KR20210154784A - 섬유질환 예방 또는 치료용 재조합 융합 단백질 - Google Patents
섬유질환 예방 또는 치료용 재조합 융합 단백질 Download PDFInfo
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- KR20210154784A KR20210154784A KR1020210176464A KR20210176464A KR20210154784A KR 20210154784 A KR20210154784 A KR 20210154784A KR 1020210176464 A KR1020210176464 A KR 1020210176464A KR 20210176464 A KR20210176464 A KR 20210176464A KR 20210154784 A KR20210154784 A KR 20210154784A
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- Medicinal Preparation (AREA)
Abstract
본 발명은 간, 췌장, 폐 등에 발생하는 섬유질환을 예방 또는 치료하기 위해 사용할 수 있는 알부민과 레티놀 결합 단백질의 융합 단백질에 관한 것으로서, 본 발명의 융합단백질은 조직의 섬유화에 원인이 되는 성상세포의 활성화를 억제 또는 불활성화를 유도하여 섬유질환의 예방 또는 치료가 가능하며, 같은 목적의 종래 융합단백질과 비교하여 낮은 농도에서 보다 우수한 효과를 나타내는바 단백질 치료제의 인체 투여 용량과 투여 빈도를 획기적으로 줄일 수 있는 범용적 기반기술이 될 수 있을 것으로 기대된다.
Description
본 발명은 간, 췌장, 폐 등에 발생하는 섬유질환을 예방 또는 치료하기 위해 사용할 수 있는 알부민과 레티놀 결합 단백질의 융합 단백질에 관한 것이다.
조직의 섬유화(fibrosis)는 조직의 기능 상실을 초래하는 치명적인 결과를 낳는다. 예를 들어, 간의 섬유화는 간 기능 상실에 이어, 간경화, 간암으로 진행되며, 만성 췌장염과 췌장암에서 공통적으로 섬유화된 조직이 관찰된다. 현재까지 이용되는 섬유질환 치료제는 없으며, 조직 이식이 가장 치료가능성이 높은 방법으로 시행되고 있다. 조직섬유화의 분자 기전은 아직 명확하게 밝혀지지 않았으며, 섬유질환 치료제의 개발 연구가 필요하다.
최근들어, 조직의 섬유화는 조직을 구성하는 세포 중 하나인 성상세포(stellate cells)의 활성화 과정(activation 및 transdifferentiation)에 의해 발생함이 밝혀졌다. 활성화된 성상세포는 콜라겐과 같은 세포외기질을 과도하게 발현하여 축적시키며, 근섬유화세포(myofibroblast)로 분화된다. 이렇게 활성화된 성상세포는 조직 섬유화 발생에 결정적인 역할을 수행한다.
성상세포(stellate cell)는 간뿐만 아니라 췌장, 폐, 신장, 그리고 장 등에 분포되어 전신에서의 레티노이드 항상성의 조절에 중심적인 역할을 한다. 식품으로부터 얻어진 비타민 A(레티놀)는 혈류에서 레티놀 결합 단백질(Retinol Binding Protein, RBP)과 결합하여 순환하다가 RBP 수용체인 STRA6를 통해 성상세포로 이동되어, 세포질 내 지방 방울 내에 레티닐 에스테르의 형태로서 저장된다. 한편, 알부민은 성상세포 내에서 그 활성화를 저해하고, 이미 활성화된 성상세포 내에서 알부민의 발현은 활성화 이전의 상태로 전환시킴이 알려져 있다.
이전의 연구에서 본 발명자들은 조직 섬유화의 시작이 되는 성상세포의 활성화 억제 또는 불활성화를 위하여 STRA6를 통해 성상세포 내로 이동할 수 있는 RBP와 알부민을 결합하여 성상세포를 타겟팅하는 융합 단백질을 제조하고 이의 섬유화 예방 및 치료 효과를 확인하였다(KR 10-1395394).
본 발명이 이루고자 하는 기술적 과제는 종래의 RBP와 알부민 융합 단백질의 성상세포의 활성화 억제 또는 불활성화 효과를 증진시키기 위한 것으로서, 본 발명은 알부민의 하나 이상의 아미노산 서열이 치환된 RBP-알부민 융합 단백질 및 서로 다른 배열로 연결된 RBP-알부민 융합 단백질을 제공하는 것이다.
또한, 본 발명은 상기 융합 단백질의 발현시킬 수 있는 벡터와 상기 벡터가 도입된 형질전환체를 제공하여 본 발명의 재조합 단백질의 대량 생산 방법을 제공하고자 한다.
또한, 본 발명의 융합 단백질은 종래의 RBP-알부민 융합 단백질보다 성상세포의 활성화 억제 또는 불활성화에 우수한 효과를 나타내는바, 본 발명은 상기 융합 단백질을 포함하는 섬유질환의 예방 또는 치료용 조성물을 제공하고자 한다.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당해 기술분야의 통상의 기술자에게 명확하게 이해될 수 있을 것이다.
상기 과제를 해결하기 위하여, 본 발명은 서열번호 1 또는 2의 아미노산 서열로 이루어지거나 이를 포함하는 융합 단백질을 제공한다.
또한, 본 발명은 서열번호 3 내지 8 중 어느 하나의 아미노산 서열로 이루어지거나 이를 포함하는 융합 단백질을 제공한다.
본 발명의 일 구현예로서, 상기 융합 단백질은 조직의 섬유화를 예방 또는 치료하기 위한 용도의 것일 수 있다.
또한, 본 발명은 상기 각 융합단백질을 암호화하는 폴리뉴클레오티드를 제공하며, 상기 폴리뉴클레오티드는 서열번호 9 내지 14 중 어느 하나의 염기서열로 이루어지거나 이를 포함할 수 있다.
또한, 본 발명은 상기 융합단백질을 암호화하는 유전자를 포함하는 재조합 벡터를 제공하며, 상기 재조합 벡터를 포함하는 형질전환체를 제공한다.
또한, 본 발명은 (1) 서열번호 1 내지 8 중 어느 하나의 아미노산 서열로 이루어진 융합 단백질을 암호화하는 유전자를 포함하는 재조합 벡터를 제조하는 단계; (2) 상기 재조합 벡터를 숙주세포에 도입하여 형질전환체를 제조하는 단계; (3) 상기 형질전환체를 배양하는 단계; 및 (4) 상기 배양액으로부터 융합 단백질을 수득하는 단계;를 포함하는 융합 단백질의 생산 방법을 제공한다.
본 발명의 일 구현예로서, 상기 융합 단백질을 암호화하는 유전자는 서열번호 9 내지 14로 이루어진 군으로부터 선택되는 1 이상의 염기서열로 이루어진 폴리뉴클레오티드를 포함할 수 있다.
본 발명의 다른 구현예로서, 상기 숙주세포는 포유류의 세포일 수 있으며, 바람직하게는 포유류의 암세포일 수 있고, 더욱 바람직하게는 CHO cell 일 수 있다.
또한, 본 발명은 서열번호 1 내지 8 중 어느 하나의 아미노산 서열로 이루어진 융합 단백질을 포함하는, 섬유질환 예방 또는 치료용 약학적 조성물을 제공할 수 있다.
또한, 본 발명은 서열번호 1 내지 8 중 어느 하나의 아미노산 서열로 이루어진 융합 단백질을 개체에 투여하는 단계를 포함하는 섬유질환의 예방 또는 치료 방법을 제공할 수 있다.
본 발명의 일 구현예로서, 상기 개체는 섬유질환의 예방 또는 치료가 필요한 인간일 수 있으며, 특히 비알코올성 지방간 질환 환자일 수 있다.
본 발명의 다른 구현예로서, 상기 방법은 상기 융합단백질의 투여 이후에 CT 촬영 또는 조직검사를 수행하여 섬유화 정도를 진단하는 단계를 추가로 포함할 수 있다.
본 발명의 일 구현예로서, 상기 섬유질환은 간 섬유증(liver fibrosis), 만성간염 (chronic hepatitis), 간경화 (cirrhosis), 간암(hepatic cancer), 화학요법-연관 지방간염(chemotherapy-associated steatohepatitis, CASH), 폐 섬유증(lung fibrosis), 신장 섬유증(renal fibrosis), 신부전(renal failure), 췌장 섬유증 (pancreatic fibrosis), 만성 췌장염(chronic pancreatitis) 및 췌장암(pancreatic cancer)으로 이루어진 군으로부터 선택되는 1 종 이상의 질환일 수 있다.
또한, 본 발명은 섬유질환의 예방 또는 치료용 약제의 제조를 위한 상기 융합단백질의 용도를 제공한다.
본 발명의 융합단백질은 조직의 섬유화에 원인이 되는 성상세포의 활성화를 억제 또는 불활성화를 유도하여 섬유질환의 예방 또는 치료가 가능하며, 같은 목적의 종래 융합단백질과 비교하여 낮은 농도에서 보다 우수한 효과를 나타내는바 단백질 치료제의 인체 투여 용량과 투여 빈도를 획기적으로 줄일 수 있는 범용적 기반기술이 될 수 있을 것으로 기대된다.
도 1은 알부민Ⅲ과 RBP가 결합된 융합단백질이 활성화된 성상세포 내에 내포되어 작용하는 기전을 도식화한 도면이다.
도 2는 본 발명의 융합단백질 1 및 2와 종래의 융합단백질 (RBP-알부민Ⅲ 및 알부민Ⅲ-RBP)의 활성화된 성상세포의 불활성화 유도 효과를 비교확인한 도면이다.
도 3은 본 발명의 융합단백질 1 및 2와 융합단백질 3 내지 6의 활성화된 성상세포의 불활성화 유도 효과를 비교확인한 도면이다.
도 4a 및 도 4b는 간 섬유화 동물모델에 융합단백질 1 및 4와 RBP-알부민² 투여에 따른 간 섬유화 정도를 시리우스 레드 조직염색을 통해 비교확인한 도면이다.
도 2는 본 발명의 융합단백질 1 및 2와 종래의 융합단백질 (RBP-알부민Ⅲ 및 알부민Ⅲ-RBP)의 활성화된 성상세포의 불활성화 유도 효과를 비교확인한 도면이다.
도 3은 본 발명의 융합단백질 1 및 2와 융합단백질 3 내지 6의 활성화된 성상세포의 불활성화 유도 효과를 비교확인한 도면이다.
도 4a 및 도 4b는 간 섬유화 동물모델에 융합단백질 1 및 4와 RBP-알부민² 투여에 따른 간 섬유화 정도를 시리우스 레드 조직염색을 통해 비교확인한 도면이다.
알부민은 주로 간세포에서 합성되는 다기능성 혈장 단백질이다. 알부민은 3개의 도메인(domain)을 가지며, 각각은 두 개의 서브도메인(subdomain) A 및 B로 구성된다. 알부민은 지방산, 레티노산을 포함한 다양한 소수성 물질과 결합하여 혈액 내에서 운반시켜 주는 분자 이동의 역할을 수행하는 것으로 알려져 있다. 결정학 분석에 따르면, 5개의 강력한 지방산 결합 부위가 알부민 내에 비대칭적으로 분포한다(서브도메인 IB에 하나, IA 과 IIA 사이에 하나, ⅢA에 둘, 그리고 ⅢB에 하나).
본 발명자들은 이전의 연구에서 알부민과 성상세포 타겟팅을 위한 레티놀 결합 단백질(Retinol Binding Protein, RBP)이 결합된 융합 단백질을 제조하였으며, 상기 융합 단백질은 활성화된 성상세포 내부로 유입되어 세포의 형태를 활성화 이전으로 역전환시키고, 이로 인하여 섬유질환의 예방 및 치료가 가능함을 확인하였다. 이에 나아가 본 발명자들은 성상세포 활성화 과정에서 레티노산(retinoic acid, RA)이 중요한 역할을 하며, 융합단백질은 이러한 RA의 세포 내 레벨을 감소시킴으로서 성상세포의 활성화를 제어함을 규명하였다. 따라서 보다 안정적으로 레티노산(retinoic acid, RA)과 결합하여 성상세포의 활성화 억제 또는 불활성화를 유도할 수 있는 융합 단백질을 개발하기 위하여, 알부민의 서열 변이와 다양한 조합의 알부민 sub-domain과 RBP의 배열을 이용하여 새로운 알부민 RBP 융합 단백질을 제조하였다.
종전 연구에서 알부민의 도메인 중에서도 도메인Ⅲ과 RBP의 융합단백질에서 성상세포의 활성화 억제 또는 불활성화 효과가 가장 우수하였는바, 이를 토대로 본 발명자들은 레티노산과의 결합력이 강화된 재조합 융합단백질을 제조하고자 하였다.
이에, 종래의 알부민 도메인 Ⅲ과 RBP 융합단백질에서 도메인 Ⅲ의 526번째와 600번째 bulky한 아미노산이 RA와의 결합에 방해가 되는 것으로 추정하고, 이를 보다 작은 크기의 아미노산으로 치환하여 RA와의 결합을 통한 활성화된 성상세포의 불활성화 효과를 확인하고자 하였다(서열번호 1 및 2 참조).
구체적으로, 본 발명자들은 알부민 도메인 Ⅲ에서 526 번째 아미노산 잔기(phenylalanine)와 600번째 아미노산 잔기(valine)를 각각 발린(valine), 알라닌(alanine)으로 교체하여 아래와 같이 융합 단백질 1 및 2를 설계하여 제조하였다.
- 융합단백질 1 : RBP(17~193) + 알부민Ⅲ{404~526(F→V)~600(V→A)~601}
- 융합단백질 2 : 알부민 [Ⅲ{404~526(F→V)~600(V→A)~601}] + RBP(17~192)
또한, 본 발명자들은 알부민의 sub-domain IB와 ⅢA가 레티노산과 매우 안정적으로 결합함을 확인하고, 아래와 같이 융합 단백질 3~6을 설계하여 제조하였다.
- 융합단백질 3 : RBP(17~193) + 알부민[ⅢA{404~517(V)} + IB(131~218)]
- 융합단백질 4: RBP(17~193) + 알부민[ⅢA{404~517(V)} + IB(134~218)]
- 융합단백질 5 : 알부민[ⅢA{404~517(V)} + IB(131~218)] + RBP(17~192)
- 융합단백질 6 : 알부민[ⅢA{404~517(V)} + IB(134~218)] + RBP(17~192)
이어서, 본 발명자들은 제작된 융합단백질 1 또는 2를 활성화된 성상세포에 처리하여 성상세포의 활성화 마커인 α-SMA(α-smooth muscle actin)와 collagen type I의 발현 수준을 측정함으로써 종래 개발된 융합단백질(RBP-알부민Ⅲ 및 알부민Ⅲ-RBP)과 성상세포의 불활성화 효과를 비교확인하였다. 그 결과, 본 발명의 융합단백질 1 및 2는 종래 개발된 융합단백질과 비교하여 낮은 농도에서 높은 수준으로 α-SMA와 collagen I의 발현을 감소시킬 수 있음을 확인하였다(실시예 1 참조).
이를 통해, 본 발명자들은 알부민Ⅲ의 526 a.a 와 600 a.a가 작은 분자량의 아미노산으로 치환되는 경우 레티노산과의 결합력이 상승되어 보다 효과적으로 성상세포의 불활성화를 유도할 수 있음을 확인하였다.
이에, 본 발명은 526 및 600번째 아미노산이 각각 독립적으로 치환된 알부민Ⅲ과 RBP가 결합된 융합단백질을 섬유질환 치료 용도에 제공할 수 있으며, 상기 융합단백질에서 알부민 Ⅲ의 526번째 아미노산 잔기로 위치할 수 있는 아미노산은 발린, 알라닌, 글리신일 수 있고, 600번째 아미노산 잔기로 위치할 수 있는 아미노산은 알라닌, 글리신 일 수 있으며, 상기 융합단백질은 서열번호 1 또는 2의 아미노산 서열로 이루어지거나 이를 포함할 수 있다.
이어서, 본 발명자들은 종래 융합단백질보다 우수한 성상세포의 활성화 과정 억제 효과를 나타낸 융합단백질 1 및 2와 융합단백질 3 내지 6의 α-SMA와 collagen I 발현 억제 효과를 비교확인하였다. 그 결과, 융합단백질 3 내지 6은 보다 낮은 농도에서도 성상세포의 활성화를 효과적으로 억제할 수 있음을 확인하였다(실시예 2).
아울러, 본 발명자들은 사염화탄소에 의한 간 섬유화 동물모델을 이용하여 융합단백질 1 및 4와 종래 융합단백질(RBP-알부민Ⅲ)의 간 섬유화 개선 효과를 확인한 결과, 융합단백질 1 및 4는 RBP-알부민Ⅲ보다 낮은 농도에서 보다 효과적으로 섬유화를 개선할 수 있음을 확인하였다(실시예 3).
이에, 본 발명자들은 상기 개발된 융합단백질 1 내지 6으로 이루어진 군으로부터 선택된 1 종 이상의 융합단백질을 포함하는 섬유질환 예방 또는 치료용 약학적 조성물을 제공할 수 있으며, 융합단백질 1 내지 6으로 이루어진 군으로부터 선택되는 2종 이상의 융합단백질을 개체에 투여하는 단계를 포함하는 섬유질환 예방 또는 치료 방법을 제공할 수 있다.
본 발명의 융합단백질 1 내지 6은 종래의 RBP과 알부민의 융합단백질 보다 낮은 농도에서 효과적으로 활성화된 성상세포의 불활성화를 유도할 수 있음을 확인하였는바, 본 발명의 약학적 조성물은 단백질 치료제의 인체 투여 용량과 투여 빈도를 획기적으로 줄일 수 있는 범용적 기반기술이 될 수 있을 것으로 기대된다.
본 발명에서 "섬유질환"이란 정상적인 조직이 파괴되면서 섬유성 결합조직으로 대체되는 섬유화(fibrosis)로 인하여 장기가 제 기능을 다하지 못하게 되어 나타나는 질병으로서, 간 섬유증(liver fibrosis), 만성간염 (chronic hepatitis), 간경화(cirrhosis), 간암(hepatic cancer), 화학요법-연관 지방 간염(chemotherapy-associated steatohepatitis, CASH), 폐 섬유증(lung fibrosis), 신장 섬유증(renal fibrosis), 신부전(renal failure), 췌장 섬유증(pancreatic fibrosis), 만성 췌장염(chronic pancreatitis) 및 췌장암(pancreatic cancer)을 포함하며, 장기의 일부가 굳는 섬유화에 의해 나타나는 질병이라면 이에 제한되지 아니한다.
본 발명에 있어서, “개체”란 포유류라면 제한되지 아니하나, 바람직하게는 인간 또는 가축일 수 있다.
본 발명에서 예방이란 본 발명에 따른 약학적 조성물의 투여에 의해 섬유질환의 발명을 지연시키거나, 조직의 섬유화를 지연시키는 모든 행위를 의미하고, 치료란 본 발명에 따른 약학적 조성물의 투여에 의해 섬유질환의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.
본 발명에 있어서 상기 융합 단백질의 형성에 사용되는 알부민은 임의의 종으로부터 유래한 것일 수 있으나, 면역원성의 위험을 피하기 위하여 투여되는 개체와 동일한 종(species)의 유래인 것이 바람직하다.
본 발명에서 약학적 조성물(pharmaceutical composition)은 융합단백질 1 내지 6 외에 조직의 섬유화를 예방 또는 치료할 수 있는 공지의 물질을 1종 이상 더 포함할 수 있으며, 약학적 조성물의 제조에 통상적으로 사용되는 적절한 담체, 부형제, 및 희석제를 더 포함할 수 있다.
본 발명에서 "담체(carrier)"란 비이클(vehicle)이라고도 불리우며, 세포 또는 조직 내로의 단백질 또는 펩타이드의 부가를 용이하게 하는 화합물을 의미하는 것으로서, 예를 들어, 디메틸술폭사이드(DMSO)는 생물체의 세포 또는 조직 내로의 많은 유기물의 투입을 용이하게 하는 통상 사용되는 담체이다.
본 발명에서 "희석제(diluent)"란 대상 단백질 또는 펩타이드의 생물학적 활성 형태를 안정화시킬 뿐만 아니라, 단백질 또는 펩타이드를 용해시키게 되는 물에서 희석되는 화합물로 정의된다. 버퍼 용액에 용해되어 있는 염은 당해 분야에서 희석제로 사용된다. 통상 사용되는 버퍼 용액은 포스페이트 버퍼 식염수이며, 이는 인간 용액의 염 상태를 모방하고 있기 때문이다. 버퍼 염은 낮은 농도에서 용액의 pH를 제어할 수 있기 때문에, 버퍼 희석제가 화합물의 생물학적 활성을 변형하는 일은 드물다. 여기에 사용된 아젤라산을 함유하는 화합물들은 인간 환자에게 그 자체로서, 또는 결합 요법에서와 같이 다른 성분들과 함께 또는 적당한 담체나 부형제와 함께 혼합된 약제학적 조성물로서, 투여될 수 있다.
또한, 본 발명에 따른 섬유질환 예방 또는 치료용 약학적 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 외용제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 본 발명의 항균용 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있으며, 예를 들어, 약학적으로 허용가능한 담체와 혼합된 형태로 약 0.001mg 내지 1000mg이 투여될 수 있다. 본 발명의 항균용 조성물은 필요에 따라 일일 1회 내지 수회로 나누어 투여할 수 있으며, 단독으로, 또는 수술, 호르몬 치료, 약물 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.
한편, 본 발명의 재조합 융합 단백질의 아미노 말단은 아세틸기, 플루오레닐 메톡시 카르보닐기, 포르밀기, 팔미토일기, 미리스틸기, 스테아릴기 및 폴리에틸렌 글리콜(PEG) 등의 보호기가 결합될 수 있으며, 펩타이드의 카르복시 말단은 히드록시기(-OH), 아미노기(-NH2), 아자이드(-NHNH2) 등으로 변형될 수 있다.
또한 본 발명의 융합 단백질 말단 또는 아미노산의 R-잔기(R-group)에 지방산(fatty acids), 당사슬(oligosaccharides chains), 모든 나노입자(골드입자, 리포솜, 헤파린, 하이드로젤 등), 아미노산, 담체 단백질(carrier proteins) 등을 결합할 수 있다. 상술한 아미노산의 변형은 본 발명의 단백질의 역가(potency)와 안정성을 개선하는 작용을 한다.
본 명세서에서 용어 “안정성”은 생체 내(in vivo) 안정성뿐만 아니라, 저장 안정성(상온, 냉장, 냉동 보관 시 저장 안정성 포함)도 의미한다.
한편, 본 발명은 하기의 단계를 포함하는 상기 융합단백질의 제조방법을 제공한다:
(1) 서열번호 3 내지 8 중 어느 하나의 아미노산 서열로 이루어진 융합 단백질을 암호화하는 유전자를 포함하는 재조합 벡터를 제조하는 단계;
(2) 상기 재조합 벡터를 숙주세포에 도입하여 형질전환체를 제조하는 단계;
(3) 상기 형질전환체를 배양하는 단계; 및
(4) 상기 배양액으로부터 융합 단백질을 수득하는 단계.
본 명세서에서 "유전자"란 최광의의 의미로 간주되어야 하며, 구조 단백질 또는 조절 단백질을 암호화하는 것으로서, 본원의 융합단백질 1 내지 6을 암호화하는 DNA 절편이라면 제한되지 아니하며, 구체적으로 서열번호 9 내지 14 중 하나의 염기서열을 포함할 수 있다.
또한, 본 발명에서 "벡터(vector)"는 적합한 숙주 내에서 DNA를 발현시킬 수 있는 적합한 조절 서열에 작동가능하게 연결된 DNA 서열을 함유하는 DNA 제조물을 의미한다. 벡터는 플라스미드, 파지 입자 또는 간단하게 잠재적 게놈 삽입물일 수 있다. 적당한 숙주로 형질전환되면, 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다. 플라스미드가 현재 벡터의 가장 통상적으로 사용되는 형태이므로, 본 발명의 명세서에서 플라스미드(plasmid)와 벡터(vector)는 때로 상호 교환적으로 사용된다.
염기서열은 다른 염기서열과 기능적 관계로 배치될 때 "작동가능하게 연결(operably linked)"된다. 이것은 적절한 분자(예를 들면, 전사 활성화 단백질)가 조절 서열(들)에 결합될 때 유전자 발현을 가능하게 하는 방식으로 연결된 유전자 및 조절 서열(들)일 수 있다. 예를 들면, 전서열(pre-sequence) 또는 분비 리더 (leader)에 대한 DNA는 폴리펩타이드의 분비에 참여하는 전단백질로서 발현되는 경우 폴리펩타이드에 대한 DNA에 작동가능 하게 연결되고; 프로모터 또는 인핸서는 서열의 전사에 영향을 끼치는 경우 코딩서열에 작동가능하게 연결되거나; 또는 리보좀 결합 부위는 서열의 전사에 영향을 끼치는 경우 코딩 서열에 작동가능하게 연결되거나; 또는 리보좀 결합 부위는 번역을 용이하게 하도록 배치되는 경우 코딩 서열에 작동가능하게 연결된다. 일반적으로, "작동가능하게 연결된"은 연결된 DNA 서열이 접촉하고, 또한 분비 리더의 경우 접촉하고 리딩 프레임 내에 존재하는 것을 의미한다. 그러나, 인핸서(enhancer)는 접촉할 필요가 없다. 이들 서열의 연결은 편리한 제한 효소 부위에서 라이게이션(연결)에 의해 수행된다. 그러한 부위가 존재하지 않는 경우, 통상의 방법에 따른 합성 올리고뉴클레오티드 어댑터(oligonucleotide adaptor) 또는 링커(linker)를 사용한다.
또한, 본 명세서에서 "형질전환" 또는 “형질주입”은 DNA를 숙주로 도입하여 DNA가 염색체 외 인자로서 또는 염색체 통합완성에 의해 복제가능하게 되는 것을 의미하며, 본 발명에서 융합단백질의 제조를 위하여 사용한 숙주 세포는 CHO cell 이나, 이에 제한되지 아니한다.
본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 이하 특정 실시예들을 도면에 예시하고 상세한 설명에 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.
[실험방법]
1. 간 성상세포(Hepatic stellate cells,
H
SCs)의 분리 및 배양
14주령의 수컷 BALB/c mice의 간을 먼저 PBS(phosphate-buffered saline)로 관류하고 이어서 콜라게나아제(collagenase), 프로나아제(pronase) 및 DNase를 함유하는 GBSS(Gey's balanced salt solution) 용액으로 관류시킨 후, 간을 추출하였다. 간에 부착된 담낭 및 결합 조직을 제거하고, 간 세포 현탁액을 상기 GBSS와 동일한 용액에 넣고 37℃ 에서 12분 동안 처리한 후, 13.4% Nycodenz 구배 상에서 1400 g 에서 20분 동안 원심분리하였다. Nycodenz 용액과 수성층의 경계면 상에 있는 성상세포를 취하고, 10% 소태아혈청(FBS)이 보충된 DMEM(Dulbecco's modified Eagle's medium) (Carlsbad, CA)에서 배양하였다. 성상세포의 순도는 현미경 관찰과 anti-tyrosine aminotransferase antibody를 이용한 웨스턴 블롯팅(western blotting)을 통해 평가하였다. 세포가 디쉬에 가득 차게(confluent) 되면 계대배양을 하고, 활성화된 성상세포로 이용하였다. 간 성상세포의 활성화는 형태학적 변화와 α-SMA와 collagen I의 발현 증가를 통해 확인하였다.
2. 융합 단백질의 설계 및 제작
2-1. 융합 단백질의 설계
레티노산과의 결합력 강화를 위하여 설계된 융합단백질 1 내지 6의 구체적인 정보는 하기 표 1에 나타내었다. RBP 유래의 영역은 밑줄로 표시하였으며, 알부민 Ⅲ에서 치환된 아미노산은 굵은 글자 및 밑줄로 표시하였다. 각 융합단백질의 N-말단에는 형질전환체 세포 밖으로 분비를 유도하기 위하여 signal peptide를 추가하였으며, mkwvwallllaawaaa 또는 mkwvtfisllflfssays 아미노산 서열로 이루어진 signal peptide를 선택하여 부가하였다.
서열번호 | 융합단백질 | 아미노산 서열 |
3 | 융합단백질1: RBP(17~193) + 알부민Ⅲ{404~526(F→V)~600(V→A)~601} | ERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE V NAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL A A |
4 | 융합단백질2: 알부민[Ⅲ{404~526(F→V)~600(V→A)~601}] + RBP(17~192) | LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE V NAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL A AERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG |
5 | 융합단백질3: RBP(17~193) + 알부민[ⅢA{404~517(V)} + IB(131~218)] | ERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA |
6 | 융합단백질4: RBP(17~193) + 알부민[ⅢA{404~517(V)} + IB(134~218)] | ERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA |
7 | 융합단백질5: 알부민[ⅢA{404~517(V)} + IB(131~218)] + RBP(17~192) | LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG |
8 | 융합단백질6: 알부민[ⅢA{404~517(V)} + IB(134~218)] + RBP(17~192) | LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG |
2-2. 융합 단백질의 제조
융합 단백질의 제조를 위하여 signal peptide를 포함하여 융합단백질 1 내지 6을 암호화하는 폴리뉴클레오티드를 제작하고 각 DNA 절편을 XbaI/KpnI cut pcDNA3.1(+) 벡터로 클로닝하여 재조합 발현 벡터를 제조하였다. 각 융합단백질을 암호화하는 DNA 절편의 구체적인 정보는 하기 표 2에 정리하였다. 상기 각 벡터를 리포펙타민 2000 (Invitrogen, Carlsbad, CA)을 이용하여 CHO cells에 도입하여 융합단백질을 발현하는 형질전환체를 제조하였다. 상기 형질전환체를 배양하여 발현 분비된 융합단백질을 친화 크로마토그래피(affinity chromatography) 및 HPLC법에 의해 정제하여 사용하였다.
서열번호 | 융합 단백질을 암호화하는 폴리뉴클레오티드 | 서열 |
9 | 융합단백질1 | ATGAAGTGGGTGTGGGCTCTGCTGCTGCTGGCTGCTTGGGCCGCTGCCGAGAGAGATTGCCGCGTGTCCAGCTTCAGAGTGAAGGAGAACTTCGACAAGGCCCGCTTTTCCGGCACCTGGTACGCTATGGCCAAGAAGGACCCCGAGGGCCTGTTCCTGCAGGACAATATCGTGGCTGAGTTTAGCGTGGATGAGACAGGCCAGATGTCTGCTACCGCCAAGGGCAGAGTGCGCCTGCTGAACAATTGGGACGTGTGCGCTGACATGGTGGGCACCTTCACAGATACCGAGGACCCAGCCAAGTTTAAGATGAAGTATTGGGGCGTGGCTAGCTTTCTGCAGAAGGGCAACGACGATCACTGGATCGTGGACACAGATTACGACACCTATGCCGTGCAGTACAGCTGCAGACTGCTGAACCTGGATGGCACATGTGCTGACTCTTATTCCTTCGTGTTTTCTCGCGATCCAAATGGCCTGCCCCCTGAGGCCCAGAAGATCGTGAGGCAGAGGCAGGAGGAGCTGTGCCTGGCTAGGCAGTACCGGCTGATCGTGCATAATGGCTATTGTGACGGCAGGCTGGTGGAGGAGCCCCAGAACCTGATCAAGCAGAATTGCGAGCTGTTCGAGCAGCTGGGCGAGTACAAGTTTCAGAACGCCCTGCTGGTGCGGTATACCAAGAAGGTGCCAGAGGTGTCCACACCCACCCTGGTGGAGGTGAGCAGGAATCTGGGCAAGGTCGGCTCTAAGTGCTGTAAGCACCCTGAGGCTAAGCGGATGCCATGCGCCGAGGATTACCTGTCTGTGGTGCTGAACCAGCTGTGCGTGCTGCATGAGAAGACACCCGTGTCCGACAGAGTGACAAAGTGCTGTACCGAGTCCCTGGTGAACAGGCGGCCCTGCTTCAGCGCCCTGGAGGTGGATGAGACCTATGTGCCTAAGGAGGTTAATGCTGAGACATTCACCTTTCACGCCGACATCTGTACACTGTCTGAGAAGGAGCGCCAGATCAAGAAGCAGACCGCTCTGGTGGAGCTGGTGAAGCATAAGCCTAAGGCTACAAAGGAGCAGCTGAAGGCCGTGATGGACGATTTCGCTGCCTTTGTGGAGAAGTGCTGTAAGGCTGACGATAAGGAGACCTGTTTCGCCGAGGAGGGCAAGAAGCTGGCGGCT |
10 | 융합단백질2 | ATGAAGTGGGTGACATTCATCAGCCTGCTGTTCCTGTTCTCCTCCGCCTACTCTCTGGTGGAGGAGCCCCAGAACCTGATCAAGCAGAATTGCGAGCTGTTCGAGCAGCTGGGCGAGTACAAGTTTCAGAACGCCCTGCTGGTGAGGTATACCAAGAAGGTGCCAGAGGTGTCCACCCCCACACTGGTGGAGGTGAGCAGGAATCTGGGCAAGGTCGGCTCTAAGTGCTGTAAGCACCCTGAGGCCAAGCGGATGCCATGCGCTGAGGATTACCTGTCCGTGGTGCTGAACCAGCTGTGCGTGCTGCATGAGAAGACACCTGTGAGCGACAGGGTGACAAAGTGCTGTACCGAGTCTCTGGTGAACAGGCGGCCCTGCTTCTCCGCTCTGGAGGTGGATGAGACCTATGTGCCTAAGGAGGTTAATGCCGAGACCTTCACATTTCACGCTGACATCTGTACACTGTCCGAGAAGGAGAGACAGATCAAGAAGCAGACCGCCCTGGTGGAGCTGGTGAAGCATAAGCCAAAGGCCACAAAGGAGCAGCTGAAGGCTGTGATGGACGATTTCGCCGCTTTTGTGGAGAAGTGCTGTAAGGCCGACGATAAGGAGACCTGCTTCGCTGAGGAGGGCAAGAAGCTGGCGGCCGAGAGAGATTGTCGCGTGTCTTCCTTTAGAGTGAAGGAGAACTTCGACAAGGCTCGCTTTAGCGGCACATGGTACGCTATGGCCAAGAAGGACCCCGAGGGCCTGTTCCTGCAGGACAATATCGTGGCCGAGTTTTCCGTGGATGAGACAGGCCAGATGAGCGCCACCGCTAAGGGAAGAGTGCGCCTGCTGAACAATTGGGACGTGTGCGCCGACATGGTGGGCACCTTCACAGATACCGAGGACCCCGCTAAGTTTAAGATGAAGTATTGGGGCGTGGCCTCTTTTCTGCAGAAGGGCAACGACGATCACTGGATCGTGGACACAGATTACGACACCTATGCTGTGCAGTACTCTTGCAGACTGCTGAACCTGGATGGCACCTGTGCCGACAGCTATTCTTTCGTGTTTTCCCGCGATCCTAATGGCCTGCCCCCTGAGGCTCAGAAGATCGTGAGGCAGAGGCAGGAGGAGCTGTGCCTGGCCAGGCAGTACCGGCTGATCGTGCATAATGGCTATTGTGACGGC |
11 | 융합단백질3 | ATGAAGTGGGTGTGGGCTCTGCTGCTGCTGGCTGCTTGGGCCGCTGCCGAGAGAGATTGCCGCGTGTCCAGCTTCAGAGTGAAGGAGAACTTCGACAAGGCCCGCTTTTCCGGCACCTGGTACGCTATGGCCAAGAAGGACCCCGAGGGCCTGTTCCTGCAGGACAATATCGTGGCTGAGTTTAGCGTGGATGAGACAGGCCAGATGTCTGCTACCGCCAAGGGCAGAGTGCGCCTGCTGAACAATTGGGACGTGTGCGCTGACATGGTGGGCACCTTCACAGATACCGAGGACCCAGCCAAGTTTAAGATGAAGTATTGGGGCGTGGCTAGCTTTCTGCAGAAGGGCAACGACGATCACTGGATCGTGGACACAGATTACGACACCTATGCCGTGCAGTACAGCTGCAGACTGCTGAACCTGGATGGCACATGTGCTGACTCTTATTCCTTCGTGTTTTCTCGCGATCCAAATGGCCTGCCCCCTGAGGCCCAGAAGATCGTGAGGCAGAGGCAGGAGGAGCTGTGCCTGGCTAGGCAGTACCGGCTGATCGTGCATAATGGCTATTGTGACGGCAGGCTGGTGGAGGAGCCCCAGAACCTGATCAAGCAGAATTGCGAGCTGTTCGAGCAGCTGGGCGAGTACAAGTTTCAGAACGCCCTGCTGGTGCGGTATACCAAGAAGGTGCCAGAGGTGTCCACACCCACCCTGGTGGAGGTGAGCAGGAATCTGGGCAAGGTCGGCTCTAAGTGCTGTAAGCACCCTGAGGCTAAGCGGATGCCATGCGCCGAGGATTACCTGTCTGTGGTGCTGAACCAGCTGTGCGTGCTGCATGAGAAGACACCCGTGTCCGACAGAGTGACAAAGTGCTGTACCGAGTCCCTGGTGAACAGGCGGCCCTGCTTCAGCGCCCTGGAGGTGgacgataacccaaatctgcccagactggtgcgcccagaggtggatgtgatgtgcacagctttccacgacaatgaggagacctttctgaagaagtacctgtatgagatcgccagacgccatccctacttttatgctcctgagctgctgttctttgccaagcggtacaaggctgccttcacagagtgctgtcaggctgctgacaaggctgcttgcctgctgccaaagctggatgagctgagggacgagggcaaggcttcttccgcc |
12 | 융합단백질4 | ATGAAGTGGGTGTGGGCTCTGCTGCTGCTGGCTGCTTGGGCCGCTGCCGAGAGAGATTGCCGCGTGTCCAGCTTCAGAGTGAAGGAGAACTTCGACAAGGCCCGCTTTTCCGGCACCTGGTACGCTATGGCCAAGAAGGACCCCGAGGGCCTGTTCCTGCAGGACAATATCGTGGCTGAGTTTAGCGTGGATGAGACAGGCCAGATGTCTGCTACCGCCAAGGGCAGAGTGCGCCTGCTGAACAATTGGGACGTGTGCGCTGACATGGTGGGCACCTTCACAGATACCGAGGACCCAGCCAAGTTTAAGATGAAGTATTGGGGCGTGGCTAGCTTTCTGCAGAAGGGCAACGACGATCACTGGATCGTGGACACAGATTACGACACCTATGCCGTGCAGTACAGCTGCAGACTGCTGAACCTGGATGGCACATGTGCTGACTCTTATTCCTTCGTGTTTTCTCGCGATCCAAATGGCCTGCCCCCTGAGGCCCAGAAGATCGTGAGGCAGAGGCAGGAGGAGCTGTGCCTGGCTAGGCAGTACCGGCTGATCGTGCATAATGGCTATTGTGACGGCAGGCTGGTGGAGGAGCCCCAGAACCTGATCAAGCAGAATTGCGAGCTGTTCGAGCAGCTGGGCGAGTACAAGTTTCAGAACGCCCTGCTGGTGCGGTATACCAAGAAGGTGCCAGAGGTGTCCACACCCACCCTGGTGGAGGTGAGCAGGAATCTGGGCAAGGTCGGCTCTAAGTGCTGTAAGCACCCTGAGGCTAAGCGGATGCCATGCGCCGAGGATTACCTGTCTGTGGTGCTGAACCAGCTGTGCGTGCTGCATGAGAAGACACCCGTGTCCGACAGAGTGACAAAGTGCTGTACCGAGTCCCTGGTGAACAGGCGGCCCTGCTTCAGCGCCCTGGAGGTGccaaatctgcccagactggtgcgcccagaggtggatgtgatgtgcacagctttccacgacaatgaggagacctttctgaagaagtacctgtatgagatcgccagacgccatccctacttttatgctcctgagctgctgttctttgccaagcggtacaaggctgccttcacagagtgctgtcaggctgctgacaaggctgcttgcctgctgccaaagctggatgagctgagggacgagggcaaggcttcttccgcc |
13 | 융합단백질5 | ATGAAGTGGGTGACATTCATCAGCCTGCTGTTCCTGTTCTCCTCCGCCTACTCTCTGGTGGAGGAGCCCCAGAACCTGATCAAGCAGAATTGCGAGCTGTTCGAGCAGCTGGGCGAGTACAAGTTTCAGAACGCCCTGCTGGTGAGGTATACCAAGAAGGTGCCAGAGGTGTCCACCCCCACACTGGTGGAGGTGAGCAGGAATCTGGGCAAGGTCGGCTCTAAGTGCTGTAAGCACCCTGAGGCCAAGCGGATGCCATGCGCTGAGGATTACCTGTCCGTGGTGCTGAACCAGCTGTGCGTGCTGCATGAGAAGACACCTGTGAGCGACAGGGTGACAAAGTGCTGTACCGAGTCTCTGGTGAACAGGCGGCCCTGCTTCTCCGCTCTGGAGGTGgacgataacccaaatctgcccagactggtgcgcccagaggtggatgtgatgtgcacagctttccacgacaatgaggagacctttctgaagaagtacctgtatgagatcgccagacgccatccctacttttatgctcctgagctgctgttctttgccaagcggtacaaggctgccttcacagagtgctgtcaggctgctgacaaggctgcttgcctgctgccaaagctggatgagctgagggacgagggcaaggcttcttccgccGAGAGAGATTGCCGCGTGTCCAGCTTCAGAGTGAAGGAGAACTTCGACAAGGCCCGCTTTTCCGGCACCTGGTACGCTATGGCCAAGAAGGACCCCGAGGGCCTGTTCCTGCAGGACAATATCGTGGCTGAGTTTAGCGTGGATGAGACAGGCCAGATGTCTGCTACCGCCAAGGGCAGAGTGCGCCTGCTGAACAATTGGGACGTGTGCGCTGACATGGTGGGCACCTTCACAGATACCGAGGACCCAGCCAAGTTTAAGATGAAGTATTGGGGCGTGGCTAGCTTTCTGCAGAAGGGCAACGACGATCACTGGATCGTGGACACAGATTACGACACCTATGCCGTGCAGTACAGCTGCAGACTGCTGAACCTGGATGGCACATGTGCTGACTCTTATTCCTTCGTGTTTTCTCGCGATCCAAATGGCCTGCCCCCTGAGGCCCAGAAGATCGTGAGGCAGAGGCAGGAGGAGCTGTGCCTGGCTAGGCAGTACCGGCTGATCGTGCATAATGGCTATTGTGACGGC |
14 | 융합단백질6 | ATGAAGTGGGTGACATTCATCAGCCTGCTGTTCCTGTTCTCCTCCGCCTACTCTCTGGTGGAGGAGCCCCAGAACCTGATCAAGCAGAATTGCGAGCTGTTCGAGCAGCTGGGCGAGTACAAGTTTCAGAACGCCCTGCTGGTGAGGTATACCAAGAAGGTGCCAGAGGTGTCCACCCCCACACTGGTGGAGGTGAGCAGGAATCTGGGCAAGGTCGGCTCTAAGTGCTGTAAGCACCCTGAGGCCAAGCGGATGCCATGCGCTGAGGATTACCTGTCCGTGGTGCTGAACCAGCTGTGCGTGCTGCATGAGAAGACACCTGTGAGCGACAGGGTGACAAAGTGCTGTACCGAGTCTCTGGTGAACAGGCGGCCCTGCTTCTCCGCTCTGGAGGTGccaaatctgcccagactggtgcgcccagaggtggatgtgatgtgcacagctttccacgacaatgaggagacctttctgaagaagtacctgtatgagatcgccagacgccatccctacttttatgctcctgagctgctgttctttgccaagcggtacaaggctgccttcacagagtgctgtcaggctgctgacaaggctgcttgcctgctgccaaagctggatgagctgagggacgagggcaaggcttcttccgccGAGAGAGATTGCCGCGTGTCCAGCTTCAGAGTGAAGGAGAACTTCGACAAGGCCCGCTTTTCCGGCACCTGGTACGCTATGGCCAAGAAGGACCCCGAGGGCCTGTTCCTGCAGGACAATATCGTGGCTGAGTTTAGCGTGGATGAGACAGGCCAGATGTCTGCTACCGCCAAGGGCAGAGTGCGCCTGCTGAACAATTGGGACGTGTGCGCTGACATGGTGGGCACCTTCACAGATACCGAGGACCCAGCCAAGTTTAAGATGAAGTATTGGGGCGTGGCTAGCTTTCTGCAGAAGGGCAACGACGATCACTGGATCGTGGACACAGATTACGACACCTATGCCGTGCAGTACAGCTGCAGACTGCTGAACCTGGATGGCACATGTGCTGACTCTTATTCCTTCGTGTTTTCTCGCGATCCAAATGGCCTGCCCCCTGAGGCCCAGAAGATCGTGAGGCAGAGGCAGGAGGAGCTGTGCCTGGCTAGGCAGTACCGGCTGATCGTGCATAATGGCTATTGTGACGGC |
3. 정량적 실시간 중합효소연쇄반응(Quantitative Real-Time PCR)
TRIzol (Ambion, Austin, TX, USA)를 이용하여 총 RNA를 준비하고 cDNA를 제작하였다. qRT-PCR은 ABI QuantStudio TM 3 Real-Time PCR System에서 수행하였다. 반응의 변화를 통제하기 위하여 PCR 산물은 GAPDH(glyceraldehyde 3-phosphate dehydrogenase)의 mRNA 수준으로 정규화하였다.
4. 간 섬유화 동물 모델 제조
미네랄 오일 중에 1:1로 용해시킨 CCl4를 BALB/c 마우스 복강 내 1 mL/kg 농도로 주 3회, 6주 동안 주사하여 간 손상을 유도한 간 섬유화 마우스 모델을 제조하였다. 대조군(control)은 동일한 양의 미네랄 오일 단독을 투여하였다. 최종 CCl4 주사 48시간 후 마우스를 희생시켰다.
희생된 마우스의 섬유화를 평가하기 위하여 간을 적출하고, 각 실험군의 간조직을 10% 완충 포르말린으로 고정 후 파라핀으로 포매하여 조직절편을 제작하였다. 각 조직 절편은 조직학적 분석을 위하여 시리우스 레드(Sirius red) 염색을 수행 후 광학현미경으로 관찰하였다.
5. 통계학적 분석
결과들은 평균±표준 편차 (SD)로 표현하였다. 통계학적 분석은 t-tests를 이용하여 수행하였다. 비교는 P<0.05 에서 유의도를 검정하였으며, P 수치는 양측검정을 수행하였다.
[실험결과]
실시예 1. 융합단백질 1 및 2와 종래의 알부민-RBP 융합 단백질의 효과 비교
종전 연구에서 성상세포의 활성화 억제 또는 불활성화에 우수한 효과를 나타낸 RBP-알부민Ⅲ 및 알부민Ⅲ-RBP과 실시예 2에서 설계한 융합단백질 1 및 2의 효과를 비교하였다. 구체적으로 활성화된 생쥐의 간 성상세포에 각 융합단백질 0.25 μM를 20시간 동안 처리하고 real-time PCR 을 이용하여 α-SMA과 collagen I의 발현 수준을 측정하였다. 종래의 융합 단백질(RBP-알부민Ⅲ 및 알부민Ⅲ-RBP)은 KR 10-1395394의 R-Ⅲ과 Ⅲ-R 융합 단백질이다.
그 결과, 도 2에 나타낸 바와 같이, 종래의 융합단백질과 비교하여 융합단백질 1 및 2는 α-SMA과 collagen I의 mRNA 발현을 더 많이 감소시킴을 확인할 수 있었다.
실시예 2. 융합단백질 1 내지 6의 효과 비교
활성화된 성상세포의 불활성화 효과에 있어서 종래의 융합단백질보다 우수한 효과를 확인한 융합단백질 1 및 2와 융합단백질 3 내지 6의 효과를 비교하여 확인하고자 하였다. 실시예 1과 동일한 방법으로 수행하되 활성화된 성상세포에 처리하는 융합단백질의 양은 0.125 μM로 하였다.
그 결과, 도 3에 나타낸 바와 같이, 융합단백질 3 내지 6은 보다 적은 양의 단백질량으로도 현저하게 높은 수준으로 α-SMA과 collagen I의 발현을 감소시킬 수 있음을 확인하였다.
실시예 3.
In vivo
에서 융합단백질 1 및 4와 종래의 알부민-RBP 융합 단백질의 효과 비교
사염화탄소(CCl4) 주사로 유도된 간 섬유화 모델을 이용하여 융합 단백질의 치료 효과를 비교 평가하였다. 구체적으로, 미네랄 오일 중에 1:1로 용해시킨 사염화탄소를 1ml/kg 농도로 주 3회, 6주 동안 BALB/c 마우스 복강 내 주사하고, 상기 융합 단백질 (25, 12.5, 6.25, 또는 3ug) 또는 식염수를 CCl4 처리 마지막 2주 동안 주당 3회 정맥 투여하였다(n=5마리).
광학현미경으로 조직을 관찰한 결과, 미네랄 오일만 투여한 대조군 마우스(control)는 정상적인 간 구조를 보여주나, CCl4 처리된 마우스에서는 심각한 간 조직 구조의 파괴 및 섬유화를 관찰할 수 있었다. 또한, 알부민과 RBP의 융합 단백질의 투여로 섬유화가 개선됨을 확인할 수 있었다(도 4a).
Image J Software (NIH)에 의한 Siruis red 염색의 정량 분석 결과, 섬유화 개선 효과는 시료에 따라 차이를 보였다(도 4b). 종래의 융합 단백질 RBP-알부민Ⅲ의 경우 25ug 투여군에서만 유의한 개선효과가 보인 반면, 융합 단백질 4는 6.25ug 투여에서도 우수한 효과를 보였다. 상기 결과로부터 융합 단백질 1 및 4는 종래의 융합 단백질과 비교하여 낮은 농도에서 우수한 효과를 나타냄을 확인할 수 있었다.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.
<110> Korea University Research and Buisness Foundation
<120> Recombinant fusion protein for preventing or treating fibrosis
disease
<130> APC-2021-0643-DIV1
<160> 14
<170> KoPatentIn 3.0
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<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein 1_varient
<220>
<221> VARIANT
<222> (374)
<223> X is alanine or glycine
<220>
<221> VARIANT
<222> (300)
<223> X is one of valine, alanine, and glycine
<400> 1
Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn Phe Asp
1 5 10 15
Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro
20 25 30
Glu Gly Leu Phe Leu Gln Asp Asn Ile Val Ala Glu Phe Ser Val Asp
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Glu Thr Gly Gln Met Ser Ala Thr Ala Lys Gly Arg Val Arg Leu Leu
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Asn Asn Trp Asp Val Cys Ala Asp Met Val Gly Thr Phe Thr Asp Thr
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Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala Ser Phe
85 90 95
Leu Gln Lys Gly Asn Asp Asp His Trp Ile Val Asp Thr Asp Tyr Asp
100 105 110
Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr
115 120 125
Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn Gly Leu
130 135 140
Pro Pro Glu Ala Gln Lys Ile Val Arg Gln Arg Gln Glu Glu Leu Cys
145 150 155 160
Leu Ala Arg Gln Tyr Arg Leu Ile Val His Asn Gly Tyr Cys Asp Gly
165 170 175
Arg Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu
180 185 190
Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg
195 200 205
Tyr Thr Lys Lys Val Pro Glu Val Ser Thr Pro Thr Leu Val Glu Val
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Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu
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Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
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Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr
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Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
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Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Xaa Asn Ala Glu Thr
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Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln
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Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys
325 330 335
Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe
340 345 350
Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu
355 360 365
Glu Gly Lys Lys Leu Xaa Ala
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<210> 2
<211> 374
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein 2_varient
<220>
<221> VARIANT
<222> (197)
<223> X is alanine or glycine
<220>
<221> VARIANT
<222> (123)
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Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe
1 5 10 15
Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
20 25 30
Thr Lys Lys Val Pro Glu Val Ser Thr Pro Thr Leu Val Glu Val Ser
35 40 45
Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
50 55 60
Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
65 70 75 80
Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys
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Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
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Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Xaa Asn Ala Glu Thr Phe
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Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile
130 135 140
Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala
145 150 155 160
Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val
165 170 175
Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
180 185 190
Gly Lys Lys Leu Xaa Ala Glu Arg Asp Cys Arg Val Ser Ser Phe Arg
195 200 205
Val Lys Glu Asn Phe Asp Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala
210 215 220
Met Ala Lys Lys Asp Pro Glu Gly Leu Phe Leu Gln Asp Asn Ile Val
225 230 235 240
Ala Glu Phe Ser Val Asp Glu Thr Gly Gln Met Ser Ala Thr Ala Lys
245 250 255
Gly Arg Val Arg Leu Leu Asn Asn Trp Asp Val Cys Ala Asp Met Val
260 265 270
Gly Thr Phe Thr Asp Thr Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr
275 280 285
Trp Gly Val Ala Ser Phe Leu Gln Lys Gly Asn Asp Asp His Trp Ile
290 295 300
Val Asp Thr Asp Tyr Asp Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu
305 310 315 320
Leu Asn Leu Asp Gly Thr Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser
325 330 335
Arg Asp Pro Asn Gly Leu Pro Pro Glu Ala Gln Lys Ile Val Arg Gln
340 345 350
Arg Gln Glu Glu Leu Cys Leu Ala Arg Gln Tyr Arg Leu Ile Val His
355 360 365
Asn Gly Tyr Cys Asp Gly
370
<210> 3
<211> 375
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein 1
<400> 3
Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn Phe Asp
1 5 10 15
Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro
20 25 30
Glu Gly Leu Phe Leu Gln Asp Asn Ile Val Ala Glu Phe Ser Val Asp
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Glu Thr Gly Gln Met Ser Ala Thr Ala Lys Gly Arg Val Arg Leu Leu
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Asn Asn Trp Asp Val Cys Ala Asp Met Val Gly Thr Phe Thr Asp Thr
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Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala Ser Phe
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Leu Gln Lys Gly Asn Asp Asp His Trp Ile Val Asp Thr Asp Tyr Asp
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Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr
115 120 125
Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn Gly Leu
130 135 140
Pro Pro Glu Ala Gln Lys Ile Val Arg Gln Arg Gln Glu Glu Leu Cys
145 150 155 160
Leu Ala Arg Gln Tyr Arg Leu Ile Val His Asn Gly Tyr Cys Asp Gly
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Arg Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu
180 185 190
Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg
195 200 205
Tyr Thr Lys Lys Val Pro Glu Val Ser Thr Pro Thr Leu Val Glu Val
210 215 220
Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu
225 230 235 240
Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
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Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr
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Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
275 280 285
Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Val Asn Ala Glu Thr
290 295 300
Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln
305 310 315 320
Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys
325 330 335
Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe
340 345 350
Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu
355 360 365
Glu Gly Lys Lys Leu Ala Ala
370 375
<210> 4
<211> 374
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein 2
<400> 4
Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe
1 5 10 15
Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
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Thr Lys Lys Val Pro Glu Val Ser Thr Pro Thr Leu Val Glu Val Ser
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Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
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Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
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Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys
85 90 95
Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
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Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Val Asn Ala Glu Thr Phe
115 120 125
Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile
130 135 140
Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala
145 150 155 160
Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val
165 170 175
Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
180 185 190
Gly Lys Lys Leu Ala Ala Glu Arg Asp Cys Arg Val Ser Ser Phe Arg
195 200 205
Val Lys Glu Asn Phe Asp Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala
210 215 220
Met Ala Lys Lys Asp Pro Glu Gly Leu Phe Leu Gln Asp Asn Ile Val
225 230 235 240
Ala Glu Phe Ser Val Asp Glu Thr Gly Gln Met Ser Ala Thr Ala Lys
245 250 255
Gly Arg Val Arg Leu Leu Asn Asn Trp Asp Val Cys Ala Asp Met Val
260 265 270
Gly Thr Phe Thr Asp Thr Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr
275 280 285
Trp Gly Val Ala Ser Phe Leu Gln Lys Gly Asn Asp Asp His Trp Ile
290 295 300
Val Asp Thr Asp Tyr Asp Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu
305 310 315 320
Leu Asn Leu Asp Gly Thr Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser
325 330 335
Arg Asp Pro Asn Gly Leu Pro Pro Glu Ala Gln Lys Ile Val Arg Gln
340 345 350
Arg Gln Glu Glu Leu Cys Leu Ala Arg Gln Tyr Arg Leu Ile Val His
355 360 365
Asn Gly Tyr Cys Asp Gly
370
<210> 5
<211> 379
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein 3
<400> 5
Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn Phe Asp
1 5 10 15
Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro
20 25 30
Glu Gly Leu Phe Leu Gln Asp Asn Ile Val Ala Glu Phe Ser Val Asp
35 40 45
Glu Thr Gly Gln Met Ser Ala Thr Ala Lys Gly Arg Val Arg Leu Leu
50 55 60
Asn Asn Trp Asp Val Cys Ala Asp Met Val Gly Thr Phe Thr Asp Thr
65 70 75 80
Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala Ser Phe
85 90 95
Leu Gln Lys Gly Asn Asp Asp His Trp Ile Val Asp Thr Asp Tyr Asp
100 105 110
Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr
115 120 125
Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn Gly Leu
130 135 140
Pro Pro Glu Ala Gln Lys Ile Val Arg Gln Arg Gln Glu Glu Leu Cys
145 150 155 160
Leu Ala Arg Gln Tyr Arg Leu Ile Val His Asn Gly Tyr Cys Asp Gly
165 170 175
Arg Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu
180 185 190
Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg
195 200 205
Tyr Thr Lys Lys Val Pro Glu Val Ser Thr Pro Thr Leu Val Glu Val
210 215 220
Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu
225 230 235 240
Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
245 250 255
Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr
260 265 270
Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
275 280 285
Leu Glu Val Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu
290 295 300
Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu
305 310 315 320
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala
325 330 335
Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu
340 345 350
Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp
355 360 365
Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala
370 375
<210> 6
<211> 376
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein 4
<400> 6
Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn Phe Asp
1 5 10 15
Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro
20 25 30
Glu Gly Leu Phe Leu Gln Asp Asn Ile Val Ala Glu Phe Ser Val Asp
35 40 45
Glu Thr Gly Gln Met Ser Ala Thr Ala Lys Gly Arg Val Arg Leu Leu
50 55 60
Asn Asn Trp Asp Val Cys Ala Asp Met Val Gly Thr Phe Thr Asp Thr
65 70 75 80
Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala Ser Phe
85 90 95
Leu Gln Lys Gly Asn Asp Asp His Trp Ile Val Asp Thr Asp Tyr Asp
100 105 110
Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr
115 120 125
Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn Gly Leu
130 135 140
Pro Pro Glu Ala Gln Lys Ile Val Arg Gln Arg Gln Glu Glu Leu Cys
145 150 155 160
Leu Ala Arg Gln Tyr Arg Leu Ile Val His Asn Gly Tyr Cys Asp Gly
165 170 175
Arg Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu
180 185 190
Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg
195 200 205
Tyr Thr Lys Lys Val Pro Glu Val Ser Thr Pro Thr Leu Val Glu Val
210 215 220
Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu
225 230 235 240
Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
245 250 255
Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr
260 265 270
Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
275 280 285
Leu Glu Val Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val
290 295 300
Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
305 310 315 320
Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu
325 330 335
Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln
340 345 350
Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg
355 360 365
Asp Glu Gly Lys Ala Ser Ser Ala
370 375
<210> 7
<211> 378
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein 5
<400> 7
Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe
1 5 10 15
Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
20 25 30
Thr Lys Lys Val Pro Glu Val Ser Thr Pro Thr Leu Val Glu Val Ser
35 40 45
Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
50 55 60
Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
65 70 75 80
Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys
85 90 95
Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
100 105 110
Glu Val Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val
115 120 125
Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys
130 135 140
Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro
145 150 155 160
Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys
165 170 175
Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu
180 185 190
Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Glu Arg Asp Cys Arg Val
195 200 205
Ser Ser Phe Arg Val Lys Glu Asn Phe Asp Lys Ala Arg Phe Ser Gly
210 215 220
Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro Glu Gly Leu Phe Leu Gln
225 230 235 240
Asp Asn Ile Val Ala Glu Phe Ser Val Asp Glu Thr Gly Gln Met Ser
245 250 255
Ala Thr Ala Lys Gly Arg Val Arg Leu Leu Asn Asn Trp Asp Val Cys
260 265 270
Ala Asp Met Val Gly Thr Phe Thr Asp Thr Glu Asp Pro Ala Lys Phe
275 280 285
Lys Met Lys Tyr Trp Gly Val Ala Ser Phe Leu Gln Lys Gly Asn Asp
290 295 300
Asp His Trp Ile Val Asp Thr Asp Tyr Asp Thr Tyr Ala Val Gln Tyr
305 310 315 320
Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr Cys Ala Asp Ser Tyr Ser
325 330 335
Phe Val Phe Ser Arg Asp Pro Asn Gly Leu Pro Pro Glu Ala Gln Lys
340 345 350
Ile Val Arg Gln Arg Gln Glu Glu Leu Cys Leu Ala Arg Gln Tyr Arg
355 360 365
Leu Ile Val His Asn Gly Tyr Cys Asp Gly
370 375
<210> 8
<211> 375
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein 6
<400> 8
Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe
1 5 10 15
Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
20 25 30
Thr Lys Lys Val Pro Glu Val Ser Thr Pro Thr Leu Val Glu Val Ser
35 40 45
Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
50 55 60
Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
65 70 75 80
Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys
85 90 95
Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
100 105 110
Glu Val Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met
115 120 125
Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
130 135 140
Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu
145 150 155 160
Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala
165 170 175
Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp
180 185 190
Glu Gly Lys Ala Ser Ser Ala Glu Arg Asp Cys Arg Val Ser Ser Phe
195 200 205
Arg Val Lys Glu Asn Phe Asp Lys Ala Arg Phe Ser Gly Thr Trp Tyr
210 215 220
Ala Met Ala Lys Lys Asp Pro Glu Gly Leu Phe Leu Gln Asp Asn Ile
225 230 235 240
Val Ala Glu Phe Ser Val Asp Glu Thr Gly Gln Met Ser Ala Thr Ala
245 250 255
Lys Gly Arg Val Arg Leu Leu Asn Asn Trp Asp Val Cys Ala Asp Met
260 265 270
Val Gly Thr Phe Thr Asp Thr Glu Asp Pro Ala Lys Phe Lys Met Lys
275 280 285
Tyr Trp Gly Val Ala Ser Phe Leu Gln Lys Gly Asn Asp Asp His Trp
290 295 300
Ile Val Asp Thr Asp Tyr Asp Thr Tyr Ala Val Gln Tyr Ser Cys Arg
305 310 315 320
Leu Leu Asn Leu Asp Gly Thr Cys Ala Asp Ser Tyr Ser Phe Val Phe
325 330 335
Ser Arg Asp Pro Asn Gly Leu Pro Pro Glu Ala Gln Lys Ile Val Arg
340 345 350
Gln Arg Gln Glu Glu Leu Cys Leu Ala Arg Gln Tyr Arg Leu Ile Val
355 360 365
His Asn Gly Tyr Cys Asp Gly
370 375
<210> 9
<211> 1173
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA encoding fusion protein 1
<400> 9
atgaagtggg tgtgggctct gctgctgctg gctgcttggg ccgctgccga gagagattgc 60
cgcgtgtcca gcttcagagt gaaggagaac ttcgacaagg cccgcttttc cggcacctgg 120
tacgctatgg ccaagaagga ccccgagggc ctgttcctgc aggacaatat cgtggctgag 180
tttagcgtgg atgagacagg ccagatgtct gctaccgcca agggcagagt gcgcctgctg 240
aacaattggg acgtgtgcgc tgacatggtg ggcaccttca cagataccga ggacccagcc 300
aagtttaaga tgaagtattg gggcgtggct agctttctgc agaagggcaa cgacgatcac 360
tggatcgtgg acacagatta cgacacctat gccgtgcagt acagctgcag actgctgaac 420
ctggatggca catgtgctga ctcttattcc ttcgtgtttt ctcgcgatcc aaatggcctg 480
ccccctgagg cccagaagat cgtgaggcag aggcaggagg agctgtgcct ggctaggcag 540
taccggctga tcgtgcataa tggctattgt gacggcaggc tggtggagga gccccagaac 600
ctgatcaagc agaattgcga gctgttcgag cagctgggcg agtacaagtt tcagaacgcc 660
ctgctggtgc ggtataccaa gaaggtgcca gaggtgtcca cacccaccct ggtggaggtg 720
agcaggaatc tgggcaaggt cggctctaag tgctgtaagc accctgaggc taagcggatg 780
ccatgcgccg aggattacct gtctgtggtg ctgaaccagc tgtgcgtgct gcatgagaag 840
acacccgtgt ccgacagagt gacaaagtgc tgtaccgagt ccctggtgaa caggcggccc 900
tgcttcagcg ccctggaggt ggatgagacc tatgtgccta aggaggttaa tgctgagaca 960
ttcacctttc acgccgacat ctgtacactg tctgagaagg agcgccagat caagaagcag 1020
accgctctgg tggagctggt gaagcataag cctaaggcta caaaggagca gctgaaggcc 1080
gtgatggacg atttcgctgc ctttgtggag aagtgctgta aggctgacga taaggagacc 1140
tgtttcgccg aggagggcaa gaagctggcg gct 1173
<210> 10
<211> 1176
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA encoding fusion protein 2
<400> 10
atgaagtggg tgacattcat cagcctgctg ttcctgttct cctccgccta ctctctggtg 60
gaggagcccc agaacctgat caagcagaat tgcgagctgt tcgagcagct gggcgagtac 120
aagtttcaga acgccctgct ggtgaggtat accaagaagg tgccagaggt gtccaccccc 180
acactggtgg aggtgagcag gaatctgggc aaggtcggct ctaagtgctg taagcaccct 240
gaggccaagc ggatgccatg cgctgaggat tacctgtccg tggtgctgaa ccagctgtgc 300
gtgctgcatg agaagacacc tgtgagcgac agggtgacaa agtgctgtac cgagtctctg 360
gtgaacaggc ggccctgctt ctccgctctg gaggtggatg agacctatgt gcctaaggag 420
gttaatgccg agaccttcac atttcacgct gacatctgta cactgtccga gaaggagaga 480
cagatcaaga agcagaccgc cctggtggag ctggtgaagc ataagccaaa ggccacaaag 540
gagcagctga aggctgtgat ggacgatttc gccgcttttg tggagaagtg ctgtaaggcc 600
gacgataagg agacctgctt cgctgaggag ggcaagaagc tggcggccga gagagattgt 660
cgcgtgtctt cctttagagt gaaggagaac ttcgacaagg ctcgctttag cggcacatgg 720
tacgctatgg ccaagaagga ccccgagggc ctgttcctgc aggacaatat cgtggccgag 780
ttttccgtgg atgagacagg ccagatgagc gccaccgcta agggaagagt gcgcctgctg 840
aacaattggg acgtgtgcgc cgacatggtg ggcaccttca cagataccga ggaccccgct 900
aagtttaaga tgaagtattg gggcgtggcc tcttttctgc agaagggcaa cgacgatcac 960
tggatcgtgg acacagatta cgacacctat gctgtgcagt actcttgcag actgctgaac 1020
ctggatggca cctgtgccga cagctattct ttcgtgtttt cccgcgatcc taatggcctg 1080
ccccctgagg ctcagaagat cgtgaggcag aggcaggagg agctgtgcct ggccaggcag 1140
taccggctga tcgtgcataa tggctattgt gacggc 1176
<210> 11
<211> 1185
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA encoding fusion protein 3
<400> 11
atgaagtggg tgtgggctct gctgctgctg gctgcttggg ccgctgccga gagagattgc 60
cgcgtgtcca gcttcagagt gaaggagaac ttcgacaagg cccgcttttc cggcacctgg 120
tacgctatgg ccaagaagga ccccgagggc ctgttcctgc aggacaatat cgtggctgag 180
tttagcgtgg atgagacagg ccagatgtct gctaccgcca agggcagagt gcgcctgctg 240
aacaattggg acgtgtgcgc tgacatggtg ggcaccttca cagataccga ggacccagcc 300
aagtttaaga tgaagtattg gggcgtggct agctttctgc agaagggcaa cgacgatcac 360
tggatcgtgg acacagatta cgacacctat gccgtgcagt acagctgcag actgctgaac 420
ctggatggca catgtgctga ctcttattcc ttcgtgtttt ctcgcgatcc aaatggcctg 480
ccccctgagg cccagaagat cgtgaggcag aggcaggagg agctgtgcct ggctaggcag 540
taccggctga tcgtgcataa tggctattgt gacggcaggc tggtggagga gccccagaac 600
ctgatcaagc agaattgcga gctgttcgag cagctgggcg agtacaagtt tcagaacgcc 660
ctgctggtgc ggtataccaa gaaggtgcca gaggtgtcca cacccaccct ggtggaggtg 720
agcaggaatc tgggcaaggt cggctctaag tgctgtaagc accctgaggc taagcggatg 780
ccatgcgccg aggattacct gtctgtggtg ctgaaccagc tgtgcgtgct gcatgagaag 840
acacccgtgt ccgacagagt gacaaagtgc tgtaccgagt ccctggtgaa caggcggccc 900
tgcttcagcg ccctggaggt ggacgataac ccaaatctgc ccagactggt gcgcccagag 960
gtggatgtga tgtgcacagc tttccacgac aatgaggaga cctttctgaa gaagtacctg 1020
tatgagatcg ccagacgcca tccctacttt tatgctcctg agctgctgtt ctttgccaag 1080
cggtacaagg ctgccttcac agagtgctgt caggctgctg acaaggctgc ttgcctgctg 1140
ccaaagctgg atgagctgag ggacgagggc aaggcttctt ccgcc 1185
<210> 12
<211> 1176
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA encoding fusion protein 4
<400> 12
atgaagtggg tgtgggctct gctgctgctg gctgcttggg ccgctgccga gagagattgc 60
cgcgtgtcca gcttcagagt gaaggagaac ttcgacaagg cccgcttttc cggcacctgg 120
tacgctatgg ccaagaagga ccccgagggc ctgttcctgc aggacaatat cgtggctgag 180
tttagcgtgg atgagacagg ccagatgtct gctaccgcca agggcagagt gcgcctgctg 240
aacaattggg acgtgtgcgc tgacatggtg ggcaccttca cagataccga ggacccagcc 300
aagtttaaga tgaagtattg gggcgtggct agctttctgc agaagggcaa cgacgatcac 360
tggatcgtgg acacagatta cgacacctat gccgtgcagt acagctgcag actgctgaac 420
ctggatggca catgtgctga ctcttattcc ttcgtgtttt ctcgcgatcc aaatggcctg 480
ccccctgagg cccagaagat cgtgaggcag aggcaggagg agctgtgcct ggctaggcag 540
taccggctga tcgtgcataa tggctattgt gacggcaggc tggtggagga gccccagaac 600
ctgatcaagc agaattgcga gctgttcgag cagctgggcg agtacaagtt tcagaacgcc 660
ctgctggtgc ggtataccaa gaaggtgcca gaggtgtcca cacccaccct ggtggaggtg 720
agcaggaatc tgggcaaggt cggctctaag tgctgtaagc accctgaggc taagcggatg 780
ccatgcgccg aggattacct gtctgtggtg ctgaaccagc tgtgcgtgct gcatgagaag 840
acacccgtgt ccgacagagt gacaaagtgc tgtaccgagt ccctggtgaa caggcggccc 900
tgcttcagcg ccctggaggt gccaaatctg cccagactgg tgcgcccaga ggtggatgtg 960
atgtgcacag ctttccacga caatgaggag acctttctga agaagtacct gtatgagatc 1020
gccagacgcc atccctactt ttatgctcct gagctgctgt tctttgccaa gcggtacaag 1080
gctgccttca cagagtgctg tcaggctgct gacaaggctg cttgcctgct gccaaagctg 1140
gatgagctga gggacgaggg caaggcttct tccgcc 1176
<210> 13
<211> 1188
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA encoding fusion protein 5
<400> 13
atgaagtggg tgacattcat cagcctgctg ttcctgttct cctccgccta ctctctggtg 60
gaggagcccc agaacctgat caagcagaat tgcgagctgt tcgagcagct gggcgagtac 120
aagtttcaga acgccctgct ggtgaggtat accaagaagg tgccagaggt gtccaccccc 180
acactggtgg aggtgagcag gaatctgggc aaggtcggct ctaagtgctg taagcaccct 240
gaggccaagc ggatgccatg cgctgaggat tacctgtccg tggtgctgaa ccagctgtgc 300
gtgctgcatg agaagacacc tgtgagcgac agggtgacaa agtgctgtac cgagtctctg 360
gtgaacaggc ggccctgctt ctccgctctg gaggtggacg ataacccaaa tctgcccaga 420
ctggtgcgcc cagaggtgga tgtgatgtgc acagctttcc acgacaatga ggagaccttt 480
ctgaagaagt acctgtatga gatcgccaga cgccatccct acttttatgc tcctgagctg 540
ctgttctttg ccaagcggta caaggctgcc ttcacagagt gctgtcaggc tgctgacaag 600
gctgcttgcc tgctgccaaa gctggatgag ctgagggacg agggcaaggc ttcttccgcc 660
gagagagatt gccgcgtgtc cagcttcaga gtgaaggaga acttcgacaa ggcccgcttt 720
tccggcacct ggtacgctat ggccaagaag gaccccgagg gcctgttcct gcaggacaat 780
atcgtggctg agtttagcgt ggatgagaca ggccagatgt ctgctaccgc caagggcaga 840
gtgcgcctgc tgaacaattg ggacgtgtgc gctgacatgg tgggcacctt cacagatacc 900
gaggacccag ccaagtttaa gatgaagtat tggggcgtgg ctagctttct gcagaagggc 960
aacgacgatc actggatcgt ggacacagat tacgacacct atgccgtgca gtacagctgc 1020
agactgctga acctggatgg cacatgtgct gactcttatt ccttcgtgtt ttctcgcgat 1080
ccaaatggcc tgccccctga ggcccagaag atcgtgaggc agaggcagga ggagctgtgc 1140
ctggctaggc agtaccggct gatcgtgcat aatggctatt gtgacggc 1188
<210> 14
<211> 1179
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA encoding fusion protein 6
<400> 14
atgaagtggg tgacattcat cagcctgctg ttcctgttct cctccgccta ctctctggtg 60
gaggagcccc agaacctgat caagcagaat tgcgagctgt tcgagcagct gggcgagtac 120
aagtttcaga acgccctgct ggtgaggtat accaagaagg tgccagaggt gtccaccccc 180
acactggtgg aggtgagcag gaatctgggc aaggtcggct ctaagtgctg taagcaccct 240
gaggccaagc ggatgccatg cgctgaggat tacctgtccg tggtgctgaa ccagctgtgc 300
gtgctgcatg agaagacacc tgtgagcgac agggtgacaa agtgctgtac cgagtctctg 360
gtgaacaggc ggccctgctt ctccgctctg gaggtgccaa atctgcccag actggtgcgc 420
ccagaggtgg atgtgatgtg cacagctttc cacgacaatg aggagacctt tctgaagaag 480
tacctgtatg agatcgccag acgccatccc tacttttatg ctcctgagct gctgttcttt 540
gccaagcggt acaaggctgc cttcacagag tgctgtcagg ctgctgacaa ggctgcttgc 600
ctgctgccaa agctggatga gctgagggac gagggcaagg cttcttccgc cgagagagat 660
tgccgcgtgt ccagcttcag agtgaaggag aacttcgaca aggcccgctt ttccggcacc 720
tggtacgcta tggccaagaa ggaccccgag ggcctgttcc tgcaggacaa tatcgtggct 780
gagtttagcg tggatgagac aggccagatg tctgctaccg ccaagggcag agtgcgcctg 840
ctgaacaatt gggacgtgtg cgctgacatg gtgggcacct tcacagatac cgaggaccca 900
gccaagttta agatgaagta ttggggcgtg gctagctttc tgcagaaggg caacgacgat 960
cactggatcg tggacacaga ttacgacacc tatgccgtgc agtacagctg cagactgctg 1020
aacctggatg gcacatgtgc tgactcttat tccttcgtgt tttctcgcga tccaaatggc 1080
ctgccccctg aggcccagaa gatcgtgagg cagaggcagg aggagctgtg cctggctagg 1140
cagtaccggc tgatcgtgca taatggctat tgtgacggc 1179
Claims (8)
- 서열번호 1 또는 2의 아미노산 서열로 이루어진 융합 단백질.
- 서열번호 3 내지 8 중 어느 하나의 아미노산 서열로 이루어진 융합 단백질.
- 제2항의 융합 단백질과 signal peptide를 암호화하는 폴리뉴클레오티드로서, 상기 폴리뉴클레오티드는 서열번호 9 내지 14 중 어느 하나의 염기서열로 이루어진 폴리뉴클레오티드.
- 제3항의 폴리뉴클레오티드를 포함하는 재조합 벡터.
- 제4항의 재조합 벡터를 포함하는 형질전환체.
- (1) 서열번호 3 내지 8 중 어느 하나의 아미노산 서열로 이루어진 융합 단백질을 암호화하는 유전자를 포함하는 재조합 벡터를 제조하는 단계;
(2) 상기 재조합 벡터를 숙주세포에 도입하여 형질전환체를 제조하는 단계;
(3) 상기 형질전환체를 배양하는 단계; 및
(4) 상기 배양액으로부터 융합 단백질을 수득하는 단계를 포함하는 융합 단백질의 생산 방법. - 서열번호 1 내지 8 중 어느 하나의 아미노산 서열로 이루어진 융합 단백질을 포함하는, 섬유질환 예방 또는 치료용 약학적 조성물.
- 제7항에 있어서,
상기 섬유질환은 간 섬유증(liver fibrosis), 만성간염 (chronic hepatitis), 간경화 (cirrhosis), 간암(hepatic cancer), 화학요법-연관 지방간염(chemotherapy-associated steatohepatitis, CASH), 폐 섬유증(lung fibrosis), 신장 섬유증(renal fibrosis), 신부전(renal failure), 췌장 섬유증 (pancreatic fibrosis), 만성 췌장염(chronic pancreatitis) 및 췌장암(pancreatic cancer)으로 이루어진 군으로부터 선택되는 1 종 이상의 질환인 것을 특징으로 하는, 약학적 조성물.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR20200044982 | 2020-04-14 | ||
KR1020200044982 | 2020-04-14 | ||
KR1020210040819A KR20210127618A (ko) | 2020-04-14 | 2021-03-30 | 섬유질환 예방 또는 치료용 재조합 융합 단백질 |
Related Parent Applications (1)
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US (1) | US20230357356A1 (ko) |
EP (1) | EP4137507A4 (ko) |
JP (1) | JP2023521906A (ko) |
KR (1) | KR102638021B1 (ko) |
CN (1) | CN115605502A (ko) |
AU (1) | AU2021255518A1 (ko) |
BR (1) | BR112022020350A2 (ko) |
CA (1) | CA3175503A1 (ko) |
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EP2277889A2 (en) * | 2001-12-21 | 2011-01-26 | Human Genome Sciences, Inc. | Fusion proteins of albumin and interferon beta |
KR20120101617A (ko) * | 2011-02-28 | 2012-09-14 | 고려대학교 산학협력단 | 알부민과 레티놀 결합 단백질의 융합 단백질 |
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- 2021-03-31 AU AU2021255518A patent/AU2021255518A1/en active Pending
- 2021-03-31 EP EP21789246.2A patent/EP4137507A4/en active Pending
- 2021-03-31 CA CA3175503A patent/CA3175503A1/en active Pending
- 2021-03-31 WO PCT/KR2021/003959 patent/WO2021210812A1/ko unknown
- 2021-03-31 CN CN202180028847.9A patent/CN115605502A/zh not_active Withdrawn
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2277889A2 (en) * | 2001-12-21 | 2011-01-26 | Human Genome Sciences, Inc. | Fusion proteins of albumin and interferon beta |
KR20120101617A (ko) * | 2011-02-28 | 2012-09-14 | 고려대학교 산학협력단 | 알부민과 레티놀 결합 단백질의 융합 단백질 |
KR101395394B1 (ko) | 2011-02-28 | 2014-05-14 | 고려대학교 산학협력단 | 알부민과 레티놀 결합 단백질의 융합 단백질 |
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EP4137507A1 (en) | 2023-02-22 |
AU2021255518A1 (en) | 2022-11-03 |
WO2021210812A1 (ko) | 2021-10-21 |
CN115605502A (zh) | 2023-01-13 |
US20230357356A1 (en) | 2023-11-09 |
BR112022020350A2 (pt) | 2022-11-22 |
EP4137507A4 (en) | 2024-04-24 |
CA3175503A1 (en) | 2021-10-21 |
KR102638021B1 (ko) | 2024-02-20 |
JP2023521906A (ja) | 2023-05-25 |
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