KR101400667B1 - Surface Activated Collagen Membrane by Peptide - Google Patents
Surface Activated Collagen Membrane by Peptide Download PDFInfo
- Publication number
- KR101400667B1 KR101400667B1 KR1020110037839A KR20110037839A KR101400667B1 KR 101400667 B1 KR101400667 B1 KR 101400667B1 KR 1020110037839 A KR1020110037839 A KR 1020110037839A KR 20110037839 A KR20110037839 A KR 20110037839A KR 101400667 B1 KR101400667 B1 KR 101400667B1
- Authority
- KR
- South Korea
- Prior art keywords
- peptide
- collagen
- seq
- binding
- shielding membrane
- Prior art date
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Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
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- C07K14/475—Growth factors; Growth regulators
- C07K14/49—Platelet-derived growth factor [PDGF]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- Materials For Medical Uses (AREA)
Abstract
본 발명은 콜라겐에 결합하는 골조직 재생능을 가지는 펩타이드가 결합된 차폐막에 관한 것으로, 더욱 자세하게는 콜라겐 결합 펩타이드와 골조직 재생 기능성 펩타이드의 융합 펩타이드가 콜라겐 표면에 고정되어 있는 콜라겐 차폐막에 관한 것이다.
본 발명의 융합 펩타이드 표면에 결합한 표면활성형 콜라겐 차폐막은 생체 내에 이식하였을 때, 국소 체류성이 증가하여 안정하게 결합할 수 있고, 골조직 재생에 관련된 세포의 이행, 증식 및 분화를 촉진하여 최종적으로 골조직 재생력을 극대화시킬 수 있어 이를 이용한 골유도재생 치료기술의 발전에 유용하다. 또한 융합 펩타이드는 콜라겐으로 제조된 다른 형태의 이식재에도 응용이 가능하고, 융합 펩타이드를 구성하는 콜라겐 결합 펩타이드는 다른 조직에 재생효과가 있는 펩타이드와도 결합하여 합성이 가능하므로, 피부와 같은 다른 목적의 조직재생용 생체재료에 응용이 가능하다.The present invention relates to a shielding membrane to which a peptide having a bone regeneration ability binding to collagen is bound, and more particularly to a collagen shielding membrane having a fusion peptide of a collagen binding peptide and a bone regeneration functional peptide immobilized on a collagen surface.
The surface active collagen shielding membrane bound to the surface of the fusion peptide of the present invention can stably bind with increased local retention when transplanted into a living body and promotes cell migration, proliferation and differentiation associated with bone regeneration, It is possible to maximize the regenerative power and is useful for the development of the bone induction regenerative therapy technique using the same. In addition, the fusion peptide can be applied to other types of grafts made of collagen, and the collagen-binding peptide constituting the fusion peptide can be synthesized by binding to a peptide having regeneration effect in other tissues. Therefore, It can be applied to biomaterials for tissue regeneration.
Description
본 발명은 콜라겐에 결합하는 골조직 재생능을 가지는 펩타이드가 결합된 차폐막에 관한 것으로, 더욱 자세하게는 콜라겐 결합 펩타이드와 골조직 재생 기능성 펩타이드의 융합 펩타이드가 콜라겐 표면에 고정되어 있는 콜라겐 차폐막에 관한 것이다.
The present invention relates to a shielding membrane to which a peptide having a bone regeneration ability binding to collagen is bound, and more particularly to a collagen shielding membrane having a fusion peptide of a collagen binding peptide and a bone regeneration functional peptide immobilized on a collagen surface.
치주질환에 의해 손상된 치조골을 재생시키는 방법으로는 자가골이식 (autografting)으로 손상된 부위를 채워 고형을 유도하는 방법이 있다. 또 다른 방법으로는 면역원성을 제거한 사람이나 동물의 뼈를 인위적인 골대체 물질로서 이용하거나 상업적으로 시판되는 수산화 아파타이트를 이용하는 방법이 있다.As a method for regenerating the alveolar bone damaged by periodontal disease, there is a method of inducing solidity by filling the damaged region by autografting. Another method is to use the bones of humans or animals that have removed immunogenicity as an artificial bone substitute, or to use commercially available apatite hydroxide.
최근에는 인공 막을 조직에 도입하여 손상된 치주조직의 치유를 증진시키고, 완전한 치주조직으로의 복원을 꾀하는 동시에 골 이식 결과를 개선시키고 새로운 치조골의 생성을 유도하려는 시도가 활발하게 이루어지고 있다. 이러한 기술에서 사용되는 차폐막은 손상 부위와 그 주위의 결체 조직을 격리 및 차단시켜 새로운 치조골 및 치주 인대조직을 생성시켜 치주조직의 재생이 원활히 일어날 수 있도록 한다. 즉, 차폐막으로 손상된 부위를 다른 주위 환경과 차단시켜 치은 섬유아세포가 침입하지 못하고 조직 중에 있는 골 및 치주 인대 재생력이 있는 세포들이 방해를 받지 않고 새로운 치주조직이 재생될 수 있도록 하는 것이다.In recent years, attempts have been made to improve the healing of damaged periodontal tissues by introducing an artificial membrane into tissues, to restore the entire periodontal tissue, to improve the bone grafting result and to induce generation of new alveolar bone. The shielding membrane used in this technique isolates and blocks the damaged area and the surrounding connective tissues to create new alveolar bone and periodontal ligament tissue so that the regeneration of periodontal tissue can be smoothly performed. In other words, the damaged area of the shielding membrane is blocked from the surrounding environment so that the gingival fibroblast can not penetrate and the new periodontal tissue can be regenerated without interfering with the bone and periodontal regenerative cells in the tissue.
또한, 여러 가지 질병이나 외상으로 인한 골 손상은 인간의 움직임에 중요한 영향력을 미치므로 골격계에서 연속성을 유지하고 기계적으로 신체를 지지할 수 있는 치료가 필요하다. 현재 골 재생 또는 골 대체 치료들이 많이 수행되고 있지만 많은 문제점이 있다.In addition, since bone damage caused by various diseases or trauma has an important influence on human motion, it is necessary to maintain continuity in the skeletal system and to support the body mechanically. Currently, bone regeneration or alternative bone replacement therapies are performed, but there are many problems.
초기에는 차폐막으로 폴리테트라플루오로에틸렌, 셀룰로오스 아세테이트, 실리콘 고무 및 폴리우레탄과 같은 비분해성 재료를 이용하였으나, 비분해성 재료로 제조된 차폐막은 생체적합성이 크지 않아 치조골이 생성된 후에 다시 차폐막을 제거하기 위한 2차 수술이 필요하고, 이러한 과정에서 불필요한 염증이나 조직 괴사가 일어날 수 있는 부작용이 있다.In the early days, non-degradable materials such as polytetrafluoroethylene, cellulose acetate, silicone rubber and polyurethane were used as shielding membranes. However, shielding membranes made of non-degradable materials are not biocompatible. After the alveolar bone is formed, , There is a side effect that unnecessary inflammation or tissue necrosis may occur in this process.
최근에는 지방족 폴리에스터나 콜라겐과 같은 생분해성 고분자를 이용한 연구가 보고되고 있는데, 생분해성 차폐막을 이용하면 차폐막을 제거하기 위한 재수술이 필요 없고 비분해성 재료로 제조된 차폐막과 비교하여 조직을 재생하는 데에는 큰 차이가 없는 것으로 보고되고 있다. 그러나, 지방족 폴리에스터로 제조된 차폐막의 경우 산성 분해물에 의해 이식된 부위에서의 염증반응을 일으킬 수 있다. 따라서 골조직을 구성하는 단백질의 대부분을 차지하는 콜라겐을 이용하여 차폐막을 제조하여 이를 임상시험에 사용되는 것이 보고되었다. Recently, biodegradable polymers such as aliphatic polyesters and collagen have been reported. Biodegradable shielding membranes do not require reoperation to remove shielding membranes. In comparison with shielding membranes made of non-degradable materials, It is reported that there is no big difference. However, shielding membranes made from aliphatic polyesters can cause an inflammatory reaction at sites implanted with acidic degradants. Therefore, it has been reported that a shielding membrane is manufactured using collagen which occupies most of the proteins constituting the bone tissue and is used in clinical trials.
종래의 기술로 한국공개특허 제2003-002224호는 천연 고분자인 키토산과 합성 고분자인 생분해성 고분자를 이용하여 제조된 조직 재생 유도용 차폐막에 관한 것으로, 이는 키토산으로 제조된 부직포, 생분해성 고분자 용액을 상기 부직포 위에 도포하여 고분자 막 형성 및 키토산으로 제조된 부직포를 상기 고분자 막에 적층하는 순서에 따라 제조과정이 진행되므로, 제조공정이 복잡한 단점이 있고, 한국등록특허 제0564366호에 따르면, 일정 강도와 생체 적합성 및 생분해성을 가지는 미세공의 공극 크기 조절이 용이하고, 간단한 제조공정으로 제조할 수 있는 천연고분자와 합성고분자의 혼합물로부터 제조되는 나노섬유가 부직포 형태로 되어 있는 조직 재생용 차폐막에 대해 제시하고 있으나, 이들 고분자 차폐막이나 지지체에 의해서는 조직성장인자가 물리적으로 혼합되어 있는 상태로서 초기 적용시 속방출이 일어나 치료기간 동안 유효 농도의 유지가 어려운 단점이 있다.Korean Unexamined Patent Publication No. 2003-002224 discloses a shielding film for inducing tissue regeneration, which is produced using chitosan, a natural polymer, and a biodegradable polymer, which is a synthetic polymer. This is because a nonwoven fabric made of chitosan, a biodegradable polymer solution The nonwoven fabric is coated on the nonwoven fabric to form a polymer film and the nonwoven fabric made of chitosan is laminated on the polymer film. Thus, the manufacturing process is complicated. According to Korean Patent No. 0564366, A nanofiber fabricated from a mixture of natural polymers and synthetic polymers, which can be easily controlled in pore sizes of biocompatible and biodegradable micropores and can be prepared by a simple manufacturing process, is presented in a nonwoven fabric-type shielding film for tissue regeneration However, depending on the polymer shielding film or the support, the tissue growth factor Physically mixed state, there is a disadvantage that it is difficult to maintain the effective concentration during the treatment period due to rapid release during initial application.
또한, 차폐막의 조직재생을 향상시키기 위해 조직성장인자(growth factor) 또는 세포 외기질 유래 물질(Extracellualr matrix)을 차폐막에 도입한 연구가 이루어졌는데, 이들은 단백질이기 때문에, 활성을 유지하기 위해서는 3차 구조를 유지해야 하지만 대부분의 세포 외기질 및 성장인자들은 온도에 영향을 받기 쉬워 생체 내에서도 쉽게 불안정해지는 단점이 있다. In order to improve the regeneration of the shielding membrane, a study has been carried out in which a growth factor or an extracellular matrix is introduced into the shielding membrane. Since they are proteins, in order to maintain the activity, However, most of the extracellular matrix and growth factors are susceptible to temperature and are easily unstable even in vivo.
따라서 원하는 치료효과를 얻기 위해서는 조직성장인자 및 세포 외기질 유래 물질을 대용량으로 투여해야 하고, 이로 인한 부작용을 유발할 수 있어, 이를 해결하기 위해 차폐막의 내부 또는 표면에 조직성장인자를 도입하려는 시도가 이루어졌다. 그러나, 조직성장인자를 차폐막에 물리적으로 흡착시키는 경우, 그 결합력이 약하므로 장기간 동안 결합을 유지할 수 없는 반면, 조직성장인자를 차폐막에 공유결합시키는 경우에는 가교제를 사용하여 장기간 동안 안정한 결합을 유지할 수 있다. 그러나 가교제로 인해 조직성장인자의 3차 구조에 변형을 초래하여 그 활성을 감소시킬 가능성이 있는 문제점이 있다.Therefore, in order to obtain a desired therapeutic effect, a large amount of a tissue growth factor and an extracellular matrix-derived substance are required to be administered. To solve this problem, an attempt has been made to introduce a tissue growth factor into the inside or the surface of a shielding membrane lost. However, when a tissue growth factor is physically adsorbed to a shielding membrane, the binding force can not be maintained for a long period of time because of its weak binding force. On the other hand, when a tissue growth factor is covalently bound to a shielding membrane, have. However, there is a possibility that the crosslinking agent may cause a change in the tertiary structure of the tissue growth factor, thereby reducing its activity.
이에 본 발명자들은 골조직 재생 기능성을 가진 표면활성형 콜라겐 차폐막을 제조하고자 예의 노력한 결과, 콜라겐에 결합력이 있는 펩타이드에 골조직 재생 기능성 펩타이드를 연결한 융합 펩타이드를 콜라겐 차폐막에 결합시키는 경우, 차폐막에 골조직 재생 기능성의 표면활성을 부여할 수 있는 것을 확인하고, 본 발명을 완성하게 되었다.
As a result of intensive efforts to produce a surface active collagen shielding membrane having bone regeneration function, the present inventors have found that when a fusion peptide comprising a peptide having a binding ability to collagen and a peptide binding to a bone regeneration functional peptide is bound to a collagen shielding membrane, And thus the present invention has been completed.
본 발명의 목적은 콜라겐에 결합력이 있는 펩타이드에 골조직 재생 기능성 펩타이드를 연결한 융합 펩타이드를 콜라겐 차폐막에 결합시켜 골조직 재생 기능성의 표면활성이 부여된 콜라겐 차폐막을 제공하는데 있다.
It is an object of the present invention to provide a collagen shielding membrane having a surface activity of bone regeneration function by binding a fusion peptide comprising a peptide having a binding force to collagen to a bone regeneration functional peptide bonded to a collagen shielding membrane.
상기 목적을 달성하기 위하여, 본 발명은 콜레겐 결합 펩타이드와 서열번호 2 내지 서열번호 23의 아미노산 서열로 구성된 군에서 선택되는 펩타이드가 결합되어 있는 콜라겐 결합능과 골조직 재생능을 가지는 융합 펩타이드 및 상기의 융합 펩타이드가 콜라겐 차폐막 표면에 고정되어 있는 것을 특징으로 하는 골조직 재생능을 가지는 콜라겐 차폐막를 제공한다.
In order to achieve the above object, the present invention provides a fusion peptide comprising a collagen binding peptide and a peptide selected from the group consisting of SEQ ID NOS: 2 to 23 and having a collagen binding ability and a bone regeneration ability, The present invention provides a collagen shielding membrane having bone regeneration ability, characterized in that the peptide is immobilized on the surface of the collagen shielding membrane.
본 발명의 융합 펩타이드 표면에 결합한 표면활성형 콜라겐 차폐막은 생체 내에 이식하였을 때, 국소 체류성이 증가하여 안정하게 결합할 수 있고, 골조직 재생에 관련된 세포의 이행, 증식 및 분화를 촉진하여 최종적으로 골조직 재생력을 극대화시킬 수 있어 이를 이용한 골유도재생 치료기술의 발전에 유용하다. 또한 융합 펩타이드는 콜라겐으로 제조된 다른 형태의 이식재에도 응용이 가능하고, 융합 펩타이드를 구성하는 콜라겐 결합 펩타이드는 다른 조직에 재생효과가 있는 펩타이드와도 결합하여 합성이 가능하므로, 피부와 같은 다른 목적의 조직재생용 생체재료에 응용이 가능하다.
The surface active collagen shielding membrane bound to the surface of the fusion peptide of the present invention can stably bind with increased local retention when transplanted into a living body and promotes cell migration, proliferation and differentiation associated with bone regeneration, It is possible to maximize the regenerative power and is useful for the development of the bone induction regenerative therapy technique using the same. In addition, the fusion peptide can be applied to other types of grafts made of collagen, and the collagen-binding peptide constituting the fusion peptide can be synthesized by binding to a peptide having regeneration effect in other tissues. Therefore, It can be applied to biomaterials for tissue regeneration.
도 1은 콜라겐 차폐막 및 3차원 지지체이다.
도 2는 콜라겐 차폐막의 전자현미경(SEM)의 표면 및 단면을 나타낸 사진이다.
도 3은 콜라겐 차폐막을 공초점 현미경으로 관찰한 것을 나타낸 사진이다
(A: 펩타이드가 결합되지 않은 콜라겐 차폐막;
B: FITC가 표지된 서열번호 24 펩타이드가 결합된 콜라겐 차폐막).
도 4는 FITC가 표지된 콜라겐 차폐막을 가토 두개골 원형 골 결손부에 이식하고, 시간의 변화에 따른 혈액을 채취하여 혈액 내로 유리된 펩타이드를 fluorometer로 측정값을 나타낸 것이다.
도 5는 콜라겐 차폐막을 가토 두개골 원형 골결손부에 이식하고 이식 4주 후 골재생 정도를 관찰한 사진이다
(A: 서열번호 1 펩타이드가 결합된 차폐막;
B: 서열번호 9 펩타이드가 결합된 차폐막; 및
C: 서열번호 24 펩타이드가 결합된 차폐막).
도 6은 콜라겐 차폐막의 대장균에 대한 성장저해 직경을 측정한 것을 나타낸 것이다.1 is a collagen shielding film and a three-dimensional support.
2 is a photograph showing a surface and a cross section of an electron microscope (SEM) of a collagen shielding film.
3 is a photograph showing a collagen shielding film observed with a confocal microscope
(A: collagen shielding membrane without peptide binding;
B: FITC-labeled collagen shielding membrane bound with peptide of SEQ ID NO: 24).
FIG. 4 shows measured values of peptides liberated into blood by transplanting a collagen-screening membrane labeled with FITC into a rabbit skull circular defect and taking blood according to time change.
FIG. 5 is a photograph showing the degree of bone regeneration after implantation of a collagen shielding layer in a rabbit skull circular defect region and 4 weeks after transplantation
(A: shielding membrane bound to peptide of SEQ ID NO: 1;
B: Shielding membrane bound with peptide of SEQ ID NO: 9; And
C: shielding film bound with peptide of SEQ ID NO: 24).
6 shows the measurement of the growth inhibition diameter of the collagen shielding membrane against E. coli.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
일 관점에서, 본 발명은 콜레겐 결합 펩타이드와 서열번호 2 내지 서열번호 23의 아미노산 서열로 구성된 군에서 선택되는 펩타이드가 결합되어 있는 콜라겐 결합능과 골조직 재생능을 가지는 융합 펩타이드에 관한 것이다.In one aspect, the present invention relates to a fusion peptide having a collagen binding ability and a bone regeneration ability, wherein a peptide selected from the group consisting of the amino acid sequence of SEQ ID NO: 2 to SEQ ID NO: 23 is combined with the collagen binding peptide.
본 발명에서 상기 콜라겐 결합 펩타이드는 콜라겐과 결합능을 가지는 펩타이드라면 제한없이 사용할 수 있으며, 본 발명의 일 실시예에서는 콜라겐 결합 펩타이드는 서열번호 1의 아미노산 서열로 표시되는 펩타이드를 사용하였으나 이에 한정되는 것은 아니다.In the present invention, the collagen-binding peptide may be used without limitation as long as it is capable of binding to collagen. In one embodiment of the present invention, the peptide having the amino acid sequence of SEQ ID NO: 1 is used as the collagen-binding peptide. .
본 발명에 있어서, 상기 융합 펩타이드는 서열번호 24의 아미노산 서열로 표시되는 것을 특징으로 할 수 있다.In the present invention, the fusion peptide may be characterized by being represented by the amino acid sequence of SEQ ID NO: 24.
본 발명에서 콜라겐에 결합하는 펩타이드는 서열번호 1(GLRSKSKKFRRPDIQYPDA)이며, 골조직 재생 기능성을 가지는 펩타이드의 N-말단에 화학적으로 부가되어 콜라겐에 대한 결합능을 증가시켜 차폐막 또는 이식재 등에 안정하게 결합할 수 있다.In the present invention, the peptide that binds to collagen is SEQ ID NO: 1 (GLRSKSKKFRRPDIQYPDA) , which is chemically added to the N-terminus of the peptide having bone regeneration function to increase the binding capacity to collagen, thereby enabling stable binding to a shielding membrane or a graft material.
본 발명에서 골조직 재생 기능성을 가지는 펩타이드는 (a)골형성 단백질(bone morphogenetic protein, BMP)-2, 4 및 6의 아미노산 서열 중 각각 2-18위치의 아미노산 서열 [BMP-2의 경우(서열번호 2), BMP-4의 경우(서열번호 3) 및 BMP-6의 경우(서열번호 4)]; BMP-2의 16-34위치의 아미노산 서열 (서열번호 5); 47-71위치의 아미노산 서열 (서열번호 6); 73-92위치의 아미노산 서열 (서열번호 7); 88-105위치의 아미노산 서열 (서열번호 8); 298-316위치의 아미노산 서열 (서열번호 9); 335-353위치의 아미노산 서열 (서열번호 10); 370-396위치의 아미노산 서열 (서열번호 11); BMP-4의 74-93위치의 아미노산 서열 (서열번호 12); 293-313위치의 아미노산 서열 (서열번호 13); 360-379위치의 아미노산 서열 (서열번호 14); 382-402위치의 아미노산 서열 (서열번호 15); BMP-6의 91-110위치의 아미노산 서열 (서열번호 16); 407-418위치의 아미노산 서열 (서열번호 17); 472-490위치의 아미노산 서열 (서열번호 18); 487-510위치의 아미노산 서열 (서열번호 19); BMP-7의 98-117위치의 아미노산 서열 (서열번호 20); 320-340위치의 아미노산 서열 (서열번호 21); 400-409위치의 아미노산 서열 (서열번호 22) 및 405-423위치의 아미노산 서열 (서열번호 23)로 구성된 군에서 선택된 하나 이상의 펩타이드에서 선택할 수 있다.
In the present invention, the peptide having the bone regeneration function has (a) an amino acid sequence of 2-18 positions in the amino acid sequence of bone morphogenetic protein (BMP) -2, 4 and 6 [in the case of BMP-2 2), in the case of BMP-4 (SEQ ID NO: 3) and in the case of BMP-6 (SEQ ID NO: 4)]; The amino acid sequence at position 16-34 of BMP-2 (SEQ ID NO: 5); The amino acid sequence at position 47-71 (SEQ ID NO: 6); The amino acid sequence at position 73-92 (SEQ ID NO: 7); The amino acid sequence at position 88-105 (SEQ ID NO: 8); The amino acid sequence at position 298-316 (SEQ ID NO: 9); The amino acid sequence at position 335-353 (SEQ ID NO: 10); The amino acid sequence at position 370-396 (SEQ ID NO: 11); The amino acid sequence at position 74-93 of BMP-4 (SEQ ID NO: 12); Amino acid sequence at position 293-313 (SEQ ID NO: 13); The amino acid sequence at position 360-379 (SEQ ID NO: 14); The amino acid sequence at position 382-402 (SEQ ID NO: 15); The amino acid sequence at position 91-110 of BMP-6 (SEQ ID NO: 16); The amino acid sequence at position 407-418 (SEQ ID NO: 17); The amino acid sequence at position 472-490 (SEQ ID NO: 18); Amino acid sequence at position 487-510 (SEQ ID NO: 19); The amino acid sequence at position 98-117 of BMP-7 (SEQ ID NO: 20); The amino acid sequence at position 320-340 (SEQ ID NO: 21); At least one peptide selected from the group consisting of the amino acid sequence at the 400-409 position (SEQ ID NO: 22) and the amino acid sequence at the 405-423 position (SEQ ID NO: 23).
상기 펩타이드의 서열번호 2~23의 아미노산 서열은 하기와 같다. The amino acid sequences of SEQ ID NOS: 2 to 23 of the above peptides are shown below.
서열번호 2: VAGTRCLLA LLLPQVLLSEQ ID NO: 2: VAGTRCLLA LLLPQVLL
서열번호 3: IPGNRMLMV VLLCQVLLSEQ ID NO: 3: IPGNRMLMV VLLCQVLL
서열번호 4: PGLGRRAQW LCWWWGLLSEQ ID NO: 4: PGLGRRAQW LCWWWGLL
서열번호 5: VLLGG AAGLVPELGR RKFASEQ ID NO: 5: VLLGG AAGLVPELGR RKFA
서열번호 6: DEVL SEFELRLLSM FGLKQRPTPS RSEQ ID NO: 6: DEVL SEFELRLLSM FGLKQRPTPS R
서열번호 7: AVVPPYML DLYRRHSGQP GSSEQ ID NO: 7: AVVPPYML DLYRRHSGQP GS
서열번호 8: GQP GSPAPDHRLE RAASRSEQ ID NO: 8: GQP GSPAPDHRLE RAASR
서열번호 9: RHPLYVDFSDVGW NDWIVASEQ ID NO: 9: RHPLYVDFSDVGW NDWIVA
서열변호 10: DHLNST NHAIVQTLVN SVN10: DHLNST NHAIVQTLVN SVN
서열번호 11: S MLYLDENEKV VLKNYQDMVV EGCGCRSEQ ID NO: 11: S MLYLDENEKV VLKNYQDMVV EGCGCR
서열번호 12: SKSAVIP DYMRDLYRLQ SGESEQ ID NO: 12: SKSAVIP DYMRDLYRLQ SGE
서열번호 13: SPKHHSQR ARKKNKNCRR HSLSEQ ID NO: 13: SPKHHSQR ARKKNKNCRR HSL
서열번호 14: L VNSVNSSIPK ACCVPTELSSEQ ID NO: 14: L VNSVNSSIPK ACCVPTELS
서열번호 15: SMLYLDEYD KVSEQ ID NO: 15: SMLYLDEYD KV
서열번호 16: RPRPLHGLQ QPQPPALRQQSEQ ID NO: 16: RPRPLHGLQ QPQPPALRQQ
서열번호 17: ELKT ACRKHELYSEQ ID NO: 17: ELKT ACRKHELY
서열번호 18: YVPKPCCAPTKLNAISVLYSEQ ID NO: 18: YVPKPCCAPTKLNAISVLY
서열번호 19: SVLY FDDNSNVILK KYRNMVVRACSEQ ID NO: 19: SVLY FDDNSNVILK KYRNMVVRAC
서열번호 20: PGG QGFSYPYKAV FSTQGPPSEQ ID NO: 20: PGG QGFSYPYKAV FSTQGPP
서열번호 21: ENSSSDQRQAC KKHELYVSFRSEQ ID NO: 21: ENSSSDQRQAC KKHELYVSFR
서열번호 22: Q LNAISVLYFSEQ ID NO: 22: Q LNAISVLYF
서열번호 23: SVLYFD DSSNVILKKY RNM
SEQ ID NO: 23: SVLYFD DSSNVILKKY RNM
다른 관점에서, 본 발명은 상기의 융합 펩타이드가 콜라겐 차폐막 표면에 고정되어 있는 것을 특징으로 하는 골조직 재생능을 가지는 콜라겐 차폐막에 관한 것이다.In another aspect, the present invention relates to a collagen shielding membrane having bone regeneration ability, characterized in that the fusion peptide is immobilized on the surface of the collagen shielding membrane.
본 발명의 일 양태에서, 서열번호 1의 콜라겐 결합 펩타이드와 서열번호 9의 골조직 재생 기능성 펩타이드를 연결하여 서열번호 24의 콜라겐에 결합하는 골조직 재생 기능성 펩타이드를 제작하고, 제작된 펩타이드가 콜라겐에 안정하게 결합하는지를 확인 및 상기 차폐막을 골결손부에 이식하여 골 재생력을 확인하였다.In one embodiment of the present invention, a collagen-binding peptide of SEQ ID NO: 1 is linked to a bone regeneration functional peptide of SEQ ID NO: 9 to produce a bone regeneration functional peptide that binds to the collagen of SEQ ID NO: 24 and the resulting peptide is stably And the shielding membrane was transplanted into the bone defect region to confirm the bone regeneration ability.
본 발명의 일 실시예에서, 콜라겐에 특이적으로 결합하는 골조직 재생능을 가지는 융합 펩타이드를 제조하기 위하여, N 말단으로부터 순서대로 콜라겐에 결합하는 서열번호 1(GLRSKSKKFRRPDIQYPDA)의 펩타이드, 골조직 재생 기능성을 가지는 서열번호 9(RHPLYVDFSDVGWNDWIVA)의 펩타이드를 함유하도록 펩타이드 합성장치를 이용하여 F-moc 고상 화학합성 방법을 이용하여 합성하였다. 블로킹 그룹으로 Fmoc-(9-Fluorenylmethoxycarbonyl)이 결합된 링크레진을 사용하여 합성기에 링크레진을 넣은 뒤, DMF로 레진을 스웰링시킨 후, 20% piperidine/DMFf를 사용하여 Fmoc-group을 제거하였다. 그리고, C 말단으로부터 서열대로 0.5M 아미노산용액(용매: DMF) 00㎖, 1.0M DIPEA(용매: DMF&NMP) 및 0.5M HBTU(용매: DMF)를 넣어 질소 기류하에서 1~2시간 동안 반응시켰다. 상기 디프로텍션(deprotection)과 커플링(coupling) 단계가 끝날 때마다 DMF로 두 번씩 세척하였으며, 마지막 아미노산을 커플링(coupling)시킨 후에도 디프로텍션(deprotection)하여 Fmoc-group를 제거하여 콜라겐에 특이적으로 결합하는 골조직 재생 기능성 융합 펩타이드(서열번호 24)를 제조하였다.
In one embodiment of the present invention, in order to prepare a fusion peptide having bone regeneration ability that specifically binds to collagen, a peptide of SEQ ID NO: 1 (GLRSKSKKFRRPDIQYPDA) binding to collagen in order from the N terminus, Was synthesized using F-moc solid phase chemical synthesis method using a peptide synthesizer to contain the peptide of SEQ ID NO: 9 (RHPLYVDFSDVGWNDWIVA). The linking resin was inserted into the synthesizer using a linking resin with Fmoc- (9-Fluorenylmethoxycarbonyl) as a blocking group, and the resin was swelled with DMF and then the Fmoc-group was removed using 20% piperidine / DMFf. Then, from the C-terminal, the reaction was carried out in a nitrogen stream for 1 to 2 hours in a sequential manner with 0.5M amino acid solution (solvent: DMF), 00M, 1.0M DIPEA (solvent: DMF & NMP) and 0.5M HBTU (solvent: DMF). After the deprotection and coupling steps were completed, the cells were washed twice with DMF. After the final amino acid was coupled, deprotection was performed to remove the Fmoc-group to remove collagen-specific (SEQ ID NO: 24). ≪ / RTI >
서열번호 24: GLRSKSKKFRRPDIQYPDARHPLYVDFSDVGWNDWIVA
SEQ ID NO: 24: GLRSKSKKFRRPDIQYPDARHPLYVDFSDVGWNDWIVA
본 발명의 다른 실시예에서, 콜라겐에 특이적으로 결합하는 골조직 재생 기능성 융합 펩타이드 합성 확인은 닌하이드린 테스트를 이용하였으며, 테스트를 거치고 합성이 완료된 레진은 DCM으로 건조시킨 후, TFA cleavage cocktail을 레진 에 넣어 3시간 동안 쉐이킹 시키고 필터링을 통해 레진과 펩타이드가 녹아 있는 cocktail을 분리하였다. 필터로 걸러진 용액을 진공증발농축기를 이용하여 제거한 후, 콜드 에테르를 넣어주거나 펩타이드가 녹아있는 TFA cocktail 용액에 직접 콜드 에테르를 넣어주어 펩타이드를 고체상으로 결정화시키고 이를 원심분리하여 분리하였다. 합성된 펩타이드 서열은 레진으로부터 절단시켜 세척과정을 거쳐 동결건조 후 액체크로마토그래피에 의해 분리 및 정제하였고, 정제된 펩타이드는 MALDI분석을 이용하여 분자량을 확인하였다. 또한, 체내에서 안정성을 시험하기 위해 서열번호 24의 제조시, N 말단에 FITC(Fluorescein isothicyanate)를 트리에틸아민을 이용하여 결합시켰으며, 이를 MALDI-TOF를 이용하여 합성된 것을 확인하였다.In another embodiment of the present invention, a ninhydrin test was used to confirm the synthesis of a bone regeneration functional fusion peptide specifically binding to collagen. After the test, the synthesized resin was dried with DCM, and the TFA cleavage cocktail For 3 hours and filtered to isolate the cocktail in which the resin and peptide were dissolved. The filtered solution was removed using a vacuum evaporator, and cold ether was added, or the peptide was dissolved in a TFA cocktail solution containing cold ether directly to crystallize the peptide into a solid phase, which was separated by centrifugation. The synthesized peptide sequence was cleaved from the resin, washed, lyophilized, separated and purified by liquid chromatography, and the purified peptide was confirmed by MALDI analysis. In order to test the stability in the body, FITC (Fluorescein isothicyanate) was bound to the N-terminus using triethylamine in the preparation of SEQ ID NO: 24, and it was confirmed that it was synthesized using MALDI-TOF.
본 발명의 다른 양태에서, 콜라겐에 특이적으로 결합하는 골조직 재생 기능성을 가진 융합 펩타이드의 콜라겐 결합력 확인하기 위하여, 돼지 피부 유래의 콜라겐을 0.1M HCl에 녹여 1%(w/v)로 만든 후, pH를 중화시키고, 용매를 휘발시켜 4%로 농축하였다. 차폐막을 제조하기 위하여, 60㎝ 지름의 dish에 농축된 콜라겐 용액을 부어 혼합하였고, 3차원 지지체를 제조하기 위하여, 콜라겐 용액을 세포배양용 24 웰 플레이트에 부어 혼합하였다. 이후, 1% 글루탈알데하이드 또는 1% 글리세랄데하이드를 가하여 가교하고, 증류수로 세척하고 동결건조시켰다. 크기 1x1㎝, 두께 250㎛로 제조된 차폐막에 서열번호 24의 융합 펩타이드 10mg/100㎕DW를 적시고 PBS로 세척하고 동결건조하였다. 펩타이드가 결합된 콜라겐 차폐막을 PBS에 넣고, 37℃에서 펩타이드 방출 테스트를 하였다. 7일 후, 처음에 넣어준 용출 용매 10㎖를 모두 제거한 후에 다시 새로운 용출 용매 10㎖를 첨가하여 37℃에서 4주 동안 용출시키고, 용출이 종료된 후 차폐막을 수거하여 펩타이드를 함량을 측정하였다. 4주 동안 용출실험을 하여 용출액에서의 펩타이드의 함량을 측정한 결과, Lot No. 1, 2 및 3에서 체류시간 13분에는 펩타이드의 최고점이 관찰되지 않았으나, 용출이 종료된 후, 측정한 펩타이드 함량은 9.2㎎, 9.43㎎ 및 9.68㎎으로, 이는 차폐막에 결합된 펩타이드가 용출액으로 방출되고 펩타이드가 차폐막에 안정하게 결합되어 있는 것을 확인할 수 있었다.In another embodiment of the present invention, in order to confirm the collagen binding ability of the fusion peptide having bone regeneration function specifically binding to collagen, collagen derived from pig skin was dissolved in 0.1 M HCl to make 1% (w / v) The pH was neutralized and the solvent was evaporated to 4%. To prepare the shielding membrane, a concentrated collagen solution was poured into a dish having a diameter of 60 cm and mixed. To prepare a three-dimensional support, the collagen solution was poured into a 24 well plate for cell culture and mixed. Then, 1% glutaraldehyde or 1% glyceraldehyde was added to crosslink, washed with distilled water and lyophilized. The fused peptide of SEQ ID NO: 24 (10 mg / 100 μlDW) was soaked in a shielding film having a size of 1 × 1 cm and a thickness of 250 μm, washed with PBS and lyophilized. Peptide-bound collagen shielding membranes were placed in PBS and peptide release test was performed at 37 < 0 > C. After 7 days, 10 ml of the first eluting solvent was removed, and 10 ml of a new eluting solvent was further added. The eluted solution was eluted at 37 ° C for 4 weeks. After the elution was completed, the shielding film was collected and the content of the peptide was measured. After elution test for 4 weeks, the content of peptide in the eluate was measured. 1, 2 and 3, peak peaks of the peptides were not observed at the residence time of 13 minutes. After the elution was completed, the peptides were measured to be 9.2 mg, 9.43 mg and 9.68 mg, And it was confirmed that the peptide was stably bound to the shielding film.
본 발명의 또 다른 양태에서, 콜라겐에 결합하는 골조직 재생 기능성을 가진 융합 펩타이드의 in vivo 안정성 확인하기 위하여, 콜라겐 차폐막에 FITC를 표지시킨 서열번호 24의 펩타이드를 결합하고, 가토의 두개골 결손부에 이식한 후, 4주 후에 희생한 후, 두개골 결손부에 이식된 차폐막을 공초점 현미경으로 관찰하였다. FITC 표지시킨 서열번호 24의 펩타이드에 결합된 차폐막을 가토의 두개골 결손부에 이식하고, 이식 전, 이식 후 1일, 3일, 7일, 14일, 21일 및 28일에 혈액을 채취하여 전신혈로 펩타이드가 유리되는지 여부를 형광광도계(Fluorometer)를 사용하여 측정하였다. 그 결과, 두개골에 이식한 차폐막의 표면에서 펩타이드가 잘 고정되어 있었으며, 주위조직에 퍼져나가지 않은 것을 확인하였고, RFI 값들이 이식 전의 값과 비교하여 차이를 보이지 않아 차폐막에 포함된 펩타이드가 혈액 내에 유리되지 않았다는 것을 확인하고, 펩타이드가 콜라겐 차페막에 잘 고정되었음을 알 수 있었다.In yet another aspect of the invention, a fusion polypeptide with a bone tissue regeneration functionality binding to collagen in In order to confirm the vivo stability, the peptides of SEQ ID NO: 24 labeled with FITC were bound to the collagen shielding membrane, and after implantation in the skull defect part of the rabbit, sacrificed after 4 weeks and the shielding membrane implanted into the skull defect part was examined with a confocal microscope Respectively. The shielding membrane bound to the peptide of SEQ ID NO: 24 labeled with FITC was transplanted into the defect of the skull of rabbit and blood was collected at 1 day, 3 days, 7 days, 14 days, 21 days, and 28 days after transplantation Whether or not the blood peptides were liberated was measured using a fluorescence spectrophotometer. As a result, it was confirmed that the peptides were well fixed on the surface of the shielding membrane implanted in the skull, and that the RFI values did not diffuse to the surrounding tissues, and the peptides included in the shielding membrane And that the peptide was well immobilized on the collagen chafed membrane.
본 발명의 또 다른 양태에서, 콜라겐 차폐막에 결합하는 골조직 재생 기능성을 가진 융합 펩타이드의 in vivo 골 재생능 확인하기 위하여, 상기 제조된 서열번호 1, 서열번호 9 및 서열번호 24의 펩타이드를 콜라겐 차폐막에 결합시킨 후, 토끼의 두개골 원형골 결손부에서 이식하여 골 재생능을 확인하였다. 마취시킨 토끼의 두개골부위에 직경 8㎜의 원형 골결손부를 형성시키고, 상기 골결손부에 콜라겐 차폐막을 덮고, 골막과 피부를 이중봉합하였다. 이식 4주 후에 동물을 희생시키고, 채취한 표본은 포르말린 용액에 넣어 고정시킨 후, 조직을 포매하여 두께 20㎛의 시편으로 제작하였다. 제작된 시편은 염기성 푹신과 톨루이딘 블루로 염색하여 비탈회 표본을 제작하였다. 제작된 표본은 광학현미경으로 관찰하여 사진 촬영을 실시하였다. 콜라겐에 특이적으로 결합하는 골조직 재생 기능성을 가진 융합 펩타이드가 포함된 차폐막에 의한 골재생 효과를 확인한 결과, 서열번호 24의 펩타이드가 결합된 콜라겐 차폐막이 서열번호 1 또는 서열번호 9의 펩타이드가 결합된 콜라겐 차폐막보다 골 재생력이 증가된 것을 확인하였으며, 이는 콜라겐에 안정하게 결합된 융합 펩타이드의 경우 이식부위에서 안정하게 체류하므로 골조직 재생능이 더 증가하였다는 것을 확인하였다. In yet another aspect of the invention, a fusion polypeptide with a bone tissue regeneration functionality binding to the collagen shield in In order to confirm vivo bone regeneration ability, peptides of SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO: 24 prepared above were bound to a collagen shielding membrane, and bone grafting ability was confirmed by transplantation from the oval bone defect region of rabbit. A circular bone defect having a diameter of 8 mm was formed in the skull region of the anesthetized rabbit. The collagen shielding membrane was covered with the bone defect, and the periosteum and skin were double sutured. Animals were sacrificed 4 weeks after transplantation. The specimens were fixed in formalin solution, and the specimens were embedded with tissue to form specimens with a thickness of 20 μm. The prepared specimens were stained with basic fuchsin and toluidine blue to prepare slice samples. The specimens were photographed with an optical microscope. As a result of confirming the bone regeneration effect by the shielding membrane containing the fusion peptide having the bone regeneration function that specifically binds to collagen, it was found that the collagen shielding membrane bound to the peptide of SEQ ID NO: 24 had a peptide binding to the peptide of SEQ ID NO: 1 or SEQ ID NO: It was confirmed that the bone regeneration ability of the fusion peptide stably bound to collagen stably stays at the transplantation site, and that the bone regeneration ability is further increased than that of the collagen shielding membrane.
본 발명의 또 다른 양태에서, 서열번호 1의 콜라겐 결합 펩타이드와 서열번호 26의 항균 기능성 펩타이드를 연결하여 서열번호 29(GLRSKSKKFRRPDIQYPDAGKCSTRGRKCCRRKK)의 콜라겐에 결합하는 항균 기능성 펩타이드를 제작하고, 제작된 펩타이드가 콜라겐에 안정하게 결합하여 항균 기능성이 있는지 세균에 대한 성장저해 정도를 측정하여 확인하였다.In another embodiment of the present invention, an antimicrobial functional peptide binding to collagen of SEQ ID NO: 29 (GLRSKSKKFRRPDIQYPDAGKCSTRGRKCCRRKK) is produced by linking the collagen peptide of SEQ ID NO: 1 with the antimicrobial peptide of SEQ ID NO: 26, And the antimicrobial function was confirmed by measuring the degree of growth inhibition against bacteria.
본 발명의 항균 기능성 펩타이드는 인간 베타 디펜신-2(human beta-defensin-2, hBD-2: 서열번호 25), 인간 베타 디펜신-3(human beta-defensin-3, hBD-3: 서열번호 26), 인간 혈소판 유래 생장인자(human platelet derived growth factor-B, PDGF-B: 서열번호 27) 및 헤파린 결합 표피 성장인자(heparin binding-epidermal growth factor, HB-EGF: 서열번호 28)로 유래되고 구성된 군에서 선택된 하나 이상의 펩타이드에서 선택할 수 있다.
The antimicrobial functional peptide of the present invention is human beta-defensin-2 (hBD-2: SEQ ID NO: 25), human beta-defensin-3 26), human platelet-derived growth factor-B (PDGF-B: SEQ ID NO: 27) and heparin binding-epidermal growth factor (HB-EGF: One or more peptides selected from the group consisting of.
상기 펩타이드의 서열번호 25~28의 아미노산 서열은 하기와 같다. The amino acid sequences of SEQ ID NOS: 25 to 28 of the above peptides are shown below.
서열번호 25(BD2-2): CPRRYKQIGTCGLPGTKCCKKPSEQ ID NO: 25 (BD2-2): CPRRYKQIGTCGLPGTKCCKKP
서열번호 26(BD3-3): GKCSTRGRKCCRRKKSEQ ID NO: 26 (BD3-3): GKCSTRGRKCCRRKK
서열번호 27(PDGF): RKIEIVRKKPIFKKATVTSEQ ID NO: 27 (PDGF): RKIEIVRKKPIFKKATVT
서열번호 28(HB-EGF): CKRKKKGKGLGKKRDPCLRKYK
SEQ ID NO: 28 (HB-EGF): CKRKKKGKGLGKKRDPCLRKYK
본 발명에 따른 콜라겐 결합 펩타이드를 포함하는 융합 펩타이드는 콜라겐에 고정됨으로써, 국소 체류성이 증가하며, 펩타이드에 의한 골재생 효과 및 항균효과가 지속될 수 있으며, 이는 골조직 및 치주조직 재생 치료에 적합한 특징이 있다.The fusion peptide comprising the collagen peptide according to the present invention is fixed to collagen, thereby increasing the local retentiveness and sustaining the bone regeneration effect and the antibacterial effect by the peptide, which is suitable for bone regeneration and periodontal regeneration treatment have.
본 발명의 또 다른 실시예에서, 콜라겐에 특이적으로 결합하는 골조직 재생능을 가지는 융합 펩타이드를 제조하기 위하여, 콜라겐에 결합하는 서열번호 1의 펩타이드와 항균기능을 가지는 서열번호 26의 펩타이드를 합성하여 상기와 동일한 방법으로 콜라겐에 특이적으로 결합하는 항균 기능성 융합 펩타이드(서열번호 29)를 제조하였다. In another embodiment of the present invention, in order to prepare a fusion peptide having bone regeneration ability that specifically binds to collagen, a peptide of SEQ ID NO: 1 binding to collagen and a peptide of SEQ ID NO: 26 having an antibacterial function are synthesized An antimicrobial functional fusion peptide (SEQ ID NO: 29) specifically binding to collagen was prepared in the same manner as described above.
본 발명의 또 다른 양태에서, 콜라겐에 특이적으로 결합하는 항균 기능성을 가진 융합 펩타이드의 항균력 확인하기 위하여, 서열번호 1의 펩타이드, 서열번호 26의 펩타이드 및 서열번호 29의 펩타이드를 사용하여 콜라겐 차폐막을 상기 실시예 2와 같은 방법으로 제조하였다. 제조된 콜라겐 차폐막을 PBS에 1시간 방치하여 세척하고, 공기 중에서 건조시켰다. 펩타이드의 항균효능을 평가하기 위하여, 아가 확산법에 따라 항균실험을 진행하였다. E. coli를 TSB 배지에서 배양시키고, 620㎚에서 흡광도가 1이 될 때까지 배양한 후 세포를 수거하여 실험에 사용하였고, PBS로 희석하여 ㎖당 105~107 개의 박테리아를 TSA플레이트에 도말하고, 37℃에서 24시간 동안 배양하였다. 균이 배양된 플레이트 위에 서열번호 1, 서열번호 26 및 서열번호 29가 결합된 콜라겐 차폐막을 올려놓고, 3일 동안 배양한 후, 균의 성장이 저해되는 직경을 측정하였다. 그 결과, 서열번호 1이 결합된 콜라겐 차폐막은 세척 전후 항균기능성이 없었으며, 서열번호 26가 결합된 콜라겐 차폐막의 경우는 세척하기 전에는 항균기능성이 높았으나, 세척 후에는 항균 기능성이 약 50%정도 감소한 것을 알 수 있었다. 또한, 서열번호 29의 경우 세척 전후 항균 기능성이 유지되었다. 이는 콜라겐 결합 펩타이드와 항균 기능성 펩타이드의 융합 펩타이드가 세척 후에도 콜라겐에 대한 결합력을 유지하였기 때문에, 항균 기능성에 변화가 없었던 것을 알 수 있었다.
In another embodiment of the present invention, in order to confirm the antimicrobial activity of a fusion peptide having antimicrobial activity that specifically binds to collagen, a peptide of SEQ ID NO: 1, a peptide of SEQ ID NO: 26, and a peptide of SEQ ID NO: 29 are used to form a collagen- Was prepared in the same manner as in Example 2 above. The prepared collagen shielding film was washed in PBS for 1 hour, and then dried in the air. In order to evaluate the antimicrobial activity of the peptides, an antimicrobial experiment was conducted according to the Agar diffusion method. E. coli was cultured in TSB medium and incubated at 620 nm until absorbance reached 1. Cells were harvested and used for experiments. 10 5 to 10 7 bacteria per ml were diluted with PBS and plated on TSA plates , And cultured at 37 DEG C for 24 hours. A collagen shielding membrane having SEQ ID NO: 1, SEQ ID NO: 26 and SEQ ID NO: 29 bound thereto was placed on a plate on which the bacteria were cultured, and after the incubation for 3 days, the diameter at which the growth of the bacteria was inhibited was measured. As a result, the collagen shielding membrane bound to SEQ ID NO: 1 had no antibacterial function before and after washing. In the case of the collagen shielding membrane bound to SEQ ID NO: 26, the antibacterial function was high before washing, but about 50% Respectively. In the case of SEQ ID NO: 29, the antibacterial function was maintained before and after washing. This indicates that the fusion peptide of the collagen-binding peptide and the antibacterial functional peptide retained the binding ability to collagen even after washing, so that the antibacterial activity was not changed.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시 예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예Example 1: 콜라겐에 특이적으로 결합하는 골조직 재생 기능성을 가진 융합 펩타이드의 제조 1: Preparation of fusion peptide having bone regeneration function that specifically binds to collagen
콜라겐에 특이적으로 결합하는 골조직 재생능을 가지는 융합 펩타이드를 제조하기 위하여, N 말단으로부터 순서대로 결합하는 콜라겐에 결합하는 서열번호 1(GLRSKSKKFRRPDIQYPDA)의 펩타이드, 골조직 재생 기능성을 가지는 서열번호 9(RHPLYVDFSDVGWNDWIVA)의 펩타이드를 함유하도록 펩타이드 합성장치를 이용하여 F-moc 고상 화학합성 방법을 이용하여 합성하였다. 블로킹 그룹(Blocking group)으로 Fmoc-(9-Fluorenylmethoxycarbonyl)이 결합된 링크레진(Rink resin, 0.075m㏖/g, 100~200 메쉬, 1% DVB crosslinking)을 사용하여 합성기에 50㎎의 링크레진(Rink resin)을 넣은 뒤, DMF로 레진(resin)을 스웰링(swelling)시킨 후, 20% piperidine/DMF 용액 20㎖를 사용하여 Fmoc-group을 제거하였다. 그리고, C 말단으로부터 서열대로 0.5M 아미노산용액(용매: DMF) 5㎖, 1.0M DIPEA(용매: DMF&NMP) 5㎖ 및 0.5M HBTU(용매: DMF) 5㎖를 넣어 질소 기류하에서 1~2시간 동안 반응시켰다. 상기 디프로텍션(deprotection)과 커플링(coupling) 단계가 끝날 때마다 DMF로 두 번씩 세척하였으며, 마지막 아미노산을 커플링(coupling)시킨 후에도 디프로텍션(deprotection)하여 Fmoc-group를 제거하여 콜라겐에 특이적으로 결합하는 골조직 재생 기능성 융합 펩타이드(서열번호 24)를 제조하였다.
A peptide of SEQ ID NO: 1 (GLRSKSKKFRRPDIQYPDA) binding to collagen which binds in order from the N-terminus, a peptide of SEQ ID NO: 9 (RHPLYVDFSDVGWNDWIVA) having bone regeneration function, Peptide synthesizer using F-moc solid phase chemical synthesis method. 50 mg of a link resin (Rink resin, 0.075 mmol / g, 100 to 200 mesh, 1% DVB crosslinking) with Fmoc- (9-Fluorenylmethoxycarbonyl) -blocking group was added to the synthesizer The resin was swelled with DMF and the Fmoc-group was removed using 20 ml of 20% piperidine / DMF solution. 5 ml of a 0.5 M amino acid solution (solvent: DMF), 5 ml of 1.0 M DIPEA (solvent: DMF & NMP) and 5 ml of 0.5 M HBTU (solvent: DMF) were placed in the nitrogen stream for 1-2 hours Lt; / RTI > After the deprotection and coupling steps were completed, the cells were washed twice with DMF. After the final amino acid was coupled, deprotection was performed to remove the Fmoc-group to remove collagen-specific (SEQ ID NO: 24). ≪ / RTI >
서열번호 24: GLRSKSKKFRRPDIQYPDARHPLYVDFSDVGWNDWIVA
SEQ ID NO: 24: GLRSKSKKFRRPDIQYPDARHPLYVDFSDVGWNDWIVA
상기 펩타이드의 합성 확인은 닌하이드린 테스트(ninhydrin test)를 이용하였으며, 테스트를 거치고 합성이 완료된 레진(resin)은 DCM으로 건조시킨 후, TFA cleavage cocktail을 레진(resin) 1g 당 20㎖의 비율로 넣어 3시간 동안 쉐이킹 시키고 필터링을 통해 레진(resin)과 펩타이드가 녹아 있는 cocktail을 분리하였다. 필터로 걸러진 용액을 진공증발농축기(rotary evaporator)를 이용하여 제거한 후, 콜드 에테르(cold ether) 1ℓ를 넣어주거나 펩타이드가 녹아있는 TFA cocktail용액에 직접 콜드 에테르를 1ℓ 넣어주어 펩타이드를 고체상으로 결정화시키고 이를 원심분리하여 분리하였다. 이때 에테르로 여러 번 세척과 원심분리 과정을 거쳐 TFA cocktail을 완전히 제거하였고, 이렇게 해서 얻어진 펩타이드는 증류수에 녹여 동결건조하였다. 합성된 펩타이드 서열은 레진으로부터 절단시켜 세척과정을 거쳐 동결건조 후 액체크로마토그래피에 의해 분리 및 정제하였고, 정제된 펩타이드는 MALDI분석을 이용하여 분자량 4534를 확인하였다. Ninhydrin test was used to confirm the synthesis of the peptide. After the resin was completely dried, the TFA cleavage cocktail was added to the resin at a ratio of 20 ml per 1 g of resin The mixture was shaken for 3 hours and filtered to remove resin and peptide cocktail. After removing the filtered solution by using a rotary evaporator, add 1 L of cold ether or add 1 L of cold ether directly to the TFA cocktail solution in which the peptide is dissolved to crystallize the peptide into a solid phase. Followed by centrifugation. At this time, the TFA cocktail was completely removed by washing and centrifuging several times with ether, and the thus obtained peptide was dissolved in distilled water and lyophilized. The synthesized peptide sequence was cleaved from the resin, washed, lyophilized and then separated and purified by liquid chromatography. The purified peptide was confirmed to have a molecular weight of 4534 using MALDI analysis.
또한, 체내에서 안정성을 시험하기 위해 서열번호 24의 제조시, N 말단에 0.3g의 FITC(Fluorescein isothicyanate)를 반응용매(pyridine:DMC:MC=12:7:5) 12㎖에서 결합시켰으며, 이를 MALDI-TOF를 이용하여 분자량 4934로 합성된 것을 확인하였다.
In order to test the stability in the body, 0.3 g of FITC (Fluorescein isothicyanate) was bound to the N-terminal in 12 ml of a reaction solvent (pyridine: DMC: MC = 12: 7: 5) It was confirmed by MALDI-TOF that it was synthesized with a molecular weight of 4934.
실시예Example 2: 콜라겐에 특이적으로 결합하는 골조직 재생 기능성을 가진 융합 펩타이드의 콜라겐 결합력 확인 2: Confirmation of collagen binding ability of fusion peptide having bone regeneration function that specifically binds to collagen
돼지 피부 유래의 콜라겐 1g을 0.1M HCl 100㎖에 녹여 1%(w/v)로 만든 후, pH를 중화시키고, 용매를 휘발시켜 4%로 농축하였다. 1 g of collagen derived from pig skin was dissolved in 100 ml of 0.1 M HCl to make 1% (w / v), the pH was neutralized, the solvent was volatilized and concentrated to 4%.
차폐막을 제조하기 위하여, 60㎝ 지름의 dish에 농축된 콜라겐 용액 8㎖를 부어 혼합하였고, 3차원 지지체를 제조하기 위하여, 콜라겐 용액 1㎖를 세포배양용 24 웰 플레이트에 부어 혼합하였다. 이후, 1% 글루탈알데하이드(glutaraldehyde) 또는 1% 글리세랄데하이드(glyceraldehyde)를 가하여 가교하고, 증류수로 세척하고 동결건조시켰다 (도 1).To prepare the shielding membrane, 8 ml of concentrated collagen solution was poured into a dish having a diameter of 60 cm. To prepare a three-dimensional support, 1 ml of the collagen solution was poured into a 24-well plate for cell culture and mixed. Then, 1% glutaraldehyde or 1% glyceraldehyde was added thereto for crosslinking, washed with distilled water and lyophilized (FIG. 1).
크기 1x1㎝, 두께 250㎛로 제조된 차폐막 0.038g에 서열번호 24의 융합 펩타이드 10mg/100㎕DW (탈이온수)를 적시고 4℃에서 8시간 동안 방치한 후, PBS로 세척하고 동결건조하였다. 펩타이드가 결합된 콜라겐 차폐막을 10㎖ PBS에 넣고, 37℃에서 펩타이드 방출 테스트를 하였다. The fused peptide of SEQ ID NO: 24 (10 mg / 100 μl of deionized water) was soaked in 0.038 g of a shielding film having a size of 1 × 1 cm and a thickness of 250 μm and allowed to stand at 4 ° C. for 8 hours, washed with PBS and lyophilized. Peptide-bound collagen shielding membranes were placed in 10 ml PBS and peptide release test was performed at 37 < 0 > C.
7일 후, 처음에 넣어준 용출 용매 10㎖를 모두 제거한 후에 다시 새로운 용출 용매 10㎖를 첨가하여 37℃에서 4주 동안 용출시키고, 용출이 종료된 후 차폐막을 수거하여 펩타이드를 함량을 측정하였다. After 7 days, 10 ml of the first eluting solvent was removed, and 10 ml of a new eluting solvent was further added. The eluted solution was eluted at 37 ° C for 4 weeks. After the elution was completed, the shielding film was collected and the content of the peptide was measured.
4주 동안 용출실험을 하여 용출액에서의 펩타이드의 함량을 측정한 결과, Lot No. 1, 2 및 3에서 체류시간 13분에는 펩타이드의 최고점(peak)이 관찰되지 않았으나, 용출이 종료된 후, 측정한 펩타이드 함량은 9.2㎎, 9.43㎎ 및 9.68㎎ 이었다 (표 2 및 표 3). 따라서, 이는 차폐막에 결합된 펩타이드가 용출액으로 방출되고 펩타이드가 차폐막에 안정하게 결합되어 있는 것을 확인할 수 있었다.
After elution test for 4 weeks, the content of peptide in the eluate was measured. In peaks 1, 2 and 3, peaks of peptides were not observed at a retention time of 13 minutes, but after elution was finished, peptide contents were 9.2 mg, 9.43 mg and 9.68 mg (Table 2 and Table 3). Thus, it was confirmed that the peptide bound to the shielding membrane was released into the eluent and the peptide was stably bound to the shielding film.
<검액제조방법> <Method of Preparing a Test Solution>
펩타이드가 결합된 차폐막 0.038g을 정밀하게 달아 이동 상의 용매 A를 5㎖ 넣고, sonication을 1시간 동안 실시하였다. 이동상의 용매 B를 5㎖ 넣고 혼합한 후, 4500rpm에서 30분간 원심분리하여 상층액 1㎖을 취하였고, 0.22 미세여과지(millipore filter)로 여과하여 검액으로 사용하였다.
0.038 g of the shielding membrane to which the peptide was bound was precisely weighed, and 5 ml of the solvent A of the mobile phase was added and the sonication was performed for 1 hour. 5 ml of solvent B in the mobile phase was added, and the mixture was centrifuged at 4500 rpm for 30 minutes to collect 1 ml of the supernatant, which was then filtered through a 0.22 microfilter filter and used as the sample solution.
<표준액제조방법><Standard solution manufacturing method>
펩타이드 표준품을 데시케이터(실리카겔)에서 5시간 건조하여 약 10㎎을 이동상의 용매 A를 넣어 녹이고, 10㎖를 표준액으로 하였다.
The peptide standard was dried in a desiccator (silica gel) for 5 hours to dissolve about 10 mg of the mobile phase A in the mobile phase, and 10 ml was used as the standard solution.
<시험방법> <Test Method>
검액 및 표준액 10㎕를 다음과 같은 액체크로마토그래프법의 조작조건으로 시험하여 검액 및 표준액의 피크면적 A T 및 A T 를 측정하였다.10 μl of the sample solution and the standard solution were tested under the operating conditions of the following liquid chromatographic method, and the peak areas A T and A T of the sample solution and the standard solution were measured.
펩타이드의 양(㎎) = 펩타이드 표준품의 양 (㎎) X A T /A T
Amount of peptide (mg) = amount of peptide standard (mg) XA T / A T
액체크로마토그래프법 분석조건은 하기와 같다.Liquid chromatographic method Analysis conditions are as follows.
기기: analytical HPLC (시마쥬사, 일본)Instrument: analytical HPLC (Shimadzu, Japan)
컬럼: C18이 결합된 5um 크기의 실리카겔로 충전되어 있음 (길이 250㎜, 안지름 4.6㎜) Column: Packed with 5um silica gel (250mm in length, 4.6mm in inner diameter) with C18 bonded.
이동상: 0.1% 트리플루오로아세트산/DDW (용매 A), 0.098% 트리플루오로아세트산/아세토나이트릴 (용매 B)Mobile phase: 0.1% trifluoroacetic acid / DDW (solvent A), 0.098% trifluoroacetic acid / acetonitrile (solvent B)
검출기: 자외부흡광광도계 (측정파장 230㎚) Detector: Ultraviolet absorptiometer (measuring wavelength 230 nm)
유속: 1㎖/minFlow rate: 1 ml / min
컬럼온도: 40℃의 일정한 온도
Column temperature: constant temperature of 40 캜
실시예Example 3: 콜라겐에 결합하는 골조직 재생 기능성을 가진 융합 3: Fusion with collagen-bound bone regeneration functionalities 펩타이드의Of peptide in in vivovivo 안정성 확인 Confirm stability
콜라겐 차폐막에 FITC를 표지시킨 서열번호 24의 펩타이드를 실시예 2와 같은 방법으로 결합시켰다. 가토의 두개골 결손부에 이식한 후, 4주 후에 희생한 후, 두개골 결손부에 이식된 차폐막을 공초점 현미경(Confocal microscopy, 올림푸스, 일본)으로 관찰하였다. FITC 표지시킨 서열번호 24의 펩타이드 10㎎이 결합된 차폐막을 가토의 두개골 결손부에 이식하고, 이식 전, 이식 후 1일, 3일, 7일, 14일, 21일 및 28일에 혈액을 채취하여 전신혈로 펩타이드가 유리되는지 여부를 형광광도계(Fluorometer)를 사용하여 측정하였다. Peptide of SEQ ID NO: 24 labeled with FITC in the collagen shielding membrane was bound in the same manner as in Example 2. [ After implantation in the skull deficient part of the rabbit, the sacrificed part was sacrificed 4 weeks later, and the shield membrane implanted into the skull defect part was observed with confocal microscopy (Olympus, Japan). The shielding membrane bound with 10 mg of the peptide of SEQ ID NO: 24 labeled with FITC was transplanted into the skull defect portion of the rabbit and blood was collected at 1 day, 3 days, 7 days, 14 days, 21 days, and 28 days after the transplantation And whether or not the whole blood peptide was liberated was measured by using a fluorescence photometer.
그 결과, 두개골에 이식한 차폐막의 표면에서 펩타이드가 잘 고정되어 있었으며, 주위조직에 퍼져나가지 않은 것을 확인하였다 (도 3). RFI 값들이 이식 전의 값과 비교하여 차이를 보이지 않아 차폐막에 포함된 펩타이드가 혈액 내에 유리되지 않았다는 것을 확인하고, 펩타이드가 콜라겐 차페막에 잘 고정되었음을 알 수 있었다 (도 4).
As a result, it was confirmed that the peptide was well fixed on the surface of the shielding membrane implanted in the skull, and did not spread to surrounding tissues (FIG. 3). The RFI values did not show any difference compared to the values before transplantation, confirming that the peptides contained in the shielding membranes were not liberated in the blood, indicating that the peptides were well immobilized on the collagen chafed membrane (FIG. 4).
실시예Example 4: 콜라겐 4: Collagen 차폐막에On shield 결합하는 골조직 재생 기능성을 가진 융합 Conjugation with Bone Regeneration Functionality 펩타이드의Of peptide inin vivovivo 골 goal 재생능Playability 확인 Confirm
실시예 1에서 제조된 서열번호 1, 서열번호 9 및 서열번호 24의 펩타이드를 각각 실시예 2의 방법으로 콜라겐 차폐막에 결합시킨 후, 토끼의 두개골 원형골 결손부에서 이식하여 골 재생능을 확인하였다. 마취시킨 토끼(Newzealand White rabbit, 종명: cuniculus)의 두개골부위에 직경 8㎜의 원형 골결손부를 형성시키고, 상기 골결손부에 콜라겐 차폐막을 덮고, 골막과 피부를 이중봉합하였다. 이식 4주 후에 동물을 희생시키고, 채취한 표본은 포르말린 용액에 넣어 고정시킨 후, 조직을 포매하여(embedding) 두께 20㎛의 시편으로 제작하였다. 제작된 시편은 염기성 푹신과 톨루이딘 블루로 염색하여 비탈회 표본을 제작하였다. 제작된 표본은 광학현미경으로 관찰하여 사진 촬영을 실시하였다. Peptides of SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO: 24 prepared in Example 1 were bound to a collagen shielding membrane by the method of Example 2, respectively, and bone grafting ability was confirmed by transplantation from the oval bone defect region of rabbit . A circular bone defect of 8 mm in diameter was formed in the skull portion of an anesthetized rabbit (Newzealand White rabbit, cuniculus). The bone defect was covered with a collagen shielding membrane, and the periosteum and skin were double sutured. Animals were sacrificed 4 weeks after transplantation. The specimens were fixed in formalin solution and embedding tissues were prepared with a thickness of 20 μm. The prepared specimens were stained with basic fuchsin and toluidine blue to prepare slice samples. The specimens were photographed with an optical microscope.
콜라겐에 특이적으로 결합하는 골조직 재생 기능성을 가진 융합 펩타이드가 포함된 차폐막에 의한 골재생 효과를 확인한 결과, 서열번호 24의 펩타이드가 결합된 콜라겐 차폐막이 서열번호 1 및 서열번호 9의 펩타이드가 결합된 콜라겐 차폐막보다 골 재생력이 증가된 것을 확인하였으며, 이는 콜라겐에 안정하게 결합된 융합 펩타이드의 경우 이식부위에서 안정하게 체류하므로 골조직 재생능이 더 증가하였다는 것을 확인하였다 (도 5). 따라서 콜라겐에 특이적으로 결합하는 골조직 재생 기능성을 가진 융합 펩타이드가 고정된 차폐막을 사용할 경우, 골조직 재생 효과가 클 것으로 예상할 수 있다.
As a result of confirming the bone regeneration effect by the shielding membrane containing the fusion peptide having the bone regeneration function that specifically binds to collagen, the collagen shielding membrane bound to the peptide of SEQ ID NO: 24 was found to have a binding property to the peptide of SEQ ID NO: 1 and SEQ ID NO: It was confirmed that the bone regeneration ability was increased more than the collagen shielding membrane, and that the fusion peptide stably bound to the collagen stably stays at the transplantation site, thereby increasing the bone regeneration ability (FIG. 5). Therefore, it is expected that the use of a shielding film having a fusion peptide having a bone-regeneration-regulating function that specifically binds to collagen is effective in regenerating bone tissue.
실시예Example 5: 콜라겐에 특이적으로 결합하는 항균 기능성을 가진 융합 5: Fusion with antimicrobial function that specifically binds to collagen 펩타이드의Of peptide 제조 Produce
콜라겐에 특이적으로 결합하는 항균 기능성을 가지는 융합 펩타이드를 제조하기 위하여, 콜라겐에 결합하는 서열번호 1(GLRSKSKKFRRPDIQYPDA)의 펩타이드와 항균기능을 가지는 서열번호 26(GKCSTRGRKCCRRKK)의 펩타이드를 합성하여 상기 실시예 1과 동일한 방법으로 콜라겐에 특이적으로 결합하는 항균 기능성 융합 펩타이드(서열번호 29)를 제조하였다.
In order to prepare a fusion peptide having an antibacterial function that specifically binds to collagen, a peptide of SEQ ID NO: 1 (GLRSKSKKFRRPDIQYPDA) binding to collagen and a peptide of SEQ ID NO: 26 (GKCSTRGRKCCRRKK) having an antibacterial function were synthesized, (SEQ ID NO: 29), which specifically binds to collagen, was prepared in the same manner as in < 1 >
서열번호 29: GLRSKSKKFRRPDIQYPDAGKCSTRGRKCCRRKK
SEQ ID NO: 29: GLRSKSKKFRRPDIQYPDAGKCSTRGRKCCRRKK
실시예Example 6: 콜라겐에 특이적으로 결합하는 항균 기능성을 가진 융합 6: Fusion with antibacterial function that specifically binds to collagen 펩타이드의Of peptide 항균력 확인 Identification of antibacterial activity
서열번호 1의 펩타이드, 서열번호 26의 펩타이드 및 서열번호 29의 펩타이드를 사용하여 콜라겐 차폐막을 상기 실시예 2와 같은 방법으로 제조하였다. 제조된 콜라겐 차폐막을 PBS 10㎖에 1시간 방치하여 세척하고, 공기 중에서 건조시켰다.A collagen shielding membrane was prepared in the same manner as in Example 2 using the peptide of SEQ ID NO: 1, the peptide of SEQ ID NO: 26, and the peptide of SEQ ID NO: 29. The prepared collagen shielding membrane was washed in 10 ml of PBS for 1 hour, and then dried in the air.
펩타이드의 항균효능을 평가하기 위하여, 아가 확산법(agar diffusion method)에 따라 항균실험을 진행하였다. E. coli를 TSB 배지(Tryptic soy broth)에서 배양시키고, 620㎚에서 흡광도가 1이 될 때까지 배양한 후 세포를 수거하여 실험에 사용하였고, PBS로 희석하여 ㎖당 105~107 개의 박테리아를 TSA(tryptic soy agar)플레이트에 도말하고, 37℃에서 24시간 동안 배양하였다. 균이 배양된 플레이트 위에 서열번호 1, 서열번호 26 및 서열번호 29가 결합된 콜라겐 차폐막을 올려놓고, 3일 동안 배양한 후, 균의 성장이 저해되는 직경을 측정하였다.In order to evaluate the antimicrobial activity of peptides, an antimicrobial experiment was conducted according to the agar diffusion method. E. coli was cultured in TSB medium (Tryptic soy broth), incubated at 620 nm until absorbance was 1, and cells were harvested and used for experiments. After dilution with PBS, 10 5 to 10 7 bacteria Was plated on a tryptic soy agar (TSA) plate and cultured at 37 占 폚 for 24 hours. A collagen shielding membrane having SEQ ID NO: 1, SEQ ID NO: 26 and SEQ ID NO: 29 bound thereto was placed on a plate on which the bacteria were cultured, and after the incubation for 3 days, the diameter at which the growth of the bacteria was inhibited was measured.
그 결과, 서열번호 1이 결합된 콜라겐 차폐막은 세척 전후 항균기능성이 없었으며, 서열번호 26이 결합된 콜라겐 차폐막의 경우는 세척하기 전에는 항균기능성이 높았으나, 세척 후에는 항균 기능성이 약 50%정도 감소한 것을 알 수 있었다. 또한, 서열번호 29의 펩타이드가 결합된 콜라겐 차폐막의 경우 세척 전후 항균 기능성이 유지되었다 (도 6). 이는 콜라겐 결합 펩타이드와 항균 기능성 펩타이드의 융합 펩타이드가 세척 후에도 콜라겐에 대한 결합력을 유지하였기 때문에, 항균 기능성에 변화가 없었던 것을 알 수 있었다.
As a result, the collagen shielding membrane bound to SEQ ID NO: 1 had no antimicrobial function before and after washing. In the case of collagen shielding membrane bound to SEQ ID NO: 26, the antimicrobial function was high before washing, but about 50% Respectively. Further, in the case of the collagen shielding membrane bound with the peptide of SEQ ID NO: 29, the antibacterial function was maintained before and after washing (Fig. 6). This indicates that the fusion peptide of the collagen-binding peptide and the antibacterial functional peptide retained the binding ability to collagen even after washing, so that the antibacterial activity was not changed.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 검은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.
Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments, and that the scope of the present invention is not limited thereto. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
서열목록 전자파일 첨부Attach an electronic file to a sequence list
Claims (4)
A fusion peptide having a collagen binding ability and a bone regeneration ability in which a peptide selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 23 is bound to a collagen-binding peptide represented by the amino acid sequence of SEQ ID NO: 1.
The fusion peptide of claim 1, wherein the fusion peptide is represented by the amino acid sequence of SEQ ID NO: 24.
A collagen shielding membrane having bone regeneration ability, characterized in that the fusion peptide of claim 1 or 3 is immobilized on the surface of the collagen shielding membrane.
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