KR20160049183A - A novel fusion protein having cell adhesion activity and osteogenic differentiation activity - Google Patents
A novel fusion protein having cell adhesion activity and osteogenic differentiation activity Download PDFInfo
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- KR20160049183A KR20160049183A KR1020140145494A KR20140145494A KR20160049183A KR 20160049183 A KR20160049183 A KR 20160049183A KR 1020140145494 A KR1020140145494 A KR 1020140145494A KR 20140145494 A KR20140145494 A KR 20140145494A KR 20160049183 A KR20160049183 A KR 20160049183A
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- South Korea
- Prior art keywords
- fusion protein
- fniii10
- activity
- bone
- fibronectin
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
Abstract
Description
본 발명은 세포 부착활성 및 골분화 촉진활성을 나타내는 피브로넥틴 융합단백질에 관한 것으로, 보다 구체적으로, 본 발명은 제3형 피브로넥틴의 10번 도메인에 자가조립서열이 결합되어, 세포 부착활성 및 골분화 촉진활성을 나타내는 피브로넥틴 융합단백질, 상기 융합 단백질을 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 융합단백질 발현용 벡터, 상기 벡터가 도입되어, 상기 융합단백질을 생산할 수 있는 형질전환체, 상기 형질전환체를 이용하여 상기 융합단백질을 생산하는 방법, 상기 융합 단백질을 사용하여 세포를 부착시키는 방법, 상기 융합 단백질을 포함하는 골질환 예방 또는 치료용 약학 조성물 및 상기 융합 단백질을 포함하는 골 또는 치아 재생용 약학 조성물에 관한 것이다.
The present invention relates to a fibronectin fusion protein exhibiting cell adhesion activity and bone mineralization promoting activity, and more particularly, the present invention relates to a fibronectin fusion protein having cell adhesion activity and promoting bone differentiation, A polynucleotide encoding the fusion protein, a vector for expressing a fusion protein comprising the polynucleotide, a transformant into which the vector is introduced to produce the fusion protein, A method for producing the fusion protein, a method for attaching cells using the fusion protein, a pharmaceutical composition for preventing or treating bone diseases including the fusion protein, and a pharmaceutical composition for bone or tooth regeneration including the fusion protein .
골-연골 계면(interface)을 포함하는 복잡한 조직의 재생은 정형외과 및 악안면 수술에 있어서 중대한 도전이 되어왔으며, 고무적인 결과들이 보고 되었음에도 불구하고, 계면을 조작하는 것과 관련된 연구는 많이 이루어지지 않았다. 이것은 계면 구조(interfacial structure)를 구성하는 조직 구성성분 및 세포가 아주 복잡하기 때문이다. 골연골 조직은 골과 연골 사이의 영구적인 계면 구조이다. 골연골 조직은 석회화되지 않은 유리질 연골과 연골하골 사이의 석회화된 연골의 얇은 층으로서 정의된다. 물리적인 특성에서의 점진적인 전이는 계면에서 스트레스 농도를 낮출 수 있으며, 손상을 예방한다. 이러한 기계적인 고려사항면에서, 계면은 높은 농도의 칼슘 염과 수직으로 움직이는 콜라겐 소섬유를 함유하는 독특한 생화학적 조성을 갖는다. Regeneration of complex tissues, including bone-cartilage interface, has been a major challenge in orthopedics and maxillofacial surgery, and although encouraging results have been reported, much research has not been done to manipulate the interface. This is because of the complexity of the tissue components and cells that make up the interfacial structure. Osteochondral tissue is the permanent interfacial structure between bone and cartilage. Osteochondral tissue is defined as a thin layer of calcified cartilage between uncalcified vitreous cartilage and cartilaginous bone. The gradual transition in physical properties can lower the stress concentration at the interface and prevent damage. In terms of these mechanical considerations, the interface has a unique biochemical composition containing high concentrations of calcium salts and collagen fibrils moving vertically.
이와 같은 골과 연골 사이의 계면 결함 영역에 도입될 수 있으며 복잡한 조직 구조를 기능적으로 조화시킬 수 있는 적절한 생체재료의 개발이 필요하다. 달리 말하면, 개별의 조직 구조를 모방하고/하거나 각각의 세포 유형의 기능을 자극하도록 고안된 생체기능적 또는 다중기능적인 구조화된 생체재료가 필요하다. 골연골 영역에서 사용하기 위한 이상성(biphasic) 스캐폴드는 골 및 연골을 동시에 재생하도록 고안되며, 다수의 그룹이 골연골 결합 회복을 위한 이상성 스캐폴드를 제안하였다. 예를 들어, Schaefer 등은 폴리글리콜릭산 망상 조직 및 폴리락틱-코글리콜릭산/폴리-에틸렌 글리콜 폼을 기반으로 한 이상성 스캐폴드를 개발하였다. 또한, 연골 상을 위하여 히알루론산 및 아텔로칼라겐을 조합하고 골성 상을 위하여 하이드록시아파타이트 및 베타-삼칼슘 포스페이트를 조합하는 이상성 스캐폴드가 골연골 결함 회복을 위하여 효과적인 것으로 증명되었다. It is necessary to develop suitable biomaterials capable of being introduced into the interface defect region between the bone and the cartilage and capable of functionally coordinating a complicated tissue structure. In other words, there is a need for biofunctional or multifunctional structured biomaterials that are designed to mimic individual tissue structures and / or stimulate the function of each cell type. The biphasic scaffold for use in the osteochondral region is designed to simultaneously regenerate bone and cartilage, and many groups have proposed an ideal scaffold for osteo-cartilage binding recovery. For example, Schaefer et al. Have developed an ideality scaffold based on polyglycolic acid network and polylactic-coglycolic acid / poly-ethylene glycol foam. It has also been demonstrated that an ideal scaffold combining hyaluronic acid and atelocalcaragen for the cartilage phase and combining hydroxyapatite and beta-tricalcium phosphate for the osseous phase is effective for the recovery of osteochondral defects.
한편, 동물 조직으로부터 분리된 연골세포, 조골세포 및 중간엽 줄기 세포(MSC)를 고안된 생체재료와 조합하여 사용하면 골-연골 복합 조직의 재생력을 향상시킬 수 있다. 예를 들어, Mahmoudifar 등은 태아 연골세포 또는 태아 조골세포 중 하나를 분리된 폴리글리콜릭산과 함께 씨딩하여 골연골 복합 구조체를 생성하였으며, Gao 등은 히알루론산에 기반한 스펀지 및 다공성 세라믹 내로 MSC-유래된 연골세포 및 MSC-유래된 조골세포를 각각 접종함으로써 골연골 이식물을 생성하였다. 또한, 한국특허공개 제2012-0036217호에는 합성 생체고분자에 젤라틴-아파타이트를 혼합하여 얻은 혼합물을 전기방사함으로써 젤라틴-아파타이트-생체고분자로 구성된 복합체 골 조직 재생용 나노섬유를 제조하는 방법이 개시되어 있고, 한국특허공개 제2012-0036218호에는 반대 상으로서 캠핀을 사용하는 상분리법을 이용하여 나노섬유상 구조의 생체 고분자를 제조하는 방법과, 반대 상으로서 캠핀을 사용하는 상분리법을 이용하고 일반적인 스캐폴딩 방법을 적용함으로써 나노섬유상 구조를 가지는 3D 스캐폴드를 제조하는 방법이 개시되어 있으며, 한국특허공개 제2012-0057429호에는 피브로넥틴 도메인 및 오스테오칼신 도메인을 포함하는 융합 단백질, 및 상기 단백질을 포함하는 뼈 또는 치아 재생용 조성물 및 뼈 질환 예방 또는 치료용 조성물이 개시되어 있고, 한국특허등록 제1317515호에는 고분자 스캐폴드 표면에 코팅된 하이드록시아파타이트 및 하이드록시아파타이트와 결합된 피브로넥틴-오스테오칼신 융합단백질을 포함하는 뼈 재생용 스캐폴드 및 이의 제조 방법이 개시되어 있다. 그러나, 종래에 개발된 기술은 골분화 자체는 효과적으로 촉진시킬 수 있었으나, 다른 조직에 비하여 높은 밀도를 갖는 골세포의 특징을 나타내는데 어려움이 있었다. 즉, 상기 기술을 이용하여 골세포의 분화를 촉진시킬 수는 있었으나, 분화된 골세포를 집중시켜서 골세포의 밀도를 증가시킬 수는 없었기 때문에, 실질적인 골형성의 효율이 매우 낮다는 단점이 있었다.
On the other hand, when chondrocytes isolated from animal tissues, osteoblasts and mesenchymal stem cells (MSCs) are used in combination with a designed biomaterial, regeneration ability of bone-cartilage complex tissue can be improved. For example, Mahmoudifar et al. Reported that one of the fetal cartilage cells or fetal osteoblast cells was seeded with a separate polyglycolic acid to produce an osteoclast complex, Gao et al. Produced MSC-derived Osteoclast < / RTI > grafts were produced by inoculating chondrocytes and MSC-derived osteoblasts, respectively. Korean Patent Laid-Open Publication No. 2012-0036217 discloses a method for preparing a composite bone tissue regeneration nanofiber composed of a gelatin-apatite-biopolymer by electrospinning a mixture obtained by mixing a synthetic biopolymer with gelatin-apatite Korean Patent Publication No. 2012-0036218 discloses a method for producing a biopolymer having a nanofiber structure by using a phase separation method using a camphin as a reverse phase and a method for producing a biopolymer having a nanofiber structure using a camphin- Discloses a method for producing a 3D scaffold having a nanofiber structure. Korean Patent Laid-Open Publication No. 2012-0057429 discloses a fusion protein comprising a fibronectin domain and an osteocalcin domain, and a bone or tooth regeneration And compositions for preventing or treating bone diseases are disclosed And, Korea Patent No. 1,317,515 discloses a fibronectin binding with the hydroxyapatite and the hydroxyapatite coating on the polymer scaffold surface, there is a method of manufacturing a bone regeneration scaffolds and their disclosed for containing a fusion protein osteocalcin. However, the conventionally developed technique has been effective in promoting bone differentiation itself, but has difficulty in characterizing bone cells having a higher density than other tissues. That is, although it was possible to promote the differentiation of osteocytes using the above-described technique, since the density of osteocytes could not be increased by concentrating the differentiated osteocytes, there was a disadvantage that the effective bone formation efficiency was very low.
이러한 배경하에서, 본 발명자들은 보다 효율적인 골 회복 또는 골재생을 촉진시킬 수 있는 방법을 개발하고자 예의 연구노력한 결과, 제3형 피브로넥틴의 10번 도메인을 포함하는 융합단백질이 세포 부착을 촉진시킬 수 있으면서도 골분화를 촉진시킬 수 있으므로, 상기 융합단백질을 사용하면 골세포로 분화될 수 있는 줄기세포를 단단하게 부착시키고, 이처럼 상호 부착된 줄기세포를 골세포로 분화시킴으로써, 높은 밀도의 골조직을 형성할 수 있음을 확인하고, 본 발명을 완성하였다.
Under these circumstances, the inventors of the present invention have made extensive efforts to develop a method capable of promoting more effective bone recovery or bone regeneration. As a result, it has been found that a fusion protein comprising the 10 domain of the type 3 fibronectin can promote cell adhesion, By using the fusion protein, the stem cells capable of differentiating into osteocytes can be firmly adhered, and the bone cells with mutually adhered stem cells can be differentiated into osteocytes to form a high density bone tissue. And completed the present invention.
본 발명의 하나의 목적은 제3형 피브로넥틴의 10번 도메인과 자가조립서열을 포함하는 융합 단백질을 제공하는 것이다.It is an object of the present invention to provide a fusion protein comprising the
본 발명의 다른 목적은 상기 융합 단백질을 코딩하는 폴리뉴클레오티드를 제공하는 것이다.Another object of the present invention is to provide a polynucleotide encoding said fusion protein.
본 발명의 또 다른 목적은 상기 폴리뉴클레오티드를 포함하는 융합단백질 발현용 벡터를 제공하는 것이다.It is still another object of the present invention to provide a vector for expressing a fusion protein comprising the polynucleotide.
본 발명의 또 다른 목적은 상기 벡터가 도입되어, 상기 융합단백질을 생산할 수 있는 형질전환체를 제공하는 것이다.It is still another object of the present invention to provide a transformant in which the vector is introduced to produce the fusion protein.
본 발명의 또 다른 목적은 상기 형질전환체를 이용하여 상기 융합단백질을 생산하는 방법을 제공하는 것이다.It is still another object of the present invention to provide a method for producing the fusion protein using the transformant.
본 발명의 또 다른 목적은 상기 융합 단백질을 포함하는 골질환 예방 또는 치료용 약학 조성물을 제공하는 것이다.It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating bone diseases comprising the fusion protein.
본 발명의 또 다른 목적은 상기 융합 단백질을 포함하는 골 또는 치아 재생용 약학 조성물을 제공하는 것이다.
It is still another object of the present invention to provide a pharmaceutical composition for bone or tooth regeneration comprising the fusion protein.
본 발명자들은 보다 효율적인 골 회복 또는 골재생을 촉진시킬 수 있는 방법을 개발하고자 다양한 연구를 수행한 결과, 골 회복 또는 골재생을 효율적으로 수행하기 위하여는 골분화를 촉진시킬 수 있을 뿐만 아니라 분화된 골세포를 결합시켜서 골밀도를 증가시키는 과정이 동시에 수행되어야 함을 알 수 있었다. 이러한 골분화 및 골세포의 결합을 효과적으로 수행하기 위한 방법을 개발하던 중, 제3형 피브로넥틴의 10번 도메인에 자가조립서열이 결합된 형태의 융합단백질을 개발하였다. 상기 융합단백질은 제3형 피브로넥틴의 10번 도메인이 내재적으로 갖고 있던 단백질 부착활성 및 세포 부착활성 보다도 더욱 향상된 단백질 또는 세포 부착활성을 나타냄을 확인하였다. 뿐만 아니라, 종래의 피브로넥틴이 갖고 있던 골분화 촉진활성 보다도 더욱 향상된 골분화 촉진활성을 나타냄을 확인하였다. 이처럼 향상된 세포 부착활성과 골분화 촉진활성을 갖는 상기 융합단백질을 사용하면, 줄기세포를 상호 부착시킨 상태에서 골분화를 촉진시킬 수 있으므로, 높은 밀도의 골조직 형성을 보조할 수 있을 것으로 분석되었다. 따라서, 상기 융합단백질은 골 회복 또는 재생 촉진용 약학 조성물의 유효성분으로서 사용될 수 있다.
The present inventors have conducted various studies to develop a more effective method for promoting bone recovery or bone regeneration. As a result, in order to efficiently perform bone recovery or bone regeneration, not only bone differentiation can be promoted, And the process of increasing the bone density by binding the cells must be performed at the same time. While developing a method for effectively performing bone differentiation and bone cell binding, a fusion protein in which a self-assembly sequence is bound to
상술한 본 발명의 목적을 달성하기 위한 일 실시양태로서, 본 발명은 제3형 피브로넥틴의 10번 도메인과 자가조립서열을 포함하는 융합 단백질을 제공한다.
In one embodiment of the present invention, the present invention provides a fusion protein comprising the
본 발명의 용어 "피브로넥틴(fibronectin, FN)"이란, 고등 동물의 세포 표면, 혈액, 세포외기질 등에 존재하고, 220 내지 250kDa의 분자량을 갖는 당단백질을 의미하는데, 이황화결합으로 인하여 이량체 또는 사량체의 형태로 존재한다. 상기 FN은 주로 간에서 합성되어 혈액으로 분비되는 이중합체 형태의 혈장 피브로넥틴과 섬유아세포에서 합성되어 세포외기질 또는 세포막으로 분비되는 사중합체 형태의 세포성 피브로넥틴으로 구별된다. 상기 FN을 코딩하는 유전자는 한 종류이지만, 상기 유전자는 RNA 스플라이싱이 발생하는 지역이 3개 부위에서 발생할 수 있어, 가능한 조합으로는 약 20종류의 단백질을 합성할 수 있다(도 1). 도 1에서 보듯이, 피브로넥틴을 코딩하는 유전자는 구조적으로 6개의 제1형 피브로넥틴 도메인, 3개의 제2형 피브로넥틴 도메인, 제3형 피브로넥틴 1 내지 14번 도메인, IIICS 도메인, 제3형 피브로넥틴 15번 도메인 및 3개의 제1형 피브로넥틴 도메인으로 구성된다. 상기 구성요소 중에서 제3형 피브로넥틴 7 내지 11번 도메인은 중앙 세포결합 도메인(central cell-binding domain, CCBD)를 구성하고, 제3형 피브로넥틴 10번 도메인은 세포 결합에 관여하는 RGD 서열을 포함하며, 제3형 피브로넥틴 12 내지 14번 도메인은 헤파린 결합 도메인(heparin-binding domain, HBD)를 구성한다고 알려져 있어, 이러한 구조로 인하여, 콜라겐, 젤라틴, 헤파린, 피브린, 인테그린 등의 물질과 결합할 수 있다고 알려져 있다. 기능적인 측면에서 FN은 세포결합, 세포증식, 세포형태 조절, 세포이동, 손상회복 등의 다양한 생리적 작용에 관여한다고 알려져 있다. 상기 피브로넥틴의 구체적인 아미노산 서열 또는 그를 코딩하는 유전자의 염기서열 정보는 NCBI의 GenBank 등 공지의 데이터베이스(GenBank Accession No. NP_002017, NP_001263337, NM_002026, NM_001276408 등)에서 얻을 수 있다. 예를 들어, 피브로넥틴으로부터 유래된 제3형 피브로넥틴 10번 도메인은 서열번호 3의 아미노산 서열로 표시될 수 있다.The term "fibronectin (FN)" of the present invention means a glycoprotein which exists in the cell surface, blood, extracellular matrix and the like of a higher animal and has a molecular weight of 220 to 250 kDa, It exists in the form of sieve. The FN is mainly classified into plasma fibronectin in the form of a duplex which is synthesized in liver and secreted into blood, and cellular fibronectin in the form of tetrapolymer synthesized in fibroblasts and secreted into extracellular matrix or cell membrane. Although the gene coding for FN is one kind, the region where RNA splicing occurs can occur at three sites, and about 20 kinds of proteins can be synthesized in a possible combination (FIG. 1). As shown in FIG. 1, the gene encoding fibronectin structurally includes 6 first type fibronectin domains, 3 second type fibronectin domains, 3
본 발명에 있어서, 상기 피브로넥틴으로부터 유래된 제3형 피브로넥틴 10번 도메인과 자가조립(self-assembled, SA) 서열을 사용하여 융합단백질을 구성할 수 있다.
In the present invention, the fusion protein can be constructed using the self-assembled (SA) sequence with the
본 발명의 용어 "자가조립(self-assembled, SA) 서열"은 친수성을 가지는 아미노산과 소수성을 가지는 아미노산을 배열하고 이들을 결합시켜 친수성과 소수성을 모두 가지는 양매성의 특징을 가지는 아미노산 서열을 의미한다. The term " self-assembled (SA) sequence "of the present invention means an amino acid sequence having both hydrophilic and hydrophobic characteristics by arranging amino acids having hydrophilicity and amino acids having hydrophobicity and bonding them.
본 발명에 있어서, 상기 자가조립 서열은 본 발명의 융합단백질을 구성할 수 있는 서열번호 1의 아미노산 서열로 표시될 수 있는 펩타이드로 해석될 수 있다.
In the present invention, the self-assembling sequence can be interpreted as a peptide which can be represented by the amino acid sequence of SEQ ID NO: 1, which can constitute the fusion protein of the present invention.
본 발명의 용어 "융합단백질"이란, 상기 제3형 피브로넥틴의 10번 도메인과 자가조립서열을 포함하도록 인위적으로 합성된 단백질을 의미한다.The term "fusion protein" of the present invention means a protein artificially synthesized to include the self-assembling sequence with the
본 발명에 있어서, 상기 융합단백질에 포함된 제3형 피브로넥틴의 10번 도메인과 자가조립서열의 배열 순서는 상기 융합단백질이 향상된 세포 부착활성 및 골분화 촉진활성을 나타내게 하는 한 특별히 이에 제한되지 않으나, 바람직하게는 제3형 피브로넥틴의 10번 도메인과 자가조립서열의 순서로 구성될 수 있고, 바람직하게는 서열번호 5의 아미노산 서열로 구성될 수 있다.
In the present invention, the sequence of the 10 domain of the type 3 fibronectin and the self-assembling sequence contained in the fusion protein is not particularly limited as long as the fusion protein exhibits improved cell adhesion activity and bone formation promoting activity, Preferably, it may consist of
또한, 상기 도메인은 상호 직접적으로 연결될 수도 있고, 링커를 통해 연결될 수도 있다. 상기 링커는 상기 융합단백질이 향상된 세포 부착활성 및 골분화 촉진활성을 나타내게 하는 한 특별히 이에 제한되지 않으나, 아미노산으로 구성된 펩타이드 링커를 사용할 수 있고, 보다 바람직하게는 잘구부러지는(flexible) 펩타이드 링커를 사용할 수 있다. 상기 펩타이드 링커는 상기 링커를 코딩하는 핵산을 각 도메인을 코딩하는 핵산 사이에 인프레임(in frame)으로 연결하여 발현벡터 내에 작동 가능하게 연결하여 발현시킬 수 있다.In addition, the domains may be directly connected to each other or may be connected through a linker. The linker is not particularly limited as long as the fusion protein exhibits improved cell adhesion activity and bone formation promoting activity, but it is possible to use a peptide linker composed of an amino acid, more preferably a flexible peptide linker . The peptide linker may be operably linked to an expression vector by linking the nucleic acid encoding the linker in-frame between the nucleic acids encoding each domain.
아울러, 상기 융합단백질은 이에 포함되는 각 도메인의 야생형의 아미노산 서열과 하나 이상의 아미노산 잔기가 상이한 서열을 가지는 폴리펩티드를 포함할 수 있다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 및 폴리펩티드에서의 아미노산 교환은 당해 분야에 공지되어 있다. 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다. 또한, 아미노산 서열상의 변이 또는 수식에 의해서 단백질의 열, pH등에 대한 구조적 안정성이 증가하거나 단백질 활성이 증가한 단백질을 포함할 수 있다.In addition, the fusion protein may include a polypeptide having a sequence that differs from the wild-type amino acid sequence of each domain contained therein by one or more amino acid residues. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, it may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, for example, heat, pH, etc. of the protein.
끝으로, 상기 융합단백질 또는 상기 융합단백질을 구성하는 각 도메인의 폴리펩티드는 당해 분야에 공지된 화학적 펩티드 합성방법으로 제조하거나, 상기 도메인을 코딩하는 유전자를 PCR (polymerase chain reaction) 에 의해 증폭하거나 공지된 방법으로 합성한 후 발현벡터에 클로닝하여 발현시켜서 제조할 수 있다.
Finally, the polypeptide of each domain constituting the fusion protein or the fusion protein may be prepared by a chemical peptide synthesis method known in the art, or may be prepared by amplifying a gene encoding the domain by PCR (polymerase chain reaction) Followed by expression and cloning into an expression vector.
본 발명의 일 실시예에 의하면, 제3형 피브로넥틴의 10번 도메인과 자가조립서열을 포함하는 융합 단백질을 발현 및 정제하고(도 1), 상기 융합단백질의 단백질 부착활성을 평가한 결과, 제3형 피브로넥틴의 10번 도메인 보다 높은 단백질 부착활성을 나타냄을 확인하였으며(도 2), 상기 융합단백질의 세포 부착활성을 평가한 결과, 제3형 피브로넥틴의 10번 도메인 보다 높은 세포 부착활성을 나타냄을 확인하였고(도 3), 상기 융합단백질의 세포 증식활성을 평가한 결과, 제3형 피브로넥틴의 10번 도메인 보다 높은 세포 증식활성을 나타냄을 확인하였으며(도 4), 상기 융합단백질의 골분화 촉진활성을 평가한 결과, 제3형 피브로넥틴의 10번 도메인 보다 높은 골분화 촉진활성을 나타냄을 확인하였다(도 5).
According to one embodiment of the present invention, a fusion protein comprising the
따라서, 본 발명에서 제공하는 융합단백질은 세포결합 활성, 세포증식 활성 및 골형성 분화활성의 측면에서 종래의 피브로넥틴 단백질 보다도 우수한 활성을 나타내는 신규한 단백질임을 알 수 있었다.
Therefore, the fusion protein provided by the present invention was found to be a novel protein exhibiting better activity than the conventional fibronectin protein in terms of cell binding activity, cell proliferation activity and osteogenesis differentiation activity.
상술한 본 발명의 목적을 달성하기 위한 다른 실시양태로서, 본 발명은 상기 융합 단백질을 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 융합단백질 발현벡터, 상기 발현벡터가 도입된 형질전환체 및 상기 형질전환체를 이용하여 상기 융합단백질을 생산하는 방법을 제공한다.
In another aspect of the present invention for achieving the object of the present invention, the present invention provides a polynucleotide encoding a fusion protein, a fusion protein expression vector comprising the polynucleotide, a transformant into which the expression vector is introduced, And a method for producing the fusion protein using a transformant.
본 발명에서 제공하는 폴리뉴클레오티드는 상기 본 발명에서 제공하는 융합단백질을 코딩하는 뉴클레오티드 서열을 포함하고, 바람직하게는 서열번호 6의 뉴클레오티드 서열이 될 수 있다.
The polynucleotide provided by the present invention includes a nucleotide sequence encoding the fusion protein provided by the present invention, preferably, the nucleotide sequence of SEQ ID NO: 6.
상기 폴리뉴클레오티드는 하나 이상의 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있다. 뉴클레오타이드 서열을 화학적으로 합성하여 제조하는 경우, 당업계에 널리 공지된 합성법, 예를 들어 문헌(Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988)에 기술된 방법을 이용할 수 있으며, 트리에스테르, 포스파이트, 포스포르아미다이트 및 H-포스페이트 방법, PCR 및 기타 오토프라이머 방법, 고체 지지체상의 올리고뉴클레오타이드 합성법 등을 들 수 있다.
The polynucleotide may be mutated by substitution, deletion, insertion, or a combination thereof, of one or more bases. When the nucleotide sequence is prepared by chemically synthesizing, it is possible to use a method well known in the art, for example, a method described in Engels and Uhlmann, Angew Chem IntEd Engl., 37: 73-127, 1988 , Triesters, phosphites, phosphoramidites and H-phosphate methods, PCR and other auto primer methods, and oligonucleotide synthesis on solid supports.
본 발명의 용어 "발현벡터"란, 목적하는 숙주세포에서 목적 펩타이드를 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 의미한다. 상기 발현벡터는 개시코돈, 종결코돈, 프로모터, 오퍼레이터 등의 발현조절 요소들을 포함하는데, 상기 개시 코돈 및 종결 코돈은 일반적으로 폴리펩타이드를 암호화하는 뉴클레오티드 서열의 일부로 간주되며, 유전자 제작물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. The term "expression vector" of the present invention refers to a recombinant vector capable of expressing a target peptide in a desired host cell, and a gene product containing an essential regulatory element operatively linked to the expression of the gene insert. The expression vector includes expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc. The initiation codon and termination codon are generally regarded as part of the nucleotide sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.
본 발명의 용어 "작동가능하게 연결(operably linked)"이란, 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질 또는 RNA를 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 상태를 의미다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동가능하게 연결되어 코딩서열의 발현에 영향을 미칠 수 있다. 발현 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다.The term "operably linked" of the present invention means a state in which a nucleic acid sequence encoding a desired protein or RNA is functionally linked to a nucleic acid expression control sequence so as to perform a general function . For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with an expression vector can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.
또한, 상기 발현벡터는 세포 배양액으로부터 단백질의 분리를 촉진하기 위하여 융합 폴리펩타이드의 배출을 위한 시그널 서열을 포함할 수 있다. 특이적인 개시 시그널은 또한 삽입된 핵산 서열의 효율적인 번역에 필요할 수도 있다. 이들 시그널은 ATG 개시코돈 및 인접한 서열들을 포함한다. 어떤 경우에는, ATG 개시 코돈을 포함할 수 있는 외인성 번역 조절 시그널이 제공되어야 한다. 이들 외인성 번역 조절 시그널들 및 개시 코돈들은 다양한 천연 및 합성 공급원일 수 있다. 발현 효율은 적당한 전사 또는 번역 강화 인자의 도입에 의하여 증가될 수 있다.In addition, the expression vector may include a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture medium. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers.
아울러, 상기 발현벡터는 융합단백질의 검출을 용이하게 하기 위하여, 임의로 엔도펩티아이제를 사용하여 제거할 수 있는 단백질 태그를 추가로 포함할 수 있다. In addition, the expression vector may further comprise a protein tag that can be removed using an endopeptidase, optionally in order to facilitate detection of the fusion protein.
본 발명의 용어 "태그(tag)"란, 정량가능한 활성 또는 특성을 나타내는 분자를 의미하며, 플로오레세인과 같은 화학적 형광물질(fluoracer), 형광 단백질(GFP) 또는 관련 단백질과 같은 폴리펩타이드 형광물질을 포함한 형광분자일 수도 있고; Myc 태그, 플래그(Flag) 태그, 히스티딘 태그, 루신 태그, IgG 태그, 스트랩타비딘 태그 등의 에피톱 태그일 수도 있다. 특히, 에피톱 태그를 사용할 경우, 바람직하게는 6개 이상의 아미노산 잔기로 구성되고, 보다 바람직하게는 8개 내지 50개의 아미노산 잔기로 구성된 펩타이드 태그를 사용할 수 있다.The term "tag " of the present invention means a molecule exhibiting quantifiable activity or property and includes a polypeptide fluorescent substance such as a fluorophore such as fluorescein, a fluorescent protein (GFP) Or may be a fluorescent molecule including; Myc tag, a Flag tag, a histidine tag, a Lucin tag, an IgG tag, or a strap tagidine tag. Particularly, in the case of using an epitope tag, a peptide tag composed of preferably 6 or more amino acid residues, more preferably 8 to 50 amino acid residues can be used.
본 발명에 있어서, 상기 발현벡터는 상술한 본 발명에서 제공하는 융합단백질을 코딩하는 뉴클레오티드 서열을 포함할 수 있는데, 이때 사용되는 벡터는 본 발명의 융합단백질을 생산할 수 있는 한, 특별히 이에 제한되지 않으나, 바람직하게는 플라스미드 DNA, 파아지 DNA 등이 될 수 있고, 보다 바람직하게는 상업적으로 개발된 플라스미드(pUC18, pBAD, pIDTSAMRT-AMP 등), 대장균 유래 플라스미드(pYG601BR322, pBR325, pUC118, pUC119 등), 바실러스 서브틸리스 유래 플라스미드(pUB110, pTP5 등), 효모-유래 플라스미드(YEp13, YEp24, YCp50 등), 파아지 DNA(Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP 등), 동물 바이러스 벡터(레트로바이러스(retrovirus), 아데노바이러스(adenovirus), 백시니아 바이러스(vaccinia virus) 등), 곤충 바이러스 벡터(배큘로바이러스(baculovirus) 등) 이 될 수 있다. 상기 발현벡터는 숙주 세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로, 목적에 가장 적합한 숙주세포를 선택하여 사용함이 바람직하다. 예를 들어, 본 발명의 실시예에서는 상기 폴리뉴클레오티드를 6개의 히스티딘 태그가 포함된 pBAD 벡터에 도입하여 발현벡터 pBAD-HisB-FNIII10-SA를 제작하였다.
In the present invention, the expression vector may include a nucleotide sequence encoding the fusion protein provided by the present invention. The vector used herein is not particularly limited as long as it can produce the fusion protein of the present invention (PUC18, pBAD, pIDTSAMRT-AMP, etc.), plasmids derived from Escherichia coli (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis plasmids Derived plasmids (e.g., pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50), phage DNAs (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11 and lambda ZAP) (eg, retrovirus, adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the amount of expression of the protein and the expression of the expression vector are different depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose. For example, in an embodiment of the present invention, the polynucleotide was introduced into a pBAD vector containing six histidine tags to prepare an expression vector pBAD-HisB-FNIII10-SA.
본 발명에서 제공하는 형질전환체는 상기 본 발명에서 제공하는 발현벡터를 숙주에 도입하여 형질전환시켜서 제작되고, 상기 발현벡터에 포함된 폴리뉴클레오티드를 발현시켜서, 본 발명의 융합단백질을 생산하는데 사용될 수 있다. 상기 형질전환은 다양한 방법에 의하여 수행될 수 있는데, 다양한 세포활성을 높은 수준으로 향상시킬 수 있는 효과를 나타내는 본 발명의 융합단백질을 생산할 수 있는 한, 특별히 이에 제한되지 않으나, CaCl2 침전법, CaCl2 침전법에 DMSO(dimethyl sulfoxide)라는 환원물질을 사용함으로써 효율을 높인 Hanahan 방법, 전기천공법(electroporation), 인산칼슘 침전법, 원형질 융합법, 실리콘 카바이드 섬유를 이용한 교반법, 아그로박테리아 매개된 형질전환법, PEG를 이용한 형질전환법, 덱스트란 설페이트, 리포펙타민 및 건조/억제 매개된 형질전환 방법 등이 사용될 수 있다. 또한, 상기 형질전환제의 제작에 사용되는 숙주역시 본 발명의 융합단백질을 생산할 수 있는 한, 특별히 이에 제한되지 않으나, 대장균(E. coli), 스트렙토마이세스, 살모넬라 티피뮤리움 등의 박테리아 세포; 사카로마이세스 세레비지애, 스키조사카로마이세스 폼베 등의 효모 세포; 피치아 파스토리스 등의 균류 세포; 드로조필라, 스포도프테라 Sf9 세포 등의 곤충 세포; CHO, COS, NSO, 293, 보우 멜라노마 세포 등의 동물 세포; 또는 식물 세포가 될 수 있다. 예를 들어, 본 발명에서는 대장균 TOP10 균주를 숙주로서 사용하였다.
The transformant provided in the present invention can be used to produce the fusion protein of the present invention by introducing the expression vector provided in the present invention into a host and transforming the same and expressing the polynucleotide contained in the expression vector have. The transformation can be carried out by various methods and is not particularly limited as long as it is capable of producing the fusion protein of the present invention showing an effect of improving various cell activities to a high level. However, CaCl 2 precipitation method, CaCl 2 2 precipitation method using a reducing material called DMSO (dimethyl sulfoxide), an electroporation method, an electroporation method, a calcium phosphate precipitation method, a protoplast fusion method, an agitation method using silicon carbide fiber, an agrobacterium-mediated trait A transformation method using PEG, a dextran sulfate, a lipofectamine, and a dry / suppression-mediated transformation method. The host used in the production of the transformant may also be a bacterial cell such as E. coli, Streptomyces or Salmonella typhimurium, as long as it can produce the fusion protein of the present invention. Yeast cells such as Saccharomyces cerevisiae, and ski-inspected caromyces pombe; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, Bowmanella cells; Or plant cells. For example, E. coli TOP10 strain was used as a host in the present invention.
상기 형질전환체는 본 발명에서 제공하는 융합단백질을 생산하는 방법에 사용될 수 있다. 구체적으로, 본 발명에서 제공하는 융합단백질을 생산하는 방법은 (a) 상기 형질전환체를 배양하여 배양물을 수득하는 단계; 및, (b) 상기 배양물로부터 본 발명의 융합단백질을 회수하는 단계를 포함한다.
The transformant may be used in a method for producing the fusion protein provided by the present invention. Specifically, the method for producing a fusion protein provided by the present invention comprises the steps of: (a) culturing the transformant to obtain a culture; And (b) recovering the fusion protein of the present invention from the culture.
본 발명의 용어 "배양"이란, 미생물을 적당히 인공적으로 조절한 환경조건에서 생육시키는 방법을 의미한다. The term "cultivation" of the present invention means a method of growing the microorganism under an appropriately artificially controlled environmental condition.
본 발명에 있어서, 상기 형질전환체를 배양하는 방법은 당업계에 널리 알려져 있는 방법을 이용하여 수행할 수 있다. 구체적으로 상기 배양은 본 발명의 융합단백질을 발현시켜서 생산할 수 있는 한 특별히 이에 제한되지 않으나, 배치 공정 또는 주입 배치 또는 반복 주입 배치 공정(fed batch or repeated fed batch process)에서 연속식으로 배양할 수 있다.In the present invention, the method for culturing the transformant may be carried out by a method well known in the art. Specifically, the culture can be continuously cultured in a batch process or an injection batch or a repeated fed batch process, as long as it can produce the fusion protein of the present invention. .
배양에 사용되는 배지는 적당한 탄소원, 질소원, 아미노산, 비타민 등을 함유한 통상의 배지 내에서 호기성 조건 하에서 온도, pH 등을 조절하면서 적절한 방식으로 특정 균주의 요건을 충족해야 한다. 사용될 수 있는 탄소원으로는 글루코즈 및 자일로즈의 혼합당을 주 탄소원으로 사용하며 이외에 수크로즈, 락토즈, 프락토즈, 말토즈, 전분, 셀룰로즈와 같은 당 및 탄수화물, 대두유, 해바라기유, 피마자유, 코코넛유 등과 같은 오일 및 지방, 팔미트산, 스테아린산, 리놀레산과 같은 지방산, 글리세롤, 에탄올과 같은 알코올, 아세트산과 같은 유기산이 포함된다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있다. 사용될 수 있는 질소원으로는 암모니아, 황산암모늄, 염화암모늄, 초산암모늄, 인산암모늄, 탄산안모늄, 및 질산암모늄과 같은 무기질소원; 글루탐산, 메티오닌, 글루타민과 같은 아미노산 및 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 맥아 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크 또는 그의 분해생성물 등 유기질소원이 사용될 수 있다. 이들 질소원은 단독 또는 조합되어 사용될 수 있다. 상기 배지에는 인원으로서 인산 제1칼륨, 인산 제2칼륨 및 대응되는 소듐-함유 염이 포함될 수 있다. 사용될 수 있는 인원으로는 인산이수소칼륨 또는 인산수소이칼륨 또는 상응하는 나트륨-함유 염이 포함된다. 또한, 무기화합물로는 염화나트륨, 염화칼슘, 염화철, 황산마그네슘, 황산철, 황산망간 및 탄산칼슘 등이 사용될 수 있다. 마지막으로, 상기 물질에 더하여 아미노산 및 비타민과 같은 필수 성장 물질이 사용될 수 있다. The medium used for the culture should meet the requirements of the specific strain in a suitable manner while controlling the temperature, pH and the like under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acid, vitamin, and the like. The carbon sources that can be used include glucose and xylose mixed sugar as main carbon sources, and sugar and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Oils and fats such as oils and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used.
또한, 배양 배지에 적절한 전구체들이 사용될 수 있다. 상기된 원료들은 배양과정에서 배양물에 적절한 방식에 의해 회분식, 유가식 또는 연속식으로 첨가될 수 있으나, 특별히 이에 제한되지는 않는다. 수산화나트륨, 수산화칼륨, 암모니아와 같은 기초 화합물 또는 인산 또는 황산과 같은 산 화합물을 적절한 방식으로 사용하여 배양물의 pH를 조절할 수 있다.In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in the culture process in a batch manner, in an oil-feeding manner or in a continuous manner by an appropriate method, but it is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture.
또한, 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 호기 상태를 유지하기 위해 배양물 내로 산소 또는 산소-함유 기체(예, 공기)를 주입한다. 배양물의 온도는 보통 27℃ 내지 37℃, 바람직하게는 30℃ 내지 35℃이다. 배양은 D형 젖산의 생성량이 최대로 얻어질 때까지 계속한다. 이러한 목적으로 보통 10 내지 100 시간에서 달성된다. 대체로 D형 젖산은 배양 배지 중으로 배출되지만, 경우에 따라서는 세포 중에 포함되어 있을 수 있다.In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. An oxygen or oxygen-containing gas (e.g., air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is usually 27 ° C to 37 ° C, preferably 30 ° C to 35 ° C. Culture continues until the amount of D-lactic acid produced is maximized. Usually for 10 to 100 hours for this purpose. Generally, D-lactic acid is released into the culture medium, but in some cases it may be contained in the cells.
아울러, 배양물로부터 상기 융합단백질을 회수하는 단계는 당업계에 공지된 방법에 의해 수행될 수 있다. 구체적으로, 상기 회수 방법은 생산된 본 발명의 융합단백질을 회수할 수 있는 한, 특별히 이에 제한되지 않으나, 바람직하게는 원심분리, 여과, 추출, 분무, 건조, 증방, 침전, 결정화, 전기영동, 분별용해(예를 들면 암모늄 설페이트 침전), 크로마토그래피(예를 들면 이온 교환, 친화성, 소수성 및 크기배제) 등의 방법을 사용할 수 있다.
In addition, the step of recovering the fusion protein from the culture can be carried out by a method known in the art. Specifically, the recovering method is not particularly limited as long as the recovered fusion protein of the present invention can be recovered. Preferably, the recovering method includes centrifugation, filtration, extraction, spraying, drying, Methods such as fractional dissolution (for example, ammonium sulfate precipitation), chromatography (for example, ion exchange, affinity, hydrophobicity and size exclusion) can be used.
상술한 본 발명의 목적을 달성하기 위한 또 다른 실시양태로서, 본 발명은 상기 융합 단백질과 줄기세포를 유효성분으로 포함하는 골질환 예방 또는 치료용 약학 조성물 또는 골 또는 치아 재생용 약학 조성물을 제공한다.
In another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating bone diseases or a pharmaceutical composition for bone or tooth regeneration, which comprises the fusion protein and the stem cell as an active ingredient .
상술한 바와 같이, 본 발명에서 제공하는 융합단백질은 세포결합 활성, 세포증식 활성 및 골형성 분화활성의 측면에서 종래의 피브로넥틴보다도 우수한 활성을 나타내는 신규한 단백질임을 확인하였으므로, 상기 융합단백질과 줄기세포를 골조직의 퇴화, 손실, 붕괴 등의 증상이 수반되는 다양한 골질환의 예방 또는 치료용 약학 조성물 또는 손상된 골 또는 치아를 재생시키기 위한 약학 조성물의 유효성분으로 사용할 수 있음을 알 수 있었다.
As described above, it was confirmed that the fusion protein provided by the present invention is a novel protein exhibiting superior activity than conventional fibronectin in terms of cell binding activity, cell proliferation activity and osteogenesis differentiation activity. Therefore, the fusion protein and the stem cell It can be used as an active ingredient of a pharmaceutical composition for preventing or treating various bone diseases accompanied by symptoms such as bone regeneration, loss or collapse, or a pharmaceutical composition for regenerating damaged bone or teeth.
본 발명의 용어 "골질환"이란, 조골세포 및 파골세포의 불균형으로 인해 유발되어 골조직의 장애를 초래하거나 또는 골조직을 파괴하는 질환을 의미하는데, 상기 골 관련 질환은 본 발명에서 제공하는 융합단백질에 의하여 증상이 예방, 개선, 경감 또는 치료될 수 있는 한 특별히 이에 제한되지 않으나, 바람직하게는 성장기 발육부진, 골절, 구루병(rickets), 골연화증(osteomalacia), 척추측만증(scoliosis), 골반변형(pelvic deformity), 골다공증(osteoprosis), 류마티스성 관절염(rheumatoid arthritis), 치주질환(periodontal disease), 갑상선기능항진증(hyperparathyroidism), 파제트병(Paget disease), 전이성 골암(metastatic bone cancers) 등이 될 수 있다.
The term "bone disease" of the present invention refers to a disease caused by imbalance of osteoblasts and osteoclasts, resulting in a disorder of bone tissue or destruction of bone tissue. Such bone disease is caused by the fusion protein The present invention is not limited to any particular one, as long as it can prevent, ameliorate, alleviate or cure the symptoms, but preferably includes growth retardation, fracture, rickets, osteomalacia, scoliosis, pelvic deformity ), Osteoporosis, rheumatoid arthritis, periodontal disease, hyperparathyroidism, Paget disease, metastatic bone cancers, and the like.
본 발명의 용어 "골질환의 예방 또는 치료"란 상기 골 관련 질환의 발병을 예방하거나 또는 상기 질환의 발병에 의한 증세를 호전하거나 이롭게 변경하는 모든 행위를 의미하는데, 골질환 증상의 예방, 감소, 개선, 그 증상의 고통 경감, 골질환 발생율 감소 또는 치료결과를 증가시키는 환자의 어떠한 다른 변화를 모두 포함한다.
The term " prevention or treatment of bone disease "of the present invention means all actions to prevent the bone disease-related disease or to improve or alleviate the symptoms caused by the disease, Improvement in pain relief, reduction in the incidence of bone disease, or any other change in the patient that increases the outcome of the treatment.
한편, 본 발명의 약학 조성물은 약학 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 구체적으로, 상기 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명에서, 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 융합단백질에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는 데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.
Meanwhile, the pharmaceutical composition of the present invention may further comprise an appropriate carrier, excipient or diluent conventionally used in the production of the pharmaceutical composition. Specifically, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the present invention, the carrier, excipient and diluent which may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 약학 조성물에 포함된 상기 융합단백질의 함량은 특별히 이에 제한되지 않으나, 최종 조성물 총중량을 기준으로 0.0001 내지 50 중량%, 보다 바람직하게는 0.01 내지 5 중량%의 함량으로 포함될 수 있다.
The content of the fusion protein contained in the pharmaceutical composition of the present invention is not particularly limited, but may be in the range of 0.0001 to 50% by weight, more preferably 0.01 to 5% by weight, based on the total weight of the final composition.
상기 본 발명의 약학 조성물은 약제학적으로 유효한 양으로 투여될 수 있는데, 본 발명의 용어 "약제학적으로 유효한 양"이란 의학적 치료 또는 예방에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료 또는 예방하기에 충분한 양을 의미하며, 유효 용량 수준은 질환의 중증도, 약물의 활성, 환자의 연령, 체중, 건강, 성별, 환자의 약물에 대한 민감도, 사용된 본 발명 조성물의 투여 시간, 투여 경로 및 배출 비율 치료기간, 사용된 본 발명의 조성물과 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적으로 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하다.
The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means a therapeutic or prophylactic treatment of a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention And the effective dose level refers to the level of the disease to be treated, the severity of the disease, the activity of the drug, the age, body weight, health, sex, sensitivity of the patient to the drug, Duration, duration of administration, factors involved in combination with or contemporaneously with the composition of the present invention, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.
본 발명의 약학조성물의 투여량은 예를 들어, 본 발명의 약학 조성물을 사람을 포함하는 포유동물에 하루 동안 0.01 내지 1 ㎎/㎏, 보다 바람직하게는 0.1 내지 0.1 ㎎/㎏으로 투여할 수 있고, 본 발명의 조성물의 투여빈도는 특별히 이에 제한되지 않으나, 1일 1회 투여하거나 또는 용량을 분할하여 수회 투여할 수 있다.
The dosage of the pharmaceutical composition of the present invention can be administered, for example, to a mammal including a human in an amount of 0.01 to 1 mg / kg, more preferably 0.1 to 0.1 mg / kg per day for a pharmaceutical composition of the present invention , The frequency of administration of the composition of the present invention is not particularly limited, but it may be administered once a day or divided into several doses.
상기 목적을 달성하기 위한 또 다른 실시양태로서, 본 발명은 상기 약학 조성물을 약제학적으로 유효한 양으로 골질환이 발병될 가능성이 있거나 또는 발병된 개체에 투여하는 단계를 포함하는 골질환을 예방 또는 치료하는 방법을 제공한다.
In another aspect of the present invention, the present invention provides a method for preventing or treating a bone disease, comprising administering the pharmaceutical composition to a subject having or likely to develop a bone disease in a pharmaceutically effective amount . ≪ / RTI >
상술한 바와 같이, 본 발명에서 제공하는 융합단백질은 골질환 예방 또는 치료용 약학 조성물의 유효성분으로서 사용할 수 있으므로, 상기 조성물은 골질환을 예방 또는 치료하거나, 또는 손상된 골 또는 치아를 재생하는데 사용될 수 있다.
As described above, since the fusion protein provided by the present invention can be used as an active ingredient of a pharmaceutical composition for preventing or treating bone diseases, the composition can be used for preventing or treating bone diseases or for regenerating damaged bone or teeth have.
본 발명에서 용어 "개체"란 골질환이 발병될 가능성이 있거나 또는 발병된 쥐, 가축, 인간 등을 포함하는 포유동물을 제한 없이 포함한다.The term "individual" as used herein includes, without limitation, mammals including rats, livestock, humans, and the like who are susceptible to or have developed bone disease.
본 발명의 골질환을 치료하는 방법에 있어서, 상기 약학 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여도 투여될 수 있다. 본 발명의 약학 조성물은 특별히 이에 제한되지 않으나, 목적하는 바에 따라 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 비내 투여, 폐내 투여, 직장내 투여될 수 있다. 다만, 경구 투여시에는 위산에 의하여 상기 융합단백질이 변성될 수 있기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 되어야 한다. 또한, 상기 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.
In the method of treating bone diseases of the present invention, the administration route of the pharmaceutical composition may be administered through any conventional route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intracorporally, or rectally, as desired, though not particularly limited thereto. However, since the fusion protein can be denatured by gastric acid upon oral administration, the oral composition should be formulated so as to coat the active agent or protect it from decomposition at the top. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.
본 발명의 융합단백질은 제3형 피브로넥틴 10번 도메인과 자가조립 서열을 포함하여, 피브로넥틴 단백질에 비하여, 세포결합, 세포증식 및 골형성 분화와 같은 다양한 세포활성을 더욱 활성화시킬 수 있음을 확인하였으므로, 골질환의 예방 또는 치료 뿐만 아니라, 손상된 골 또는 치아를 재생하는데에도 널리 활용될 수 있을 것이다.
Since the fusion protein of the present invention includes the type 3
도 1의 A는 본 발명에서 제공하는 FNIII10-SA를 발현시킬 수 있는 발현벡터 pBAD-HisB-FNIII10-SA의 구조를 나타내는 개열지도이고, 도 1의 B는 FNIII10와 FNIII10-SA를 웨스턴블럿 분석한 결과를 나타내는 사진으로서, 1번 레인은 FNIII10를 나타내고, 2번 레인은 FNIII10-SA를 나타낸다.
도 2는 FNIII10와 FNIII10-SA의 단백질 부착활성을 상기 각 단백질의 코팅농도의 변화에 따라 측정한 결과를 나타내는 그래프로서, (□)는 FNIII10을 나타내고, (■)는 FNIII10-SA를 나타낸다.
도 3은 FNIII10와 FNIII10-SA의 rMSCs 부착활성을 상기 각 단백질의 코팅농도의 변화에 따라 측정한 결과를 나타내는 그래프로서, (□)는 FNIII10을 나타내고, (■)는 FNIII10-SA를 나타낸다.
도 4는 FNIII10와 FNIII10-SA의 rMSCs 증식촉진 활성을 상기 각 단백질의 코팅농도의 변화에 따라 측정한 결과를 나타내는 그래프로서, (□)는 FNIII10을 나타내고, (■)는 FNIII10-SA를 나타낸다.
도 5는 rMSCs 골분화 관련 유전자의 발현수준에 미치는 FNIII10와 FNIII10-SA의 효과를 비교한 결과를 나타내는 그래프로서, (□)는 FNIII10을 나타내고, (■)는 FNIII10-SA를 나타낸다.FIG. 1A is a cleavage map showing the structure of an expression vector pBAD-HisB-FNIII10-SA capable of expressing FNIII10-SA provided by the present invention, and FIG. 1B is a Western blot analysis of FNIII10 and FNIII10- As a photograph showing the result,
FIG. 2 is a graph showing the results of measurement of protein adhesion activity of FNIII10 and FNIII10-SA according to the change of the coating concentration of each protein, wherein (□) represents FNIII10 and (■) represents FNIII10-SA.
FIG. 3 is a graph showing the results of measurement of rMSCs adhesion activity of FNIII10 and FNIII10-SA according to the change of coating concentration of each protein, wherein (□) represents FNIII10 and (■) represents FNIII10-SA.
FIG. 4 is a graph showing the results of measuring the activity of promoting the proliferation of rMSCs of FNIII10 and FNIII10-SA according to the change of the coating concentration of each protein, wherein (□) represents FNIII10 and (■) represents FNIII10-SA.
FIG. 5 is a graph showing the results of comparing the effects of FNIII10 and FNIII10-SA on the expression level of the rMSCs bone differentiation-related gene, wherein (□) represents FNIII10 and (■) represents FNIII10-SA.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실시예Example
1: One:
FNIII10FNIII10
--
SASA
의 발현 및 정제≪ / RTI >
자가조립서열 SA(Self-assembly sequance, 서열번호 1)이 FNIII10(서열번호 3)에 결합된 형태의 융합단백질인 FNIII10-SA(서열번호 5)를 제조하기 위하여, 먼저, 상기 SA를 코딩하는 폴리뉴클레오티드(서열번호 2)를 하기의 프라이머를 사용한 PCR을 수행하여 합성하였다. 이때, PCR은 30사이클(55 ℃ 1분, 72 ℃ 1분 및 94 ℃ 1분) 조건으로 수행하였다.
In order to prepare a fusion protein FNIII10-SA (SEQ ID NO: 5) in which the self-assembly sequence SA (SEQ ID NO: 1) is bound to FNIII10 (SEQ ID NO: 3) Nucleotide (SEQ ID NO: 2) was synthesized by performing PCR using the following primers. At this time, PCR was carried out under the conditions of 30 cycles (55 ° C for 1 minute, 72 ° C for 1 minute, and 94 ° C for 1 minute).
pBAD F: 5'-TAG CAT GAC TGG TGG ACA GC-3'(서열번호 7)pBAD F: 5'-TAG CAT GAC TGG TGG ACA GC-3 '(SEQ ID NO: 7)
SA R: 5'-TTA GAA TTC TTA AAA CAG CAG CAG CAG CAG AAA CAG CAG GCA GCA GCA GCC GCC ATC GCT ATC GCT GCC GGT ACC GCC CTC GAG-3'(서열번호 8)
SA (SEQ ID NO: 8) 5'-TTA GAA TTC TTA AAA CAG CAG CAG CAG CAG AAA CAG CAG GCA GCA GCA GCC GCC ATC GCT ATC GCT GCC GGT ACC GCC CTC GAG-3 '
상기 합성된 폴리뉴클레오티드에 제한효소 XhoI/EcoRI을 처리하여 절단산물을 수득하고, 상기 수득한 절단산물을 FNIII10 발현벡터 pBAD-HisB-FNIII10에 도입하여, FNIII10-SA를 발현시킬 수 있는 발현벡터 pBAD-HisB-FNIII10-SA를 제작하였다(도 1의 A).The cleavage product was obtained by treating the synthesized polynucleotide with restriction enzyme XhoI / EcoRI, and the cleavage product thus obtained was introduced into FNIII10 expression vector pBAD-HisB-FNIII10 to obtain an expression vector pBAD- HisB-FNIII10-SA was prepared (Fig. 1, A).
도 1의 A는 본 발명에서 제공하는 FNIII10-SA를 발현시킬 수 있는 발현벡터 pBAD-HisB-FNIII10-SA의 구조를 나타내는 개열지도이다.FIG. 1A is a cleavage map showing the structure of an expression vector pBAD-HisB-FNIII10-SA capable of expressing FNIII10-SA provided by the present invention.
상기 제작된 pBAD-HisB-FNIII10-SA를 숙주세포인 TOP10 대장균에 도입하여 형질전환체를 수득하고, 상기 수득한 형질전환체를 앰피실린이 포함된 LB 배지에 접종한 다음, 37 ℃에서 하룻밤동안 배양하였다. 배양물의 흡광도(A600)가 0.6이 될 때, 0.1% (w/v) L-아라비노스를 배지에 처리하여 목적하는 융합단백질인 FNIII10-SA의 발현을 유도하고, 20 ℃에서 6시간 동안 배양하였다. 배양이 종료된 후, 배양물을 원심분리(6,000 ×g, 10분)하여 침전된 균체를 수득하고, 이를 용해시킨 후, 초음파처리하여 세포파쇄물을 수득하였다. 상기 수득한 세포파쇄물을 원심분리(14,000 ×g, 30분)하여, 상등액을 수득하고, 이를 니켈-니트릴로트리아세트산 레진(nickel-nitrilotriacetic acid resin)이 충진된 컬럼크로마토그래피에 적용하여 목적하는 융합단백질인 FNIII10-SA을 정제하였다. 정제된 FNIII10-SA을 12% (v/v) SDS-PAGE에 적용한 다음, 항-폴리히스티딘 항체를 사용한 웨스턴블럿 분석을 수행하여 FNIII10-SA의 발현여부 및 순도를 결정하였다(도 1의 B). 이때, 대조군으로는 정제된 FNIII10을 사용하였다.The prepared pBAD-HisB-FNIII10-SA was introduced into TOP10 E. coli as a host cell to obtain a transformant. The obtained transformant was inoculated into LB medium containing ampicillin, Lt; / RTI > When the absorbance (A600) of the culture reached 0.6, 0.1% (w / v) L-arabinose was treated in the medium to induce the expression of the desired fusion protein FNIII10-SA and cultured at 20 DEG C for 6 hours . After completion of the culture, the culture was centrifuged (6,000 x g, 10 minutes) to obtain precipitated cells, which were dissolved and sonicated to obtain cell lysates. The obtained cell lysate was centrifuged (14,000 x g, 30 minutes) to obtain a supernatant. This supernatant was applied to column chromatography packed with nickel-nitrilotriacetic acid resin to obtain the desired fusion protein Gt; FNIII10-SA < / RTI > was purified. Purified FNIII10-SA was applied to 12% (v / v) SDS-PAGE followed by western blot analysis using anti-polyhistidine antibody to determine the expression and purity of FNIII10-SA (Figure 1 B) . At this time, purified FNIII10 was used as a control group.
도 1의 B는 FNIII10와 FNIII10-SA를 웨스턴블럿 분석한 결과를 나타내는 사진으로서, 1번 레인은 FNIII10를 나타내고, 2번 레인은 FNIII10-SA를 나타낸다. 도 1의 B에서 보듯이, FNIII10는 약 25 kDa의 분자량을 나타내고, FNIII10-SA는 약 27 kDa의 분자량을 나타냄을 확인하였다. 이로부터 SA에 의하여 약 2kDa의 분자량이 증가함을 알 수 있었다.FIG. 1B is a photograph showing the result of Western blot analysis of FNIII10 and FNIII10-SA, wherein
아울러, 상기 FNIII10와 FNIII10-SA의 정제수율은 각각 배양물 1ℓ당 1.2 및 1.3 mg/㎖이며, 정제된 FNIII10-SA의 순도는 약 98%임을 확인하였다.
In addition, the purification yields of FNIII10 and FNIII10-SA were 1.2 and 1.3 mg / ml per liter of the culture, respectively, and the purified FNIII10-SA had a purity of about 98%.
실시예Example
2: 2:
FNIII10FNIII10
--
SASA
의 단백질 부착활성 분석Of protein adhesion activity
상기 정제된 FNIII10-SA의 단백질 부착활성을 평가하기 위하여, 24웰 플레이트에 0 내지 10 ㎍/㎖의 농도로 FNIII10 또는 FNIII10-SA를 가하고 4 ℃에서 하룻밤 동안 방치하여 코팅시킨 다음, 각 웰을 PBS로 세척하고, 상기 세척된 각 웰에 1% (w/v) BSA 용액을 가하고 실온에서 1시간 동안 반응시킨켜서 비특이적결합을 방지하였다. 상기 BSA를 제거하고, TBS-T로 세척한 다음, 200 ㎕ Turbo TMB-ELISA를 가하고 실온에서 30분 동안 반응시켰으며, H2SO4(100 ㎕, 2M)를 부가하여 반응을 종료시키고, 각 웰의 흡광도를 450 nm에서 측정하였다(도 2).To evaluate the protein-binding activity of the purified FNIII10-SA, FNIII10 or FNIII10-SA was added to a 24-well plate at a concentration of 0 to 10 μg / ml and allowed to stand overnight at 4 ° C., , And 1% (w / v) BSA solution was added to each of the washed wells to react for 1 hour at room temperature to prevent nonspecific binding. The BSA was removed and washed with TBS-T, followed by addition of 200 μl of Turbo TMB-ELISA, reaction at room temperature for 30 minutes, completion of reaction by adding H 2 SO 4 (100 μl, 2M) The absorbance of the wells was measured at 450 nm (Fig. 2).
도 2는 FNIII10와 FNIII10-SA의 단백질 부착활성을 상기 각 단백질의 코팅농도의 변화에 따라 측정한 결과를 나타내는 그래프로서, (□)는 FNIII10을 나타내고, (■)는 FNIII10-SA를 나타낸다. 도 2에서 보듯이, 대조군인 FNIII10은 농도가 증가하여도 단백질 부착활성이 증가하지 않고, 1 ㎍/㎖ 이상의 농도로 처리한 후에 단백질 부착활성이 증가하는 양상을 나타낸 반면, FNIII10-SA는 0.5 ㎍/㎖ 이상의 농도로 처리할 경우 용량의존적으로 단백질 부착활성이 증가하고, 5 ㎍/㎖의 농도로 처리한 경우에는 가장 우수한 단백질 부착활성을 나타내었으나, 10 ㎍/㎖의 농도로 처리한 경우에는 단백질 부착활성이 감소함을 확인하였다.FIG. 2 is a graph showing the results of measurement of protein adhesion activity of FNIII10 and FNIII10-SA according to the change of the coating concentration of each protein, wherein (□) represents FNIII10 and (■) represents FNIII10-SA. As shown in FIG. 2, FNIII10 showed an increase in protein adherence activity after treatment at a concentration of 1 μg / ml or more without increasing protein adhesion activity even when the concentration increased, whereas FNIII10-SA showed 0.5 μg / Ml, the protein adherence activity was increased in a dose - dependent manner. When the concentration was 5 ㎍ / ㎖, the protein attachment activity was the highest. However, when the concentration was 10 ㎍ / It was confirmed that the adhesion activity was decreased.
통상적으로, FNIII10을 포함하는 피브로넥틴(FN)은 우수한 단백질 부착활성을 나타낸다고 알려져 있으나, 본 발명에서 제공하는 FNIII10-SA는 FNIII10 보다도 더욱 우수한 단백질 부착활성을 나타내었으므로, 조직공학적인 측면에서 활용성이 높은 것으로 분석되었다.
Generally, it is known that fibronectin (FN) containing FNIII10 exhibits excellent protein adhesion activity. However, since FNIII10-SA provided by the present invention exhibits more excellent protein binding activity than FNIII10, its applicability in terms of tissue engineering Respectively.
실시예Example
3: 3:
FNIII10FNIII10
--
SASA
의 세포 부착활성 분석Of cell adhesion activity
상기 실시예 2의 결과로부터 본 발명에서 제공하는 FNIII10-SA가 우수한 단백질 부착활성을 나타냄을 확인하였으므로, 상기 FNIII10-SA가 세포 부착활성을 나타내는지를 확인하고자 하였다.
From the results of Example 2, it was confirmed that FNIII10-SA provided by the present invention exhibits excellent protein-adhering activity. Therefore, it was examined whether FNIII10-SA exhibits cell adhesion activity.
실시예Example
3-1: 줄기세포 배양 3-1: Stem cell culture
수컷 Sprague-Dawley 랫트(4-5 주령, 180-200 g)의 골수로부터 랫트의 중간엽 줄기세포(Rat mesenchymal stem cells, rMSCs)를 분리하였다. 상기 분리된 rMSCs를 10% FBS, 100 units/㎖ 페니실린, 100 mg/㎖ 스트렙토마이신 및 0.25 mg/㎖ 암포테리신 B를 포함하는 α-MEM 배지에 접종하고, 5% CO2 및 37 ℃ 조건에서 배양하였다. 배양이 종료된 세포에 0.25% 트립신-EDTA를 5분 동안 처리하여 배양된 세포를 회수하고, 이를 다시 계대배양하는 방식을 3회 반복하여, 계대배양된 rMSCs를 수득하였다.
Rat mesenchymal stem cells (rMSCs) were isolated from bone marrow of male Sprague-Dawley rats (4-5 weeks old, 180-200 g). The separated rMSCs were inoculated into? -MEM medium containing 10% FBS, 100 units / ml penicillin, 100 mg / ml streptomycin and 0.25 mg / ml amphotericin B, and cultured at 5% CO 2 and 37 ° C Lt; / RTI > The cultured cells were treated with 0.25% trypsin-EDTA for 5 minutes to collect the cultured cells, and then subcultured again to obtain subcultured rMSCs.
실시예Example
3-2: 3-2:
FNIII10FNIII10
--
SASA
의 of
rMSCsrMSCs
부착활성 분석 Attachment activity analysis
먼저, 24웰 플레이트에 0 내지 10 ㎍/㎖의 농도로 FNIII10 또는 FNIII10-SA를 가하고 4 ℃에서 하룻밤 동안 방치하여 코팅시킨 다음, 각 웰을 DPBS(Dulbecco's phosphate buffered saline)로 세척하고, 상기 세척된 각 웰에 1% (w/v) BSA 용액을 가하고 실온에서 1시간 동안 반응시킨켜서 비특이적결합을 방지하였다. 상기 BSA를 제거하고, TBS-T로 세척한 다음, 상기 실시예 3-1에서 수득한 rMSCs를 웰당 1 × 105 세포수의 밀도로 접종하고, 30분 동안 방치한 다음, DPBS로 세척하고, 3.7% (w/v) 포르말린을 가하여 실온에서 15분 동안 반응시켜서, 세포를 고정시켰다. 상기 고정된 rMSCs에 0.25% (w/v) 크리스탈 바이올렛(crystal violet)을 가하여 37 ℃에서 1시간 동안 염색하고, DPBS로 3회 세척하였다. 상기 세척된 세포에 2% SDS 용액을 가하여 용해시켜서 세포용해물을 수득하고, 상기 수득한 세포용해물을 96웰 플레이트로 옮긴다음, 570 nm에서 흡광도를 측정하였다(도 3).
First, FNIII10 or FNIII10-SA was added to a 24-well plate at a concentration of 0 to 10 μg / ml and allowed to stand overnight at 4 ° C., and then each well was washed with DPBS (Dulbecco's phosphate buffered saline) A 1% (w / v) BSA solution was added to each well and reacted at room temperature for 1 hour to prevent nonspecific binding. After the BSA was removed and washed with TBS-T, the rMSCs obtained in Example 3-1 were inoculated at a density of 1 × 10 5 cells per well, left for 30 minutes, washed with DPBS, The cells were fixed by adding 3.7% (w / v) formalin for 15 minutes at room temperature. 0.25% (w / v) crystal violet was added to the fixed rMSCs, stained at 37 ° C for 1 hour, and washed three times with DPBS. 2% SDS solution was added to the washed cells to dissolve the cell lysate, and the resulting cell lysate was transferred to a 96-well plate and absorbance was measured at 570 nm (FIG. 3).
도 3은 FNIII10와 FNIII10-SA의 rMSCs 부착활성을 상기 각 단백질의 코팅농도의 변화에 따라 측정한 결과를 나타내는 그래프로서, (□)는 FNIII10을 나타내고, (■)는 FNIII10-SA를 나타낸다. 도 3에서 보듯이, FNIII10 또는 FNIII10-SA 모두, 0.5 ㎍/㎖ 이상의 농도로 코팅할 경우에 rMSCs 부착활성이 증가하였다. 다만, FNIII10는 처리농도가 증가할 수록 rMSCs 부착활성이 꾸준하게 증가하여, 10 ㎍/㎖의 농도로 코팅할 경우에 최대값을 나타내었으나, FNIII10-SA는 5 ㎍/㎖의 농도로 코팅할 경우에 rMSCs 부착활성이 최대 값을 나타내었고, 10 ㎍/㎖의 농도로 코팅할 경우에는 rMSCs 부착활성이 감소함을 확인하였다. 그러나, 5 ㎍/㎖의 농도로 코팅할 경우에 FNIII10이 나타내는 rMSCs 부착활성 보다 FNIII10-SA이 나타내는 rMSCs 부착활성의 값이 현저히 증가하고, 10 ㎍/㎖의 농도로 코팅할 경우에 FNIII10이 나타내는 rMSCs 부착활성이 최대값과 FNIII10-SA이 나타내는 rMSCs 부착활성의 감소된 값이 유사하였으므로, FNIII10 보다도 FNIII10-SA이 rMSCs 부착활성의 측면에서 우수한 효과를 나타냄을 알 수 있었다.FIG. 3 is a graph showing the results of measurement of rMSCs adhesion activity of FNIII10 and FNIII10-SA according to the change of coating concentration of each protein, wherein (□) represents FNIII10 and (■) represents FNIII10-SA. As shown in FIG. 3, when FNIII10 or FNIII10-SA was coated at a concentration of 0.5 μg / ml or more, rMSCs adhesion activity was increased. However, as the concentration of FNIII10 increased, the activity of rMSCs adhered increased steadily and reached a maximum value when coated at a concentration of 10 μg / ml. When FNIII10-SA was coated at a concentration of 5 μg / ml The rMSCs adhesion activity showed the maximum value, and when it was coated at the concentration of 10 ㎍ / ㎖, the rMSCs adhesion activity decreased. However, when coated at a concentration of 5 / / ml, the value of the rMSCs adhesion activity exhibited by FNIII10-SA was significantly higher than the rMSCs adhesion activity exhibited by FNIII10, and when coated at a concentration of 10 / / It was found that FNIII10-SA showed more excellent effect on rMSCs adhesion activity than FNIII10 because the maximum value of adhesion activity was similar to the reduced value of rMSCs adhesion activity exhibited by FNIII10-SA.
이처럼, 본 발명에서 제공하는 FNIII10-SA는 rMSCs 부착활성의 측면에서도 FNIII10 보다도 더욱 우수한 부착활성을 나타내었으므로, 조직세포의 결합에 소요되는 시간을 단축시킬 수 있다는 점에서, 조직공학적인 측면에서 활용성이 우수함을 다시한번 확인할 수 있었다.
As described above, the FNIII10-SA provided by the present invention exhibits a better adhesion activity than the FNIII10 in terms of the activity of rMSCs attachment, and thus can be used in terms of tissue engineering in that it can shorten the time required for binding of tissue cells I was able to confirm once again that sex is excellent.
실시예Example
4: 4:
FNIII10FNIII10
--
SASA
의 세포증식 촉진활성 분석Of cell proliferation promoting activity
0 또는 5 ㎍/㎖의 농도로 FNIII10 또는 FNIII10-SA가 코팅된 24웰 플레이트를 사용하고, rMSCs를 5일 동안 배양하는 것을 제외하고는, 상기 실시예 3-2와 동일한 방법으로 rMSCs를 배양하였다. 배양이 종료된 rMSCs를 DPBS로 3회 세척하고, 상기 세척된 rMSCs가 포함된 각 웰에 500 ㎕의 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 용액(5 mg/㎖ in PBS)을 포함하는 배지를 가하여 4시간 동안 반응시킨 후, 상기 배지를 제거하고, 200 ㎕ DMSO를 가하여 포르마잔 결정을 용해시켰으며, 540 nm에서 흡광도를 측정하였다(도 4).RMSCs were cultured in the same manner as in Example 3-2 except that 24 well plates coated with FNIII10 or FNIII10-SA at a concentration of 0 or 5 占 퐂 / ml were used and rMSCs were cultured for 5 days . The cultured rMSCs were washed three times with DPBS and 500 μl of a solution of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) in each well containing the washed rMSCs (5 mg / ml in PBS) was added and reacted for 4 hours. Then, the medium was removed, 200 μl of DMSO was added to dissolve the formazan crystals, and the absorbance was measured at 540 nm ).
도 4는 FNIII10와 FNIII10-SA의 rMSCs 증식촉진 활성을 상기 각 단백질의 코팅농도의 변화에 따라 측정한 결과를 나타내는 그래프로서, (□)는 FNIII10을 나타내고, (■)는 FNIII10-SA를 나타낸다. 도 4에서 보듯이, FNIII10와 FNIII10-SA 모두 rMSCs의 증식을 촉진시켰으며, FNIII10 보다는 FNIII10-SA가 더욱 향상된 효과를 나타냄을 확인하였다.
FIG. 4 is a graph showing the results of measuring the activity of promoting the proliferation of rMSCs of FNIII10 and FNIII10-SA according to the change of the coating concentration of each protein, wherein (□) represents FNIII10 and (■) represents FNIII10-SA. As shown in FIG. 4, both FNIII10 and FNIII10-SA promoted the proliferation of rMSCs, and FNIII10-SA showed more improved effect than FNIII10.
실시예Example
5: 5:
FNIII10FNIII10
--
SASA
의 of
골분화Bone differentiation
촉진활성 분석 Accelerated activity assay
5 ㎍/㎖의 농도로 FNIII10 또는 FNIII10-SA가 코팅된 24웰 플레이트를 사용하고, rMSCs를 7일 동안 배양하는 것을 제외하고는, 상기 실시예 3-2와 동일한 방법으로 rMSCs를 배양하였다. 상기 배양된 rMSCs를 Easy-spin RNA Extraction kit(iNtRON, Korea)에 적용하여 총 RNA를 수득하고, 이로부터 cDNA를 합성하였으며, 합성된 cDNA를 주형으로 하고, 하기의 프라이머를 사용하며, ABI Step One real-time PCR system을 사용한 PCR을 수행하여 골분화에 관여하는 마커 유전자인 Col I(collagen type I), OC(osteocalcin), OPN(osteopontin) 및 Runx 2(runt-related transcription factor 2) 유전자의 상대적인 발현수준을 평가하였다(도 5).
RMSCs were cultured in the same manner as in Example 3-2 except that 24 well plates coated with FNIII10 or FNIII10-SA at a concentration of 5 / / ml were used and rMSCs were cultured for 7 days. The cultured rMSCs were applied to an Easy-spin RNA Extraction kit (iNtRON, Korea) to obtain total RNAs, and cDNAs were synthesized therefrom. Using the synthesized cDNA as a template, the following primers were used and ABI Step One PCR was performed using real-time PCR system to compare the relative activities of the marker genes Col I (collagen type I), OC (osteocalcin), OPN (osteopontin) and Runx 2 (runt-related transcription factor 2) Expression levels were assessed (Figure 5).
GAPDH F: 5'-TGGAAGGACTCATGACCACA-3'(서열번호 9)GAPDH F: 5'-TGGAAGGACTCATGACCACA-3 '(SEQ ID NO: 9)
GAPDH R: 5'-TTCAGCTCAGGGATGACCTT-3'(서열번호 10)
GAPDH R: 5'-TTCAGCTCAGGGATGACCTT-3 '(SEQ ID NO: 10)
Col I F: 5'-CTGGCAAGAACGGAGATGAT-3'(서열번호 11)Col I F: 5'-CTGGCAAGAACGGAGATGAT-3 '(SEQ ID NO: 11)
Col I R: 5'-TTAGGACCAGCAGGACCAGT-3'(서열번호 12)
Col IR: 5'-TTAGGACCAGCAGGACCAGT-3 '(SEQ ID NO: 12)
OC F: 5'-CATCACTGCCACCCAGAAGAC-3'(서열번호 13)OC F: 5'-CATCACTGCCACCCAGAAGAC-3 '(SEQ ID NO: 13)
OC R: 5'-CAGTGGATGCAGGGATGATGT-3'(서열번호 14)
OC R: 5'-CAGTGGATGCAGGGATGATGT-3 '(SEQ ID NO: 14)
OPN F: 5'-CCAATGAAAGCCATGACCAC-3'(서열번호 15)OPN F: 5'-CCAATGAAAGCCATGACCAC-3 '(SEQ ID NO: 15)
OPN R: 5'-CGACTGTAGGGACGATTGGA-3'(서열번호 16)
OPN R: 5'-CGACTGTAGGGACGATTGGA-3 '(SEQ ID NO: 16)
Runx 2 F: 5'-CGCCCCTCCCTGAACTCT-3'(서열번호 17)Runx 2 F: 5'-CGCCCCTCCCTGAACTCT-3 '(SEQ ID NO: 17)
Runx 2 R: 5'-TGCCTGCCTGGGATCTGTA-3'(서열번호 18)
Runx 2 R: 5'-TGCCTGCCTGGGATCTGTA-3 '(SEQ ID NO: 18)
도 5는 rMSCs 골분화 관련 유전자의 발현수준에 미치는 FNIII10와 FNIII10-SA의 효과를 비교한 결과를 나타내는 그래프로서, (□)는 FNIII10을 나타내고, (■)는 FNIII10-SA를 나타낸다. 도 5에서 보듯이, FNIII10 보다는 FNIII10-SA가 골분화 관련 유전자의 발현수준을 상대적으로 촉진시키는 효과를 나타냄을 확인하였다.FIG. 5 is a graph showing the results of comparing the effects of FNIII10 and FNIII10-SA on the expression level of the rMSCs bone differentiation-related gene, wherein (□) represents FNIII10 and (■) represents FNIII10-SA. As shown in FIG. 5, it was confirmed that FNIII10-SA rather than FNIII10 has an effect of relatively promoting the expression level of bone morphogenesis-related genes.
공지된 바에 의하면, FNIII10을 포함하는 피브로넥틴(FN)은 골분화를 촉진시키는 활성을 나타낸다고 알려져 있으나, 본 발명에서 제공하는 FNIII10-SA는 FNIII10 보다도 더욱 우수한 골분화 촉진활성을 나타내었으므로, 골손상 질환을 치료하기 위한 줄기세포 치료방법을 수행함에 있어서, 상기 FNIII10-SA는 골분화 보조제로서 사용할 수 있음을 알 수 있었다.
It is known that fibronectin (FN) containing FNIII10 exhibits activity of promoting bone differentiation. However, since FNIII10-SA provided by the present invention exhibits better bone-differentiation promoting activity than FNIII10, It was found that FNIII10-SA can be used as a bone-differentiation auxiliary in performing a stem cell treatment method for treating bone marrow stem cells.
<110> Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> A novel fusion protein having cell adhesion activity and osteogenic differentiation activity <130> KPA140870-KR <160> 18 <170> KopatentIn 2.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> self-assembled sequence <400> 1 Ser Asp Ser Asp Gly Gly Cys Cys Cys Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 <210> 2 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> self-assembled sequence <400> 2 tctgattctg atggcggctg ctgctgcctg ctgctgctgc tgctgctg 48 <210> 3 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> FNIII10 <400> 3 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Cys Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 <210> 4 <211> 279 <212> DNA <213> Artificial Sequence <220> <223> FNIII10 <400> 4 gtgtctgatg tgccgcgtga tctggaagtg gtggcggcga ccccgacctc tctgctgatt 60 tcttgggatg cgccggcggt gaccgtgcgt tattatcgta ttacctatgg cgaaaccggc 120 ggcaactctc cggtgcagga atttaccgtg ccgggctcta aatctaccgc gaccatttct 180 ggcctgaaac cgggcgtgga ttgcaccatt accgtgtatg cggtgaccgg ccgtggcgat 240 tctccggcgt cttctaaacc gatttctatt aactatcgt 279 <210> 5 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> FNIII10-SA <400> 5 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Cys Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95 Asp Lys Pro Glu Leu Gly Leu Glu Gly Gly Thr Val Ser Asp Ser Asp 100 105 110 Gly Gly Cys Cys Cys Leu Leu Leu Leu Leu Leu Leu 115 120 <210> 6 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> FNIII10-SA <400> 6 gtgtctgatg tgccgcgtga tctggaagtg gtggcggcga ccccgacctc tctgctgatt 60 tcttgggatg cgccggcggt gaccgtgcgt tattatcgta ttacctatgg cgaaaccggc 120 ggcaactctc cggtgcagga atttaccgtg ccgggctcta aatctaccgc gaccatttct 180 ggcctgaaac cgggcgtgga ttgcaccatt accgtgtatg cggtgaccgg ccgtggcgat 240 tctccggcgt cttctaaacc gatttctatt aactatcgta ccgaaattga taaaccggaa 300 ctgggcctgg aaggcggcac cgtgtctgat tctgatggcg gctgctgctg cctgctgctg 360 ctgctgctgc tg 372 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tagcatgact ggtggacagc 20 <210> 8 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ttagaattct taaaacagca gcagcagcag aaacagcagg cagcagcagc cgccatcgct 60 atcgctgccg gtaccgccct cgag 84 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tggaaggact catgaccaca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ttcagctcag ggatgacctt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctggcaagaa cggagatgat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ttaggaccag caggaccagt 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 catcactgcc acccagaaga c 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cagtggatgc agggatgatg t 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ccaatgaaag ccatgaccac 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cgactgtagg gacgattgga 20 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 cgcccctccc tgaactct 18 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tgcctgcctg ggatctgta 19 <110> Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> A novel fusion protein having cell adhesion activity and 오테이티아티아이오 <130> KPA140870-KR <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> self-assembled sequence <400> 1 Ser Asp Ser Asp Gly Gly Cys Cys Cys Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 <210> 2 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> self-assembled sequence <400> 2 tctgattctg atggcggctg ctgctgcctg ctgctgctgc tgctgctg 48 <210> 3 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> FNIII10 <400> 3 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Cys Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 <210> 4 <211> 279 <212> DNA <213> Artificial Sequence <220> <223> FNIII10 <400> 4 gtgtctgatg tgccgcgtga tctggaagtg gtggcggcga ccccgacctc tctgctgatt 60 tcttgggatg cgccggcggt gaccgtgcgt tattatcgta ttacctatgg cgaaaccggc 120 ggcaactctc cggtgcagga atttaccgtg ccgggctcta aatctaccgc gaccatttct 180 ggcctgaaac cgggcgtgga ttgcaccatt accgtgtatg cggtgaccgg ccgtggcgat 240 tctccggcgt cttctaaacc gatttctatt aactatcgt 279 <210> 5 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> FNIII10-SA <400> 5 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Cys Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95 Asp Lys Pro Glu Leu Gly Leu Glu Gly Gly Thr Val Ser Asp Ser Asp 100 105 110 Gly Gly Cys Cys Cys Leu Leu Leu Leu Leu Leu Leu 115 120 <210> 6 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> FNIII10-SA <400> 6 gtgtctgatg tgccgcgtga tctggaagtg gtggcggcga ccccgacctc tctgctgatt 60 tcttgggatg cgccggcggt gaccgtgcgt tattatcgta ttacctatgg cgaaaccggc 120 ggcaactctc cggtgcagga atttaccgtg ccgggctcta aatctaccgc gaccatttct 180 ggcctgaaac cgggcgtgga ttgcaccatt accgtgtatg cggtgaccgg ccgtggcgat 240 tctccggcgt cttctaaacc gatttctatt aactatcgta ccgaaattga taaaccggaa 300 ctgggcctgg aaggcggcac cgtgtctgat tctgatggcg gctgctgctg cctgctgctg 360 ctgctgctgc tg 372 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tagcatgact ggtggacagc 20 <210> 8 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ttagaattct taaaacagca gcagcagcag aaacagcagg cagcagcagc cgccatcgct 60 atcgctgccg gtaccgccct cgag 84 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tggaaggact catgaccaca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ttcagctcag ggatgacctt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctggcaagaa cggagatgat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ttaggaccag caggaccagt 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 catcactgcc acccagaaga c 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cagtggatgc agggatgatg t 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ccaatgaaag ccatgaccac 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cgactgtagg gacgattgga 20 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 cgcccctccc tgaactct 18 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tgcctgcctg ggatctgta 19
Claims (12)
A fusion protein comprising domain 10 of the type 3 fibronectin and the self-assembling sequence.
상기 제3형 피브로넥틴의 10번 도메인은 서열번호 1의 아미노산 서열로 구성되는 것인 융합단백질.
The method according to claim 1,
Wherein the 10-th domain of said type 3 fibronectin is comprised of the amino acid sequence of SEQ ID NO: 1.
상기 자가조립서열은 서열번호 3의 아미노산 서열로 구성되는 것인 융합단백질.
The method according to claim 1,
Wherein the self-assembled sequence is comprised of the amino acid sequence of SEQ ID NO: 3.
상기 융합단백질은 서열번호 5의 아미노산 서열로 구성되는 것인 융합단백질
The method according to claim 1,
Wherein said fusion protein is a fusion protein comprising the amino acid sequence of SEQ ID NO:
상기 융합단백질은 세포결합 활성, 세포증식 활성 및 골분화 촉진활성을 나타내는 것인 융합단백질.
The method according to claim 1,
Wherein the fusion protein exhibits cell binding activity, cell proliferation activity, and bone resorption promoting activity.
A polynucleotide comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 5.
서열번호 6의 뉴클레오티드 서열을 포함하는 폴리뉴클레오티드.
The method according to claim 6,
A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6.
An expression vector comprising the polynucleotide of claim 6.
A transformant transformed by the introduction of the expression vector of claim 8.
(b) 상기 배양물로부터 제1항의 융합단백질을 회수하는 단계를 포함하는, 제1항의 융합단백질의 생산방법.
(a) culturing the transformant of claim 9 to obtain a culture; And
(b) recovering the fusion protein of claim 1 from the culture.
A pharmaceutical composition for preventing or treating bone diseases, which comprises the fusion protein of any one of claims 1 to 5 and stem cells as an active ingredient.
A pharmaceutical composition for bone or tooth regeneration comprising the fusion protein of any one of claims 1 to 5 and stem cells as an active ingredient.
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| KR20190041946A (en) | 2017-10-13 | 2019-04-23 | 순천향대학교 산학협력단 | Method for differentiating mesenchymal stem cells to bone cell using multi-channel granules |
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| KR101241667B1 (en) * | 2012-07-20 | 2013-03-11 | 명지대학교 산학협력단 | Novel peptide tag and uses thereof |
| KR20140095556A (en) * | 2011-11-11 | 2014-08-01 | 배리에이션 바이오테크놀로지스 아이엔씨. | Compositions and methods for treatment of cytomegalovirus |
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| KR20180135689A (en) * | 2017-06-13 | 2018-12-21 | 인하대학교 산학협력단 | Composition for promoting differentiation of stem cell into osteoblasts containing fibulin-1 |
| KR20190041946A (en) | 2017-10-13 | 2019-04-23 | 순천향대학교 산학협력단 | Method for differentiating mesenchymal stem cells to bone cell using multi-channel granules |
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