KR102462168B1 - Dental implant coated with mussel adhesive protein - Google Patents

Dental implant coated with mussel adhesive protein Download PDF

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KR102462168B1
KR102462168B1 KR1020200102400A KR20200102400A KR102462168B1 KR 102462168 B1 KR102462168 B1 KR 102462168B1 KR 1020200102400 A KR1020200102400 A KR 1020200102400A KR 20200102400 A KR20200102400 A KR 20200102400A KR 102462168 B1 KR102462168 B1 KR 102462168B1
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김명호
강혜원
임무학
박현정
서재란
최봉혁
원동환
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0012—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools characterised by the material or composition, e.g. ceramics, surface layer, metal alloy
    • A61C8/0013—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools characterised by the material or composition, e.g. ceramics, surface layer, metal alloy with a surface layer, coating
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C13/00—Dental prostheses; Making same
    • A61C13/0003—Making bridge-work, inlays, implants or the like
    • A61C13/0006—Production methods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C13/00—Dental prostheses; Making same
    • A61C13/01—Palates or other bases or supports for the artificial teeth; Making same
    • A61C13/02—Palates or other bases or supports for the artificial teeth; Making same made by galvanoplastic methods or by plating; Surface treatment; Enamelling; Perfuming; Making antiseptic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0003—Not used, see subgroups
    • A61C8/0009—Consolidating prostheses or implants, e.g. by means of stabilising pins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0018—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools characterised by the shape
    • A61C8/0037—Details of the shape
    • A61C2008/0046—Textured surface, e.g. roughness, microstructure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/02—Methods for coating medical devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses

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  • Health & Medical Sciences (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Dentistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Transplantation (AREA)
  • Dermatology (AREA)
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  • Orthopedic Medicine & Surgery (AREA)
  • Engineering & Computer Science (AREA)
  • Ceramic Engineering (AREA)
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Abstract

본 발명은 (1) 임플란트를 입자 분사 후 산처리법으로 표면처리하는 단계; (2) 홍합접착단백질 및 아스코르브산을 포함하는 코팅 수용액에 상기 (1) 단계의 임플란트를 침지시켜 임플란트 표면에 홍합접착단백질-아스코르브산 코팅막을 형성시키는 단계; 및 (3) 알칼리금속 또는 알칼리토금속의 무기염을 포함하는 pH 8~9의 완충용액에 상기 (2) 단계의 코팅막이 형성된 임플란트를 침지시키는 단계;를 포함하는 치과용 임플란트의 제조방법에 관한 것이다. The present invention comprises the steps of (1) surface-treating the implant with an acid treatment method after particle injection; (2) immersing the implant of step (1) in an aqueous coating solution containing mussel adhesive protein and ascorbic acid to form a mussel adhesive protein-ascorbic acid coating film on the implant surface; and (3) immersing the implant on which the coating film of step (2) is formed in a buffer solution of pH 8 to 9 containing an inorganic salt of an alkali metal or alkaline earth metal; .

Description

홍합접착단백질로 코팅된 치과용 임플란트{Dental implant coated with mussel adhesive protein}Dental implant coated with mussel adhesive protein

본 발명은 홍합접착단백질로 코팅된 치과용 임플란트 및 이의 제조방법에 관한 것이다. The present invention relates to a dental implant coated with mussel adhesive protein and a method for manufacturing the same.

치과용 임플란트는 인간의 턱뼈 위에 인공치아를 영구적으로 이식시키기 위해 사용하기 때문에 인간의 생체조직에 대하여 매우 안정적인 생체친화 재료를 사용하여야 하며 부작용 및 기타 화학, 생화학적 반응성이 없는 것이어야 한다. 또한 반복되는 하중 및 순간적인 압력의 부과에도 변형 및 파괴되지 않도록 기계적 강도가 매우 높아야하기 때문에 임플란트의 적절한 소재로서 티타늄의 생체적합성이 우수하다는 사실은 상당히 예전부터 알려져 왔으며, 현재까지 활발히 사용되고 있다. 임플란트 표면의 진화된 양상을 살펴보면 1세대는 선반 가공에 의한 매끈한 표면처리 방식으로 오랜 기간 동안 임플란트 술식에 이용되어 왔다. 그러나 골 밀도가 낮은 골에서도 임플란트 시술의 성공 여부는 인조조직인 임플란트가 뼈 조직 안에서 결합되는 골융합(Osseointegration)이 얼마나 견고하게, 그리고 빨리 완성되는가에 달려있다고 할 수 있기 때문에, 골융합에 있어서 중요한 결정인자로 임플란트의 표면처리 방식이 지적되어 왔다. Because dental implants are used to permanently implant artificial teeth on human jawbone, biocompatible materials that are very stable for human tissue must be used, and they must have no side effects and other chemical and biochemical reactivity. In addition, the fact that titanium has excellent biocompatibility as an appropriate material for implants has been known for quite some time, and is actively used until now, because mechanical strength must be very high to prevent deformation and destruction even under repeated loads and instantaneous pressure. Looking at the evolution of the implant surface, the first generation has been used for a long time in implant surgery as a smooth surface treatment method by lathe processing. However, even in bone with low bone density, the success of the implant procedure depends on how firmly and quickly the osseointegration, where the artificial tissue implant is combined in the bone tissue, is completed. Surface treatment methods of implants have been pointed out.

기존의 통상의 가공 방법으로 처리된 피막 표면을 형성한 표면에 비해 표면 거칠기를 증가시켜 생체조직과 접촉하는 표면적을 넓힌 2세대 임플란트에는 흡수성 분사 매질법인 RBM법(Resorbable blasting media)과 임플란트의 표면에 Al2O3 입자를 분사(blasting)하고, 이후 강산(H2SO4/HCl)을 처리하는 방법으로서 크레이터(crater)와 마이크로-피트(micropit)를 생성키는 화학적 가공 방법인 입자 분사 후 산처리법(Sandblasting with large grit and Acid etching, SLA법 또는 SA법)이 있다. RBM법은 저렴하지만 시술 기간이 길다는 단점이 있고, RBM법에서 발전된 형태인 SLA법은 현재 대표적으로 사용되고 있는 제품군으로. 기존의 RBM법에 비하여 표면적이 40% 이상 증가하는 효과를 보였기 때문에 조골세포의 분화에 유리하며, 골과 임플란트 사이의 골유착능을 증가시킬 수 있다는 장점이 있다. 이에 따라 임플란트 시술 후 평균 치유 기간을 12주에서 6～8주로 단축할 수 있다.In the second-generation implant, which increases the surface area in contact with living tissue by increasing the surface roughness compared to the surface on which the surface of the coating film treated with the conventional processing method is formed, the resorbable blasting media (RBM) method, which is a resorbable blasting medium, and Al 2 O 3 particles are sprayed (blasting) and then a strong acid (H 2 SO 4 /HCl) is treated, which is a chemical processing method that creates craters and micro-pits. There is a treatment method (Sandblasting with large grit and acid etching, SLA method or SA method). Although the RBM method is inexpensive, it has the disadvantage of a long treatment period. Compared to the conventional RBM method, it has the advantage of increasing the surface area by more than 40%, which is advantageous for the differentiation of osteoblasts, and has the advantage of increasing the osseointegration capacity between the bone and the implant. Accordingly, the average healing period after implant surgery can be shortened from 12 weeks to 6-8 weeks.

하지만 SLA법에 의해 표면 처리된 친수성 티타늄 표면은 공기 중에 노출될 경우, 임플란트 표면의 산화층이 성장하고 공기 중 탄소 오염원의 비가역적 흡착에 의해 빠르게 소수화되는 단점을 가진다. 소수화된 표면은 골세포의 임플란트 표면으로의 유입을 방해하여 초기 골과 임플란트 사이의 접촉률을 감소시키기 때문에 골 유착 성능이 감소되어 초기 임플란트 시술 실패에 대한 잠재적인 이유가 된다.However, when the hydrophilic titanium surface treated by the SLA method is exposed to air, an oxide layer on the implant surface grows and it is rapidly hydrophobized by irreversible adsorption of carbon contaminants in the air. The hydrophobized surface prevents the influx of osteocytes to the implant surface and reduces the contact rate between the initial bone and the implant.

따라서 SLA법으로 형성된 표면이 대기 중에서 소수화되는 것을 방지하고자 물이나 불활성 기체가 충진된 용기에 포장함으로써 공기와의 접촉을 차단하여 친수성을 유지시키기 위한 방식이 사용되었다. 하지만 위와 같은 포장 방법도 친수성 유지에는 효과적이지만, 임플란트 표면의 미세 구조(표면적의 증가, 거칠기의 향상)를 변화시키는 표면처리만으로는 골 유착 성능을 더욱 향상시켜 임플란트 시술 기간을 단축하기에는 한계가 있다.Therefore, in order to prevent the surface formed by the SLA method from being hydrophobized in the atmosphere, a method for maintaining hydrophilicity by blocking contact with air by packaging in a container filled with water or an inert gas was used. However, although the above packaging method is effective in maintaining hydrophilicity, there is a limit to shortening the implant operation period by further improving the osseointegration performance only by surface treatment that changes the microstructure (increase of surface area, improvement of roughness) of the implant surface.

이에, 본 발명은 기존의 SLA법으로 형성된 표면과 같은 물리적인 표면 처리 방법이 갖는 한계를 극복하기 위하여 홍합접착단백질을 치과용 임플란트 표면에 코팅시키는 방법과 초친수성을 갖게 하는 표면처리 방법으로 제조된 치과용 임플란트 및 이의 제조 방법을 제공한다.Therefore, in order to overcome the limitations of the physical surface treatment method such as the surface formed by the conventional SLA method, the present invention is prepared by a method of coating a mussel adhesive protein on the surface of a dental implant and a surface treatment method to have superhydrophilicity. A dental implant and a method for manufacturing the same are provided.

본 발명이 해결하고자 하는 과제는 표면이 SLA법으로 처리된 치과용 임플란트 표면의 소수성을 개선하기 위한 것으로, 초친수성이며, 혈액친화성이 향상되어 조골세포의 분화 및 증식을 유도 또는 촉진할 수 있는 빠른 골형성 및 골유착 특성을 갖는 치과용 임플란트 및 이의 제조방법을 제공하는 것이다. The problem to be solved by the present invention is to improve the hydrophobicity of the surface of a dental implant whose surface is treated with the SLA method, which is super hydrophilic and has improved blood affinity to induce or promote the differentiation and proliferation of osteoblasts. An object of the present invention is to provide a dental implant having rapid osseointegration and osseointegration properties and a method for manufacturing the same.

상기한 과제를 해결하기 위하여, 본 발명은 (1) 임플란트를 입자 분사 후 산처리법(Sandblasting with large grit and Acid etching, SLA법)으로 표면처리하는 단계;In order to solve the above problems, the present invention comprises the steps of (1) surface-treating the implant with an acid treatment method (Sandblasting with large grit and Acid etching, SLA method) after particle injection;

(2) 홍합접착단백질 및 아스코르브산을 포함하는 코팅 수용액에 상기 (1) 단계의 임플란트를 침지시켜 임플란트 표면에 홍합접착단백질-아스코르브산 코팅막을 형성시키는 단계; 및(2) immersing the implant of step (1) in an aqueous coating solution containing mussel adhesive protein and ascorbic acid to form a mussel adhesive protein-ascorbic acid coating film on the implant surface; and

(3) 알칼리금속 또는 알칼리토금속의 무기염을 포함하는 pH 8~9의 완충용액에 상기 (2) 단계의 코팅막이 형성된 임플란트를 침지시키는 단계;를 포함하는 치과용 임플란트의 제조방법을 제공한다. (3) immersing the implant on which the coating film of step (2) is formed in a buffer solution of pH 8 to 9 containing an inorganic salt of an alkali metal or alkaline earth metal; provides a method for manufacturing a dental implant comprising a.

상기 홍합접착단백질은 홍합에서 유래한 접착단백질로, 바람직하게는 미틸러스 에둘리스(Mytilus edulis), 미틸러스 갈로프로빈시얼리스(Mytilus galloprovincialis) 또는 미틸러스 코루스커스(Mytilus coruscus) 에서 유래한 홍합접착단백질 또는 이의 변이체를 포함하나, 이에 제한되지 않는다.The mussel adhesive protein is an adhesive protein derived from mussels, preferably Mytilus edulis ( Mytilus ). edulis ), Mytilus galloprovincialis ( Mytilus galloprovincialis ) or Mytilus coruscus ( Mytilus ) coruscus ) derived from mussel adhesive protein or a variant thereof, but is not limited thereto.

본 발명의 홍합접착단백질은 상기 홍합 종에서 각각 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-5 또는 이의 변이체를 포함할 수 있으며, 바람직하게는 fp(foot protein)-1 (서열번호 1), fp-2 (서열번호 4), fp-3 (서열번호 5), fp-4 (서열번호 6), fp-5 (서열번호 7), 및 fp-6 (서열번호 8)로 이루어진 군에서 선택된 단백질, 또는 2종 이상의 단백질이 연결되어 있는 융합 단백질, 또는 상기 단백질의 변이체를 포함하나, 이에 제한되지 않는다. The mussel adhesive protein of the present invention is Mefp (Mytilus edulis foot protein)-1, Mgfp (Mytilus galloprovincialis foot protein)-1, Mcfp (Mytilus coruscus foot protein)-1, Mefp-2, Mefp- 3, may include Mgfp-3 and Mgfp-5 or a variant thereof, preferably fp (foot protein)-1 (SEQ ID NO: 1), fp-2 (SEQ ID NO: 4), fp-3 (SEQ ID NO: 5) ), a protein selected from the group consisting of fp-4 (SEQ ID NO: 6), fp-5 (SEQ ID NO: 7), and fp-6 (SEQ ID NO: 8), or a fusion protein in which two or more proteins are linked, or the above including, but not limited to, variants of proteins.

또한, 본 발명의 홍합접착단백질은 국제공개번호 제 WO2006/107183호 또는 제WO2005/092920호에 기재된 모든 홍합 접착 단백질을 포함한다. 바람직하게는, 상기 홍합접착단백질은 fp-151(서열번호 9), fp-131(서열번호 10), fp-353(서열번호 11), fp-153(서열번호 12), fp-351(서열번호 13) 등의 융합 단백질을 포함할 수 있으나, 이에 제한되지 않는다. 또한, 본 발명의 홍합 접착단백질은 fp-1 에서 80번 정도 반복되는 데카펩타이드(서열번호 2)가 1 내지 12회 또는 그 이상으로 연속하여 연결된 폴리펩타이드를 포함할 수 있다.In addition, the mussel adhesive protein of the present invention includes all mussel adhesive proteins described in International Publication No. WO2006/107183 or WO2005/092920. Preferably, the mussel adhesive protein is fp-151 (SEQ ID NO: 9), fp-131 (SEQ ID NO: 10), fp-353 (SEQ ID NO: 11), fp-153 (SEQ ID NO: 12), fp-351 (SEQ ID NO: 12) No. 13) and the like, but are not limited thereto. In addition, the mussel adhesive protein of the present invention may include a polypeptide in which a decapeptide (SEQ ID NO: 2) repeated about 80 times in fp-1 is continuously linked 1 to 12 times or more.

또한, 본 발명의 홍합 접착 단백질은 fp-1에서 80번 정도 반복되는 데카펩타이드(서열번호 2)가 1 내지 12회 또는 그 이상으로 연속하여 연결된 폴리펩타이드를 포함할 수 있다. 바람직하게, 상기 서열번호 2의 데카펩타이드가 12회 연속하여 연결된 fp-1 variant 폴리펩타이드(서열번호 3)일 수 있으나, 이에 제한되지 않는다.In addition, the mussel adhesive protein of the present invention may include a polypeptide in which a decapeptide (SEQ ID NO: 2) repeated about 80 times in fp-1 is continuously linked 1 to 12 times or more. Preferably, the decapeptide of SEQ ID NO: 2 may be an fp-1 variant polypeptide (SEQ ID NO: 3) linked 12 times in a row, but is not limited thereto.

또한, 본 발명의 홍합접착단백질은 fp-151의 변이체(서열번호 15)일 수 있으나, 이에 제한되지 않는다. 서열번호 15의 단백질 서열은 서열번호 9와 대비하여 링커 서열 등이 제외된 서열이다. 구체적으로, 서열번호 14로 표시되는 fp-1 변이체 서열 사이에 서열번호 16으로 표시되는 Mgfp-5의 서열을 융합한 융합 단백질 서열이다. In addition, the mussel adhesive protein of the present invention may be a variant of fp-151 (SEQ ID NO: 15), but is not limited thereto. The protein sequence of SEQ ID NO: 15 is a sequence in which a linker sequence and the like are excluded in comparison with SEQ ID NO: 9. Specifically, it is a fusion protein sequence in which the sequence of Mgfp-5 shown in SEQ ID NO: 16 is fused between the fp-1 mutant sequences shown in SEQ ID NO: 14.

본 발명은 또한 위 언급된 홍합접착단백질들의 특성을 유지할 수 있는 보존적 아미노산 서열을 포함하는 범위에서 홍합접착단백질은 변형될 수 있다. 즉, 실질적으로 동등한 효과를 나타내는 상기 서열번호들의 아미노산 서열과 70% 이상, 바람직하게는 80% 이상, 보다 더 바람직하게는 90%이상, 즉, 95%, 96%, 97%, 98%, 99% 또는 그 이상의 서열 동일성을 가지는 아미노산 서열은 또한 본 발명의 범주에 포함될 수 있다.According to the present invention, the mussel adhesive protein may be modified within the range including a conservative amino acid sequence capable of maintaining the properties of the mussel adhesive protein mentioned above. That is, 70% or more, preferably 80% or more, even more preferably 90% or more, that is, 95%, 96%, 97%, 98%, 99 of the amino acid sequence of the SEQ ID NOs showing a substantially equivalent effect. Amino acid sequences having % or more sequence identity are also included within the scope of the present invention.

상기 홍합접착단백질은 티로신 잔기가 카테콜 화합물로 변환된 것이 바람직하고, 전체 티로신 잔기의 10 ~ 100%가 카테콜 화합물로 변환된 것이 바람직하다. 대부분의 홍합접착단백질의 전체 아미노산 서열에서 티로신이 차지하는 비중은 약 1 ~ 50 %일 수 있다. 홍합접착단백질 내의 티로신은 수화과정을 통하여 OH기가 첨가되어 카테콜 화합물인 도파(DOPA)로 변환될 수 있다. 그러나 대장균에서 생산된 홍합접착단백질은 티로신 잔기들이 변환되어 있지 않으므로, 별도의 효소 및 화학적 처리 방법에 의하여 티로신을 도파로 변환시키는 수정 반응을 수행하는 것이 바람직하다. 홍합접착단백질에 포함된 티로신 잔기를 도파로 수정하는 방법은 당업계에 알려진 방법을 사용할 수 있으며 특별히 제한되지 않는다.In the mussel adhesive protein, the tyrosine residue is preferably converted into a catechol compound, and 10 to 100% of the total tyrosine residue is preferably converted into a catechol compound. The proportion of tyrosine in the total amino acid sequence of most mussel adhesive proteins may be about 1 to 50%. Tyrosine in the mussel adhesive protein can be converted into a catechol compound, DOPA, by adding an OH group through the hydration process. However, since tyrosine residues are not converted in the mussel adhesive protein produced in Escherichia coli, it is preferable to perform a fertilization reaction for converting tyrosine into waveguide by a separate enzymatic and chemical treatment method. A method for modifying the tyrosine residue included in the mussel adhesive protein with waveguide may be a method known in the art and is not particularly limited.

상기 카테콜 화합물은 디하이드록시기를 포함하는 화합물로, 가교작용을 통해 홍합접착단백질에 접착력을 부여하는 화합물을 의미한다. 구체적으로 도파(3,4-dihydroxyphenylalanine, DOPA), 도파 o-퀴논(Dopa o-quinone), 토파(2,4,5-trihydroxyphenylalanine, TOPA), 토파 퀴논(Topa quinone) 및 이들의 유도체로 이루어진 군에서 선택된 어느 하나 이상인 것일 수 있다.The catechol compound is a compound containing a dihydroxy group, and refers to a compound that imparts adhesion to the mussel adhesive protein through a crosslinking action. Specifically, dopa (3,4-dihydroxyphenylalanine, DOPA), dopa o-quinone (Dopa o-quinone), topa (2,4,5-trihydroxyphenylalanine, TOPA), topa quinone (Topa quinone) and the group consisting of derivatives thereof It may be any one or more selected from.

상기 홍합접착단백질은 서열번호 1, 서열번호 2, 서열번호 3, 서열번호 4, 서열번호 5, 서열번호 6, 서열번호7, 서열번호 8, 서열번호 9, 서열번호 10, 서열번호 11, 서열번호 12, 서열번호 13, 서열번호 14, 서열번호 15 및 서열번호 16으로 이루어진 군으로부터 선택된 1종 이상의 아미노산 서열로 이루어진 것일 수 있다.The mussel adhesive protein is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, sequence Number 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and may consist of one or more amino acid sequences selected from the group consisting of SEQ ID NO: 16.

본 발명에서 홍합접착단백질의 변이체(mutants)는 바람직하게는 홍합접착단백질의 접착력을 유지하는 전제하에 상기 홍합접착단백질의 카르복실말단이나 아미노말단에 추가적인 서열을 포함하거나 일부 아미노산이 다른 아미노산으로 치환된 것일 수 있다. 보다 바람직하게는 상기 홍합접착단백질의 카르복실말단 또는 아미노말단에 RGD를 포함하는 3 내지 25개의 아미노산으로 이루어진 폴리펩타이드가 연결된 것이거나 홍합접착단백질을 이루는 타이로신 잔기 총수의 1 내지 100%, 바람직하게는 5 내지 100%가 3,4-디하이드록시페닐-L-알라닌(DOPA)로 치환된 것일 수 있다.In the present invention, the mutants of the mussel adhesive protein preferably include an additional sequence at the carboxyl or amino terminus of the mussel adhesive protein under the premise of maintaining the adhesion of the mussel adhesive protein, or some amino acids are substituted with other amino acids. it could be More preferably, a polypeptide consisting of 3 to 25 amino acids including RGD is linked to the carboxyl or amino terminus of the mussel adhesive protein, or 1 to 100% of the total number of tyrosine residues constituting the mussel adhesive protein, preferably 5 to 100% may be substituted with 3,4-dihydroxyphenyl-L-alanine (DOPA).

상기 홍합접착단백질은 이에 한정되지 않지만 바람직하게는 외부 유전자를 발현할 수 있는 용도로 제작된 통상의 벡터에 발현 가능하도록 삽입하여, 유전공학적인 방법으로 대량 생산할 수 있다. 상기 벡터는 단백질을 생산하기 위한 숙주세포의 종류 및 특성에 따라 적절히 선택하거나, 신규로 제작할 수 있다. 상기 벡터를 숙주세포에 형질전환하는 방법 및 형질전환체로부터 재조합 단백질을 생산하는 방법은 통상의 방법으로 용이하게 실시할 수 있다. 상기한 벡터의 선택, 제작, 형질전환 및 재조합 단백질의 발현 등의 방법은, 본 발명이 속하는 기술분야의 당업자라면 용이하게 실시할 수 있으며, 통상의 방법에서 일부의 변형도 본 발명에 포함된다.The mussel adhesive protein is not limited thereto, but is preferably inserted so as to be expressed in a conventional vector prepared for the purpose of expressing a foreign gene, and can be mass-produced by a genetic engineering method. The vector may be appropriately selected or newly constructed according to the type and characteristics of a host cell for producing the protein. A method of transforming the vector into a host cell and a method of producing a recombinant protein from a transformant can be easily carried out by a conventional method. Methods such as selection, construction, transformation and expression of the recombinant protein described above can be easily carried out by those skilled in the art to which the present invention pertains, and some modifications in conventional methods are also included in the present invention.

본 발명에 의하면, 상기 (2) 단계의 코팅 수용액은 pH가 2 내지 3일 수 있으며, 용매로 증류수 또는 생리식염수를 사용할 수 있나 이에 제한되지는 않는다..According to the present invention, the aqueous coating solution in step (2) may have a pH of 2 to 3, and distilled water or physiological saline may be used as a solvent, but is not limited thereto.

본 발명에 의하면, 상기 코팅 수용액 중의 홍합접착단백질 대 아스코르브산의 함량비는 1 : 10 내지 200 몰비로 포함되는 것일 수 있으며, 바람직하게는 1 : 10 내지 25 몰비로 포함되는 것일 수 있으며, 더욱 바람직하게는 1 : 12.5 몰비로 포함되는 것일 수 있다. 코팅막의 성분으로 홍합접착단백질과 아스코르브산을 함께 사용하는 경우, 홍합접착단백질만을 사용하는 경우에 비하여 혈액친화성 및 친수성이 향상되고, 생체적합성 및 생리활성이 우수하다. 뿐만 아니라, 아스코르브산의 첨가는 코팅안정성을 증가시켜 장기간 보관 또는 사용에도 안정적으로 코팅층이 유지된다. According to the present invention, the content ratio of mussel adhesive protein to ascorbic acid in the aqueous coating solution may be included in a molar ratio of 1: 10 to 200, preferably, in a molar ratio of 1: 10 to 25, more preferably Preferably, it may be included in a molar ratio of 1:12.5. When mussel adhesive protein and ascorbic acid are used together as components of the coating film, blood affinity and hydrophilicity are improved, and biocompatibility and physiological activity are excellent compared to the case where only mussel adhesive protein is used. In addition, the addition of ascorbic acid increases the coating stability, so that the coating layer is stably maintained even for long-term storage or use.

본 발명에 의하면, 상기 코팅 수용액 중의 홍합접착단백질의 농도는 0.01 내지 100 mM일 수 있으며, 아스코르브산의 농도는 홍합접착단백질 1 몰에 대하여 아스코르브산 10 내지 200 몰비로 포함된다. According to the present invention, the concentration of the mussel adhesive protein in the coating aqueous solution may be 0.01 to 100 mM, and the concentration of ascorbic acid is included in a molar ratio of 10 to 200 ascorbic acid with respect to 1 mol of the mussel adhesive protein.

본 발명에 의하면, 상기 알칼리금속 또는 알칼리토금속의 무기염은 소듐카보네이트, 소듐바이카보네이트, 쇼듐포스페이트, 칼슘 카보네이트, 포타슘카보네이트, 마그네슘카보네이트, 칼슘포스페이트, 포타슘포스페이트 및 마그네슘포스페이트로 이루어진 군으로부터 선택되는 어느 하나일 수 있으며, 예컨대 쇼듐바이카보네이트일 수 있다.According to the present invention, the inorganic salt of the alkali metal or alkaline earth metal is any one selected from the group consisting of sodium carbonate, sodium bicarbonate, sodium phosphate, calcium carbonate, potassium carbonate, magnesium carbonate, calcium phosphate, potassium phosphate and magnesium phosphate. It may be, for example, may be shodium bicarbonate.

본 발명에 의하면, 홍합접착단백질-아스코르브산의 코팅막은 홍합접착단백질 및 아스코르브산이 치과용 임플란트의 표면에 분산되어 물리적으로 흡착된 것, 화학적으로 결합된 것 또는 이들 모두일 수 있다. According to the present invention, the mussel adhesive protein-ascorbic acid coating film may be physically adsorbed, chemically bonded, or both of the mussel adhesive protein and ascorbic acid dispersed on the surface of the dental implant.

본 발명에 의하면, 상기 (2) 단계는 임플란트를 코팅 수용액에 6 내지 18시간, 바람직하게는 9 내지 15시간, 예컨대 12시간 동안 침지시키는 것일 수 있으며, 침지 온도는 상온 또는 20 내지 37℃인 것이 바람직하다. 침지 온도가 상기 범위 미만이면 코팅층 형성에 시간이 많이 소요되고, 상기 범위를 초과하면 단백질의 변성이 일어날 수 있다. According to the present invention, the step (2) may be to immerse the implant in the aqueous coating solution for 6 to 18 hours, preferably 9 to 15 hours, for example, 12 hours, and the immersion temperature is room temperature or 20 to 37°C. desirable. If the immersion temperature is less than the above range, it takes a lot of time to form the coating layer, and if it exceeds the above range, denaturation of the protein may occur.

본 발명에 의하면, 상기 알칼리금속 또는 알칼리토금속의 무기염을 포함하는 pH 8~9의 완충용액에 상기 (2) 단계의 코팅막이 형성된 임플란트를 침지시키는 단계는 홍합접착단백질 및 아스코르브산을 임플란트의 표면에 고밀도로 강력하게 흡착시키기 위해 수행되는 것일 수 있다. 상기 pH 범위에서의 처리는 코팅막 중의 홍합접착단백질 및 아스코르브산의 밀도를 더욱 높일 수 있으므로, 코팅막의 두께가 얇으면서도 내구성이 우수하며, 친수성 및 혈액친화성이 뛰어나고, 골형성 및 골유착 특성이 뛰어난 치과용 임플란트를 제공할 수 있으므로 바람직하다. According to the present invention, the step of immersing the implant on which the coating film of step (2) is formed in a buffer solution of pH 8 to 9 containing an inorganic salt of an alkali metal or alkaline earth metal comprises applying mussel adhesive protein and ascorbic acid to the surface of the implant. It may be performed to strongly adsorb at high density to the Since the treatment in the above pH range can further increase the density of mussel adhesive protein and ascorbic acid in the coating film, the coating film has a thin thickness and excellent durability, has excellent hydrophilicity and blood affinity, and has excellent bone formation and osseointegration properties. It is preferable because it can provide a dental implant.

본 발명에 의하면, 상기 (3) 단계는 완충용액에 6 내지 18시간, 바람직하게는 9 내지 15시간, 예컨대 12시간 동안 침지시키는 것일 수 있으며, 침지 온도는 상온 또는 20 내지 37℃인 것이 바람직하다.According to the present invention, the step (3) may be immersed in a buffer solution for 6 to 18 hours, preferably 9 to 15 hours, for example, 12 hours, and the immersion temperature is preferably room temperature or 20 to 37°C. .

본 발명에 의하면, 상기 (3) 단계의 완충용액 중의 알칼리금속 또는 알칼리토금속의 무기염의 농도는 2 내지 200 mM일 수 있으며, 치과용 임플란트 코팅막 중의 홍합접착단백질의 농도에 따라 완충용액 중의 알칼리금속 또는 알칼리토금속의 무기염의 농도를 조절할 수 있다. According to the present invention, the concentration of the inorganic salt of alkali metal or alkaline earth metal in the buffer solution of step (3) may be 2 to 200 mM, and depending on the concentration of the mussel adhesive protein in the dental implant coating film, alkali metal or alkaline metal in the buffer solution It is possible to control the concentration of inorganic salts of alkaline earth metals.

본 발명에 의하면, 상기 방법은 치과용 임플란트의 표면 상에 홍합접착단백질을 1 내지 500 μg/cm2의 농도로 코팅시키기 위함일 수 있다. According to the present invention, the method may be for coating the mussel adhesive protein at a concentration of 1 to 500 μg/cm 2 on the surface of the dental implant.

또한 본 발명은 상기 제조방법으로 제조된 홍합접착단백질 및 아스코르브산 코팅막을 포함하는 치과용 임플란트를 제공한다.The present invention also provides a dental implant comprising the mussel adhesive protein and ascorbic acid coating film prepared by the above manufacturing method.

본 발명에 의하면, 상기 코팅막은 친수성, 바람직하게는 초친수성일 수 있다. 코팅막의 우수한 친수성은 혈액 친화성을 향상시킬 수 있으며, 이를 통해 세포 부착능이 향상되고, 임플란트 표면에서 세포 활성도가 증가되어 빠른 골형성 및 골분화가 유도되므로 바람직하다.According to the present invention, the coating film may be hydrophilic, preferably superhydrophilic. The excellent hydrophilicity of the coating film can improve blood affinity, thereby improving cell adhesion and increasing cell activity on the implant surface, which is preferable because rapid bone formation and bone differentiation are induced.

본 발명에 의하면, 상기 코팅막은 홍합접착단백질 대 아스코르브산이 1 : 10 내지 200 몰비, 바람직하게는 1 : 10 내지 25 몰비로 포함된 것일 수 있으며, 코팅막 중의 홍합접착단백질의 함량은 1 내지 500 μg/cm2 일 수 있으나 이에 제한되는 것은 아니다. According to the present invention, the coating film may contain mussel adhesive protein to ascorbic acid in a molar ratio of 1: 10 to 200, preferably 1: 10 to 25, and the content of the mussel adhesive protein in the coating film is 1 to 500 μg/ cm 2 may be, but is not limited thereto.

본 발명은 소수성 SLA법으로 처리된 임플란트 표면에 홍합접착단백질을 코팅함으로써, 기존의 SLA법으로 처리된 표면의 단점으로 제시되고 있는 소수성 표면을 친수성 표면으로 바꾸며, 혈액 친화성 및 표면 접착력이 향상된 표면으로 개질하는 치과용 임플란트 및 이의 제조방법을 제공한다. 본 발명에 따른 홍합접착단백질 및 아스코르브산의 코팅막을 포함하는 치과용 임플란트의 제조방법은 기존의 SLA표면이 지니는 기계적 성능은 유지하면서도 친수성과 혈액친화성이 뛰어나 골과 임플란트 계면 사이의 친화성이 향상되고, 뛰어난 표면 접착력으로 임플란트 식립 후 초기 골 고정력을 개선시키는 효과가 있다. 뿐만 아니라, 조골세포의 분화와 증식을 유도 및 촉진하여 임플란트의 골형성 및 골유착을 크게 향상시킬 수 있으며, 임플란트 표면의 소수화를 일으키는 오염원의 흡착을 억제하여 초친수성이 장시간 유지되므로 산업상 유리하다. The present invention changes the hydrophobic surface, which is presented as a disadvantage of the existing SLA-treated surface, into a hydrophilic surface by coating the mussel adhesive protein on the surface of the implant treated with the hydrophobic SLA method, and the surface with improved blood affinity and surface adhesion Provided are a dental implant modified with a , and a method for manufacturing the same. The method for manufacturing a dental implant comprising a coating film of mussel adhesive protein and ascorbic acid according to the present invention improves the affinity between the bone and implant interface due to excellent hydrophilicity and blood affinity while maintaining the mechanical performance of the existing SLA surface. It has the effect of improving initial bone fixation after implant placement with excellent surface adhesion. In addition, by inducing and promoting the differentiation and proliferation of osteoblasts, it is possible to greatly improve the osseointegration and osseointegration of the implant, and it is advantageous for industry because it suppresses the adsorption of contaminants that cause hydrophobicity of the implant surface and maintains the superhydrophilicity for a long time. .

도 1은 홍합접착단백질이 코팅된 SLA disc 표면의 혈액 친화성을 평가한 결과이다.
도 2는 40 ℃에서 1년의 가속 노화된 홍합접착단백질이 코팅된 치과용 임플란트의 표면 접촉각을 평가한 결과이다.
도 3은 홍합접착단백질이 코팅된 치과용 임플란트의 X-선 광전자 분광법 (X-ray photoelectron spectroscopy, XPS)을 통해 표면 홍합접착단백질 코팅층의 형성을 확인한 결과를 나타낸 결과이다.
도 4는 홍합접착단백질 코팅에 따른 SLA disc 표면의 조골세포 부착성 및 부착 형태를 형광현미경 이미지 및 이의 조합 이미지로 나타낸 것이다.
도 5는 홍합접착단백질 코팅에 따른 조골세포 부착 능력 및 세포 증식 정도를 나타낸 결과이다.
도 6은 홍합접착단백질 코팅에 따른 조골세포의 분화정도를 분석하기 위한 alizarin red S 염색 및 분화 결과 세포 내 칼슘 침착 능력을 분석하기 위한 침착 칼슘의 정량 결과이다.
도 7은 홍합접착단백질이 코팅된 치과용 임플란트의 혈액 친화성을 평가한 결과이다.1 is a result of evaluating the blood affinity of the mussel adhesive protein-coated SLA disc surface.
Figure 2 is a result of evaluating the surface contact angle of the dental implant coated with mussel adhesive protein accelerated aging at 40 ℃ 1 year.
3 is a result showing the result of confirming the formation of a surface mussel adhesive protein coating layer through X-ray photoelectron spectroscopy (X-ray photoelectron spectroscopy, XPS) of the dental implant coated with mussel adhesive protein.
4 is a fluorescence microscope image and a combination image showing the osteoblast adhesion and adhesion form on the surface of the SLA disc according to the mussel adhesive protein coating.
5 is a result showing the osteoblast adhesion ability and cell proliferation according to the mussel adhesive protein coating.
6 is alizarin red S staining for analyzing the degree of differentiation of osteoblasts according to the mussel adhesive protein coating and quantitative results of calcium deposited to analyze the intracellular calcium deposition ability.
7 is a result of evaluating the blood affinity of the dental implant coated with mussel adhesive protein.

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 examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the examples.

실시예Example

실시예Example 1. 치과용 임플란트 표면의 1. Dental implant surface 홍합접착단백질Mussel Adhesive Protein 코팅 coating

(1) 임플란트 세척(1) Implant cleaning

홍합접착단백질을 표면에 코팅하기 전, SLA(입자 분사후 산처리법)공정을 거친 치과용 임플란트를 에탄올로 10분간, 그리고 멸균 증류수로 10분간 2회 초음파세척 한 후, 건조시켰다. Before coating the mussel adhesive protein on the surface, the dental implant that had undergone the SLA (particle spraying acid treatment) process was ultrasonically cleaned twice with ethanol for 10 minutes and with sterile distilled water for 10 minutes, and then dried.

(2) 홍합접착단백질 코팅(2) Mussel adhesive protein coating

본 발명의 홍합접착단백질의 코팅은 티로신 잔기가 카테콜 화합물로 변환된 홍합접착단백질이 포함된 산성 코팅 수용액을 사용하여 임플란트 면에 홍합접착단백질을 코팅하는 단계 및 홍합접착단백질이 코팅된 임플란트를 금속염의 중성 완충용액에 침지하여 표면의 홍합접착단백질을 집적시키는 단계를 포함한다. The coating of the mussel adhesive protein of the present invention includes the steps of coating the mussel adhesive protein on the implant surface using an acidic coating aqueous solution containing the mussel adhesive protein in which the tyrosine residue is converted into a catechol compound, and applying the mussel adhesive protein-coated implant to a metal salt. It includes the step of immersing in a neutral buffer solution of the mussel adhesion protein on the surface.

먼저, 코팅 단계는 0.8 mM 농도의 홍합접착단백질 및 10 mM 농도의 아스코르브산을 혼합하여 증류수에 pH 2~3으로 용해하여 코팅 수용액을 제조하고, 치과용 임플란트를 상기 수용액 2 ml에 12시간 동안 침지한 후 상온에서 2시간 동안 건조하여 수행하였다. First, in the coating step, 0.8 mM concentration of mussel adhesive protein and 10 mM concentration of ascorbic acid are mixed and dissolved in distilled water at pH 2-3 to prepare a coating solution, and the dental implant is immersed in 2 ml of the aqueous solution for 12 hours. After drying, it was carried out at room temperature for 2 hours.

2차 코팅은 소듐바이카보네이트를 26 mM의 농도범위로 포함하는 pH 8~9의 완충용액 2 ml에 상기 1차 코팅된 임플란트를 12시간 동안 침지함으로써 표면의 단백질의 흡착(adsorption)을 진행시켜 수행하였으며, 치과용 임플란트 표면에 제대로 흡착되지 않은 홍합접착단백질의 제거를 위해 증류수로 세척하고, 상온에서 3시간 동안 건조하였다. The secondary coating is carried out by adsorption of proteins on the surface by immersing the first coated implant in 2 ml of a buffer solution of pH 8-9 containing sodium bicarbonate in a concentration range of 26 mM for 12 hours. In order to remove the mussel adhesive protein that was not properly adsorbed to the dental implant surface, it was washed with distilled water and dried at room temperature for 3 hours.

실시예Example 2. 2. SLASLA 디스크 표면의 of the disk surface 홍합접착단백질Mussel Adhesive Protein 코팅 coating

(1) 디스크(disc) 세척(1) cleaning the disc

홍합접착단백질을 코팅하기 전, SLA(입자 분사 후 산처리법) 공정을 거친 지름이 8mm인 디스크를 에탄올로 10분간, 그리고 멸균 증류수로 10분간 2회 초음파 세척한 후 건조시켰다.Before coating the mussel adhesive protein, the 8mm-diameter disk that had undergone the SLA (particle spraying and acid treatment) process was ultrasonically washed twice with ethanol for 10 minutes and with sterile distilled water for 10 minutes, and then dried.

(2) 홍합접착단백질 코팅(2) Mussel adhesive protein coating

본 발명의 코팅은 디스크 표면에 티로신 잔기가 카테콜 화합물로 변환된 홍합접착단백질을 포함하는 코팅 수용액을 이용하여 1차 코팅하는 단계, 및 1차 코팅된 임플란트를 완충용액에 침지하여 표면의 홍합접착단백질을 흡착시켜 2차 코팅하는 단계를 포함한다. The coating of the present invention comprises the steps of first coating using a coating aqueous solution containing a mussel adhesive protein in which a tyrosine residue is converted into a catechol compound on the surface of the disc, and immersing the first coated implant in a buffer solution to adhere the mussels on the surface It includes the step of adsorbing the protein and secondary coating.

1차 코팅은 0.8 mM 농도의 홍합접착단백질 및 10 mM 농도의 아스코르브산을 혼합하여 증류수, 생리식염수 등에 pH 2~3으로 용해하여 코팅 수용액을 제조하고, 제조된 코팅 수용액 50 μl을 상기 디스크 표면위에 균일하게 도포하여 12시간 동안 건조하였다. For the first coating, a coating solution is prepared by mixing mussel adhesive protein with a concentration of 0.8 mM and ascorbic acid with a concentration of 10 mM, dissolving it in distilled water or physiological saline, etc. It was applied uniformly and dried for 12 hours.

2차 코팅은 알칼리 또는 알칼리토금속이 혼합된 중탄산염, 탄산염, 인산염으로 이루어진 군에서 선택된 적어도 어느 하나 이상의 물질을 26 mM의 농도범위로 포함하는 pH 8 ~ 9의 완충용액 1 ml에 상기 1차 코팅된 디스크를 12시간 동안 침지함으로써 표면의 단백질의 흡착(adsorption)을 진행시켜 수행하였으며, 디스크 표면에 제대로 흡착되지 않은 홍합접착단백질의 제거를 위해 증류수로 세척하고, 상온에서 3시간 동안 건조하였다. The secondary coating is first coated in 1 ml of a buffer solution of pH 8 to 9 containing at least one material selected from the group consisting of bicarbonate, carbonate, and phosphate mixed with alkali or alkaline earth metal in a concentration range of 26 mM. The disc was immersed for 12 hours to proceed with adsorption of proteins on the surface, washed with distilled water to remove the mussel adhesive protein that was not properly adsorbed to the disc surface, and dried at room temperature for 3 hours.

실시예Example 3. 혈액 친화성 평가 3. Blood Compatibility Assessment

실시예 2에서 제조된 홍합접착단백질이 코팅된 SLA 디스크 표면의 혈액 친화성을 확인하기 위하여 래빗(Rabbit) 혈액 5 μl을 디스크 중앙에 투하한 후, 측면 사진 분석을 통해 혈액과 디스크 표면과의 접촉각을 측정하였으며, 이를 도 1에 나타내었다. 이때, 대조군으로는 홍합접착단백질 코팅을 하지 않은 디스크를 사용하였다.To check the blood affinity of the mussel adhesive protein-coated SLA disk surface prepared in Example 2, 5 μl of rabbit blood was dropped into the center of the disk, and the contact angle between the blood and the disk surface was analyzed through side photo analysis. was measured, and it is shown in FIG. 1 . At this time, as a control, a disc not coated with mussel adhesive protein was used.

도 1에 나타낸 바와 같이, 실시예 2의 디스크의 접촉각은 약 20°이고, 대조군 디스크의 접촉각은 약 110°로 측정되었다. 상기 결과로부터 본 발명에 따라 홍합접착단백질의 코팅은 대조군에 비해 디스크의 접촉각을 현저하게 감소시킬 수 있음을 확인할 수 있다. 또한, 이러한 표면처리 공정은 혈액친화성이 높음을 시사한다. As shown in FIG. 1 , the contact angle of the disk of Example 2 was about 20°, and the contact angle of the control disk was about 110°. From the above results, it can be confirmed that the coating of the mussel adhesive protein according to the present invention can significantly reduce the contact angle of the disc compared to the control. In addition, this surface treatment process suggests that blood affinity is high.

실시예Example 4. 가속노화 실험을 통한 친수성 표면 형성 평가 4. Evaluation of hydrophilic surface formation through accelerated aging experiments

실시예 1의 홍합접착단백질 코팅된 치과용 임플란트를 가속노화 조건(약 40℃)에서 18주간 방치하여 보관기간 1년의 조건을 조성한 후, 젖음성을 확인하기 위해 증류수 5 μl를 임플란트 중앙에 투하한 후, 측면 사진 분석을 통해 증류수와 SLA 표면과의 접촉각을 측정하였다. 이때, 음성 대조군으로는 SLA 임플란트를 사용하였으며, 양성 대조군으로는 가속노화 조건을 거치지 않은 홍합접착단백질 코팅 임플란트를 사용하였다.The mussel adhesive protein-coated dental implant of Example 1 was left under accelerated aging conditions (about 40° C.) for 18 weeks to create a storage period of 1 year, and then 5 μl of distilled water was dropped into the center of the implant to check wettability. Then, the contact angle between distilled water and the SLA surface was measured through side photo analysis. At this time, as a negative control, an SLA implant was used, and as a positive control, a mussel adhesive protein-coated implant that did not undergo accelerated aging was used.

도 2에 나타낸 바와 같이, 음성 대조군의 경우 접촉각이 110°인 것에 비하여 18주간 가속노화 처리된 경우 접촉각이 0°로 낮아졌다. 이러한 보관기간 1년의 가속노화 조건을 거친 후에도 여전히 가속노화 이전과 동등 수준 이상으로 접촉각을 유지시키는 결과는 본 발명의 홍합접착단백질로 코팅된 표면처리 공정이 초친수성 표면 형성에 우수한 효과가 있음을 보여준다. As shown in FIG. 2 , in the case of accelerated aging for 18 weeks, the contact angle was lowered to 0° compared to the case of the negative control group having a contact angle of 110°. The result of maintaining the contact angle at the same level or higher as before accelerated aging even after the accelerated aging condition of 1 year of storage shows that the surface treatment process coated with the mussel adhesive protein of the present invention has an excellent effect in forming a superhydrophilic surface. show

실시예Example 5. 표면 성분 분석 5. Surface composition analysis

실시예 1의 홍합접착단백질로 코팅된 치과용 임플란트의 화학적 특성 분석 및 코팅에 따른 임플란트 표면의 성분 변화를 확인하기 위하여 X-선 광전자 분광법 (X-ray photoelectron spectroscopy, XPS)을 수행하였다. XPS 분석은 임플란트 소재와 코팅물질에 대한 Ti, C, O, N 원소에 대해 시편의 중앙부를 측정하였으며, Survey scan, atomic %, 각 원소에 대한 Narrow scan 결과를 토대로 해석하였다. 이때, 대조군으로는 홍합접착단백질 코팅을 하지 않은 임플란트를 사용하였다.X-ray photoelectron spectroscopy (XPS) was performed to analyze the chemical properties of the dental implant coated with the mussel adhesive protein of Example 1 and to confirm the component change of the implant surface according to the coating. For XPS analysis, the center of the specimen was measured for Ti, C, O, and N elements for implant material and coating material, and it was analyzed based on the results of survey scan, atomic %, and narrow scan for each element. In this case, as a control, an implant not coated with mussel adhesive protein was used.

도 3에 나타낸 바와 같이, 대조군의 표면은 평균적으로 Ti 17.3%, C 29.6%, N 0.6%, O 48.6%의 조성을 갖는 것으로 측정되었다. 반면, 실시예 1의 홍합접착단백질로 코팅된 임플란트 표면은 평균적으로 C 62.1%, N 8.62%, O 25.3%의 조성을 갖는 표면이 형성되는 것이 관찰되었고, 특히, 홍합접착단백질로 코팅으로 인하여 Ti 피크는 검출되지 않고, C, N 피크의 강도(intensity) 및 영역(Area)이 증가하는 것으로 관찰되었다. 이러한 결과는 치과용 임플란트 표면에 홍합접착단백질 코팅층이 잘 형성되었음을 보여준다. As shown in FIG. 3 , the surface of the control group was measured to have a composition of 17.3% Ti, 29.6% C, 0.6% N, and 48.6% O on average. On the other hand, the surface of the implant coated with the mussel adhesive protein of Example 1 was observed to have an average composition of C 62.1%, N 8.62%, and O 25.3%, and in particular, the Ti peak due to the coating with the mussel adhesive protein was not detected, and it was observed that the intensity and area of the C and N peaks increased. These results show that the mussel adhesive protein coating layer was well formed on the surface of the dental implant.

실시예Example 6. 6. 홍합접착단백질Mussel Adhesive Protein 코팅 coating SLASLA 디스크의 of disk In vitroin vitro 세포 cell 부착성adherence 및 부착 형태 확인 and check the attachment form

in vito 에서 홍합접착단백질 코팅 여부에 따른 세포 부착성 및 부착 형태를 확인하였다. 이를 위해 실시예 2의 홍합접착단백질로 코팅된 SLA 디스크 표면에 쥐 조골세포(MC3T3-E1)를 배양하였다. 실험에 사용된 세포의 농도는 2×103 cell/cm2 이었으며, 1시간 동안 배양한 후 파라포름알데히드 (4%)를 이용하여 고정화 하였다. 고정화된 세포는 DAPI 및 팔로이딘(phalloidin)을 이용하여 각각 세포질과 핵을 염색한 후 형광 염색을 진행하였다. 염색된 세포의 형상은 공초점 레이저 현미경 (Confocal Laser Scanning Microscope, CLSM)을 이용하여 관찰하였다. 이때, 대조군으로는 홍합접착단백질 코팅을 하지 않은 SLA 디스크를 사용하였다.Cell adhesion and adhesion morphology were confirmed according to whether or not the mussel adhesive protein was coated in vito . To this end, mouse osteoblasts (MC3T3-E1) were cultured on the surface of the SLA disc coated with the mussel adhesive protein of Example 2. The concentration of cells used in the experiment was 2×10 3 cell/cm 2 , and after culturing for 1 hour, it was immobilized using paraformaldehyde (4%). The immobilized cells were stained with cytoplasm and nucleus using DAPI and phalloidin, respectively, and then fluorescence staining was performed. The shape of the stained cells was observed using a Confocal Laser Scanning Microscope (CLSM). In this case, as a control, an SLA disk not coated with mussel adhesive protein was used.

도 4에 나타낸 바와 같이, 홍합접착단백질이 코팅된 표면에서 조골세포의 morphology가 길쭉하게 뻗어 근접한 세포끼리 붙어있는 경향이 보였다. 또한 세포핵(nucleus) 염색 결과, 부착된 세포의 수가 훨씬 더 많이 관찰되는 경향을 확인할 수 있었다. 이는 SLA 표면에 도입된 홍합접착단백질이 안정적으로 조골세포의 부착능을 향상시킨 것을 시사한다. 상기와 같은 결과로부터, 홍합접착단백질이 코팅된 SLA 디스크 표면은 대조군인 홍합접착단백질이 코팅되지 않은 SLA 디스크 표면에 비해 활성이 유지되고, 세포의 부착능이 향상되는 것을 확인하였다. As shown in FIG. 4 , the morphology of osteoblasts stretched elongated on the surface coated with mussel adhesive protein, and the adjacent cells tended to stick together. In addition, as a result of cell nucleus (nucleus) staining, it was confirmed that the number of attached cells was observed to be much higher. This suggests that the mussel adhesive protein introduced to the SLA surface stably improved the ability to adhere to osteoblasts. From the above results, it was confirmed that the SLA disc surface coated with mussel adhesive protein maintained activity and improved cell adhesion compared to the control SLA disc surface not coated with mussel adhesive protein.

실시예Example 7. 7. 홍합접착단백질의mussel adhesive protein In vitroin vitro 세포 cell 부착성adherence 및 and 증식능proliferative capacity 확인 Confirm

임플란트 코팅 물질인 홍합접착단백질의 in vitro 단계에서 세포 기능 향상 능력을 분석하였다. 실시예 2와 동일한 방법으로 표면처리되지 않은 폴리스티렌(PS) 플레이트에 홍합접착단백질을 0, 10, 25, 50 μg/cm2의 농도로 코팅하고, 쥐 조골세포(MC3T3-E1)를 배양하였다. 실험에 사용된 세포의 농도는 2×104 cell/cm2 이었으며, 3시간 동안 배양한 후 CCK-8 분석으로 세포 부착 및 증식을 확인하였다.The ability of mussel adhesive protein, an implant coating material, to improve cell function in vitro was analyzed. In the same manner as in Example 2, non-surface-treated polystyrene (PS) plates were coated with mussel adhesive protein at concentrations of 0, 10, 25, and 50 μg/cm 2 , and mouse osteoblasts (MC3T3-E1) were cultured. The concentration of cells used in the experiment was 2×10 4 cell/cm 2 , and after culturing for 3 hours, cell adhesion and proliferation were confirmed by CCK-8 analysis.

도 5에 나타낸 바와 같이, 홍합접착단백질을 코팅한 폴리스티렌 표면에서 표면 처리하지 않은 표면에 비해 높은 세포 부착 및 증식을 확인하였다. 홍합접착단백질은 초기 세포 부착능을 향상시켜 홍합접착단백질로 표면처리되지 않은 표면에 비하여 세포를 효과적으로 증식시키는 것으로 관찰되었다. 따라서, 본 발명에 따른 홍합접착단백질은 세포 활성도를 증가시키는데 유용하게 활용될 수 있음을 확인하였다. As shown in FIG. 5 , high cell adhesion and proliferation were confirmed on the polystyrene surface coated with the mussel adhesive protein compared to the surface not treated with the surface. It was observed that the mussel adhesive protein improved the initial cell adhesion ability and effectively proliferated the cells compared to the surface that was not surface-treated with the mussel adhesive protein. Therefore, it was confirmed that the mussel adhesive protein according to the present invention can be usefully used to increase cell activity.

실시예Example 8. 8. 홍합접착단백질의mussel adhesive protein In vitroin vitro 조골세포 osteoblasts 분화능pluripotency 확인 Confirm

임플란트 코팅 물질인 홍합접착단백질의 in vitro 단계에서 조골세포의 분화능을 확인하였다. 실시예 2와 동일한 방법으로 표면처리되지 않은 폴리스티렌(PS) 플레이트에 홍합접착단백질을 0, 10, 50 μg/cm2의 농도로 코팅하고, 쥐 조골세포(MC3T3-E1)를 7일 동안 배양하였다. 조골세포의 골분화능 지표인 석회질(calcium deposit)을 색상으로 확인할 수 있는 ARS 염색(Alizarin red S staining) 수행을 위해 배양된 조골세포를 PBS로 세척하고 파라포름알데히드 (4%)로 15분간 고정하였다. 40 mM alizarin red S 용액으로 20분간 염색한 후 증류수로 여러 번 세척하여 여분의 염색액을 제거하고 염색 양상을 관찰하였다. 다음으로, 정량적으로 확인하기 위하여 10% 아세트산을 처리하여 칼슘을 얻고, 칼슘량을 측정하였다. The differentiation ability of osteoblasts was confirmed in the in vitro stage of mussel adhesive protein, which is an implant coating material. In the same manner as in Example 2, non-surface-treated polystyrene (PS) plates were coated with mussel adhesive protein at concentrations of 0, 10, and 50 μg/cm 2 , and mouse osteoblasts (MC3T3-E1) were cultured for 7 days. . In order to perform ARS staining (Alizarin red S staining), which can confirm the color of calcium deposit, which is an indicator of osteogenic potential of osteoblasts, cultured osteoblasts were washed with PBS and fixed with paraformaldehyde (4%) for 15 minutes. . After staining with 40 mM alizarin red S solution for 20 minutes, it was washed several times with distilled water to remove excess staining solution and the staining pattern was observed. Next, in order to confirm quantitatively, calcium was obtained by treatment with 10% acetic acid, and the amount of calcium was measured.

도 6에 나타낸 바와 같이, 홍합접착단백질이 코팅되지 않은 폴리스티렌 표면에 비하여 홍합접착단백질을 코팅한 폴리스티렌 표면은 진한 붉은색으로 염색되었다. 다음으로 염색 정도를 정량화하기 위해 기질 내 칼슘량을 비교하였으며, 홍합접착단백질의 경우 홍합접착단백질이 코팅되지 않은 경우(대조군) 대비 흡광값이 약 97% 증가하는 것으로 관찰되었다. 이를 통해 홍합접착단백질이 조골세포의 골분화능이 있음이 확인되었다. (*P<0.05, ***P<0.005)As shown in FIG. 6 , the polystyrene surface coated with the mussel adhesive protein was stained dark red compared to the polystyrene surface not coated with the mussel adhesive protein. Next, the amount of calcium in the matrix was compared to quantify the degree of staining. In the case of mussel adhesive protein, it was observed that the absorbance value increased by about 97% compared to the case where the mussel adhesive protein was not coated (control group). Through this, it was confirmed that the mussel adhesive protein has the osteogenic differentiation ability of osteoblasts. (*P<0.05, ***P<0.005)

실시예Example 9. 혈액 친화성 평가 9. Blood Compatibility Assessment

실시예 1의 홍합접착단백질이 코팅된 치과용 임플란트 표면의 혈액친화성을 확인하였다. 토끼(Rabbit) 혈액에 실시예 1의 임플란트를 15초 동안 약 6 mm를 담지한 후, 임플란트 표면을 타고 올라오는 혈액의 높이를 측정하여 혈액 친화성을 평가하였다. 대조군으로는 홍합접착단백질을 코팅하지 않은 임플란트를 사용하였다. The blood affinity of the dental implant surface coated with the mussel adhesive protein of Example 1 was confirmed. After loading about 6 mm of the implant of Example 1 in rabbit blood for 15 seconds, the blood affinity was evaluated by measuring the height of the blood rising up the implant surface. As a control, implants not coated with mussel adhesive protein were used.

도 7에 나타낸 바와 같이, 홍합접착단백질 코팅을 하지 않은 대조군보다 실시예 1의 임플란트에서 표면에 달라붙은 혈액의 높이가 높아진 것이 육안으로 관측되었다. 대조군의 임플란트에서는 혈액이 표면을 타고 올라오지 않은 반면, 실시예 1의 홍합접착단백질이 코팅된 임플란트의 경우 침지 5초 경과 시점에서 이미 임플란트의 끝까지 혈액이 상승하였다. 이를 통해 본 발명에 따라 홍합접착단백질의 코팅이 임플란트 표면의 혈액 친화성을 크게 향상시키는 것을 확인하였다. As shown in FIG. 7 , it was visually observed that the height of the blood adhering to the surface of the implant of Example 1 was higher than that of the control not coated with the mussel adhesive protein. In the implant of the control group, blood did not rise on the surface, whereas in the case of the implant coated with the mussel adhesive protein of Example 1, blood had already risen to the end of the implant at the time of immersion for 5 seconds. Through this, it was confirmed that the coating of the mussel adhesive protein according to the present invention greatly improved the blood affinity of the implant surface.

<110> Nature Gluetech Co., Ltd. <120> Dental implant coated with mussel adhesive protein <130> NATUREGLUETECH1-1P <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 800 <212> PRT <213> Artificial Sequence <220> <223> fp-1 <400> 1 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 130 135 140 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 145 150 155 160 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 165 170 175 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 180 185 190 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 195 200 205 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 210 215 220 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 225 230 235 240 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 245 250 255 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 260 265 270 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 275 280 285 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 290 295 300 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 305 310 315 320 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 325 330 335 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 340 345 350 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 355 360 365 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 370 375 380 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 385 390 395 400 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 405 410 415 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 420 425 430 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 435 440 445 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 450 455 460 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 465 470 475 480 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 485 490 495 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 500 505 510 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 515 520 525 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 530 535 540 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 545 550 555 560 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 565 570 575 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 580 585 590 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 595 600 605 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 610 615 620 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 625 630 635 640 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 645 650 655 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 660 665 670 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 675 680 685 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 690 695 700 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 705 710 715 720 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 725 730 735 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 740 745 750 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 755 760 765 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 770 775 780 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 785 790 795 800 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant <400> 2 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 1 5 10 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant <400> 3 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys 115 120 <210> 4 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> fp-2 <400> 4 Leu Phe Ser Phe Phe Leu Leu Leu Thr Cys Thr Gln Leu Cys Leu Gly 1 5 10 15 Thr Asn Arg Pro Asp Tyr Asn Asp Asp Glu Glu Asp Asp Tyr Lys Pro 20 25 30 Pro Val Tyr Lys Pro Ser Pro Ser Lys Tyr Arg Pro Val Asn Pro Cys 35 40 45 Leu Lys Lys Pro Cys Lys Tyr Asn Gly Val Cys Lys Pro Arg Gly Gly 50 55 60 Ser Tyr Lys Cys Phe Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn Cys Asn 65 70 75 80 Leu Lys Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn Lys Ser Arg Cys 85 90 95 Val Pro Val Gly Lys Thr Phe Lys Cys Val Cys Arg Asn Gly Asn Phe 100 105 110 Gly Arg Leu Cys Glu Lys Asn Val Cys Ser Pro Asn Pro Cys Lys Asn 115 120 125 Asn Gly Lys Cys Ser Pro Leu Gly Lys Thr Gly Tyr Lys Cys Thr Cys 130 135 140 Ser Gly Gly Tyr Thr Gly Pro Arg Cys Glu Val His Ala Cys Lys Pro 145 150 155 160 Asn Pro Cys Lys Asn Lys Gly Arg Cys Phe Pro Asp Gly Lys Thr Gly 165 170 175 Tyr Lys Cys Arg Cys Val Asp Gly Tyr Ser Gly Pro Thr Cys Gln Glu 180 185 190 Asn Ala Cys Lys Pro Asn Pro Cys Ser Asn Gly Gly Thr Cys Ser Ala 195 200 205 Asp Lys Phe Gly Asp Tyr Ser Cys Glu Cys Arg Pro Gly Tyr Phe Gly 210 215 220 Pro Glu Cys Glu Arg Tyr Val Cys Ala Pro Asn Pro Cys Lys Asn Gly 225 230 235 240 Gly Ile Cys Ser Ser Asp Gly Ser Gly Gly Tyr Arg Cys Arg Cys Lys 245 250 255 Gly Gly Tyr Ser Gly Pro Thr Cys Lys Val Asn Val Cys Lys Pro Thr 260 265 270 Pro Cys Lys Asn Ser Gly Arg Cys Val Asn Lys Gly Ser Ser Tyr Asn 275 280 285 Cys Ile Cys Lys Gly Gly Tyr Ser Gly Pro Thr Cys Gly Glu Asn Val 290 295 300 Cys Lys Pro Asn Pro Cys Gln Asn Arg Gly Arg Cys Tyr Pro Asp Asn 305 310 315 320 Ser Asp Asp Gly Phe Lys Cys Arg Cys Val Gly Gly Tyr Lys Gly Pro 325 330 335 Thr Cys Glu Asp Lys Pro Asn Pro Cys Asn Thr Lys Pro Cys Lys Asn 340 345 350 Gly Gly Lys Cys Asn Tyr Asn Gly Lys Ile Tyr Thr Cys Lys Cys Ala 355 360 365 Tyr Gly Trp Arg Gly Arg His Cys Thr Asp Lys Ala Tyr Lys Pro Asn 370 375 380 Pro Cys Val Val Ser Lys Pro Cys Lys Asn Arg Gly Lys Cys Ile Trp 385 390 395 400 Asn Gly Lys Ala Tyr Arg Cys Lys Cys Ala Tyr Gly Tyr Gly Gly Arg 405 410 415 His Cys Thr Lys Lys Ser Tyr Lys Lys Asn Pro Cys Ala Ser Arg Pro 420 425 430 Cys Lys Asn Arg Gly Lys Cys Thr Asp Lys Gly Asn Gly Tyr Val Cys 435 440 445 Lys Cys Ala Arg Gly Tyr Ser Gly Arg Tyr Cys Ser Leu Lys Ser Pro 450 455 460 Pro Ser Tyr Asp Asp Asp Glu Tyr 465 470 <210> 5 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> fp-3 <400> 5 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Gly Ser 50 <210> 6 <211> 750 <212> PRT <213> Artificial Sequence <220> <223> fp-4 <400> 6 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 Asn 65 70 75 80 His Val His Arg His Ser Val Leu His Gly His Val His Arg His Arg 85 90 95 Val Leu His Arg His Val His Arg His Asn Val Leu His Gly His Val 100 105 110 His Arg His Arg Val Leu His Lys His Val His Asn His Arg Val Leu 115 120 125 His Lys His Leu His Lys His Gln Val Leu His Gly His Val His Arg 130 135 140 His Gln Val Leu His Lys His Val His Asn His Arg Val Leu His Lys 145 150 155 160 His Leu His Lys His Gln Val Leu His Gly His Val His Thr His Arg 165 170 175 Val Leu His Lys His Val His Lys His Arg Val Leu His Lys His Leu 180 185 190 His Lys His Gln Val Leu His Gly His Ile His Thr His Arg Val Leu 195 200 205 His Lys His Leu His Lys His Gln Val Leu His Gly His Val His Thr 210 215 220 His Arg Val Leu His Lys His Val His Lys His Arg Val Leu His Lys 225 230 235 240 His Leu His Lys His Gln Val Leu His Gly His Val His Met His Arg 245 250 255 Val Leu His Lys His Val His Lys His Arg Val Leu His Lys His Val 260 265 270 His Lys His His Val Val His Lys His Val His Ser His Arg Val Leu 275 280 285 His Lys His Val His Lys His Arg Val Glu His Gln His Val His Lys 290 295 300 His His Val Leu His Arg His Val His Ser His His Val Val His Ser 305 310 315 320 His Val His Lys His Arg Val Val His Ser His Val His Lys His Asn 325 330 335 Val Val His Ser His Val His Arg His Gln Ile Leu His Arg His Val 340 345 350 His Arg His Gln Val Val His Arg His Val His Arg His Leu Ile Ala 355 360 365 His Arg His Ile His Ser His Gln Ala Ala Val His Arg His Val His 370 375 380 Thr His Phe Glu Gly Asn Phe Asn Asp Asp Gly Thr Asp Val Asn Leu 385 390 395 400 Arg Ile Arg His Gly Ile Ile Tyr Phe Gly Gly Asn Thr Tyr Arg Leu 405 410 415 Ser Gly Gly Arg Arg Arg Phe Met Thr Leu Trp Gln Glu Cys Leu Glu 420 425 430 Ser Tyr Gly Asp Ser Asp Glu Cys Phe Val Gln Leu Leu Glu Gly Asn 435 440 445 Gln His Leu Phe Thr Val Val Gln Gly His His Ser Thr Ser Phe Arg 450 455 460 Ser Asp Leu Ser Asn Asp Leu His Pro Asp Asn Asn Ile Glu Gln Ile 465 470 475 480 Ala Asn Asp His Val Asn Asp Ile Ala Gln Ser Thr Asp Gly Asp Ile 485 490 495 Asn Asp Phe Ala Asp Thr His Tyr Asn Asp Val Ala Pro Ile Ala Asp 500 505 510 Val His Val Asp Asn Ile Ala Gln Thr Ala Asp Asn His Val Lys Asn 515 520 525 Ile Ala Gln Thr Ala His His His Val Asn Asp Val Ala Gln Ile Ala 530 535 540 Asp Asp His Val Asn Asp Ile Gly Gln Thr Ala Tyr Asp His Val Asn 545 550 555 560 Asn Ile Gly Gln Thr Ala Asp Asp His Val Asn Asp Ile Ala Gln Thr 565 570 575 Ala Asp Asp His Val Asn Ala Ile Ala Gln Thr Ala Asp Asp His Val 580 585 590 Asn Ala Ile Ala Gln Thr Ala Asp Asp His Val Asn Asp Ile Gly Asp 595 600 605 Thr Ala Asn Ser His Ile Val Arg Val Gln Gly Val Ala Lys Asn His 610 615 620 Leu Tyr Gly Ile Asn Lys Ala Ile Gly Lys His Ile Gln His Leu Lys 625 630 635 640 Asp Val Ser Asn Arg His Ile Glu Lys Leu Asn Asn His Ala Thr Lys 645 650 655 Asn Leu Leu Gln Ser Ala Leu Gln His Lys Gln Gln Thr Ile Glu Arg 660 665 670 Glu Ile Gln His Lys Arg His Leu Ser Glu Lys Glu Asp Ile Asn Leu 675 680 685 Gln His Glu Asn Ala Met Lys Ser Lys Val Ser Tyr Asp Gly Pro Val 690 695 700 Phe Asn Glu Lys Val Ser Val Val Ser Asn Gln Gly Ser Tyr Asn Glu 705 710 715 720 Lys Val Pro Val Leu Ser Asn Gly Gly Gly Tyr Asn Gly Lys Val Ser 725 730 735 Ala Leu Ser Asp Gln Gly Ser Tyr Asn Glu Gly Tyr Ala Tyr 740 745 750 <210> 7 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> fp-5 <400> 7 Lys His His His His His His Ser Ser Glu Glu Tyr Lys Gly Gly Tyr 1 5 10 15 Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly 20 25 30 Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys 35 40 45 Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys 50 55 60 Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly 65 70 75 80 Ser Ser <210> 8 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> fp-6 <400> 8 Ile Ala Ala Leu Cys Gly Ile Val Lys Ser Ile Asp Ser Asp Asp Ser 1 5 10 15 Asp Tyr Asp Tyr Lys Gly Arg Gly Tyr Cys Thr Asn Lys Gly Cys Arg 20 25 30 Ser Gly Tyr Asn Tyr Phe Gly Asn Lys Gly Tyr Cys Lys Tyr Gly Glu 35 40 45 Lys Ser Tyr Thr Tyr Asn Cys Asn Ser Tyr Ala Gly Cys Cys Leu Pro 50 55 60 Arg Asn Pro Tyr Gly Lys Leu Lys Tyr Tyr Cys Thr Asn Lys Tyr Gly 65 70 75 80 Cys Pro Asn Asn Tyr Tyr Phe Tyr Asn Asn Lys Gly Tyr Tyr Tyr Leu 85 90 95 Glu His His His His His His 100 <210> 9 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> fp-151 <400> 9 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ser Ser 50 55 60 Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His 65 70 75 80 Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly 85 90 95 Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser 100 105 110 Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly 115 120 125 Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro 130 135 140 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 145 150 155 160 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 165 170 175 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 180 185 190 Tyr Pro Pro Thr Tyr Lys Leu 195 <210> 10 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> fp-131 <400> 10 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala Asp 50 55 60 Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn 65 70 75 80 Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn 85 90 95 Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 130 135 140 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 145 150 155 160 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu 165 170 <210> 11 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> fp-353 <400> 11 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Pro Trp Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr 50 55 60 Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly 65 70 75 80 Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys 85 90 95 Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr 100 105 110 His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala 115 120 125 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly 130 135 140 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn 145 150 155 160 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser 165 170 175 <210> 12 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> fp-153 <400> 12 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ser Ser 50 55 60 Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His 65 70 75 80 Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly 85 90 95 Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser 100 105 110 Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly 115 120 125 Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala Asp Tyr Tyr Gly 130 135 140 Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn Tyr Asn Arg 145 150 155 160 Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn Gly Trp Lys 165 170 175 Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser 180 185 <210> 13 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> fp-351 <400> 13 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Pro Trp Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr 50 55 60 Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly 65 70 75 80 Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys 85 90 95 Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr 100 105 110 His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser 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 180 185 <210> 14 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant <400> 14 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 50 55 60 <210> 15 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> fp-151 variant <400> 15 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu 50 55 60 Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly 65 70 75 80 Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr 85 90 95 Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys 100 105 110 Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys 115 120 125 Lys Tyr Tyr Gly 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> 16 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> mgfp-5 <400> 16 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 <110> Nature Gluetech Co., Ltd. <120> Dental implant coated with mussel adhesive protein <130> NATUREGLUETECH1-1P <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 800 <212> PRT <213> Artificial Sequence <220> <223> fp- 1 <400> 1 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 130 135 140 Tyr Pro Pro Thr Tyr Lys Ala L ys Pro Ser Tyr Pro Pro Thr Tyr Lys 145 150 155 160 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 165 170 175 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 180 185 190 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 195 200 205 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 210 215 220 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 225 230 235 240 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 245 250 255 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 260 265 270 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Thr 275 280 285 Tyr Lys Ala Lys P ro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 290 295 300 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 305 310 315 320 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 325 330 335 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 340 345 350 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 355 360 365 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 370 375 380 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 385 390 395 400 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 405 410 415 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 420 425 430 Pro S er Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 435 440 445 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 450 455 460 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 465 470 475 480 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 485 490 495 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 500 505 510 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 515 520 525 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 530 535 540 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 545 550 555 560 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 565 570 575 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 580 585 590 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 595 600 605 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 610 615 620 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 625 630 635 640 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 645 650 655 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 660 665 670 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 675 680 685 Tyr Lys Ala Lys Pro Ser Tyr Pro Thr Tyr Lys Ala Lys Pro Ser 690 695 700 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 705 710 715 720 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 725 730 735 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 740 745 750 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 755 760 765 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 770 775 780 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 785 790 795 800 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant <400> 2 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 1 5 10 <210> 3 <211> 120 < 212> PRT <213> Artificial Sequence <220> <223> fp-1 variant <400> 3 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Ty r Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys 115 120 <210> 4 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> fp-2 <400> 4 Leu Phe Ser Phe Phe Leu Leu Leu Thr Cys Thr Gln Leu Cys Leu Gly 1 5 10 15 Thr Asn Arg Pro Asp Tyr Asn Asp Asp Glu Glu Asp Asp Tyr Lys Pro 20 25 30 Pro Val Tyr Lys Pro Ser Pro Ser Lys Tyr Arg Pro Val Asn Pro Cys 35 40 45 Leu Lys Lys Pro Cys Lys Tyr Asn Gly Val Cys Lys Pro Arg Gly Gly 50 55 60 Ser Tyr Lys Cys Phe Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn Cys Asn 65 70 75 80 Leu Lys Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn Lys Ser Arg Cys 85 90 95 Val Pro Val Gly Lys Thr Phe Lys Cys Val Cys Arg Asn Gly Asn Phe 100 105 110 Gly Arg Leu Cys Glu Lys Asn Val Cys Ser Pro Asn Pro Cys Lys Asn 115 120 125 Asn Gly Lys Cys Ser Pro Leu Gly Lys Thr Gly Tyr Lys Cys Thr Cys 130 135 140 Ser Gly Gly Tyr Thr Gly Pro Arg Cys Glu Val His Ala Cys Lys Pro 145 150 155 160 Asn Pro Cys Lys Asn Lys Gly Arg Cys Phe Pro Asp Gly Lys Thr Gly 165 170 175 Tyr Lys Cys Arg Cys Val Asp Gly Tyr Ser Gly Pro Thr Cys Gln Glu 180 185 190 Asn Ala Cys Lys Pro Asn Pro Cys Ser Asn Gly Gly Thr Cys Ser Ala 195 200 205 Asp Lys Phe Gly Asp Tyr Ser Cys Glu Cys Arg Pro Gly Tyr Phe Gly 210 215 220 Pro Glu Cys Glu Arg Tyr Val Cys Ala Pro Asn Pro Cys Lys Asn Gly 225 230 235 240 Gly Ile Cys Ser Ser Asp Gly Ser Gly Gly Tyr Arg Cys Arg Cys Lys 245 250 255 Gly Gly Tyr Ser Gly Pro Thr Cys Lys Val Asn Val Cys Lys Pro Thr 260 265 270 Pro Cys Lys Asn Ser Gly Arg Cys Val Asn Lys Gly Ser Ser Tyr Asn 275 280 285 Cys Ile Cys Lys Gly Gly Tyr Ser Gly Pro Thr Cys Gly Glu Asn Val 290 295 300 Cys Lys Pro Asn Pro Cys Gln Asn Arg Gly Arg Cys Tyr Pro Asp Asn 305 310 315 320 Ser Asp Asp Gly Phe Lys Cys Arg Cys Val Gly Gly Tyr Lys Gly Pro 325 330 335 Thr Cys Glu Asp Lys Pro Asn Pro Cys Asn Thr Lys Pro Cys Lys Asn 340 345 350 Gly Gly Lys Cys Asn Tyr Asn Gly Lys Ile Tyr Thr Cys Lys Cys Ala 355 360 365 Tyr Gly Trp Arg Gly Arg His Cys Thr Asp Lys Ala Tyr Lys Pro Asn 370 375 380 Pro Cys Val Val Ser Lys Pro Cys Lys Asn Arg Gly Lys Cys Ile Trp 385 390 395 400 Asn Gly Lys Ala Tyr Arg Cys Lys Cys Ala Tyr Gly Tyr Gly Gly Arg 405 410 415 His Cys Thr Lys Lys Ser Tyr Lys Lys Asn Pro Cys Ala Ser Arg Pro 420 425 430 Cys Lys Asn Arg Gly Lys Cys Thr Asp Lys Gly Asn Gly Tyr Val Cys 435 440 445 Lys Cys Ala Arg Gly Tyr Ser Gly Arg Tyr Cys Ser Leu Lys Ser Pro 450 455 460 Pro Ser Tyr Asp Asp Asp Glu Tyr 465 470 < 210> 5 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> fp-3 <400> 5 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Gly Ser 50 <210> 6 <211> 750 <212 > PRT <213> Artificial Sequence <220> <223> fp-4 <400> 6 Tyr Gly Ar g 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 Asn 65 70 75 80 His Val His Arg His Ser Val Leu His Gly His Val His Arg His Arg 85 90 95 Val Leu His Arg His Val His Arg His Asn Val Leu His Gly His Val 100 105 110 His Arg His Arg Val Leu His Lys His Val His Asn His Arg Val Leu 115 120 125 His Lys His Leu His Lys His Gln Val Leu His Gly His Val His Arg 130 135 140 His Gln Val Leu His Lys His Val His Asn His Arg Val Leu His Lys 145 150 155 160 His Leu His Lys His Gln Val Leu His Gly His Val His Thr His Arg 165 170 175 Val Leu His Lys His Val His Lys His Arg Val Leu His Lys His Leu 180 185 190 His Lys His Gln Val Leu His Gly His Ile His Thr His Arg Val Leu 195 200 205 His Lys His Leu His Lys His Gln Val Leu His Gly His Val His Thr 210 215 220 His Arg Val Leu His Lys His Val His Lys His Arg Val Leu His Lys 225 230 235 240 His Leu His Lys His Gln Val Leu His Gly His Val His Met His Arg 245 250 255 Val Leu His Lys His Val His Lys His Arg Val Leu His Lys His Val 260 265 270 His Lys His His Val Val His Lys His Val His Ser His Arg Val Leu 275 280 285 His Lys His Val His Lys His Arg Val Glu His Gln His Val His Lys 290 295 300 His His Val Leu His Arg His Val His Ser His His Val Val His Ser 305 310 315 320 His Val His Lys His Arg Val Val His Ser His Val His Lys His Asn 325 330 335 Val Val His Ser His Val His Arg His Gln Ile Leu His Arg His Val 340 345 350 His Arg His Gln Val Val His Arg His Val His Arg His Leu Ile Ala 355 360 365 His Arg His Ile His Ser His Gln Ala Ala Val His Arg His Val His 370 375 380 Thr His Phe Glu Gly Asn Phe Asn Asp Asp Gly Thr Asp Val Asn Leu 385 390 395 400 Arg Ile Arg His Gly Ile Ile Tyr Phe Gly Gly Asn Thr Tyr Arg Leu 405 410 415 Ser Gly Gly Arg Arg Arg Phe Met Thr Leu Trp Gln Glu Cys Leu Glu 420 425 430 Ser Tyr Gly Asp Ser Asp Glu Cys Phe Val Gln Leu Leu Glu Gly Asn 435 440 445 Gln His Leu Phe Thr Val Val Gln Gly His His Ser Thr Ser Phe Arg 450 455 460 Ser Asp Leu Ser Asn Asp Leu His Pro Asp Asn Asn Ile Glu Gln Ile 465 470 475 480 Ala Asn Asp His Val Asn Asp Ile Ala Gln Ser Thr Asp Gly Asp Ile 485 490 495 Asn Asp Phe Ala Asp Thr His Tyr Asn Asp Val Ala Pro Ile Ala Asp 500 505 510 Val His Val Asp Asn Ile Ala Gln Thr Ala Asp Asn His Val Lys Asn 515 520 525 Ile Ala Gln Thr Ala His His His Val Asn Asp Val Ala Gln Ile Ala 530 535 540 Asp Asp His Val Asn Asp Ile Gly Gln Thr Ala Tyr Asp His Val Asn 545 550 555 560 Asn Ile Gly Gln Thr Ala Asp Asp His Val Asn Asp Ile Ala Gln Thr 565 570 575 Ala Asp Asp His Val Asn Ala Ile Ala Gln Thr Ala Asp Asp His Val 580 585 590 Asn Ala Ile Ala Gln Thr Ala Asp Asp His Val Asn Asp Ile Gly Asp 595 60 0 605 Thr Ala Asn Ser His Ile Val Arg Val Gln Gly Val Ala Lys Asn His 610 615 620 Leu Tyr Gly Ile Asn Lys Ala Ile Gly Lys His Ile Gln His Leu Lys 625 630 635 640 Asp Val Ser Asn Arg His Ile Glu Lys Leu Asn Asn His Ala Thr Lys 645 650 655 Asn Leu Leu Gln Ser Ala Leu Gln His Lys Gln Gln Thr Ile Glu Arg 660 665 670 Glu Ile Gln His Lys Arg His Leu Ser Glu Lys Glu Asp Ile Asn Leu 675 680 685 Gln His Glu Asn Ala Met Lys Ser Lys Val Ser Tyr Asp Gly Pro Val 690 695 700 Phe Asn Glu Lys Val Ser Val Val Ser Asn Gln Gly Ser Tyr Asn Glu 705 710 715 720 Lys Val Pro Val Leu Ser Asn Gly Gly Gly Tyr Asn Gly Lys Val Ser 725 730 735 Ala Leu Ser Asp Gln Gly Ser Tyr Asn Glu Gly Tyr Ala Tyr 740 745 750 <210> 7 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> fp-5 <400> 7 Lys His His His His His His Ser Ser Glu Glu Tyr Lys Gly Gly Tyr 1 5 10 15 Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly 20 25 30 Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys 35 40 45 Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys 50 55 60 Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly 65 70 75 80 Ser Ser <210> 8 <211> 103 <212> PRT <213> Artificial Sequence <220 > <223> fp-6 <400> 8 Ile Ala Ala Leu Cys Gly Ile Val Lys Ser Ile Asp Ser Asp Asp Ser 1 5 10 15 Asp Tyr Asp Tyr Lys Gly Arg Gly Tyr Cys Thr Asn Lys Gly Cys Arg 20 25 30 Ser Gly Tyr Asn Tyr Phe Gly Asn Lys Gly Tyr Cys Lys Tyr Gly Glu 35 40 45 Lys Ser Tyr Thr Tyr Asn Cys Asn Ser Tyr Ala Gly Cys Cys Leu Pro 50 55 60 Arg Asn Pro Tyr Gly Lys Leu Lys Tyr Tyr Cys Thr Asn Lys Tyr Gly 65 70 75 80 Cys Pro Asn Asn Tyr Tyr Phe Tyr Asn Asn Lys Gly Tyr Tyr Tyr Leu 85 90 95 Glu His His His His His His 100 <210> 9 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> fp-151 <400> 9 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ser Ser 50 55 60 Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His 65 70 75 80 Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly 85 90 95 Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser 100 105 110 Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly 115 120 125 Tyr Lys Lys Tyr Tyr Tyr Gly Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro 130 135 140 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 145 150 155 160 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 165 170 175 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 180 185 190 Tyr Pro Pro Thr Tyr Lys Leu 195 <210> 10 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> fp-131 <400> 10 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala Asp 50 55 60 Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn 65 70 75 80 Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn 85 90 95 Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 130 135 140 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 145 150 155 160 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu 165 170 <210> 11 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> fp-353 <400> 11 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Pro Trp Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr 50 55 60 Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly 65 70 75 80 Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys 85 90 95 Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr 100 105 110 His Arg Lys Gly Tyr Lys Lys Tyr Tyr Tyr Gly Gly Ser Ser Gly Ser Ala 115 120 125 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly 130 135 140 Asn Tyr As n Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn 145 150 155 160 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser 165 170 175 <210> 12 <211> 187 <212> PRT <213 > Artificial Sequence <220> <223> fp-153 <400> 12 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ser Ser 50 55 60 Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His 65 70 75 80 Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly 85 90 95 Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Tyr Lys Tyr Lys Asn Ser 100 105 110 Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly 115 120 125 Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala Asp Tyr Tyr Gly 130 135 140 Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn Tyr Asn Arg 145 150 155 160 Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn Gly Trp Lys 165 170 175 Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser 180 185 <210> 13 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> fp-351 <400> 13 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Pro Trp Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr 50 55 60 Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly 65 70 75 80 Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys 85 90 95 Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr 100 105 110 His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser 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 180 185 <210> 14 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant <400> 14 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 50 55 60 <210> 15 <211> 196 <212> PRT <213> Artificial Sequence <220> <223 > fp-151 variant <400> 15 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu 50 55 60 Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly 65 70 75 80 Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr 85 90 95 Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys 100 105 110 Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys 115 120 125 Lys Tyr Tyr Gly 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 16 5 170 175 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> 16 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> mgfp-5 <400> 16 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 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 60Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser 65 70 75

Claims (14)

(1) 임플란트를 입자 분사 후 산처리법으로 표면처리하는 단계;
(2) 홍합접착단백질 및 아스코르브산을 포함하는 코팅 수용액에 상기 (1) 단계의 임플란트를 침지시켜 임플란트 표면에 홍합접착단백질-아스코르브산 코팅막을 형성시키는 단계; 및
(3) 알칼리금속 또는 알칼리토금속의 무기염을 포함하는 pH 8~9의 완충용액에 상기 (2) 단계의 코팅막이 형성된 임플란트를 침지시키는 단계;를 포함하며,
상기 홍합접착단백질은 서열번호 1, 서열번호 2, 서열번호 3, 서열번호 4, 서열번호 5, 서열번호 6, 서열번호7, 서열번호 8, 서열번호 9, 서열번호 10, 서열번호 11, 서열번호 12, 서열번호 13, 서열번호 14 및 서열번호 15로 이루어진 군으로부터 선택된 1종 이상의 아미노산 서열 또는 홍합접착단백질의 변이체인, 치과용 임플란트의 제조방법.(1) surface-treating the implant with an acid treatment method after particle injection;
(2) immersing the implant of step (1) in an aqueous coating solution containing mussel adhesive protein and ascorbic acid to form a mussel adhesive protein-ascorbic acid coating film on the implant surface; and
(3) immersing the implant on which the coating film of step (2) is formed in a buffer solution of pH 8 to 9 containing an inorganic salt of an alkali metal or alkaline earth metal;
The mussel adhesive protein is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, sequence No. 12, SEQ ID NO: 13, SEQ ID NO: 14 and one or more amino acid sequences selected from the group consisting of SEQ ID NO: 15 or a variant of the mussel adhesive protein, a method for producing a dental implant. 삭제delete 제1항에 있어서,
홍합접착단백질은 티로신잔기가 카테콜 화합물로 변환된 것인, 치과용 임플란트의 제조방법. According to claim 1,
The mussel adhesive protein is a tyrosine residue converted into a catechol compound, a method of manufacturing a dental implant. 제3항에 있어서,
카테콜 화합물은 도파(3,4-dihydroxyphenylalanine, DOPA), 도파 o-퀴논(Dopa o-quinone), 토파(2,4,5-trihydroxyphenylalanine, TOPA), 토파 퀴논(Topa quinone) 및 이들의 유도체로 이루어진 군에서 선택된 어느 하나 이상인, 치과용 임플란트의 제조방법.4. The method of claim 3,
Catechol compounds are dopa (3,4-dihydroxyphenylalanine, DOPA), dopa o-quinone (Dopa o-quinone), topa (2,4,5-trihydroxyphenylalanine, TOPA), topa quinone (Topa quinone) and derivatives thereof. Any one or more selected from the group consisting of, a method of manufacturing a dental implant. 제1항에 있어서,
상기 (2) 단계의 코팅 수용액은 pH가 2 내지 3인, 치과용 임플란트의 제조방법.According to claim 1,
The aqueous coating solution of step (2) has a pH of 2 to 3, a method of manufacturing a dental implant. 제1항에 있어서,
코팅 수용액 중의 홍합접착단백질 대 아스코르브산의 함량비는 1 : 10 내지 200 몰비로 포함되는 것인, 치과용 임플란트의 제조방법.According to claim 1,
The content ratio of mussel adhesive protein to ascorbic acid in the coating aqueous solution is 1: 10 to 200 molar ratio, the method for producing a dental implant. 삭제delete 제1항에 있어서,
알칼리금속 또는 알칼리토금속의 무기염은 소듐카보네이트, 소듐바이카보네이트, 쇼듐포스페이트, 칼슘 카보네이트, 포타슘카보네이트, 마그네슘카보네이트, 칼슘포스페이트, 포타슘포스페이트 및 마그네슘포스페이트로 이루어진 군으로부터 선택되는 어느 하나인, 치과용 임플란트의 제조방법.According to claim 1,
The inorganic salt of an alkali metal or alkaline earth metal is any one selected from the group consisting of sodium carbonate, sodium bicarbonate, sodium phosphate, calcium carbonate, potassium carbonate, magnesium carbonate, calcium phosphate, potassium phosphate and magnesium phosphate, of a dental implant. manufacturing method. 제1항에 있어서,
홍합접착단백질은 임플란트 표면 상에 1 내지 500 μg/cm2의 농도로 코팅되는 것인, 치과용 임플란트의 제조방법.According to claim 1,
The mussel adhesive protein is coated on the implant surface at a concentration of 1 to 500 μg/cm 2 , A method for producing a dental implant. 제1항에 따른 제조방법으로 제조된 홍합접착단백질 및 아스코르브산의 코팅막을 포함하는 치과용 임플란트.A dental implant comprising a coating film of mussel adhesive protein and ascorbic acid prepared by the method according to claim 1 . 제10항에 있어서,
상기 코팅막은 친수성인, 치과용 임플란트.11. The method of claim 10,
The coating film is hydrophilic, dental implant. 제10항에 있어서,
상기 코팅막은 홍합접착단백질 대 아스코르브산이 1 : 10 내지 200 몰비로 포함되는 것인, 치과용 임플란트.11. The method of claim 10,
The coating film is mussel adhesive protein to ascorbic acid 1: 10 to 200 will be included in the molar ratio, dental implant. 삭제delete 제10항에 있어서,
홍합접착단백질은 임플란트 표면에 1 내지 500 μg/cm2의 양으로 코팅된 것인, 치과용 임플란트.11. The method of claim 10,
The mussel adhesive protein is coated on the implant surface in an amount of 1 to 500 μg/cm 2 , a dental implant.
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