분자일 수 있다. 또는, 제1 성분 및 제2 성분에 음이온성 고분자가 더 첨가된 것일 수 있다. 음이온성 고분자는 페레독신(ferredoxin), 폴리스티렌술폰산(poly styrene sulfonic acid), 아라비아 검(gum arabic), 카보폴(carbopol), 고 또는 저 메톡실 펙틴(high or low methoxyl pectin), 카르복시메틸 구아검 나트륨(sodium carboxymethyl guar gum), 잔탄 검(xanthan gum), 유청 단백질(whey protein), 레구민(faba bean legumin), 카르복시메틸 셀룰로오스(carboxymethyl cellulose), 알긴산(alginate), 캐러지넌(carrageenan), 헥사메타인산나트륨(sodium hexametaphosphate), 카제인 나트륨(sodium casinate), 헤모글로빈(hemoglobin), 헤파린(heparin) 및 세포외 다당체 B40(exopolysaccharide B40)으로 구성된 군에서 선택되는 것일 수 있다. 본 발명에 따른 콜로이드 용액 형태의 지혈용 조성물은 제1 성분 및 제2 성분을 1:0.25 내지 1:2.33의 중량비로 포함할 수 있다. 중량비가 이 범위를 벗어나는 경우 코아세르베이트 수율이 낮아져 충분한 양의 코아세르베이트를 얻을 수 없게 된다.When the composition of the present invention is provided in the form of a colloidal solution, the colloidal solution may include coacervate formed from a first component and a second component. In this case, the second component may be an anionic polymer. Alternatively, anionic polymers may be further added to the first component and the second component. Anionic polymers include ferredoxin, poly styrene sulfonic acid, gum arabic, carbopol, high or low methoxyl pectin, carboxymethyl guar gum Sodium (carboxymethyl guar gum), xanthan gum, whey protein, legamin (faba bean legumin), carboxymethyl cellulose, carboxymethyl cellulose, alginate, carrageenan, hexa Sodium metaphosphate (sodium hexametaphosphate), sodium caseinate (sodium casinate), hemoglobin (hemoglobin), heparin (heparin) and may be selected from the group consisting of extracellular polysaccharide B40 (exopolysaccharide B40). The composition for hemostasis in the form of a colloidal solution according to the present invention may include the first component and the second component in a weight ratio of 1: 0.25 to 1: 2.33. When the weight ratio is outside this range, the yield of coacervate is lowered, so that a sufficient amount of coacervate cannot be obtained.

본 발명의 조성물이 나노섬유 형태로 제공되는 경우, 나노섬유는 제1 성분 및 제2 성분의 혼합물을 전기방사하여 제조한 것이거나, 제2 성분을 전기방사하여 제조한 나노섬유에 제1 성분을 흡착시키거나 코팅한 것일 수 있다.When the composition of the present invention is provided in the form of nanofibers, the nanofibers are prepared by electrospinning a mixture of the first component and the second component, or the first component in the nanofibers prepared by electrospinning the second component. It may be adsorbed or coated.

본 발명의 조성물이 용액 형태로 제공되는 경우, 용액은 제1 성분 및 제2 성분을 멸균수, 식염수, 완충액 및 초산나트륨 수용액으로 이루어진 군에서 선택되는 용매와 혼합한 것일 수 있다. 상기 용액은 약학적으로 허용되는 첨가제를 더 포함할 수 있고, 투여 부위 및 경로에 따라 주사액, 드레싱, 실란트, 농축액, 젤 등의 형태로 활용될 수 있다.When the composition of the present invention is provided in the form of a solution, the solution may be a mixture of a first component and a second component with a solvent selected from the group consisting of sterile water, saline, buffer, and aqueous sodium acetate solution. The solution may further include a pharmaceutically acceptable additive, and may be utilized in the form of an injection solution, dressing, sealant, concentrate, gel, etc. depending on the site and route of administration.

본 발명의 조성물이 분말 형태로 제공되는 경우, 분말은 제1 성분 및 제2 성분의 혼합물을 1 mm 이상의 입체, 1 μm 내지 1 mm의 분체, 0.1 내지 1 μm의 미분체 또는 1 μm 미만의 초미분체로 파쇄한 것일 수 있다. 상기 혼합물은 약학적으로 허용되는 첨가제를 더 포함할 수 있다.When the composition of the present invention is provided in the form of a powder, the powder comprises a mixture of the first component and the second component of 1 mm or more solid, 1 μm to 1 mm powder, 0.1 to 1 μm fine powder, or less than 1 μm ultrafine It may be crushed into powder. The mixture may further include a pharmaceutically acceptable additive.

본 발명의 조성물이 스프레이 형태로 제공되는 경우, 스프레이는 제1 성분 및 제2 성분의 혼합물을 외상 및 외과 수술 부위에 스프레이로 도포하는 것일 수 있다.When the composition of the present invention is provided in the form of a spray, the spray may be a mixture of the first component and the second component applied to the trauma and surgical surgical site as a spray.

실시예Example 및  And 실험예Experimental example

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

실시예 1. 홍합 접착 단백질/말미잘 실크 단백질이 광가교된 하이드로젤Example 1. Hydrogel with mussel adhesive protein / anemone silk protein photocrosslinked

300 mg/mL의 fp-151 단백질 포름산 용액 2 mL와 300 mg/mL 서열번호 12의 말미잘 실크 단백질 포름산 용액 1 mL를 상온에서 혼합하고 3시간 동안 교반하였다. 혼합물에 가교제로 트리스비피리딘루테늄디클로라이드 1 mM 및 과황산나트륨 30 mM를 첨가하고, 치과용 LED 조사 램프를 이용하여 (FORZA4, 1200 mW/cm2) 460 nm 파장의 청색광을 3분간 조사하였다. 이를 상온에서 1시간 동안 건조시키고, 증류수로 5회 이상 세척해 용매와 가교제를 제거하여 하이드로젤을 수득하였다.2 mL of 300 mg / mL fp-151 protein formic acid solution and 1 mL of 300 mg / mL anemone silk protein formic acid solution of SEQ ID NO: 12 were mixed at room temperature and stirred for 3 hours. Trisbipyridine ruthenium dichloride 1 mM and sodium persulfate 30 mM were added to the mixture as a crosslinking agent, and blue light having a wavelength of 460 nm was irradiated for 3 minutes using a dental LED irradiation lamp (FORZA4, 1200 mW / cm 2 ). It was dried at room temperature for 1 hour, washed with distilled water 5 or more times to remove the solvent and the crosslinking agent to obtain a hydrogel.

비교예 1. 홍합 접착 단백질을 포함하는 하이드로젤Comparative Example 1. Hydrogel containing mussel adhesive protein

실시예 1에 따라 fp-151 단백질 포름산 용액만을 사용하여 하이드로젤을 제조하였다.A hydrogel was prepared according to Example 1 using only the fp-151 protein formic acid solution.

비교예 2. 말미잘 실크 단백질을 포함하는 하이드로젤Comparative Example 2. Hydrogel containing anemone silk protein

실시예 1에 따라 서열번호 12의 말미잘 실크 단백질 포름산 용액만을 사용하여 하이드로젤을 제조하였다.A hydrogel was prepared using only the anemone silk protein formic acid solution of SEQ ID NO: 12 according to Example 1.

기계적 물성 확인Check mechanical properties

실시예 1, 비교예 1 및 2에 따라 제조한 하이드로젤의 기계적 물성을 확인하였다. 추가적인 대조군으로는 시중에서 판매되는 써지셀 (Surgicel®) 및 바이오글루 (Bioglue®)를 사용하였다.The mechanical properties of the hydrogels prepared according to Example 1 and Comparative Examples 1 and 2 were confirmed. As a further control, commercially available surge cells (Surgicel ® ) and bioglue (Bioglue ® ) were used.

각 샘플을 0.5 × 2 cm의 직사각형으로 절단하여 인장강도계에 걸고 0.83 mm/s 의 속력으로 인장하여 샘플이 끊어지기 직전에 걸린 힘을 샘플의 단면적으로 나누어 각 샘플의 최대 인장 응력을 확인하였다 (도 2).Each sample was cut into 0.5 × 2 cm rectangles, hung on a tensile strength meter, and pulled at a speed of 0.83 mm / s to divide the force applied just before the sample was cut by the cross-section of the sample to determine the maximum tensile stress of each sample ( Figure 2).

각 샘플의 초기 인장 응력을 길이 변형률로 나누어 각 샘플의 영 계수를 확인하였다 (도 3).The initial tensile stress of each sample was divided by the length strain to confirm the Young's modulus of each sample (FIG. 3).

실시예 2. 홍합 접착 단백질 및 히알루론산을 포함하는 콜로이드 용액Example 2. Colloidal solution comprising mussel adhesive protein and hyaluronic acid

10 mg/mL의 fp-151 단백질 포름산 용액과 10 mg/mL의 히알루론산 수용액을 1:0.25의 중량비로 혼합하여 코아세르베이트를 포함하는 콜로이드 용액을 얻었다. 그 다음 원심분리기를 이용해 코아세르베이트 상을 분리하였다.A 10 mg / mL fp-151 protein formic acid solution and a 10 mg / mL hyaluronic acid aqueous solution were mixed at a weight ratio of 1: 0.25 to obtain a colloidal solution containing coacervate. The coacervate phase was then separated using a centrifuge.

비교예 3. 홍합 접착 단백질을 포함하는 용액Comparative Example 3. Solution containing mussel adhesive protein

fp-151 단백질을 인산 완충액에 300 g/L의 농도로 혼합하여 홍합 접착 단백질 용액을 준비하였다.A mussel adhesive protein solution was prepared by mixing fp-151 protein in a phosphate buffer at a concentration of 300 g / L.

실시예 3. 홍합 접착 단백질 및 말미잘 실크 단백질을 포함하는 나노섬유Example 3. Nanofiber comprising mussel adhesive protein and anemone silk protein

헥사플루오로이소프로판올(HFIP)과 트리플루오로아세트산(TFA)를 9:1의 부피비로 혼합한 혼합물 1 mL에 150 mg의 서열번호 12의 말미잘 실크 단백질을 첨가하여 혼합하고, 상온에서 3시간 동안 교반하였다. 23 gauge 바늘 주사기에 상기 혼합물을 넣고 3 kV/cm의 전기장 내에서 1 mL/hr의 속도로 사출하고, 방사된 나노섬유를 상온에서 3시간 동안 건조한 후 90% 에탄올 용액에 넣어 경화시켰다.150 mg of anemone silk protein of SEQ ID NO: 12 was added to 1 mL of a mixture of hexafluoroisopropanol (HFIP) and trifluoroacetic acid (TFA) in a volume ratio of 9: 1, mixed and stirred at room temperature for 3 hours. Did. The mixture was put in a 23 gauge needle syringe and injected at a rate of 1 mL / hr in an electric field of 3 kV / cm, and the spun nanofiber was dried at room temperature for 3 hours and then cured by putting it in a 90% ethanol solution.

말미잘 실크 단백질을 포함하는 나노섬유의 표면에 10 mg/mL fp-151 단백질 포름산 용액 1 mL를 부어 fp-151 단백질을 6시간 동안 흡착시키고, 동결 건조하여 홍합 접착 단백질 및 말미잘 실크 단백질을 포함하는 나노섬유를 제조하였다.The surface of the nanofibers containing anemone silk protein is poured with 1 mL of 10 mg / mL fp-151 protein formic acid solution to adsorb fp-151 protein for 6 hours, and freeze-dried to include mussel adhesive protein and anemone silk protein nano Fibers were prepared.

비교예 4. 말미잘 실크 단백질을 포함하는 나노섬유 제조Comparative Example 4. Preparation of nanofibers containing anemone silk protein

실시예 3에 따른 방법으로 말미잘 실크 단백질을 포함하는 나노섬유를 제조하였다.Nanofibers containing sea anemone silk proteins were prepared by the method according to Example 3.

실험예 1. 하이드로젤 소재의 혈액 응고 효과Experimental Example 1. Blood clotting effect of hydrogel material

실시예 1에 따라 제조한 하이드로젤을 각 10 mg 크기로 캐스팅하여 동결 건조한 후 96-웰 플레이트에 넣고, 혈액 0.1 mL를 첨가하여 37 ℃ 인큐베이터에서 45분 동안 방치하였다. 대조군으로는 비교예 1, 2 및 바이오글루를 사용하였다.The hydrogels prepared according to Example 1 were cast to a size of 10 mg each, freeze-dried, placed in a 96-well plate, and 0.1 mL of blood was added and left in an incubator at 37 ° C for 45 minutes. As a control, Comparative Examples 1 and 2 and bio glue were used.

각 샘플과 반응한 후의 잔류 혈액을 증류수에 200 배로 희석하고, 마이크로플레이트 리더기를 이용하여 540 nm 파장의 흡광도를 측정하여, 각 샘플과 응고되지 않은 적혈구의 농도를 측정함으로써, 혈액 응고 효과를 확인하였다(도 4). The blood clotting effect was confirmed by diluting the residual blood after reacting with each sample 200 times in distilled water and measuring the absorbance at a wavelength of 540 nm using a microplate reader to measure the concentration of each sample and uncoagulated red blood cells. (Figure 4).

실험예 2. 하이드로젤 소재가 PF4 분비에 미치는 영향Experimental Example 2. Effect of hydrogel material on PF4 secretion

실험예 1에 따라 반응시킨 혈액을 12,000 rpm에서 30분 동안 원심 분리하여 혈장을 분리하고 증류수로 200 배 희석한 후 이 샘플에 존재하는 PF4의 농도를 ELISA로 분석하였다. PF4 캡쳐 항체로 코팅하고 BSA로 블로킹한 96-웰 마이크로플레이트에 각 샘플을 100 μm씩 넣어 상온에서 2시간 동안 반응시키고, 각 웰을 300 μm 워시 버퍼로 3회 세척하였다. 이후 검출 항체 용액 100 μm를 넣어 상온에서 2시간 반응한 후 마찬가지로 각 웰을 세척하였다. 이후 100 μm 스트렙타비딘-HRP 용액을 넣고 암실에서 20분간 반응시키고, 각 웰을 세척하였다. 이후 100 μm 과산화수소수-테트라메틸벤지딘 용액을 넣어 암실에서 20분 반응한 후 50 μm 1M 황산 용액으로 발색을 중지하여 마이크로플레이트 리더로 각 웰의 450 nm 흡광도를 측정해 PF4 분비량(ng/mL)을 확인하였다(도 5). 실시예 1에 따른 하이드로젤 소재를 사용하는 경우에는 혈소판이 활성화되어 응집이 일어났을 때 분비되는 PF4가 600ng/mL 이상 분비된 것을 확인하였다.The blood reacted according to Experimental Example 1 was centrifuged at 12,000 rpm for 30 minutes to separate plasma, diluted 200 times with distilled water, and the concentration of PF4 present in this sample was analyzed by ELISA. Each sample was put in 100 μm in a 96-well microplate coated with PF4 capture antibody and blocked with BSA for 2 hours at room temperature, and each well was washed 3 times with 300 μm wash buffer. Thereafter, 100 μm of the detection antibody solution was added and reacted at room temperature for 2 hours, and then each well was washed. Then, 100 μm streptavidin-HRP solution was added, and the reaction was performed for 20 minutes in the dark, and each well was washed. Then, after adding 100 μm hydrogen peroxide-tetramethylbenzidine solution for 20 minutes in the dark, stopping color development with 50 μm 1M sulfuric acid solution, measuring the absorbance at 450 nm of each well with a microplate reader to determine the amount of PF4 secretion (ng / mL). It was confirmed (Fig. 5). In the case of using the hydrogel material according to Example 1, it was confirmed that PF4 secreted when platelets are activated and aggregated, and secreted more than 600 ng / mL.

실험예 3. 하이드로젤 소재의 지혈 효과Experimental Example 3. Hemostatic effect of hydrogel material

마우스의 간에 22 gauge로 구멍을 내고, 300 mg/mL의 fp-151 단백질 포름산 용액 2 mL와 300 mg/mL 서열번호 12의 말미잘 실크 단백질 포름산 용액 1 mL를 상온에서 혼합하고 3시간 동안 교반하여 얻은 혼합물 0.2 mL를 직접 주사하고, 460 nm 파장으로 광가교하여 젤화시켰다. 출혈이 멎을 때까지의 출혈량을 계량하였다(도 6). 본 발명의 하이드로젤을 도입하는 경우 음성 대조군에 비해 출혈량이 약 60% 이상 감소한 것을 확인하였다.The mouse liver was punctured at 22 gauge and obtained by mixing 2 mL of 300 mg / mL fp-151 protein formic acid solution and 1 mL of 300 mg / mL anemone silk protein formic acid solution of SEQ ID NO: 12 at room temperature and stirring for 3 hours. 0.2 mL of the mixture was directly injected and gelled by photocrosslinking at a wavelength of 460 nm. The amount of bleeding until bleeding was stopped was measured (Fig. 6). When introducing the hydrogel of the present invention, it was confirmed that the bleeding amount was reduced by about 60% or more compared to the negative control.

실험예 4. 콜로이드 용액의 혈액 응고 효과Experimental Example 4. Blood clotting effect of colloidal solution

실시예 2에 따라 제조한 콜로이드 용액 10 μL을 혈액 100 μL에 첨가하고 0.2 M의 염화칼슘 10 μL와 혼합시켜 37 ℃에서 10분 동안 반응시켰다. 대조군으로는 전혈, 음성 대조군(생리 식염수) 및 비교예 3을 사용하였다. 도 7은 음성 대조군과 실시예 2 및 비교예 3에 따른 콜로이드 용액을 혈액에 처리하였을 때 혈소판 활성(화살표)을 확인한 전자현미경 사진이다.10 μL of the colloidal solution prepared according to Example 2 was added to 100 μL of blood, mixed with 10 μL of 0.2 M calcium chloride, and reacted at 37 ° C. for 10 minutes. As a control group, whole blood, a negative control group (physiological saline) and Comparative Example 3 were used. 7 is an electron micrograph confirming platelet activity (arrow) when the negative control and the colloidal solutions according to Example 2 and Comparative Example 3 were treated with blood.

각 샘플에서 실험예 1과 같은 방법으로 전혈을 기준으로 상대적인 혈액응고능력을 확인하였다(도 8). 실시예 2에 따른 콜로이드 용액을 사용하는 경우 전혈에 비해 약 1.7배 이상의 혈액응고능력이 있음을 확인하였다.In each sample, relative blood clotting ability was confirmed based on whole blood in the same manner as in Experimental Example 1 (FIG. 8). When using the colloidal solution according to Example 2, it was confirmed that it has a blood clotting ability of about 1.7 times or more compared to whole blood.

실험예 5. 콜로이드 용액이 PF4 분비에 미치는 영향Experimental Example 5. Effect of colloidal solution on PF4 secretion

실시예 2에 따라 제조한 콜로이드 용액 10 μL을 혈액 100 μL에 첨가하고 상온에서 45분 동안 혼합하였다. 대조군으로는 음성 대조군(생리 식염수), 양성 대조군(5 μg/mL 콜라겐 용액) 및 비교예 3을 사용하였다.10 μL of the colloidal solution prepared according to Example 2 was added to 100 μL of blood and mixed for 45 minutes at room temperature. As a control, a negative control (physiological saline), a positive control (5 μg / mL collagen solution) and Comparative Example 3 were used.

각 샘플에서 반응한 혈액을 2,500 rpm에서 30분 동안 원심 분리하여 혈장을 분리하고 증류수로 200 배로 희석한 후 이 샘플에 존재하는 PF4의 농도를 ELISA로 분석하였다. PF4 캡쳐 항체로 코팅하고 BSA로 블로킹한 96-웰 마이크로플레이트에 각 샘플을 100 μm 넣고 4 ℃에서 하룻밤 동안 반응시킨 후, 각 웰을 300 μm 워시 버퍼로 4회 세척하였다. 이후 100 μm 검출 항체 용액을 첨가하고 상온에서 1시간 반응시킨 후 각 웰을 세척하였다. 이후 100 μm 스트렙타비딘-HRP 용액을 넣어 암실에서 45분 반응한 후 각 웰을 세척하였다. 이후 100 μm 과산화수소수-테트라메틸벤지딘 용액을 넣어 암실에서 30분 반응 시킨 후 50 μm 0.45 M 황산 용액으로 발색을 중지하여 마이크로플레이트 리더로 각 웰의 450 nm 흡광도를 측정하여, 전혈을 기준으로한 상대적인 PF4 분비량을 확인하였다(도 9). 이때, 실시예 2에 따른 콜로이드 용액을 사용하는 경우 약 3배 이상의 PF4가 분비되는 것을 확인하였다. 각 샘플에서 혈소판이 활성화되어 응집된 것(화살표)을 전자현미경으로 확인하였다(도 10).The blood reacted in each sample was centrifuged at 2,500 rpm for 30 minutes to separate plasma, diluted 200-fold with distilled water, and the concentration of PF4 present in this sample was analyzed by ELISA. 100 μm of each sample was placed in a 96-well microplate coated with PF4 capture antibody and blocked with BSA, and reacted overnight at 4 ° C., and then each well was washed 4 times with 300 μm wash buffer. Then, 100 μm detection antibody solution was added and reacted at room temperature for 1 hour, and then each well was washed. Thereafter, 100 μm streptavidin-HRP solution was added to react for 45 minutes in the dark, and then each well was washed. Thereafter, 100 μm hydrogen peroxide-tetramethylbenzidine solution was added and reacted in the dark for 30 minutes, and then color development was stopped with a 50 μm 0.45 M sulfuric acid solution to measure absorbance at 450 nm of each well using a microplate reader, and relative relative to whole blood. The amount of PF4 secretion was confirmed (Fig. 9). At this time, when using the colloidal solution according to Example 2, it was confirmed that PF4 is secreted by about 3 times or more. In each sample, platelets were activated and aggregated (arrows) were confirmed with an electron microscope (FIG. 10).

실험예Experimental example 6. 나노섬유의 혈액 응고 효과 6. Blood coagulation effect of nanofibers

실시예 3에 따라 제조한 나노섬유를 5 mm 생검 펀치로 잘라내어 옐로 마이크로파이펫 팁 상단에 끼워 고정하고, 그 위에 혈액 0.1 mL를 얹었다. 대조군으로는 비교예 4의 나노섬유를 사용하였다. 5분 후 홍합 접착 단백질 및 말미잘 실크 단백질을 포함하는 나노섬유의 경우 혈액이 응고되어 나노섬유를 통과하는 혈액이 관찰되지 않았으나, 말미잘 실크 단백질만을 포함하는 나노섬유의 경우에는 혈액이 응고되지 못하고 나노섬유를 통과하여 팁 아래쪽으로 이동하는 것을 확인하였다(도 11).The nanofibers prepared according to Example 3 were cut with a 5 mm biopsy punch and fixed by inserting them on the top of a yellow micropipette tip, and 0.1 mL of blood was placed thereon. As a control, the nanofiber of Comparative Example 4 was used. In the case of nanofibers containing mussel adhesive protein and anemone silk protein after 5 minutes, blood was clot and blood passing through the nanofiber was not observed, but in the case of nanofibers containing only anemone silk protein, blood did not coagulate and nanofibers It was confirmed to move downward through the tip (FIG. 11).

이상과 같이 실시예를 통하여 본 발명을 설명하였다. 본 발명이 속하는 기술분야의 통상의 기술자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 그러므로 상술한 실시예들은 모든 면에 예시적인 것이며 한정적인 것이 아닌 것으로서 이해되어야 한다. 본 발명의 범위는 상세한 설명보다는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The present invention has been described through the examples as described above. Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. Therefore, the above-described embodiments are to be understood as illustrative in all respects and not restrictive. The scope of the present invention is indicated by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted to be included in the scope of the present invention.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Novel hemostatic agent using mussel adhesive protein and method for preparing the same <130> 1-37P-1 <150> KR 10-2016-0152646 <151> 2016-11-16 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 1695 <212> PRT <213> Artificial Sequence <220> <223> fp-1 <400> 1 Met Glu Thr Gly Leu Ala Gly Leu Tyr Ile Leu Glu Leu Tyr Ser Leu 1 5 10 15 Glu Ala Ala Ser Asn Leu Glu Ala Cys Tyr Ser Leu Glu Ala Leu Glu 20 25 30 Ala Cys Tyr Ser Ile Leu Glu Pro His Glu Thr His Arg Pro His Glu 35 40 45 Ala Ser Pro Val Ala Leu Leu Glu Ala Gly Leu Tyr Pro His Glu Ser 50 55 60 Glu Arg Ala Ser Asn Gly Leu Tyr Ala Ser Asn Ile Leu Glu Thr Tyr 65 70 75 80 Arg Ala Ser Asn Ala Leu Ala His Ile Ser Val Ala Leu Ser Glu Arg 85 90 95 Ser Glu Arg Thr Tyr Arg Ala Leu Ala Gly Leu Tyr Ala Leu Ala Ser 100 105 110 Glu Arg Ala Leu Ala Gly Leu Tyr Ala Leu Ala Thr Tyr Arg Leu Tyr 115 120 125 Ser Leu Tyr Ser Leu Glu Ala Pro Arg Ala Ala Ser Asn Ala Leu Ala 130 135 140 Thr Tyr Arg Pro Arg Ala Thr Tyr Arg Gly Leu Tyr Thr His Arg Leu 145 150 155 160 Tyr Ser Pro Arg Ala Gly Leu Ala Pro Arg Ala Val Ala Leu Thr Tyr 165 170 175 Arg Leu Tyr Ser Pro Arg Ala Val Ala Leu Leu Tyr Ser Thr His Arg 180 185 190 Ser Glu Arg Thr Tyr Arg Ser Glu Arg Ala Leu Ala Pro Arg Ala Thr 195 200 205 Tyr Arg Leu Tyr Ser Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr 210 215 220 Arg Gly Leu Asn Gly Leu Asn Leu Glu Ala Leu Tyr Ser Leu Tyr Ser 225 230 235 240 Leu Tyr Ser Val Ala Leu Ala Ser Pro Thr Tyr Arg Ala Arg Gly Pro 245 250 255 Arg Ala Thr His Arg Leu Tyr Ser Ser Glu Arg Thr Tyr Arg Pro Arg 260 265 270 Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Gly Leu Tyr Ser Glu Arg 275 280 285 Leu Tyr Ser Thr His Arg Ala Ser Asn Thr Tyr Arg Leu Glu Ala Pro 290 295 300 Arg Ala Leu Glu Ala Ala Leu Ala Leu Tyr Ser Leu Tyr Ser Leu Glu 305 310 315 320 Ala Ser Glu Arg Ser Glu Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala 325 330 335 Ile Leu Glu Leu Tyr Ser Thr His Arg Thr His Arg Thr Tyr Arg Ala 340 345 350 Ser Asn Ala Leu Ala Leu Tyr Ser Thr His Arg Ala Ser Asn Thr Tyr 355 360 365 Arg Pro Arg Ala Pro Arg Ala Val Ala Leu Thr Tyr Arg Leu Tyr Ser 370 375 380 Pro Arg Ala Leu Tyr Ser Met Glu Thr Thr His Arg Thr Tyr Arg Pro 385 390 395 400 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg 405 410 415 Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala 420 425 430 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ser Glu Arg Leu 435 440 445 Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg 450 455 460 Ala Leu Tyr Ser Ile Leu Glu Thr His Arg Cys Tyr Ser Pro Arg Ala 465 470 475 480 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu 485 490 495 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg 500 505 510 Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser 515 520 525 Leu Tyr Ser Thr His Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr 530 535 540 His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser Val Ala 545 550 555 560 Leu Thr His Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg 565 570 575 Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser Pro Arg Ala Ser 580 585 590 Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Ile Leu Glu Thr Tyr 595 600 605 Arg Leu Tyr Ser Ser Glu Arg Leu Tyr Ser Pro Arg Ala Thr His Arg 610 615 620 Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser Ile Leu Glu Thr 625 630 635 640 His Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr 645 650 655 Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg 660 665 670 Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu 675 680 685 Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr 690 695 700 Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser 705 710 715 720 Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr Arg Leu 725 730 735 Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr 740 745 750 Arg Pro Arg Ala Ser Glu Arg Thr His Arg Thr Tyr Arg Leu Tyr Ser 755 760 765 Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr Arg Pro 770 775 780 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu 785 790 795 800 Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala 805 810 815 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu 820 825 830 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg 835 840 845 Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser 850 855 860 Pro Arg Ala Thr His Arg Thr Tyr Arg Ile Leu Glu Ala Leu Ala Leu 865 870 875 880 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg 885 890 895 Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser 900 905 910 Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr 915 920 925 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg 930 935 940 Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg 945 950 955 960 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Ser Glu Arg Ser 965 970 975 Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr 980 985 990 Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg 995 1000 1005 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr 1010 1015 1020 His Arg Thr Tyr Arg Pro Arg Ala Ser Glu Arg Thr His Arg Thr Tyr 1025 1030 1035 1040 Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg 1045 1050 1055 Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu 1060 1065 1070 Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr 1075 1080 1085 Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg 1090 1095 1100 Thr Tyr Arg Pro Arg Ala Ser Glu Arg Thr His Arg Thr Tyr Arg Leu 1105 1110 1115 1120 Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr 1125 1130 1135 Arg Pro Arg Ala Ser Glu Arg Thr His Arg Thr Tyr Arg Leu Tyr Ser 1140 1145 1150 Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro 1155 1160 1165 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg 1170 1175 1180 Ala Leu Tyr Ser Ile Leu Glu Ser Glu Arg Thr Tyr Arg Pro Arg Ala 1185 1190 1195 1200 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu 1205 1210 1215 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Ser Glu 1220 1225 1230 Arg Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser 1235 1240 1245 Ser Glu Arg Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr 1250 1255 1260 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg 1265 1270 1275 1280 Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg 1285 1290 1295 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr 1300 1305 1310 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg 1315 1320 1325 Ala Thr His Arg Thr Tyr Arg Pro Arg Ala Ser Glu Arg Thr His Arg 1330 1335 1340 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr 1345 1350 1355 1360 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg 1365 1370 1375 Ala Thr His Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg 1380 1385 1390 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser 1395 1400 1405 Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr 1410 1415 1420 Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg 1425 1430 1435 1440 Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu 1445 1450 1455 Tyr Ser Ser Glu Arg Leu Tyr Ser Ser Glu Arg Ser Glu Arg Thr Tyr 1460 1465 1470 Arg Pro Arg Ala Ser Glu Arg Ser Glu Arg Thr Tyr Arg Leu Tyr Ser 1475 1480 1485 Pro Arg Ala Leu Tyr Ser Leu Tyr Ser Thr His Arg Thr Tyr Arg Pro 1490 1495 1500 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg 1505 1510 1515 1520 Ala Leu Tyr Ser Leu Glu Ala Thr His Arg Thr Tyr Arg Pro Arg Ala 1525 1530 1535 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu 1540 1545 1550 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg 1555 1560 1565 Ala Ser Glu Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser 1570 1575 1580 Ile Leu Glu Thr His Arg Thr Tyr Arg Pro Arg Ala Ser Glu Arg Thr 1585 1590 1595 1600 His Arg Thr Tyr Arg Leu Tyr Ser Leu Glu Ala Leu Tyr Ser Pro Arg 1605 1610 1615 Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg 1620 1625 1630 Thr Tyr Arg Leu Tyr Ser Ser Glu Arg Leu Tyr Ser Thr His Arg Ser 1635 1640 1645 Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr 1650 1655 1660 Arg Ala Ser Asn Leu Tyr Ser Leu Tyr Ser Ile Leu Glu Ser Glu Arg 1665 1670 1675 1680 Thr Tyr Arg Pro Arg Ala Ser Glu Arg Gly Leu Asn Thr Tyr Arg 1685 1690 1695 <210> 2 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> fp-2 <400> 2 Thr Asn Arg Pro Asp Tyr Asn Asp Asp Glu Glu Asp Asp Tyr Lys Pro 1 5 10 15 Pro Val Tyr Lys Pro Ser Pro Ser Lys Tyr Arg Pro Val Asn Pro Cys 20 25 30 Leu Lys Lys Pro Cys Lys Tyr Asn Gly Val Cys Lys Pro Arg Gly Gly 35 40 45 Ser Tyr Lys Cys Phe Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn Cys Asn 50 55 60 Leu Lys Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn Lys Ser Arg Cys 65 70 75 80 Val Pro Val Gly Lys Thr Phe Lys Cys Val Cys Arg Asn Gly Asn Phe 85 90 95 Gly Arg Leu Cys Glu Lys Asn Val Cys Ser Pro Asn Pro Cys Lys Asn 100 105 110 Asn Gly Lys Cys Ser Pro Leu Gly Lys Thr Gly Tyr Lys Cys Thr Cys 115 120 125 Ser Gly Gly Tyr Thr Gly Pro Arg Cys Glu Val His Ala Cys Lys Pro 130 135 140 Asn Pro Cys Lys Asn Lys Gly Arg Cys Phe Pro Asp Gly Lys Thr Gly 145 150 155 160 Tyr Lys Cys Arg Cys Val Asp Gly Tyr Ser Gly Pro Thr Cys Gln Glu 165 170 175 Asn Ala Cys Lys Pro Asn Pro Cys Ser Asn Gly Gly Thr Cys Ser Ala 180 185 190 Asp Lys Phe Gly Asp Tyr Ser Cys Glu Cys Arg Pro Gly Tyr Phe Gly 195 200 205 Pro Glu Cys Glu Arg Tyr Val Cys Ala Pro Asn Pro Cys Lys Asn Gly 210 215 220 Gly Ile Cys Ser Ser Asp Gly Ser Gly Gly Tyr Arg Cys Arg Cys Lys 225 230 235 240 Gly Gly Tyr Ser Gly Pro Thr Cys Lys Val Asn Val Cys Lys Pro Thr 245 250 255 Pro Cys Lys Asn Ser Gly Arg Cys Val Asn Lys Gly Ser Ser Tyr Asn 260 265 270 Cys Ile Cys Lys Gly Gly Tyr Ser Gly Pro Thr Cys Gly Glu Asn Val 275 280 285 Cys Lys Pro Asn Pro Cys Gln Asn Arg Gly Arg Cys Tyr Pro Asp Asn 290 295 300 Ser Asp Asp Gly Phe Lys Cys Arg Cys Val Gly Gly Tyr Lys Gly Pro 305 310 315 320 Thr Cys Glu Asp Lys Pro Asn Pro Cys Asn Thr Lys Pro Cys Lys Asn 325 330 335 Gly Gly Lys Cys Asn Tyr Asn Gly Lys Ile Tyr Thr Cys Lys Cys Ala 340 345 350 Tyr Gly Trp Arg Gly Arg His Cys Thr Asp Lys Ala Tyr Lys Pro Asn 355 360 365 Pro Cys Val Val Ser Lys Pro Cys Lys Asn Arg Gly Lys Cys Ile Trp 370 375 380 Asn Gly Lys Ala Tyr Arg Cys Lys Cys Ala Tyr Gly Tyr Gly Gly Arg 385 390 395 400 His Cys Thr Lys Lys Ser Tyr Lys Lys Asn Pro Cys Ala Ser Arg Pro 405 410 415 Cys Lys Asn Arg Gly Lys Cys Thr Asp Lys Gly Asn Gly Tyr Val Cys 420 425 430 Lys Cys Ala Arg Gly Tyr Ser Gly Arg Tyr Cys Ser Leu Lys Ser Pro 435 440 445 Pro Ser Tyr Asp Asp Asp Glu Tyr 450 455 <210> 3 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> fp-3 <400> 3 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly 1 5 10 15 Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp 20 25 30 Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 <210> 4 <211> 787 <212> PRT <213> Artificial Sequence <220> <223> fp-4 <400> 4 Tyr Gly Arg Arg Tyr Gly Glu Pro Ser Gly Tyr Ala Asn Ile Gly His 1 5 10 15 Arg Arg Tyr Tyr Glu Arg Ala Ile Ser Phe His Arg His Ser His Val 20 25 30 His Gly His His Leu Leu His Arg His Val His Arg His Ser Val Leu 35 40 45 His Gly His Val His Met His Arg Val Ser His Arg Ile Met His Arg 50 55 60 His Arg Val Leu His Gly His Val His Arg His Arg Val Leu His Arg 65 70 75 80 His Val His Arg His Arg Val Leu His Gly His Val His Arg His Arg 85 90 95 Val Leu His Arg His Leu His Arg His Arg Val Leu His Gly His Val 100 105 110 His Arg His Arg Val Leu His Asn His Val His Arg His Ser Val Leu 115 120 125 His Gly His Val His Arg His Arg Val Leu His Arg His Val His Arg 130 135 140 His Asn Val Leu His Gly His Val His Arg His Arg Val Leu His Lys 145 150 155 160 His Val His Asp His Arg Val Leu His Lys His Leu His Lys His Gln 165 170 175 Val Leu His Gly His Val His Arg His Gln Val Leu His Lys His Val 180 185 190 His Asn His Arg Val Leu His Lys His Leu His Lys His Gln Val Leu 195 200 205 His Gly His Val His Thr His Arg Val Leu His Lys His Val His Lys 210 215 220 His Arg Val Leu His Lys His Leu His Lys His Gln Val Leu His Gly 225 230 235 240 His Ile His Thr His Arg Val Leu His Lys His Leu His Lys His Gln 245 250 255 Val Leu His Gly His Val His Thr His Arg Val Leu His Lys His Val 260 265 270 His Lys His Arg Val Leu His Lys His Leu His Lys His Gln Val Leu 275 280 285 His Gly His Val His Met His Arg Val Leu His Lys His Val His Lys 290 295 300 His Arg Val Leu His Lys His Val His Lys His His Val Val His Lys 305 310 315 320 His Val His Ser His Arg Val Leu His Lys His Val His Lys His Arg 325 330 335 Val Glu His Gln His Val His Lys His His Val Leu His Arg His Val 340 345 350 His Ser His His Val Val His Ser His Val His Lys His Arg Val Val 355 360 365 His Ser His Val His Lys His Asn Val Val His Ser His Val His Arg 370 375 380 His Gln Ile Leu His Arg His Val His Arg His Gln Val Val His Arg 385 390 395 400 His Val His Arg His Leu Ile Ala His Arg His Ile His Ser His Gln 405 410 415 Ala Ala Val His Arg His Val His Thr His Val Phe Glu Gly Asn Phe 420 425 430 Asn Asp Asp Gly Thr Asp Val Asn Leu Arg Ile Arg His Gly Ile Ile 435 440 445 Tyr Gly Gly Asn Thr Tyr Arg Leu Ser Gly Gly Arg Arg Arg Phe Met 450 455 460 Thr Leu Trp Gln Glu Cys Leu Glu Ser Tyr Gly Asp Ser Asp Glu Cys 465 470 475 480 Phe Val Gln Leu Gly Asn Gln His Leu Phe Thr Val Val Gln Gly His 485 490 495 His Ser Thr Ser Phe Arg Ser Asp Leu Ser Asn Asp Leu His Pro Asp 500 505 510 Asn Asn Ile Glu Gln Ile Ala Asn Asp His Val Asn Asp Ile Ala Gln 515 520 525 Ser Thr Asp Gly Asp Ile Asn Asp Phe Ala Asp Thr His Tyr Asn Asp 530 535 540 Val Ala Pro Ile Ala Asp Val His Val Asp Asn Ile Ala Gln Thr Ala 545 550 555 560 Asp Asn His Val Lys Asn Ile Ala Gln Thr Ala His His His Val Asn 565 570 575 Asp Val Ala Gln Ile Ala Asp Asp His Val Asn Asp Ile Gly Gln Thr 580 585 590 Ala Tyr Asp His Val Asn Asn Ile Gly Gln Thr Ala Asp Asp His Val 595 600 605 Asn Asp Ile Ala Gln Thr Ala Asp Asp His Val Asn Ala Ile Ala Gln 610 615 620 Thr Ala Asp Asp His Val Asn Ala Ile Ala Gln Thr Ala Asp His Val 625 630 635 640 Asn Asp Ile Gly Asp Thr Ala Asn Ser His Ile Val Arg Val Gln Gly 645 650 655 Val Ala Lys Asn His Leu Tyr Gly Ile Asn Lys Ala Ile Gly Lys His 660 665 670 Ile Gln His Leu Lys Asp Val Ser Asn Arg His Ile Glu Lys Leu Asn 675 680 685 Asn His Ala Thr Lys Asn Leu Leu Gln Ser Ala Leu Gln His Lys Gln 690 695 700 Gln Thr Ile Glu Arg Glu Ile Gln His Lys Arg His Leu Ser Glu Lys 705 710 715 720 Glu Asp Ile Asn Leu Gln His Glu Asn Ala Met Lys Ser Lys Val Ser 725 730 735 Tyr Asp Gly Pro Val Phe Asn Glu Lys Val Ser Val Val Ser Asn Gln 740 745 750 Gly Ser Tyr Asn Glu Lys Val Pro Val Leu Ser Asn Gly Gly Gly Tyr 755 760 765 Asn Gly Lys Val Ser Ala Leu Ser Asp Gln Gly Ser Tyr Asn Glu Gly 770 775 780 Tyr Ala Tyr 785 <210> 5 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> fp-5 <400> 5 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His 1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr 20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys 35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg 50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser 65 70 75 <210> 6 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> fp-6 <400> 6 Gly Gly Gly Asn Tyr Arg Gly Tyr Cys Ser Asn Lys Gly Cys Arg Ser 1 5 10 15 Gly Tyr Ile Phe Tyr Asp Asn Arg Gly Phe Cys Lys Tyr Gly Ser Ser 20 25 30 Ser Tyr Lys Tyr Asp Cys Gly Asn Tyr Ala Gly Cys Cys Leu Pro Arg 35 40 45 Asn Pro Tyr Gly Arg Val Lys Tyr Tyr Cys Thr Lys Lys Tyr Ser Cys 50 55 60 Pro Asp Asp Phe Tyr Tyr Tyr Asn Asn Lys Gly Tyr Tyr Tyr Tyr Asn 65 70 75 80 Asp Lys Asp Tyr Phe Asn Cys Gly Ser Tyr Asn Gly Cys Cys Leu Arg 85 90 95 Ser Gly Tyr <210> 7 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> fp-151 <400> 7 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala 20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu 50 55 60 Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser 65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys 85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly 100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr 115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr 130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 180 185 190 Pro Thr Tyr Lys 195 <210> 8 <211> 172 <212> PRT <213> Artificial Sequence <220> <223> fp-131 <400> 8 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala 20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala 50 55 60 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly 65 70 75 80 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn 85 90 95 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala 100 105 110 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 115 120 125 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 130 135 140 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 145 150 155 160 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu 165 170 <210> 9 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> fp-353 <400> 9 Met Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly 1 5 10 15 Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly 20 25 30 Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Glu 35 40 45 Phe Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn 50 55 60 His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly 65 70 75 80 Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Trp Lys Tyr Lys 85 90 95 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg 100 105 110 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Lys Leu Ala Asp 115 120 125 Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn 130 135 140 Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn 145 150 155 160 Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Leu Glu 165 170 <210> 10 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> fp-153 <400> 10 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala 20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Phe Pro 35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Glu Phe Ser 50 55 60 Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn His Tyr 65 70 75 80 His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys 85 90 95 Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn 100 105 110 Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys 115 120 125 Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Lys Leu Ala Asp Tyr Tyr 130 135 140 Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn Tyr Asn 145 150 155 160 Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn Gly Trp 165 170 175 Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Leu Glu His His His His 180 185 190 His His <210> 11 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fp-351 <400> 11 Met Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly 1 5 10 15 Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly 20 25 30 Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Glu 35 40 45 Phe Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn 50 55 60 His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly 65 70 75 80 Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr 85 90 95 Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His 100 105 110 Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Lys Leu Ala 115 120 125 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 130 135 140 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 145 150 155 160 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 165 170 175 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu Glu 180 185 <210> 12 <211> 319 <212> PRT <213> sea anemone <400> 12 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly 1 5 10 15 His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro 20 25 30 Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro 35 40 45 Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn 50 55 60 Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln 65 70 75 80 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly 85 90 95 Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln Gly Pro 100 105 110 Gly Asn Thr Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly 115 120 125 Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly 130 135 140 Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr 145 150 155 160 Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly 165 170 175 Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys 180 185 190 Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly 195 200 205 Gln Gly Pro Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr 210 215 220 Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp 225 230 235 240 Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys 245 250 255 Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly 260 265 270 Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr 275 280 285 Gly Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln Gly 290 295 300 Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly 305 310 315 <210> 13 <211> 327 <212> PRT <213> sea anemone <400> 13 Met Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr 1 5 10 15 Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp 20 25 30 Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr 35 40 45 Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly 50 55 60 Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly 65 70 75 80 Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr 85 90 95 Gly Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln Gly 100 105 110 Pro Gly Asn Thr Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro 115 120 125 Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro 130 135 140 Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn 145 150 155 160 Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln 165 170 175 Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly 180 185 190 Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro 195 200 205 Gly Gln Gly Pro Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn 210 215 220 Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln 225 230 235 240 Asp Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly 245 250 255 Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser 260 265 270 Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn 275 280 285 Thr Gly Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln 290 295 300 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Leu 305 310 315 320 Glu His His His His His His 325 <210> 14 <211> 340 <212> PRT <213> sea anemone <400> 14 His Met Lys Ala Ile Phe Val Leu Lys Asp Asp Asp Asp Lys Gly Pro 1 5 10 15 Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly His Pro 20 25 30 Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn 35 40 45 Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln 50 55 60 Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly 65 70 75 80 Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro 85 90 95 Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro 100 105 110 Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln Gly Pro Gly Asn 115 120 125 Thr Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr 130 135 140 Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp 145 150 155 160 Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Cys 165 170 175 Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly 180 185 190 Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly 195 200 205 Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly 210 215 220 Pro Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr 225 230 235 240 Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly 245 250 255 Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly 260 265 270 Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr 275 280 285 Gly Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr 290 295 300 Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly 305 310 315 320 Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Leu Glu His His 325 330 335 His His His His 340 <110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Novel hemostatic agent using mussel adhesive protein and method          for preparing the same <130> 1-37P-1 <150> KR 10-2016-0152646 <151> 2016-11-16 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 1695 <212> PRT <213> Artificial Sequence <220> <223> fp-1 <400> 1 Met Glu Thr Gly Leu Ala Gly Leu Tyr Ile Leu Glu Leu Tyr Ser Leu   1 5 10 15 Glu Ala Ala Ser Asn Leu Glu Ala Cys Tyr Ser Leu Glu Ala Leu Glu              20 25 30 Ala Cys Tyr Ser Ile Leu Glu Pro His Glu Thr His Arg Pro His Glu          35 40 45 Ala Ser Pro Val Ala Leu Leu Glu Ala Gly Leu Tyr Pro His Glu Ser      50 55 60 Glu Arg Ala Ser Asn Gly Leu Tyr Ala Ser Asn Ile Leu Glu Thr Tyr  65 70 75 80 Arg Ala Ser Asn Ala Leu Ala His Ile Ser Val Ala Leu Ser Glu Arg                  85 90 95 Ser Glu Arg Thr Tyr Arg Ala Leu Ala Gly Leu Tyr Ala Leu Ala Ser             100 105 110 Glu Arg Ala Leu Ala Gly Leu Tyr Ala Leu Ala Thr Tyr Arg Leu Tyr         115 120 125 Ser Leu Tyr Ser Leu Glu Ala Pro Arg Ala Ala Ser Asn Ala Leu Ala     130 135 140 Thr Tyr Arg Pro Arg Ala Thr Tyr Arg Gly Leu Tyr Thr His Arg Leu 145 150 155 160 Tyr Ser Pro Arg Ala Gly Leu Ala Pro Arg Ala Val Ala Leu Thr Tyr                 165 170 175 Arg Leu Tyr Ser Pro Arg Ala Val Ala Leu Leu Tyr Ser Thr His Arg             180 185 190 Ser Glu Arg Thr Tyr Arg Ser Glu Arg Ala Leu Ala Pro Arg Ala Thr         195 200 205 Tyr Arg Leu Tyr Ser Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr     210 215 220 Arg Gly Leu Asn Gly Leu Asn Leu Glu Ala Leu Tyr Ser Leu Tyr Ser 225 230 235 240 Leu Tyr Ser Val Ala Leu Ala Ser Pro Thr Tyr Arg Ala Arg Gly Pro                 245 250 255 Arg Ala Thr His Arg Leu Tyr Ser Ser Glu Arg Thr Tyr Arg Pro Arg             260 265 270 Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Gly Leu Tyr Ser Glu Arg         275 280 285 Leu Tyr Ser Thr His Arg Ala Ser Asn Thr Tyr Arg Leu Glu Ala Pro     290 295 300 Arg Ala Leu Glu Ala Ala Leu Ala Leu Tyr Ser Leu Tyr Ser Leu Glu 305 310 315 320 Ala Ser Glu Arg Ser Glu Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala                 325 330 335 Ile Leu Glu Leu Tyr Ser Thr His Arg Thr His Arg Thr Tyr Arg Ala             340 345 350 Ser Asn Ala Leu Ala Leu Tyr Ser Thr His Arg Ala Ser Asn Thr Tyr         355 360 365 Arg Pro Arg Ala Pro Arg Ala Val Ala Leu Thr Tyr Arg Leu Tyr Ser     370 375 380 Pro Arg Ala Leu Tyr Ser Met Glu Thr Thr His Arg Thr Tyr Arg Pro 385 390 395 400 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg                 405 410 415 Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala             420 425 430 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ser Glu Arg Leu         435 440 445 Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg     450 455 460 Ala Leu Tyr Ser Ile Leu Glu Thr His Arg Cys Tyr Ser Pro Arg Ala 465 470 475 480 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu                 485 490 495 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg             500 505 510 Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser         515 520 525 Leu Tyr Ser Thr His Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr     530 535 540 His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser Val Ala 545 550 555 560 Leu Thr His Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg                 565 570 575 Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser Pro Arg Ala Ser             580 585 590 Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Ile Leu Glu Thr Tyr         595 600 605 Arg Leu Tyr Ser Ser Glu Arg Leu Tyr Ser Pro Arg Ala Thr His Arg     610 615 620 Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser Ile Leu Glu Thr 625 630 635 640 His Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr                 645 650 655 Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg             660 665 670 Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu         675 680 685 Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr     690 695 700 Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser 705 710 715 720 Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr Arg Leu                 725 730 735 Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr             740 745 750 Arg Pro Arg Ala Ser Glu Arg Thr His Arg Thr Tyr Arg Leu Tyr Ser         755 760 765 Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr Arg Pro     770 775 780 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu 785 790 795 800 Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala                 805 810 815 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu             820 825 830 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg         835 840 845 Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser     850 855 860 Pro Arg Ala Thr His Arg Thr Tyr Arg Ile Leu Glu Ala Leu Ala Leu 865 870 875 880 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg                 885 890 895 Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser             900 905 910 Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr         915 920 925 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg     930 935 940 Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg 945 950 955 960 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Ser Glu Arg Ser                 965 970 975 Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr             980 985 990 Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg         995 1000 1005 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr    1010 1015 1020 His Arg Thr Tyr Arg Pro Arg Ala Ser Glu Arg Thr His Arg Thr Tyr 1025 1030 1035 1040 Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg                1045 1050 1055 Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu            1060 1065 1070 Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr        1075 1080 1085 Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg    1090 1095 1100 Thr Tyr Arg Pro Arg Ala Ser Glu Arg Thr His Arg Thr Tyr Arg Leu 1105 1110 1115 1120 Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr His Arg Thr Tyr                1125 1130 1135 Arg Pro Arg Ala Ser Glu Arg Thr His Arg Thr Tyr Arg Leu Tyr Ser            1140 1145 1150 Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro        1155 1160 1165 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg    1170 1175 1180 Ala Leu Tyr Ser Ile Leu Glu Ser Glu Arg Thr Tyr Arg Pro Arg Ala 1185 1190 1195 1200 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu                1205 1210 1215 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Ser Glu            1220 1225 1230 Arg Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser        1235 1240 1245 Ser Glu Arg Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr    1250 1255 1260 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg 1265 1270 1275 1280 Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg                1285 1290 1295 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr            1300 1305 1310 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg        1315 1320 1325 Ala Thr His Arg Thr Tyr Arg Pro Arg Ala Ser Glu Arg Thr His Arg    1330 1335 1340 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Thr 1345 1350 1355 1360 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg                1365 1370 1375 Ala Thr His Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg            1380 1385 1390 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser        1395 1400 1405 Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr    1410 1415 1420 Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg 1425 1430 1435 1440 Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu                1445 1450 1455 Tyr Ser Ser Glu Arg Leu Tyr Ser Ser Glu Arg Ser Glu Arg Thr Tyr            1460 1465 1470 Arg Pro Arg Ala Ser Glu Arg Ser Glu Arg Thr Tyr Arg Leu Tyr Ser        1475 1480 1485 Pro Arg Ala Leu Tyr Ser Leu Tyr Ser Thr His Arg Thr Tyr Arg Pro    1490 1495 1500 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg 1505 1510 1515 1520 Ala Leu Tyr Ser Leu Glu Ala Thr His Arg Thr Tyr Arg Pro Arg Ala                1525 1530 1535 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu            1540 1545 1550 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg        1555 1560 1565 Ala Ser Glu Arg Thr Tyr Arg Leu Tyr Ser Pro Arg Ala Leu Tyr Ser    1570 1575 1580 Ile Leu Glu Thr His Arg Thr Tyr Arg Pro Arg Ala Ser Glu Arg Thr 1585 1590 1595 1600 His Arg Thr Tyr Arg Leu Tyr Ser Leu Glu Ala Leu Tyr Ser Pro Arg                1605 1610 1615 Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg            1620 1625 1630 Thr Tyr Arg Leu Tyr Ser Ser Glu Arg Leu Tyr Ser Thr His Arg Ser        1635 1640 1645 Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr    1650 1655 1660 Arg Ala Ser Asn Leu Tyr Ser Leu Tyr Ser Ile Leu Glu Ser Glu Arg 1665 1670 1675 1680 Thr Tyr Arg Pro Arg Ala Ser Glu Arg Gly Leu Asn Thr Tyr Arg                1685 1690 1695 <210> 2 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> fp-2 <400> 2 Thr Asn Arg Pro Asp Tyr Asn Asp Asp Glu Glu Asp Asp Tyr Lys Pro   1 5 10 15 Pro Val Tyr Lys Pro Ser Pro Ser Lys Tyr Arg Pro Val Asn Pro Cys              20 25 30 Leu Lys Lys Pro Cys Lys Tyr Asn Gly Val Cys Lys Pro Arg Gly Gly          35 40 45 Ser Tyr Lys Cys Phe Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn Cys Asn      50 55 60 Leu Lys Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn Lys Ser Arg Cys  65 70 75 80 Val Pro Val Gly Lys Thr Phe Lys Cys Val Cys Arg Asn Gly Asn Phe                  85 90 95 Gly Arg Leu Cys Glu Lys Asn Val Cys Ser Pro Asn Pro Cys Lys Asn             100 105 110 Asn Gly Lys Cys Ser Pro Leu Gly Lys Thr Gly Tyr Lys Cys Thr Cys         115 120 125 Ser Gly Gly Tyr Thr Gly Pro Arg Cys Glu Val His Ala Cys Lys Pro     130 135 140 Asn Pro Cys Lys Asn Lys Gly Arg Cys Phe Pro Asp Gly Lys Thr Gly 145 150 155 160 Tyr Lys Cys Arg Cys Val Asp Gly Tyr Ser Gly Pro Thr Cys Gln Glu                 165 170 175 Asn Ala Cys Lys Pro Asn Pro Cys Ser Asn Gly Gly Thr Cys Ser Ala             180 185 190 Asp Lys Phe Gly Asp Tyr Ser Cys Glu Cys Arg Pro Gly Tyr Phe Gly         195 200 205 Pro Glu Cys Glu Arg Tyr Val Cys Ala Pro Asn Pro Cys Lys Asn Gly     210 215 220 Gly Ile Cys Ser Ser Asp Gly Ser Gly Gly Tyr Arg Cys Arg Cys Lys 225 230 235 240 Gly Gly Tyr Ser Gly Pro Thr Cys Lys Val Asn Val Cys Lys Pro Thr                 245 250 255 Pro Cys Lys Asn Ser Gly Arg Cys Val Asn Lys Gly Ser Ser Tyr Asn             260 265 270 Cys Ile Cys Lys Gly Gly Tyr Ser Gly Pro Thr Cys Gly Glu Asn Val         275 280 285 Cys Lys Pro Asn Pro Cys Gln Asn Arg Gly Arg Cys Tyr Pro Asp Asn     290 295 300 Ser Asp Asp Gly Phe Lys Cys Arg Cys Val Gly Gly Tyr Lys Gly Pro 305 310 315 320 Thr Cys Glu Asp Lys Pro Asn Pro Cys Asn Thr Lys Pro Cys Lys Asn                 325 330 335 Gly Gly Lys Cys Asn Tyr Asn Gly Lys Ile Tyr Thr Cys Lys Cys Ala             340 345 350 Tyr Gly Trp Arg Gly Arg His Cys Thr Asp Lys Ala Tyr Lys Pro Asn         355 360 365 Pro Cys Val Val Ser Lys Pro Cys Lys Asn Arg Gly Lys Cys Ile Trp     370 375 380 Asn Gly Lys Ala Tyr Arg Cys Lys Cys Ala Tyr Gly Tyr Gly Gly Arg 385 390 395 400 His Cys Thr Lys Lys Ser Tyr Lys Lys Asn Pro Cys Ala Ser Arg Pro                 405 410 415 Cys Lys Asn Arg Gly Lys Cys Thr Asp Lys Gly Asn Gly Tyr Val Cys             420 425 430 Lys Cys Ala Arg Gly Tyr Ser Gly Arg Tyr Cys Ser Leu Lys Ser Pro         435 440 445 Pro Ser Tyr Asp Asp Asp Glu Tyr     450 455 <210> 3 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> fp-3 <400> 3 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly   1 5 10 15 Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp              20 25 30 Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr          35 40 45 <210> 4 <211> 787 <212> PRT <213> Artificial Sequence <220> <223> fp-4 <400> 4 Tyr Gly Arg Arg Tyr Gly Glu Pro Ser Gly Tyr Ala Asn Ile Gly His   1 5 10 15 Arg Arg Tyr Tyr Glu Arg Ala Ile Ser Phe His Arg His Ser His Val              20 25 30 His Gly His His Leu Leu His Arg His Val His Arg His Ser Val Leu          35 40 45 His Gly His Val His Met His Arg Val Ser His Arg Ile Met His Arg      50 55 60 His Arg Val Leu His Gly His Val His Arg His Arg Val Leu His Arg  65 70 75 80 His Val His Arg His Arg Val Leu His Gly His Val His Arg His Arg                  85 90 95 Val Leu His Arg His Leu His Arg His Arg Val Leu His Gly His Val             100 105 110 His Arg His Arg Val Leu His Asn His Val His Arg His Ser Val Leu         115 120 125 His Gly His Val His Arg His Arg Val Leu His Arg His Val His Arg     130 135 140 His Asn Val Leu His Gly His Val His Arg His Arg Val Leu His Lys 145 150 155 160 His Val His Asp His Arg Val Leu His Lys His Leu His Lys His Gln                 165 170 175 Val Leu His Gly His Val His Arg His Gln Val Leu His Lys His Val             180 185 190 His Asn His Arg Val Leu His Lys His Leu His Lys His Gln Val Leu         195 200 205 His Gly His Val His Thr His Arg Val Leu His Lys His Val His Lys     210 215 220 His Arg Val Leu His Lys His Leu His Lys His Gln Val Leu His Gly 225 230 235 240 His Ile His Thr His Arg Val Leu His Lys His Leu His Lys His Gln                 245 250 255 Val Leu His Gly His Val His Thr His Arg Val Leu His Lys His Val             260 265 270 His Lys His Arg Val Leu His Lys His Leu His Lys His Gln Val Leu         275 280 285 His Gly His Val His Met His Arg Val Leu His Lys His Val His Lys     290 295 300 His Arg Val Leu His Lys His Val His Lys His His Val Val His Lys 305 310 315 320 His Val His Ser His Arg Val Leu His Lys His Val His Lys His Arg                 325 330 335 Val Glu His Gln His Val His Lys His His Val Leu His Arg His Val             340 345 350 His Ser His His Val Val His Ser His Val His Lys His Arg Val Val         355 360 365 His Ser His Val His Lys His Asn Val Val His Ser His Val His Arg     370 375 380 His Gln Ile Leu His Arg His Val His Arg His Gln Val Val His Arg 385 390 395 400 His Val His Arg His Leu Ile Ala His Arg His Ile His Ser His Gln                 405 410 415 Ala Ala Val His Arg His Val His Thr His Val Phe Glu Gly Asn Phe             420 425 430 Asn Asp Asp Gly Thr Asp Val Asn Leu Arg Ile Arg His Gly Ile Ile         435 440 445 Tyr Gly Gly Asn Thr Tyr Arg Leu Ser Gly Gly Arg Arg Arg Phe Met     450 455 460 Thr Leu Trp Gln Glu Cys Leu Glu Ser Tyr Gly Asp Ser Asp Glu Cys 465 470 475 480 Phe Val Gln Leu Gly Asn Gln His Leu Phe Thr Val Val Gln Gly His                 485 490 495 His Ser Thr Ser Phe Arg Ser Asp Leu Ser Asn Asp Leu His Pro Asp             500 505 510 Asn Asn Ile Glu Gln Ile Ala Asn Asp His Val Asn Asp Ile Ala Gln         515 520 525 Ser Thr Asp Gly Asp Ile Asn Asp Phe Ala Asp Thr His Tyr Asn Asp     530 535 540 Val Ala Pro Ile Ala Asp Val His Val Asp Asn Ile Ala Gln Thr Ala 545 550 555 560 Asp Asn His Val Lys Asn Ile Ala Gln Thr Ala His His His Val Asn                 565 570 575 Asp Val Ala Gln Ile Ala Asp Asp His Val Asn Asp Ile Gly Gln Thr             580 585 590 Ala Tyr Asp His Val Asn Asn Ile Gly Gln Thr Ala Asp Asp His Val         595 600 605 Asn Asp Ile Ala Gln Thr Ala Asp Asp His Val Asn Ala Ile Ala Gln     610 615 620 Thr Ala Asp Asp His Val Asn Ala Ile Ala Gln Thr Ala Asp His Val 625 630 635 640 Asn Asp Ile Gly Asp Thr Ala Asn Ser His Ile Val Arg Val Gln Gly                 645 650 655 Val Ala Lys Asn His Leu Tyr Gly Ile Asn Lys Ala Ile Gly Lys His             660 665 670 Ile Gln His Leu Lys Asp Val Ser Asn Arg His Ile Glu Lys Leu Asn         675 680 685 Asn His Ala Thr Lys Asn Leu Leu Gln Ser Ala Leu Gln His Lys Gln     690 695 700 Gln Thr Ile Glu Arg Glu Ile Gln His Lys Arg His Leu Ser Glu Lys 705 710 715 720 Glu Asp Ile Asn Leu Gln His Glu Asn Ala Met Lys Ser Lys Val Ser                 725 730 735 Tyr Asp Gly Pro Val Phe Asn Glu Lys Val Ser Val Val Ser Asn Gln             740 745 750 Gly Ser Tyr Asn Glu Lys Val Pro Val Leu Ser Asn Gly Gly Gly Tyr         755 760 765 Asn Gly Lys Val Ser Ala Leu Ser Asp Gln Gly Ser Tyr Asn Glu Gly     770 775 780 Tyr Ala Tyr 785 <210> 5 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> fp-5 <400> 5 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His   1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr              20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys          35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg      50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser  65 70 75 <210> 6 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> fp-6 <400> 6 Gly Gly Gly Asn Tyr Arg Gly Tyr Cys Ser Asn Lys Gly Cys Arg Ser   1 5 10 15 Gly Tyr Ile Phe Tyr Asp Asn Arg Gly Phe Cys Lys Tyr Gly Ser Ser              20 25 30 Ser Tyr Lys Tyr Asp Cys Gly Asn Tyr Ala Gly Cys Cys Leu Pro Arg          35 40 45 Asn Pro Tyr Gly Arg Val Lys Tyr Tyr Cys Thr Lys Lys Tyr Ser Cys      50 55 60 Pro Asp Asp Phe Tyr Tyr Tyr Asn Asn Lys Gly Tyr Tyr Tyr Tyr Asn  65 70 75 80 Asp Lys Asp Tyr Phe Asn Cys Gly Ser Tyr Asn Gly Cys Cys Leu Arg                  85 90 95 Ser Gly Tyr             <210> 7 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> fp-151 <400> 7 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu      50 55 60 Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser  65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys                  85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly             100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr         115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr     130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys                 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             180 185 190 Pro Thr Tyr Lys         195 <210> 8 <211> 172 <212> PRT <213> Artificial Sequence <220> <223> fp-131 <400> 8 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala      50 55 60 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly  65 70 75 80 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn                  85 90 95 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala             100 105 110 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro         115 120 125 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro     130 135 140 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 145 150 155 160 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu                 165 170 <210> 9 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> fp-353 <400> 9 Met Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly   1 5 10 15 Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly              20 25 30 Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Glu          35 40 45 Phe Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn      50 55 60 His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly  65 70 75 80 Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Trp Lys Tyr Lys                  85 90 95 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg             100 105 110 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Lys Leu Ala Asp         115 120 125 Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn     130 135 140 Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn 145 150 155 160 Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Leu Glu                 165 170 <210> 10 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> fp-153 <400> 10 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Phe Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Glu Phe Ser      50 55 60 Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn His Tyr  65 70 75 80 His Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys                  85 90 95 Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn             100 105 110 Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys         115 120 125 Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Lys Leu Ala Asp Tyr Tyr     130 135 140 Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn Tyr Asn 145 150 155 160 Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn Gly Trp                 165 170 175 Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Leu Glu His His His His             180 185 190 His His         <210> 11 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fp-351 <400> 11 Met Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly   1 5 10 15 Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly              20 25 30 Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Glu          35 40 45 Phe Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn      50 55 60 His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly  65 70 75 80 Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr                  85 90 95 Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His             100 105 110 Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Lys Leu Ala         115 120 125 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro     130 135 140 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 145 150 155 160 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr                 165 170 175 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu Glu             180 185 <210> 12 <211> 319 <212> PRT <213> sea anemone <400> 12 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly   1 5 10 15 His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro              20 25 30 Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro          35 40 45 Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn      50 55 60 Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln  65 70 75 80 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly                  85 90 95 Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln Gly Pro             100 105 110 Gly Asn Thr Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly         115 120 125 Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly     130 135 140 Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr 145 150 155 160 Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly                 165 170 175 Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys             180 185 190 Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly         195 200 205 Gln Gly Pro Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr     210 215 220 Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp 225 230 235 240 Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys                 245 250 255 Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly             260 265 270 Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr         275 280 285 Gly Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln Gly     290 295 300 Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly 305 310 315 <210> 13 <211> 327 <212> PRT <213> sea anemone <400> 13 Met Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr   1 5 10 15 Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp              20 25 30 Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr          35 40 45 Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly      50 55 60 Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly  65 70 75 80 Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr                  85 90 95 Gly Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln Gly             100 105 110 Pro Gly Asn Thr Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro         115 120 125 Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro     130 135 140 Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn 145 150 155 160 Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln                 165 170 175 Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly             180 185 190 Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro         195 200 205 Gly Gln Gly Pro Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn     210 215 220 Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln 225 230 235 240 Asp Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly                 245 250 255 Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser             260 265 270 Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn         275 280 285 Thr Gly Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln     290 295 300 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Leu 305 310 315 320 Glu His His His His His His                 325 <210> 14 <211> 340 <212> PRT <213> sea anemone <400> 14 His Met Lys Ala Ile Phe Val Leu Lys Asp Asp Asp Asp Lys Gly Pro   1 5 10 15 Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly His Pro              20 25 30 Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn          35 40 45 Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln      50 55 60 Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly  65 70 75 80 Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro                  85 90 95 Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro             100 105 110 Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln Gly Pro Gly Asn         115 120 125 Thr Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr     130 135 140 Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp 145 150 155 160 Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Cys                 165 170 175 Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly             180 185 190 Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly         195 200 205 Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly     210 215 220 Pro Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr 225 230 235 240 Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly                 245 250 255 Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly             260 265 270 Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr         275 280 285 Gly Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr     290 295 300 Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly 305 310 315 320 Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Leu Glu His His                 325 330 335 His His His His             340

Claims (12)

제1 성분으로서 홍합 접착 단백질; 및
제2 성분으로서 홍합 접착 단백질 이외의 생체 유래 고분자;
를 포함하고, 상기 생체 유래 고분자는 말미잘 실크 단백질, 히알루론산, 콜라젠, 젤라틴, 알부민, 알긴산, 셀룰로오스, 트롬빈 및 피브린으로 이루어진 군에서 1종 이상 선택되는 것이며,
제1 성분 및 제2 성분을 2:8 내지 8:2의 중량비로 포함하며,
하이드로젤, 나노섬유, 분말 및 스프레이로 이루어진 군에서 선택되는 형태로 제공되는 지혈용 조성물로서,
상기 조성물의 형태가 하이드로젤 형태로 제공되는 경우, 제1 성분 및 제2 성분의 혼합물이 광가교되어 형성된 것인, 지혈용 조성물.Mussel adhesion protein as a first component; And
As a second component, a bio-derived polymer other than mussel adhesive protein;
Including, the bio-derived polymer is one or more selected from the group consisting of anemone silk protein, hyaluronic acid, collagen, gelatin, albumin, alginic acid, cellulose, thrombin and fibrin,
It includes the first component and the second component in a weight ratio of 2: 8 to 8: 2,
As a composition for hemostasis provided in a form selected from the group consisting of hydrogel, nanofiber, powder and spray,
When the form of the composition is provided in the form of a hydrogel, the mixture of the first component and the second component is formed by cross-linking, hemostatic composition. 제1항에 있어서,
홍합 접착 단백질은 Mefp-1, Mgfp-1, Mcfp-1, Mefp-2, Mefp-3, Mgfp-3 및 Mgfp-5로 이루어진 군에서 1종 이상 선택되는 것인, 지혈용 조성물.According to claim 1,
The mussel adhesion protein is selected from the group consisting of Mefp-1, Mgfp-1, Mcfp-1, Mefp-2, Mefp-3, Mgfp-3 and Mgfp-5, and a composition for hemostasis. 제1항에 있어서,
홍합 접착 단백질은 fp-1, fp-2, fp-3, fp-4, fp-5 및 fp-6로 이루어진 군에서 1종 이상 선택되는 것인, 지혈용 조성물.According to claim 1,
The mussel adhesive protein is selected from the group consisting of fp-1, fp-2, fp-3, fp-4, fp-5 and fp-6, one or more, hemostatic composition. 제1항에 있어서,
홍합 접착 단백질은 fp-151, fp-131, fp-353, fp-153 및 fp-351로 이루어진 군에서 1종 이상 선택되는 것인, 지혈용 조성물.According to claim 1,
The mussel adhesive protein is selected from the group consisting of fp-151, fp-131, fp-353, fp-153, and fp-351, one or more, hemostatic composition. 제1항에 있어서,
홍합 접착 단백질 20 내지 80 중량%를 포함하는 지혈용 조성물.According to claim 1,
Hemostatic composition comprising 20 to 80% by weight of mussel adhesive protein. 삭제delete 삭제delete 제1항에 있어서,
염화칼슘, 아데노신이인산, 비타민 K, 트롬보플라스틴, 포도당, 탈크, 탄산나트륨, 스테아린산마그네슘 및 폴리비닐알코올로 이루어진 군에서 1종 이상 선택되는 첨가제를 더 포함하는 지혈용 조성물.According to claim 1,
A composition for hemostasis further comprising at least one additive selected from the group consisting of calcium chloride, adenosine diphosphate, vitamin K, thromboplastin, glucose, talc, sodium carbonate, magnesium stearate and polyvinyl alcohol. 삭제delete 삭제delete 삭제delete 제1항 내지 제5항 및 제8항 중 어느 한 항의 지혈용 조성물을 포함하는 지혈제.A hemostatic agent comprising the composition for hemostasis according to any one of claims 1 to 5 and 8.
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