KR102554313B1 - Therapeutic protein CTP delta for treating coronavirus disease COVID-19 and use thereof - Google Patents
Therapeutic protein CTP delta for treating coronavirus disease COVID-19 and use thereof Download PDFInfo
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- KR102554313B1 KR102554313B1 KR1020200157149A KR20200157149A KR102554313B1 KR 102554313 B1 KR102554313 B1 KR 102554313B1 KR 1020200157149 A KR1020200157149 A KR 1020200157149A KR 20200157149 A KR20200157149 A KR 20200157149A KR 102554313 B1 KR102554313 B1 KR 102554313B1
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Abstract
본 발명은 코로나바이러스 감염증 COVID-19 치료제 단백질 CTP 델타 및 이의 용도에 관한 것으로서, 기존에 알려진 SARS-CoV의 RBD와 ACE2의 결합부위를 모사하는 펩타이드(P6)에 비해, 본 발명의 치료제 단백질 CTP 델타는 SARS-CoV2 RBD의 새로운 에피토프와의 결합을 더욱 강하게 만들기 위해 아미노산을 구성하는 원자들 차원의 상호작용을 원천적으로 디자인한, 아미노산의 새로운 서열을 추가한 새로운 부분으로 구성되어 있다. 본 발명에서는, RBD와 hACE2 사이의 기존에 알려진 결합 경계면의 후면에 있는 R454, K458, D467, E471의 전하를 띄는(Charged) 아미노산들과 추가적으로 상호작용할 수 있는, 확장된 치료제 단백질 CTP 델타를 독창적으로 디자인하여 기존 알려진 펩타이드 보다 강하게 결합할 수 있는 새로운 디자인의 치료제 단백질 CTP 델타를 제시하였으며, 본 발명의 치료제 단백질 CTP 델타는 향후 COVID-19 치료제로서 높은 활용 가능성을 나타낸다.The present invention relates to the therapeutic protein CTP delta for coronavirus infection COVID-19 and its use, compared to the previously known peptide (P6) that mimics the binding site of RBD and ACE2 of SARS-CoV, the therapeutic protein CTP delta of the present invention consists of a new part with the addition of a new sequence of amino acids, originally designed for interactions at the atomic level constituting amino acids to make the binding with the new epitope of SARS-CoV2 RBD stronger. In the present invention, an extended therapeutic protein, CTP delta, that can additionally interact with charged amino acids of R454, K458, D467, and E471 on the back side of the previously known binding interface between RBD and hACE2 has been developed. We designed and presented a new design therapeutic protein CTP delta that can bind more strongly than previously known peptides, and the therapeutic protein CTP delta of the present invention shows high potential for use as a future COVID-19 treatment.
Description
본 발명은 코로나바이러스 감염증 COVID-19 치료제 단백질 CTP 델타(COVID-19 Therapeutic Protein Delta) 및 이의 용도에 관한 것이다.The present invention relates to a coronavirus infection COVID-19 therapeutic protein CTP delta (COVID-19 Therapeutic Protein Delta) and its use.
COVID-19 질병을 일으키는 코로나 바이러스 SARS-CoV2는 인간 폐 상피세포에 주로 존재하는 안지오텐신 변환 효소 2(Angiotensin-converting enzyme 2; hACE2) 단백질에 결합하여 세포 내로 침입하게 되며 바이러스의 유전물질을 인간 세포 내부에서 복제시켜 바이러스를 재생산한다. 코로나 바이러스가 인간 세포로 침입하는 것을 방지하는 전략을 찾는 것이 COVID-19 감염병을 치료하는 근본적인 방법이다.SARS-CoV2, the coronavirus that causes the COVID-19 disease, binds to the Angiotensin-converting enzyme 2 (hACE2) protein, which is mainly present in human lung epithelial cells, invades cells and transfers the genetic material of the virus into human cells. replicates and reproduces the virus. Finding strategies to prevent the coronavirus from entering human cells is a fundamental way to treat the COVID-19 pandemic.
코로나 바이러스의 표면에 존재하는 스파이크(Spike) 단백질 S1-RBD(Receptor Binding Domain)은 hACE2 단백질에 결합하며, 이때 결합된 3차원 구조에 대해서는 최근 연구들에 의하여 밝혀져 있다(Jun Lan, et al., Nature, 2020).The spike protein S1-RBD (Receptor Binding Domain) present on the surface of the corona virus binds to the hACE2 protein, and the three-dimensional structure of the binding at this time has been revealed by recent studies (Jun Lan, et al., Nature, 2020).
RBD와 hACE2의 결합을 차단할 수 있으면 근본적으로 코로나 바이러스의 세포 침입을 막을 수 있으며 코로나 바이러스의 활성을 극단적으로 낮추거나 감염력을 없애 COVID-19 감염병을 치료할 수 있다. 코로나 바이러스의 RBD를 중화하는 방법 등이 현재 치료제 개발의 원리 중 하나로써 활발하게 연구되고 있다.If you can block the combination of RBD and hACE2, you can fundamentally prevent the cell invasion of the coronavirus and treat the COVID-19 infectious disease by extremely lowering the activity of the coronavirus or eliminating its infectivity. A method of neutralizing the RBD of the coronavirus is currently being actively studied as one of the principles of therapeutic development.
RBD를 중화하는 방식에는 RBD가 세포 표면의 hACE2와 결합하지 못하도록 hACE2 대신 RBD에 다른 물질이 미리 결합하도록 하는 방법들이 연구되고 있다. 기존 hACE2 기반으로 개발되거나 제안된 RBD 중화 치료제로써 폐암치료제로 쓰이는 인간 재조합 수용성 ACE2(human recombinant soluble ACE2; hrsACE2=APN01)가 있다. 또한 아이디어 차원의 펩타이드 치료제들이 제안되고 있으며 대부분 hACE2의 결합부위만을 모사하는 것에 초점을 두어 디자인되고 있다. 이에, 기존에 제시된 펩타이드 치료제에 비해 보다 강하게 RBD와 결합할 수 있는 단백질 치료제의 개발이 필요하다.As a method of neutralizing RBD, methods of pre-binding other substances to RBD instead of hACE2 to prevent RBD from binding to hACE2 on the cell surface are being studied. There is human recombinant soluble ACE2 (hrsACE2=APN01) used as a lung cancer treatment as an RBD neutralizing treatment developed or proposed based on the existing hACE2. In addition, idea-level peptide therapeutics are being proposed, and most of them are designed with a focus on mimicking only the binding site of hACE2. Accordingly, it is necessary to develop a protein therapeutic agent capable of binding to RBD more strongly than previously proposed peptide therapeutic agents.
본 발명은 코로나바이러스 감염증 COVID-19 치료제 단백질 CTP 델타 및 이의 용도에 대한 것으로, 서열번호 1로 표시되는 아미노산 서열로 이루어진, 코로나바이러스의 수용체 결합 도메인(Receptor Binding Domain; RBD)에 특이적으로 결합하는 단백질 및 상기 단백질을 유효성분으로 포함하는 코로나바이러스 감염 진단용 조성물, 코로나바이러스 감염증 예방 또는 치료용 약학조성물, 코로나바이러스 감염증 예방 또는 개선용 건강기능식품 조성물 및 약물 전달용 조성물을 제공하고자 한다.The present invention relates to a coronavirus infection COVID-19 therapeutic protein CTP delta and its use, which specifically binds to the Receptor Binding Domain (RBD) of coronavirus, consisting of the amino acid sequence represented by SEQ ID NO: 1. It is intended to provide a protein and a composition for diagnosing coronavirus infection containing the protein as an active ingredient, a pharmaceutical composition for preventing or treating coronavirus infection, a health functional food composition for preventing or improving coronavirus infection, and a composition for drug delivery.
상기 과제의 해결을 위해, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진, 코로나바이러스의 수용체 결합 도메인(Receptor Binding Domain; RBD)에 특이적으로 결합하는 단백질을 제공한다. In order to solve the above problems, the present invention provides a protein that specifically binds to the Receptor Binding Domain (RBD) of coronavirus, consisting of the amino acid sequence represented by SEQ ID NO: 1.
또한, 본 발명은 상기 단백질을 코딩하는 폴리뉴클레오타이드, 상기 폴리뉴클레오타이드를 포함하는 재조합벡터 및 상기 재조합벡터로 형질전환된 형질전환체(단, 인간은 제외함)를 제공한다.In addition, the present invention provides a polynucleotide encoding the protein, a recombinant vector containing the polynucleotide, and a transformant (except for humans) transformed with the recombinant vector.
또한, 본 발명은 상기 단백질을 유효성분으로 포함하는 코로나바이러스 감염 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing coronavirus infection comprising the protein as an active ingredient.
또한, 본 발명은 상기 단백질을 유효성분으로 포함하는 코로나바이러스 감염증 예방 또는 치료용 약학조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the protein as an active ingredient.
또한, 본 발명은 상기 단백질을 유효성분으로 포함하는 코로나바이러스 감염증 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving coronavirus infection comprising the protein as an active ingredient.
또한, 본 발명은 상기 단백질을 유효성분으로 포함하는 약물 전달용 조성물을 제공한다.In addition, the present invention provides a drug delivery composition comprising the protein as an active ingredient.
본 발명은 코로나바이러스 감염증 COVID-19 치료제 단백질 CTP 델타 및 이의 용도에 관한 것으로서, 기존에 알려진 SARS-CoV의 RBD와 ACE2의 결합부위를 모사하는 펩타이드(P6)에 비해, 본 발명의 치료제 단백질 CTP 델타는 SARS-CoV2 RBD의 새로운 에피토프와의 결합을 더욱 강하게 만들기 위해 아미노산을 구성하는 원자들 차원의 상호작용을 원천적으로 디자인한, 아미노산의 새로운 서열을 추가한 새로운 부분으로 구성되어 있다. 본 발명에서는, RBD와 hACE2 사이의 기존에 알려진 결합 경계면의 후면에 있는 R454, K458, D467, E471 등의 전하를 띄는(Charged) 아미노산들과 추가적으로 상호작용할 수 있는, 확장된 치료제 단백질 CTP 델타를 독창적으로 디자인하여 기존 알려진 펩타이드 보다 강하게 결합할 수 있는 새로운 디자인의 치료제 단백질 CTP 델타를 제시하였으며, 본 발명의 치료제 단백질 CTP 델타는 향후 COVID-19 치료제로서 높은 활용 가능성을 나타낸다.The present invention relates to the therapeutic protein CTP delta for coronavirus infection COVID-19 and its use, compared to the previously known peptide (P6) that mimics the binding site of RBD and ACE2 of SARS-CoV, the therapeutic protein CTP delta of the present invention consists of a new part with the addition of a new sequence of amino acids, originally designed for interactions at the atomic level constituting amino acids to make the binding with the new epitope of SARS-CoV2 RBD stronger. In the present invention, an extended therapeutic protein CTP delta that can additionally interact with charged amino acids such as R454, K458, D467, and E471 on the back side of the previously known binding interface between RBD and hACE2 is uniquely prepared. and presented a new design therapeutic protein CTP delta that can bind more strongly than previously known peptides, and the therapeutic protein CTP delta of the present invention shows high potential for use as a future COVID-19 treatment.
도 1은 RBD(Pink)와 CTP 델타의 결합구조를 나타낸다.
도 2는 대장균에서 발현된 치료제 단백질 CTP-δ1의 분리 및 정제 결과를 나타낸다. (A) BL21(DE3) RIL 셀에서 정제된 CTP-δ1. M: Molecular weight markers, T: IPTG 발현 후 총 셀, S: 셀 파쇄 후 수용성 용액 그리고 P: 불용성 용액, Lane 1 ~ 10: Ni-IMAC으로 정제된 CTP-δ1 결과, (B) 이온 교환 크로마토그래피(Ion Exchange Chromatography; IEX)로 정제된 CTP-δ1 결과. (C) CTP-δ1을 순수하게 분리 정제한 SDS-PAGE 결과, (D) CTP-δ1을 순도 98%로 최종 정제한 SDS-PAGE 결과.
도 3은 미손상(intact) 치료제 단백질 CTP-δ1의 질량 분석 결과를 나타낸다.
도 4는 LC-MS/MS 분석을 이용하여 확인된 치료제 단백질 CTP-δ1의 서열 및 N, C-말단의 MS/MS 스펙트럼을 나타낸다.
도 5는 hACE2, RBD, 치료제 단백질 CTP 델타의 Far UV CD 스펙트럼을 나타낸다.
도 6은 RBD에 hACE2와 CTP 델타를 적정하여 측정한 MST의 형광의 세기 변화를 나타낸다.Figure 1 shows the bonding structure of RBD (Pink) and CTP delta.
Figure 2 shows the results of isolation and purification of the therapeutic protein CTP-δ1 expressed in E. coli. (A) CTP-δ1 purified from BL21(DE3) RIL cells. M: Molecular weight markers, T: Total cells after expression of IPTG, S: Aqueous solution after cell disruption and P: Insoluble solution,
Figure 3 shows the mass spectrometry results of intact therapeutic protein CTP-δ1.
Figure 4 shows the MS/MS spectrum of the sequence and N, C-terminus of the therapeutic protein CTP-δ1 identified using LC-MS/MS analysis.
Figure 5 shows the Far UV CD spectra of hACE2, RBD, and therapeutic protein CTP delta.
6 shows the change in fluorescence intensity of MST measured by titrating RBD with hACE2 and CTP delta.
본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진, 코로나바이러스의 수용체 결합 도메인(Receptor Binding Domain; RBD)에 특이적으로 결합하는 단백질을 제공한다. The present invention provides a protein that specifically binds to the Receptor Binding Domain (RBD) of coronavirus, consisting of the amino acid sequence represented by SEQ ID NO: 1.
상세하게는, 상기 단백질은 코로나바이러스의 RBD와 안지오텐신 변환 효소 2(Angiotensin-converting enzyme 2; ACE2)의 결합을 억제할 수 있으나, 이에 한정되는 것은 아니다.In detail, the protein may inhibit the binding of RBD of coronavirus and angiotensin-converting enzyme 2 (ACE2), but is not limited thereto.
상세하게는, 상기 코로나바이러스는 SARS-CoV2일 수 있으나, 이에 한정되는 것은 아니다.Specifically, the coronavirus may be SARS-CoV2, but is not limited thereto.
상세하세는, 상기 단백질은 SARS-CoV2 RBD의 R454, K458, D467 및 E471과 결합할 수 있으나, 이에 한정되는 것은 아니다.In detail, the protein may bind to R454, K458, D467 and E471 of SARS-CoV2 RBD, but is not limited thereto.
본 발명의 단백질은 당업계에 공지된 화학적 합성(Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983)에 의해 쉽게 제조될 수 있다. 대표적인 방법으로서 액체 또는 고체상 합성, 단편 응축, F-MOC 또는 T-BOC 화학법이 포함되지만(Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989), 이들로 한정되는 것은 아니다.The protein of the present invention can be easily prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997 A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989), but is not limited thereto.
또한, 본 발명의 단백질은 유전공학적 방법에 의해 제조될 수 있다. 우선, 통상적인 방법에 따라 상기 단백질을 코딩하는 DNA 서열을 합성한다. DNA 서열은 적절한 프라이머를 사용하여 PCR 증폭함으로써 합성할 수 있다. 다른 방법으로 당업계에 공지된 표준 방법에 의해, 예컨대, 자동 DNA 합성기(예: Biosearch 또는 AppliedBiosystems사에서 판매하는 것)를 사용하여 DNA 서열을 합성할 수도 있다. 제작된 DNA 서열은 이 DNA 서열에 작동가능하게 연결되어(operatively linked) 그 DNA 서열의 발현을 조절하는 하나 또는 그 이상의 발현 조절 서열(expression control sequence)(예: 프로모터, 인핸서 등)을 포함하는 벡터에 삽입시키고, 이로부터 형성된 재조합 발현 벡터로 숙주세포를 형질전환시킨다. 생성된 형질전환체를 상기 DNA 서열이 발현되도록 적절한 배지 및 조건 하에서 배양하여, 배양물로부터 상기 DNA 서열에 의해 코딩된 실질적으로 순수한 단백질을 회수한다. 상기 회수는 이 기술분야에서 공지된 방법(예컨대, 크로마토그래피)을 이용하여 수행할 수 있다. 상기에서 '실질적으로 순수한 단백질'이라 함은 본 발명에 따른 단백질이 숙주로부터 유래된 어떠한 다른 단백질도 실질적으로 포함하지 않는 것을 의미한다. In addition, the protein of the present invention can be produced by genetic engineering methods. First, a DNA sequence encoding the protein is synthesized according to a conventional method. DNA sequences can be synthesized by PCR amplification using appropriate primers. Alternatively, the DNA sequence may be synthesized by standard methods known in the art, such as using an automated DNA synthesizer (eg, sold by Biosearch or AppliedBiosystems). The constructed DNA sequence is a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) operatively linked to the DNA sequence to control the expression of the DNA sequence. and the host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are cultured under appropriate media and conditions to allow expression of the DNA sequence, and a substantially pure protein encoded by the DNA sequence is recovered from the culture. The recovery may be performed using a method known in the art (eg, chromatography). As used herein, 'substantially pure protein' means that the protein according to the present invention does not substantially contain any other host-derived proteins.
본 발명에서 상기 서열번호 1로 표시되는 아미노산 서열로 이루어진 단백질은 이의 기능적 변이체를 포함하는 개념이다. “기능적 변이체”란 본 발명의 단백질의 성질에는 영향을 미치지 않는 아미노산 위치에서 일부 아미노산의 치환이 발생된 모든 유사한 서열을 의미한다.In the present invention, the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is a concept including a functional variant thereof. "Functional variant" means any similar sequence in which substitution of some amino acids occurs at amino acid positions that do not affect the properties of the protein of the present invention.
또한, 본 발명은 상기 단백질을 코딩하는 폴리뉴클레오타이드를 제공한다.In addition, the present invention provides a polynucleotide encoding the protein.
상기 “폴리뉴클레오타이드(polynucleotide)”는 단일가닥 또는 이중가닥 형태로 존재하는 디옥시리보뉴클레오티드 또는 리보뉴클레오티드의 중합체이다. RNA 게놈 서열, DNA(gDNA 및 cDNA) 및 이로부터 전사되는 RNA 서열을 포괄하며, 특별하게 다른 언급이 없는 한 천연의 폴리뉴클레오타이드의 유사체를 포함한다.The "polynucleotide" is a polymer of deoxyribonucleotides or ribonucleotides that exists in single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified.
상기 폴리뉴클레오타이드는 상기 단백질을 코딩하는 뉴클레오타이드 서열뿐만 아니라, 그 서열에 상보적인(complementary) 서열도 포함한다. 상기 상보적인 서열은 완벽하게 상보적인 서열뿐만 아니라, 실질적으로 상보적인 서열도 포함한다.The polynucleotide includes not only a nucleotide sequence encoding the protein, but also a sequence complementary to the sequence. The complementary sequences include sequences that are substantially complementary as well as sequences that are perfectly complementary.
또한, 상기 폴리뉴클레오타이드는 변형될 수 있다. 상기 변형은 뉴클레오타이드의 추가, 결실 또는 비보존적 치환 또는 보존적 치환을 포함한다. 상기 아미노산 서열을 코딩하는 폴리뉴클레오타이드는 상기 뉴클레오타이드 서열에 대하여 실질적인 동일성을 나타내는 뉴클레오타이드 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기 뉴클레오타이드 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 80%의 상동성, 최소 90%의 상동성 또는 최소 95%의 상동성을 나타내는 서열일 수 있다.In addition, the polynucleotide may be modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides. A polynucleotide encoding the amino acid sequence is also construed to include a nucleotide sequence exhibiting substantial identity to the nucleotide sequence. The substantial identity is at least 80% homology when the nucleotide sequence and any other sequence are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art, It may be a sequence exhibiting at least 90% homology or at least 95% homology.
또한, 본 발명은 상기 폴리뉴클레오타이드를 포함하는 재조합벡터를 제공한다.In addition, the present invention provides a recombinant vector comprising the polynucleotide.
또한, 본 발명은 상기 재조합벡터로 형질전환된 형질전환체(단, 인간은 제외함)를 제공한다.In addition, the present invention provides transformants (except for humans) transformed with the recombinant vector.
본 발명에 있어서,“벡터”는 클론유전자(또는 클론 DNA의 다른 조각)를 운반하는데 사용되는 스스로 복제되는 DNA 분자를 의미한다.In the present invention, “vector” refers to a self-replicating DNA molecule used to transfer clonal genes (or other fragments of clonal DNA).
본 발명에서 있어서, “재조합벡터”는 숙주 세포 내에서 삽입된 핵산을 발현할 수 있는 당 분야에 공지된 플라스미드, 바이러스 벡터 또는 기타 매개체를 의미하는 것으로서, 당업계에 공지된 통상의 발현벡터에 본 발명의 단백질을 암호화하는 폴리뉴클레오타이드가 작동가능하게 연결된 것일 수 있다. 상기 재조합벡터는 일반적으로 숙주세포에서 증식할 수 있는 복제원점, 발현을 조절하는 하나 이상의 발현 조절 서열(예. 프로모터, 인핸서 등), 선별 마커(selective marker) 및 발현 조절 서열과 작동가능하게 연결된 본 발명의 단백질을 암호화하는 폴리뉴클레오타이드를 포함할 수 있다. 형질전환체는 상기 재조합벡터에 의해 형질전환된 것일 수 있다.In the present invention, "recombinant vector" refers to a plasmid, viral vector or other medium known in the art capable of expressing an inserted nucleic acid in a host cell, and is compared to conventional expression vectors known in the art. Polynucleotides encoding the proteins of the invention may be operably linked. The recombinant vector is generally operably linked to an origin of replication capable of proliferating in a host cell, one or more expression control sequences (eg, promoter, enhancer, etc.) for controlling expression, a selective marker, and an expression control sequence. It may contain polynucleotides encoding the proteins of the invention. A transformant may be one transformed by the recombinant vector.
바람직하게는 형질전환체는 본 발명의 단백질을 암호화하는 폴리뉴클레오타이드를 포함하는 재조합벡터를 당업계에 공지된 방법, 예를 들어 이에 한정되지는 않으나, 일시적 형질감염(transient transfection), 미세주사, 형질도입(transduction), 세포융합, 칼슘 포스페이트 침전법, 리포좀 매개된 형질감염(liposome-mediated transfection), DEAE 덱스트란-매개된 형질감염(DEAE Dextran- mediated transfection), 폴리브렌-매개된 형질 감염(polybrene-mediated transfection), 전기침공법(electropora tion), 유전자 총(gene gun) 및 세포 내로 핵산을 유입시키기 위한 다른 공지의 방법에 의해 숙주세포에 도입하여 수득할 수 있다(Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).Preferably, the transformant is a recombinant vector containing a polynucleotide encoding the protein of the present invention by methods known in the art, such as, but not limited to, transient transfection, microinjection, and transfection. Transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran- mediated transfection, polybrene-mediated transfection -mediated transfection), electroporation (electropora tion), gene gun (gene gun), and can be obtained by introducing into a host cell by other known methods for introducing nucleic acids into cells (Wu et al., J. Bio.Chem., 267:963-967, 1992; Wu and Wu, J. Bio.Chem., 263:14621-14624, 1988).
또한, 본 발명은 상기 단백질을 유효성분으로 포함하는 코로나바이러스 감염 진단용 조성물을 제공한다. In addition, the present invention provides a composition for diagnosing coronavirus infection comprising the protein as an active ingredient.
바람직하게는, 상기 코로나바이러스는 SARS-CoV2일 수 있으나, 이에 한정되는 것은 아니다.Preferably, the coronavirus may be SARS-CoV2, but is not limited thereto.
본 발명에서 "진단"이란 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단은 코로나바이러스 감염의 존재 또는 특징을 확인하는 것이다. In the present invention, “diagnosis” means confirming the presence or character of a pathological condition. For purposes of this invention, diagnosis is the confirmation of the presence or characteristics of a coronavirus infection.
본 발명의 단백질을 이용한 코로나바이러스 감염의 진단은 혈액, 소변이나 바이옵시(biopsy)에 의해 직접 얻은 해당 조직 또는 세포에 본 발명의 단백질을 반응시켜 이들의 결합을 검출함으로써 진단할 수 있다. Diagnosis of coronavirus infection using the protein of the present invention can be diagnosed by reacting the protein of the present invention with blood, urine, or directly obtained tissue or cells obtained by biopsy and detecting their binding.
또한, 본 발명의 단백질이 코로나바이러스의 RBD에 결합하였는지 여부를 용이하게 확인, 검출 및 정량하기 위하여, 본 발명의 단백질은 표지된 상태로 제공될 수 있다. 즉, 검출가능한 표지에 링크(예: 공유 결합 또는 가교)되어 제공될 수 있다. 상기 검출 가능한 표지는 발색효소(예: 퍼옥시다제(peroxidase), 알칼라인 포스파타제(alkaline phosphatase)), 방사성 동위원소(예: 124I, 125I, 111In, 99mTc, 32P, 35S), 크로모포어(chromophore), 발광물질 또는 형광물질(예: FITC, RITC, 로다민(rhodamine), 시아닌(cyanine), 텍사스레드(Texas Red), 플로레신(fluorescein), 피코에리트린(phycoerythrin), 퀀텀닷(quantum dots))등 일 수 있다.In addition, in order to easily identify, detect and quantify whether the protein of the present invention binds to the RBD of the coronavirus, the protein of the present invention may be provided in a labeled state. That is, it may be provided by being linked (eg, covalent bond or cross-linking) to a detectable label. The detectable label is a color enzyme (eg, peroxidase, alkaline phosphatase), a radioactive isotope (eg, 124 I, 125 I, 111 In, 99 mTc, 32 P, 35 S), Chromophores, luminescent or fluorescent substances (e.g. FITC, RITC, rhodamine, cyanine, Texas Red, fluorescein, phycoerythrin, Quantum dots may be the like.
유사하게, 상기 검출 가능한 표지는 항체 에피토프(epitope), 기질(substrate), 보조인자(cofactor), 저해제 또는 친화 리간드일 수 있다. 이러한 표지는 본 발명의 단백질을 합성하는 과정 중에 수행할 수도 있고, 이미 합성된 단백질에 추가로 수행될 수도 있다. 만약 검출가능한 표지로 형광물질을 이용하는 경우에는 형광단층촬영(Fluorescence mediated tomography: FMT)으로 코로나바이러스 감염을 진단할 수 있다. 예컨대, 형광물질로 표지된 본 발명의 단백질을 혈액 내로 순환시키고 형광단층촬영으로 단백질에 의한 형광을 관찰할 수 있다. 형광이 관찰된다면, 코로나바이러스 감염으로 진단된다.Similarly, the detectable label may be an antibody epitope, substrate, cofactor, inhibitor or affinity ligand. Such labeling may be performed during the process of synthesizing the protein of the present invention or may be additionally performed on already synthesized proteins. If a fluorescent material is used as a detectable label, coronavirus infection can be diagnosed by fluorescence mediated tomography (FMT). For example, the protein of the present invention labeled with a fluorescent material can be circulated in the blood, and fluorescence by the protein can be observed by fluorescence tomography. If fluorescence is observed, a coronavirus infection is diagnosed.
또한, 본 발명은 상기 단백질을 유효성분으로 포함하는 코로나바이러스 감염증 예방 또는 치료용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the protein as an active ingredient.
바람직하게는, 상기 코로나바이러스 감염증은 COVID-19일 수 있으나, 이에 한정되는 것은 아니다.Preferably, the coronavirus infection may be COVID-19, but is not limited thereto.
본 발명의 약학 조성물은 유효 성분 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등의 가용화제를 사용할 수 있다. 본 발명의 약학 조성물은 투여를 위해서 유효 성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1 종 이상 포함하여 약학 조성물로 바람직하게 제제화할 수 있다. 액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. The pharmaceutical composition of the present invention can be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, and a glidant. Alternatively, a solubilizer such as a flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
본 발명의 약학 조성물의 약제 제제 형태는 과립제, 산제, 피복정, 정제, 캡슐제, 좌제, 시럽, 즙, 현탁제, 유제, 점적제 또는 주사 가능한 액제 및 활성 화합물의 서방출형 제제 등이 될 수 있다. 본 발명의 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다. 본 발명의 약학 조성물의 유효성분의 유효량은 질환의 예방 또는 치료 요구되는 양을 의미한다. 따라서, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효 성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 이에 제한되는 것은 아니나, 예컨대, 본 발명의 조성물은 성인의 경우, 1일 1회 내지 수회 투여시, 0.1ng/kg~10g/kg 용량으로 투여할 수 있다.The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release preparations of active compounds. can The pharmaceutical composition of the present invention can be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered with An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for preventing or treating a disease. Therefore, the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health condition, sex and diet, administration time, administration route and composition It can be controlled by various factors including secretion rate, duration of treatment, and drugs used concurrently. Although not limited thereto, for example, the composition of the present invention can be administered at a dose of 0.1 ng/kg to 10 g/kg when administered once to several times a day for adults.
또한, 본 발명은 상기 단백질을 유효성분으로 포함하는 코로나바이러스 감염증 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving coronavirus infection comprising the protein as an active ingredient.
본 발명의 건강기능식품 조성물은 분말, 과립, 정제, 캡슐, 시럽 또는 음료의 형태로 제공될 수 있으며, 상기 건강기능식품 조성물은 유효성분 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health functional food composition of the present invention may be provided in the form of powder, granule, tablet, capsule, syrup or beverage, and the health functional food composition is used with other food or food additives in addition to the active ingredient, and according to a conventional method can be used appropriately. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.
상기 건강기능식품 조성물에 함유된 유효성분의 유효용량은 상기 약학조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the active ingredient contained in the health functional food composition can be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it is less than the above range. It is certain that the active ingredient can be used in an amount greater than the above range because there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다.There is no particular limitation on the type of health food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, vitamin complexes, and the like are exemplified.
또한, 본 발명은 상기 단백질을 유효성분으로 포함하는 약물 전달용 조성물을 제공한다. In addition, the present invention provides a drug delivery composition comprising the protein as an active ingredient.
바람직하게는 상기 약물은 항바이러스제일 수 있고, 보다 바람직하게는 상기 항바이러스제는 코로나바이러스에 대한 항바이러스제일 수 있으며, 상기 코로나바이러스는 SARS-CoV2일 수 있으나, 이에 한정되는 것은 아니다.Preferably, the drug may be an antiviral agent, more preferably, the antiviral agent may be an antiviral agent for coronavirus, and the coronavirus may be SARS-CoV2, but is not limited thereto.
본 발명에 따른 단백질은 약물을 코로나바이러스에 선택적으로 전달하는 지능형 약물 전달체로서 사용될 수 있다. 본 발명의 단백질은 종래 공지의 약물과 연결하여 코로나바이러스 감염증 치료에 이용한다면 본 발명의 단백질에 의해 약물이 코로나바이러스에만 선택적으로 전달되기 때문에 약물의 효력을 증가시킬 수 있고 동시에 약물의 부작용을 현저히 줄일 수 있다.The protein according to the present invention can be used as an intelligent drug delivery system that selectively delivers drugs to coronaviruses. If the protein of the present invention is used for the treatment of coronavirus infection in connection with a conventionally known drug, the protein of the present invention selectively delivers the drug only to the coronavirus, thereby increasing the efficacy of the drug and significantly reducing the side effects of the drug. can
상기 약물은 항바이러스제로서, 본 발명의 단백질에 연결될 수 있는 항바이러스제로는 종래 코로나바이러스 감염증의 치료에 사용되는 것이라면 제한 없이 사용될 수 있다. 항바이러스제와 본 발명의 단백질 간의 연결은 당업계에 공지된 방법, 예컨대, 공유 결합, 가교 등을 통해 수행될 수 있다. 이를 위해 본 발명의 단백질은 필요하다면 그 활성이 소실되지 않는 범위에서 화학적으로 수식(modification)될 수 있다.The drug is an antiviral agent, and any antiviral agent that can be linked to the protein of the present invention can be used without limitation as long as it is conventionally used for the treatment of coronavirus infection. Linkage between the antiviral agent and the protein of the present invention may be performed through methods known in the art, such as covalent linkage, crosslinking, and the like. To this end, if necessary, the protein of the present invention may be chemically modified to the extent that its activity is not lost.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
<< 실시예Example 1> 코로나바이러스 감염증 1> Coronavirus infection COVIDCOVID -19 치료제 단백질 -19 therapeutic protein CTPCTP 델타의 단백질 3차원 구조 분석 3D structure analysis of delta protein
1. 단백질 3차원 구조 준비1. Preparation of three-dimensional structures of proteins
Protein Data Bank(PDB, https://www.rcsb.org/)에 등록된 hACE2 - RBD의 결합 구조(PDB ID: 6M0J)를 사용하였다. P6 부분의 구조는 hACE2의 22~44-G-351~357의 서열을 사용하여 hACE2-RBD의 결합 구조를 바탕으로 MODELLER를 사용하여 모델링하였다. The binding structure (PDB ID: 6M0J) of hACE2-RBD registered in Protein Data Bank (PDB, https://www.rcsb.org/) was used. The structure of the P6 part was modeled using MODELLER based on the binding structure of hACE2-RBD using the sequence of 22~44-G-351~357 of hACE2.
2. 분자동역학 시뮬레이션 (Molecular Dynamic Simulation)2. Molecular Dynamics Simulation
모델링 된 hACE2-RBD complex, P6-RBD complex, CTP 델타 monomer-RBD complex, P6, CTP 델타 monomer에 대해서 AMBER18 패키지를 사용하여 ff14SB force field를 적용한 분자 동역학 시뮬레이션을 수행하였다. AMBER18에 포함된 pmemd.cuda 프로그램으로 시뮬레이션이 실행되었으며, 시뮬레이션을 위한 초기 구조는 준비된 단백질의 외곽에서 15Å의 길이를 더한 octahedron TIP3P 물 박스를 적용하였다. 물 박스를 기준으로 periodic boundary 조건이 사용되었으며 net charge를 중화(neutralization) 시키기 위해 Na+, Cl- 이온들이 첨가되었다. 9Å의 거리를 기준으로 먼 거리의 전자기력은 Particle-mesh Ewald(PME) 방법이 적용되었다. 수소 분자의 공유결합 거리를 고정시키는 SHAKE 알고리즘을 사용하였으며 2fs/step의 time step으로 시뮬레이션이 진행되었다. 먼저, 단백질의 backbone 구조에 0.5 kcal/mol의 position restraint를 적용하여 5000 step의 minimization 시뮬레이션을 하였으며 0.1 kcal/mol의 position restraint을 적용하여 10K에서 300K의 온도까지 NVT 앙상블 조건에서 25 ps 동안 heating 시뮬레이션을 수행하였다. 300K의 온도에서 1ns 동안 NPT 앙상블 조건으로 equilibrium 시뮬레이션이 수행되었다. Production run은 300K의 온도와 1bar의 압력으로 NPT 앙상블 조건에서 수행되었으며 RBD와의 complex의 경우 500ns 동안, CTP 델타 단독의 경우 1μs 동안 시뮬레이션이 수행되었다. 시뮬레이션은 각 케이스에 대해서 5개의 독립적인 trajectory가 수행되었다.Molecular dynamics simulations were performed using the AMBER18 package for the modeled hACE2-RBD complex, P6-RBD complex, CTP delta monomer-RBD complex, P6, and CTP delta monomer using the ff14SB force field. The simulation was performed with the pmemd.cuda program included in AMBER18, and the initial structure for the simulation was an octahedron TIP3P water box with a length of 15 Å added to the outside of the prepared protein. Periodic boundary conditions were used based on the water box, and Na+ and Cl- ions were added to neutralize the net charge. The Particle-mesh Ewald (PME) method was applied to the electromagnetic force at a distance of 9 Å. The SHAKE algorithm, which fixes the covalent bond distance of hydrogen molecules, was used, and the simulation was conducted with a time step of 2fs/step. First, a 5000-step minimization simulation was performed by applying a position restraint of 0.5 kcal/mol to the backbone structure of the protein, and a heating simulation was performed for 25 ps under NVT ensemble conditions from 10 K to 300 K by applying a position restraint of 0.1 kcal/mol. performed. Equilibrium simulations were performed under NPT ensemble conditions for 1 ns at a temperature of 300 K. The production run was performed under NPT ensemble conditions at a temperature of 300 K and a pressure of 1 bar. Simulations were performed for 500 ns in the case of complex with RBD and 1 μs in the case of CTP delta alone. Simulations were performed on 5 independent trajectories for each case.
RBD와 CTP 델타의 결합 에너지는 300~500ns 사이의 시뮬레이션 스냅샷들을 사용하여서 MMGBSA 알고리즘으로 계산되었고, normal mode analysis 방식을 통해 엔트로피가 계산되었다. 결합 자유에너지 계산에는 AMBER18 패키지의 MMPBSA.py 프로그램이 사용되었다.The binding energies of RBD and CTP delta were calculated with the MMGBSA algorithm using simulation snapshots between 300 and 500 ns, and entropy was calculated through normal mode analysis. The MMPBSA.py program of the AMBER18 package was used to calculate the binding free energy.
3. 3. CTPCTP 델타의 구조 structure of delta
모델링 된 CTP-δ1은 HTH 46AA형 구조를 가진다. 모델링 된 구조의 monomer form을 분자동역학 시뮬레이션을 통해 용액(solution) 내에서의 구조 앙상블을 분석해보았을 때, 도 1의 구조 패턴을 보인다. The modeled CTP-δ1 has an HTH 46AA type structure. When the structural ensemble in the solution was analyzed through molecular dynamics simulation of the monomer form of the modeled structure, the structural pattern of FIG. 1 was shown.
CTP-δ1는 N-말단 부분에 Helix-turn-Helix 구조를 추가하여 RBD의 R454, K458, D467, E471 등과 상호작용할 E3, D6, K7, K9, E11를 배치한 유연한 turn 구조를 가진 치료제 단백질이다. RBD와 강한 결합이 가능하지만 구조적 안정성이 떨어진다.CTP-δ1 is a therapeutic protein with a flexible turn structure in which E3, D6, K7, K9, and E11 interact with R454, K458, D467, and E471 of RBD by adding a Helix-turn-Helix structure to the N-terminus. . Strong bonding with RBD is possible, but structural stability is poor.
4. 4. CTPCTP 델타- delta- RBD의RBD's 결합 자유 에너지 예측 Binding free energy prediction
CTP 델타가 RBD와 얼마나 잘 결합하는지를 확인하기 위해 결합 에너지와 결합 자유에너지를 계산하였다(표 1). 분자동역학 시뮬레이션을 사용하여 hACE2, P6, CTP 델타가 각각 RBD와 결합한 구조 앙상블 수집하고, 구조 앙상블로부터 MMGBSA 알고리즘을 사용하여 결합 에너지(ΔE)와 결합 자유에너지(ΔG)를 분석한다.To confirm how well the CTP delta binds to RBD, the binding energy and binding free energy were calculated (Table 1). Using molecular dynamics simulation, the structural ensemble of hACE2, P6, and CTP delta bound to RBD, respectively, is collected, and the binding energy (ΔE) and binding free energy (ΔG) are analyzed from the structural ensemble using the MMGBSA algorithm.
CTP들은 음의 결합 자유에너지를 가지며 RBD와 결합하였을 때 안정하다는 것을 알 수 있으며, 새로운 에피토프와의 상호작용으로 시뮬레이션 상 hACE2, P6보다 에너지(ΔE)적으로 더욱 강하게 결합하며 자유에너지(ΔG)적으로도 비슷하거나 더욱 강하게 결합한다.It can be seen that CTPs have negative binding free energies and are stable when bound to RBD, and by interaction with the new epitope, they bind more strongly in terms of energy (ΔE) than hACE2 and P6 in simulation, and their free energy (ΔG) also binds similarly or more strongly.
5. 5. CTPCTP 델타의 In- Delta's In- silicoin silico 면역원성 분석 (In- Immunogenicity assay (In- silicoin silico ImmunogenecityImmunogenecity Analysis) Analysis)
In-silico 면역원성은 세계적으로 잘 구축된 NetMHC-4.0 서버(http://www.cbs.dtu.dk/services/NetMHC/)를 사용하여 분석하였다. CTP 델타의 아미노산 서열에 대하여 NetMHC4.0 서버에 있는 81개의 서로 다른 사람의 MHC allele들과 동물(Monkey, Cattle, Pig와 Mouse)의 41개의 allele에 대해서 peptide-MHC class I의 결합에 대해 예측하였다. 13~14개의 아미노산 길이의 펩타이드 단위로 검색하여 전체 검색된 펩타이드의 수(Total)와 그 중에서 강한 결합(SB), 약한 결합(WB)이 예상되는 펩타이드의 개수를 세어서 그 비율을 (SB+WB)/Total로 계산하여 면역원성을 검증하였다.In-silico immunogenicity was analyzed using the world-famous NetMHC-4.0 server (http://www.cbs.dtu.dk/services/NetMHC/). For the amino acid sequence of CTP delta, peptide-MHC class I binding was predicted for 81 different human MHC alleles in the NetMHC4.0 server and 41 alleles of animals (Monkey, Cattle, Pig and Mouse). . By searching in peptide units of 13 to 14 amino acids in length, counting the total number of peptides searched (Total) and the number of peptides expected to have strong binding (SB) and weak binding (WB) among them, and the ratio (SB + WB ) / Total was calculated to verify immunogenicity.
전체에 대한 비율 (SB+WB)/Total이 낮을수록 본 발명의 중화 치료제 단백질 단백질이 체내에 투입되었을 때에 타 단백질들과 항원-항체 반응을 적게 한다는 것을 나타낸다(표 2 내지 표 7).The lower the ratio to the total (SB+WB)/Total, the lower the antigen-antibody reaction with other proteins when the protein of the present invention is introduced into the body (Tables 2 to 7).
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<< 실시예Example 2> 코로나바이러스 감염증 2> Coronavirus infection COVIDCOVID -19 치료제 단백질 -19 therapeutic protein CTPCTP 델타의 delta 클로닝cloning
1. 균주 및 배지1. Strains and Media
유전자 클로닝에 사용된 화학품은 모두 분석용 등급을 사용하였다. 완료된 클론은 증식을 위해 DH5α 대장균(E. coli) 균주를 사용하여 형질전환을 수행하였고, ampicillin (엘피에스, 100 μg/ml)을 넣은 LB 배지로 선별하였다. 세포배양은 37℃에서 교반하였다.All chemicals used for gene cloning were of analytical grade. Completed clones were transformed using DH5α Escherichia coli ( E. coli ) strains for growth, and were selected in LB medium containing ampicillin (LPS, 100 μg/ml). Cell culture was stirred at 37°C.
2. 플라스미드 제작2. Plasmid Construction
In-silico 상에서 디자인된 단백질의 아미노산 서열을 Sequence Manipulation Suite (https://www.bioinformatics.org/sms2/rev_trans.html) tool을 사용하여 뉴클레오타이드(nucleotide)로 변환시킨 후, hACE2에 존재하는 서열과 비교하여 DNA 서열을 결정하였다. 합성된 DNA 단편 (바이오니아, 유전자 합성)은 forward primer 5'-GGAGATATACATATGGGCGAATTTGCGGATAAGTTG와 reverse primer 5'-GTGGTGCTCGAGCCTGAAGTCGCCCTTCC로 PCR 증폭시킨 후 EZ-Fusion™ HT Cloning Kit (엔지노믹스)를 사용한 ligation independent cloning 방법으로 Nde I 과 Xho I 제한효소 처리 한 pET-21a 벡터에 삽입되었다.After converting the amino acid sequence of the protein designed in silico into nucleotides using the Sequence Manipulation Suite (https://www.bioinformatics.org/sms2/rev_trans.html) tool, the sequences present in hACE2 and DNA sequences were determined by comparison. The synthesized DNA fragment (Bioneer, Gene Synthesis) was PCR amplified with forward primer 5'-GGAGATATACATATGGGCGAATTTGCGGATAAGTTG and reverse primer 5'-GTGGTGCTCGAGCCTGAAGTCGCCCTTCC, and Nde I and Xho It was inserted into the pET-21a vector treated with I restriction enzyme.
3. 실험결과3. Experimental results
본 발명의 치료제 단백질 CTP 델타는 S1-RBD 단백질이 hACE2 단백질에 결합하지 못하도록 중화(neutralization) 작용 혹은 저해(inhibition) 작용을 하는 새로운 아미노산 서열의 치료제 단백질로서, In-silico 상에서 디자인된 치료제 단백질 CTP 델타의 아미노산 서열은 표 8과 같다.The therapeutic protein CTP delta of the present invention is a therapeutic protein with a novel amino acid sequence that neutralizes or inhibits S1-RBD protein from binding to hACE2 protein, and is a therapeutic protein CTP delta designed in silico. The amino acid sequence of is shown in Table 8.
<< 실시예Example 3> 코로나바이러스 감염증 3> Coronavirus infection COVIDCOVID -19 치료제 단백질 -19 therapeutic protein CTPCTP 델타의 발현 및 정제 Expression and purification of delta
1. 단백질 발현 및 정제 재료1. Protein expression and purification materials
단백질 발현 및 정제에 사용된 화학품은 모두 분석용 등급을 사용하였다. 단백질 발현 및 정제를 위해 사용된 암피실린(Ampicillin), 1-티오-δ-d-갈락토피라노시드(1-thio-δ-d-galactopyranoside; IPTG), 소듐 포스페이트-모노베이직(Sodium Phosphate-Monobasic), 소듐 포스페이트-다이베이직(Sodium Phosphate-Dibasic), TRIS는 LPS(대전, 한국)에서 구매하였고, 쿠마시 브릴런트 블루(Coomassie brilliant blue) R-250, 2-머캡토에탄올(2-mercaptoethanol), 클로람페니콜(Chloramphenicol) 그리고 이미다졸(Imidazole)은 Biosesang(성남, 한국)에서 구매하였으며, 소듐 클로라이드(Sodium Chloride)는 덕산(대전, 한국)에서 구매하였고, 모든 컬럼은 GE Healthcare(Piscataway, NJ)에서 구매하였다. 페닐메탄설포닐 플루오라이드(Phenylmethanesulfonyl Fluoride; PMSF), 트리즈마 하이드로클로라이드(trizma hydrochloride), Amicon Ultra-15 Centrifugal Filter Units은 Millipore(Billerica, MA)에서 구매하여 사용하였다.All chemicals used for protein expression and purification were of analytical grade. Ampicillin, 1-thio-δ-d-galactopyranoside (IPTG), Sodium Phosphate-Monobasic used for protein expression and purification ), Sodium Phosphate-Dibasic, TRIS were purchased from LPS (Daejeon, Korea), Coomassie brilliant blue R-250, 2-mercaptoethanol , Chloramphenicol and Imidazole were purchased from Biosesang (Seongnam, Korea), Sodium Chloride was purchased from Deoksan (Daejeon, Korea), and all columns were purchased from GE Healthcare (Piscataway, NJ). Purchased. Phenylmethanesulfonyl Fluoride (PMSF), trizma hydrochloride, and Amicon Ultra-15 Centrifugal Filter Units were purchased from Millipore (Billerica, MA) and used.
2. 단백질 발현 및 정제 기기2. Protein Expression and Purification Instruments
단백질 발현 위해 진탕 배양기(NBS/Innova 42R) 그리고 고성능 대용량 원심분리기(Beckman Coulter. Inc./AVANTI JXN-26)를 사용하였고, 단백질 고속 분리 시스템(GE Healthcare/AKTA Pure M and Start System), 단백질 고속 분리 시스템(GE Healthcare/FPLC Accessory System), 초음파 파쇄기(Q700-Sonicator), 냉장 원심 분리기(Epoendorf-5810R)를 사용하여 단백질을 분리 정제하였다. For protein expression, a shaker (NBS/Innova 42R) and a high-capacity high-capacity centrifuge (Beckman Coulter. Inc./AVANTI JXN-26) were used, and a protein high-speed separation system (GE Healthcare/AKTA Pure M and Start System) and protein high-speed Proteins were separated and purified using a separation system (GE Healthcare/FPLC Accessory System), an ultrasonic disruptor (Q700-Sonicator), and a refrigerated centrifuge (Epoendorf-5810R).
3. 치료제 단백질 3. Therapeutic proteins CTPCTP 델타 발현 및 배양 Delta expression and culture
pET-21a 벡터에 클로닝 된 유전자를 BL21(DE3) RIL 대장균(E. Coli) 균주를 사용하여 발현을 위한 형질전환을 수행하였고, 배지는 암피실린(Ampicillin; 100mg/ml) 그리고 클로람페니콜(Chloramphenicol; 50mg/ml)을 넣은 LB 배지를 이용하여 37℃에서 세포배양을 하였다. OD600=0.6~0.8이 되도록 배양한 후 1M IPTG를 최종 농도 0.5 mM이 되도록 첨가한 후 37℃에서 4시간 발현하였다. 7000RPM, 4℃로 설정된 고성능 대용량 원심분리기(Beckman Coulter. Inc./AVANTI JXN-26)를 이용하여 20분 동안 셀을 수확한 후 즉시 사용하거나 -80℃에 보관하였다. The gene cloned into the pET-21a vector was transformed for expression using the BL21(DE3) RIL E. Coli strain, and the medium was ampicillin (100 mg/ml) and chloramphenicol (50 mg/ml). ml) was used for cell culture at 37°C using LB medium. After culturing to OD 600 =0.6-0.8, 1M IPTG was added to a final concentration of 0.5 mM, and then expressed at 37°C for 4 hours. Cells were harvested for 20 minutes using a high-capacity centrifuge (Beckman Coulter. Inc./AVANTI JXN-26) set at 7000 RPM and 4° C. and then used immediately or stored at -80° C.
4. 치료제 단백질 4. Therapeutic proteins CTPCTP 델타 분리 정제 delta separation purification
셀 1g 기준 10ml Lysis buffer(pH 8.0, 50 mM Tris-HCl, 500 mM NaCl, 1 mM PMSF, 0.25% Tween-20(v/v))에 녹인 후 초음파 파쇄기(Q700-Sonicator)를 사용하여 셀을 파쇄하였다. 용액과 펠렛(pellet)을 분리하기 위해 18000 RPM 그리고 4℃로 설정된 고속 원심 분리기를 이용하여 30분 진행하였다. 수용성 용액에 있는 불순물을 제거하기 위하여 0.45μm syringe filter를 수행하였다. 불순물이 제거된 용액을 Wash Buffer (pH 8.0, 50 mM Tris-HCl 500 mM NaCl)로 안정화된 HisTrap HP 5ml (GE Healthcare) 컬럼에 흘려 준 후 Elution Buffer (pH 8.0, 50 mM Tris-HCl 500 mM NaCl, 500 mM Imidazole)를 0 에서 100% Gradient Elution으로 설정하여 진행하였다. 그 결과, 100 에서 300 mM Imidazole 농도에서 치료제 단백질 CTP 델타가 분리된 것을 확인하였다. 분리된 치료제 단백질 CTP 델타를 15시간 동안 Cold room에서 버퍼 교환(pH 8.0, 20 mM Tris-HCl)을 진행하였다. Binding Buffer (pH 8.0, 20 mM Tris-HCl)로 안정화된 HiTrap Q HP 5ml (GE Healthcare) 컬럼에 NaCl이 제거된 CTP 델타 용액을 흘려 준 후 Elution Buffer (pH 8.0, 20 mM Tris-HCl 1.0 M NaCl)를 0 에서 100% Gradient Elution으로 설정하여 진행하였다. 그 결과, 120에서 250 mM NaCl 농도에서 치료제 단백질 CTP 델타가 분리된 것을 확인하였다. 최종적으로 pH 7.4, 10 mM Phosphate buffer, 150 mM NaCl로 안정화된 HiLoad Superdex 75 16/600 컬럼을 이용하여 Dimer 구조를 가지는 치료제 단백질 CTP 델타를 순수하게 분리 정제하였다. NanoDrop를 이용하여 A280nm에서 정량하였으며, ImageJ 프로그램을 사용하여 순도를 계산한 결과 >96% 고순도 치료제 단백질 CTP 델타 단백질을 획득하였다(도 2).After dissolving in 10 ml Lysis buffer (pH 8.0, 50 mM Tris-HCl, 500 mM NaCl, 1 mM PMSF, 0.25% Tween-20 (v/v)) based on 1 g of cells, use a sonicator (Q700-Sonicator) to break the cells. shredded. In order to separate the solution and the pellet, it was conducted for 30 minutes using a high-speed centrifuge set at 18000 RPM and 4°C. A 0.45 μm syringe filter was applied to remove impurities in the aqueous solution. The solution from which impurities were removed was flowed through a HisTrap HP 5ml (GE Healthcare) column stabilized with Wash Buffer (pH 8.0, 50 mM Tris-HCl, 500 mM NaCl), and then Elution Buffer (pH 8.0, 50 mM Tris-HCl, 500 mM NaCl). , 500 mM Imidazole) was set at 0 to 100% Gradient Elution. As a result, it was confirmed that the therapeutic protein CTP delta was isolated at concentrations of 100 to 300 mM imidazole. The separated therapeutic protein CTP delta was subjected to buffer exchange (pH 8.0, 20 mM Tris-HCl) in a cold room for 15 hours. After flowing NaCl-free CTP delta solution on HiTrap Q HP 5ml (GE Healthcare) column stabilized with Binding Buffer (pH 8.0, 20 mM Tris-HCl), Elution Buffer (pH 8.0, 20 mM Tris-HCl, 1.0 M NaCl ) was set from 0 to 100% Gradient Elution. As a result, it was confirmed that the therapeutic protein CTP delta was isolated at concentrations of 120 to 250 mM NaCl. Finally, using a HiLoad Superdex 75 16/600 column stabilized with pH 7.4, 10 mM Phosphate buffer, and 150 mM NaCl, the therapeutic protein CTP delta having a dimer structure was purified and purified. A was quantified at 280 nm using NanoDrop, and as a result of calculating the purity using the ImageJ program, >96% high purity therapeutic protein CTP delta protein was obtained (FIG. 2).
<< 실시예Example 4> 코로나바이러스 감염증 4> Coronavirus infection COVIDCOVID -19 치료제 단백질 -19 therapeutic protein CTPCTP 델타의 질량 분석 delta mass spectrometry
1. 질량 분석(Mass Spectrometry Analysis)1. Mass Spectrometry Analysis
단백질의 전체 질량 및 아미노산 서열 정보를 알기 위해 질량분석기를 사용하였다. 전체 단백질의 질량 분석은 고성능 매트릭스 레이저 탈착 이온화 질량분석기(Bruker, UltrafleXtreme MALDI TOF/TOF)를 사용하였다. 단백질 아미노산 서열 분석에는 하이브리드 3중4중극자 선형 이온트랩 질량분석기(Sciex, QTRAP), 사중극자 오비트랩 질량분석기(Thermo scientific, Q Exactive PLUS) 가 사용되었으며, QTRAP에는 마이크로 액체크로마토그래피(Sciex, M5 MicroLC), Q Exactive PLUS에는 나노-마이크로 액체크로마토그래피(Waters, ACQUITY UPLC M-Class system)를 사용하였다. A mass spectrometer was used to determine the total mass and amino acid sequence information of the protein. Mass spectrometry of total protein was performed using a high-performance matrix laser desorption ionization mass spectrometer (Bruker, UltrafleXtreme MALDI TOF/TOF). For protein amino acid sequence analysis, a hybrid triple quadrupole linear ion trap mass spectrometer (Sciex, QTRAP) and quadrupole orbit trap mass spectrometer (Thermo scientific, Q Exactive PLUS) were used, and micro liquid chromatography (Sciex, M5 MicroLC), Q Exactive PLUS, nano-micro liquid chromatography (Waters, ACQUITY UPLC M-Class system) was used.
2. 2. MALDIMALDI TOF를TOF 위한 샘플 준비 및 Sample preparation for and MALDIMALDI TOF를TOF 이용한 미손상(intact) 단백질 분석 Intact protein analysis using
ACE2, RBD, CTP 델타 단백질의 전체 질량 분석은 10㎍의 단백질을 Microcon centrifugal filter(Merck, MRCPRT010)를 사용하여 저분자의 화합물들을 제거한 단백질과 sinapinic acid(Bruker, 8201345) 매트릭스를 1:1 비율로 혼합한 뒤 target plate에 올려 건조한 후, 고성능 매트릭스 레이저 탈착 이온화 질량분석기의 flexcontrol 프로그램으로 실험을 진행하였다. Starter kit for MALDI-TOF MS(Bruker, 8208241)을 사용하여 질량 값을 보정하였으며, flexcontrol에서 제공하는 방법을 바탕으로 평균 1000 레이져샷으로 스펙트럼을 얻었다. 각각의 스펙트럼 데이터는 flexanalysis 프로그램을 이용하여 결과 값을 얻었다.For total mass spectrometry of ACE2, RBD, and CTP delta proteins, 10 μg of protein was mixed with a protein from which small molecular compounds were removed using a Microcon centrifugal filter (Merck, MRCPRT010) and a sinapinic acid (Bruker, 8201345) matrix at a 1:1 ratio. After drying on the target plate, the experiment was conducted with the flexcontrol program of the high-performance matrix laser desorption ionization mass spectrometer. The mass value was calibrated using the starter kit for MALDI-TOF MS (Bruker, 8208241), and the spectrum was obtained with an average of 1000 laser shots based on the method provided by flexcontrol. Each spectral data was obtained using the flexanalysis program.
3. LC-MS/MS 분석을 위한 샘플 준비3. Sample preparation for LC-MS/MS analysis
단일 단백질의 아미노산 서열 분석을 위하여 RBD, hACE2, CTP 델타 단백질 20㎍을 100 mM triethylammonium bicarbonate(Sigma, T7408-100ML) 와 2% sodium dodecyl sulfate(Duksan, 6645) 농도가 되도록 하였다. 10 mM의 dithiothreitol(BIO-RAD, 161-0611)을 가하고 56℃에서 30분 처리 후, 20 mM의 iodoaetamide(Sigma, T1503)를 가하고 차광시킨 후 실온에서 30분 처리하였다. 이후 S-trap mini columns(PROTIFI, C02-mini-80)을 사용하여 저분자의 화합물들을 제거하고 단백질 분해효소 Trypsin(Promega, V5111), Glu-C(Promega, V1651)를 2㎍을 가한 후 37℃에서 16시간 효소분해 반응을 시켰다. 반응이 끝난 후 원심분리기로 분리하여 진공건조하고 0.1% trifluoroacetic acid를 가하여 시료를 준비하였다. 그리고, RBD 와 ACE2 단백질은 탈당화(deglycosylation) 실험을 추가하였다. 탈당화(deglycosylation) 실험을 위해 단백질 분해효소 처리한 시료에 100 mM triethylammonium bicarbonate 와 2μg의 PNGase F(Promega, V4831)를 가한 후 37℃에서 4시간 효소분해 반응을 시켰다. 반응이 끝난 이후 S-trap mini columns을 사용하여 단백질을 원심분리기로 분리했다. 이를 진공건조하고 0.1% trifluoroacetic acid를 가하여 시료를 준비하였다. RBD 와 hACE2 단백질은 사중극자 오비트랩 질량분석기를 사용하였으며 나노-마이크로 액체크로마토그래피로 실험을 진행하였다. CTP 델타 단백질은 하이브리드 3중4중극자 선형 이온트랩질량분석기를 사용하였으며 마이크로 액체크로마토그래피를 이용하여 실험을 진행하였다.For amino acid sequence analysis of a single protein, 20 μg of RBD, hACE2, and CTP delta proteins were mixed with 100 mM triethylammonium bicarbonate (Sigma, T7408-100ML) and 2% sodium dodecyl sulfate (Duksan, 6645). After adding 10 mM of dithiothreitol (BIO-RAD, 161-0611) and processing at 56° C. for 30 minutes, 20 mM of iodoaetamide (Sigma, T1503) was added, shielded from light, and treated for 30 minutes at room temperature. Afterwards, small molecules were removed using S-trap mini columns (PROTIFI, C02-mini-80), and 2 μg of proteolytic enzymes Trypsin (Promega, V5111) and Glu-C (Promega, V1651) were added, followed by 37°C. Enzymatic digestion was performed for 16 hours. After the reaction was completed, the mixture was separated by a centrifuge, vacuum dried, and a sample was prepared by adding 0.1% trifluoroacetic acid. And, RBD and ACE2 proteins were added to the deglycosylation experiment. For the deglycosylation experiment, 100 mM triethylammonium bicarbonate and 2 μg of PNGase F (Promega, V4831) were added to the proteolytic enzyme-treated sample, followed by enzymatic digestion at 37°C for 4 hours. After the reaction was completed, proteins were separated by centrifugation using S-trap mini columns. Samples were prepared by vacuum drying and adding 0.1% trifluoroacetic acid. RBD and hACE2 proteins were tested using a quadrupole orbital mass spectrometer and nano-micro liquid chromatography. The CTP delta protein was tested using a hybrid triple quadrupole linear ion trap mass spectrometer and micro liquid chromatography.
4. LC-MS/MS 분석: 4. LC-MS/MS Analysis: ACQUITYACQUITY UPLCUPLC M-Class system - Q M-Class system - Q ExactiveExactive PLUS PLUS
액체 크로마토그래피에 사용된 용매는 모두 HPLC 급 용매를 사용하였다. Water(Duksan, 7732-18-5), acetonitrile(Honeywell, AH015-4)을 용매로 사용하였으며 dimethyl sulfoxide(Sigma, 472301-1L), trifluoroacetic acid(Thermo scientific, 28904), formic acid(Thermo scientific, 28905)를 실험용액 내의 매질로 사용하였다. 나노-마이크로 액체크로마토그래피는 solvent A(0.1% formic acid, 5% dimethyl sulfoxide in water) 와 solvent B(0.1% formic acid, 5% dimethyl sulfoxide in 80% acetonitrile)로 구성하였다. Trapping column으로 Acclaim™ PepMap™ 100 C18 LC Column(Thermo scientific, 164197)를 analytical column으로 EASY-Spray™ HPLC Columns(Thermo scientific, ES803A)를 사용하였다. 액체크로마토그래피는 시료 0.5㎍ 주입 후 trapping 단계에서 solvent A 95%, solvent B 5% 조건과 유속 8μl/min으로 5분간 진행하였다. Analytical 단계에서는 유속 250nl/min으로 진행하였다. Solvent A 95%, solvent B 5% 조건으로 5분 후 solvent B 5%에서 40%까지 순차적으로 올려 140분간 진행하며 시료를 분석했으며, 다시 5분간 95%까지 올린 후 15분간 컬럼을 활성화하고 solvent B를 5%까지 5분간 내린 후 40분간 이를 유지하며 재평형화했다. 사중극자 오비트랩 질량분석기는 Pierce™ LTQ Velos ESI Positive Ion Calibration Solution(Thermo scientific, 88323)으로 질량 값을 보정하였다. 분석법은 full-MS 와 data dependent MS/MS 방법으로 실험을 진행하였다. 산출된 데이터는 proteome discoverer(Thermo scientific)를 이용하여 결과를 얻었다.All solvents used in liquid chromatography were HPLC grade solvents. Water (Duksan, 7732-18-5) and acetonitrile (Honeywell, AH015-4) were used as solvents, dimethyl sulfoxide (Sigma, 472301-1L), trifluoroacetic acid (Thermo scientific, 28904), formic acid (Thermo scientific, 28905 ) was used as a medium in the experimental solution. Nano-micro liquid chromatography was composed of solvent A (0.1% formic acid, 5% dimethyl sulfoxide in water) and solvent B (0.1% formic acid, 5% dimethyl sulfoxide in 80% acetonitrile). Acclaim
5. LC-MS/MS 분석: M5 5. LC-MS/MS Analysis: M5 MicroLCMicroLC - - QTRAPQTRAP
분석에 사용한 컬럼은 Kinetex 2.6u XB-C18 100A(Phenomenex, 00B-4496-AC)를 사용하였다. 마이크로 액체크로마토그래피는 solvent A(0.1% formic acid in water) 와 solvent B(0.1% formic acid in acetonitrile)로 구성하였다. 액체크로마토그래피 조건은 시료 1.5㎍ 주입 후 유속 20μl/min으로 진행하였다. Solvent A 95%, solvent B 5% 조건으로 5분 후 solvent B 5%에서 40%까지 순차적으로 올려 45분간 진행하며 시료를 분석했으며, 다시 5분간 95%까지 올린 후 4분간 컬럼을 활성화하고 solvent B를 5%까지 3분간 내린 후 3분간 이를 유지하며 재평형화했다. 하이브리드 3중4중극자 선형 이온트랩 질량분석기는 standard chemical kit(Sciex, 4406127)로 질량 값을 보정하였다. 분석법은 Q3 MS 와 information dependent acquisition을 이용한 enhanced product ion 방법으로 실험을 진행하였다. 산출된 데이터는 convert MS(proteowizard.sourceforge.net)를 이용하여 wiff 파일을 mgf 파일로 변환한 뒤 searchGUI 와 peptide-shaker(compomics.github.io)를 이용하여 결과를 얻었다.The column used for analysis was Kinetex 2.6u XB-C18 100A (Phenomenex, 00B-4496-AC). Micro liquid chromatography was composed of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Liquid chromatography conditions were performed at a flow rate of 20 μl/min after injecting 1.5 μg of the sample. Solvent A 95%,
6. 미손상(intact) 단백질 질량 분석 결과6. Intact protein mass spectrometry results
MALDI-TOF를 이용하여 미손상(intact) 단백질의 질량을 측정하였다. RBD는 30kDa으로 측정되었다. 30kDa 외에 피크는 dimer로 60kDa의 peak과 전하(z)가 2인 monomer (m/z=15kDa), 전하(z)가 2인 trimer(m/z= 45kDa) 등이 함께 나타났다. 단백질의 서열로부터 계산한 RBD의 질량이 25.9kDa인 것을 고려하면, 4.1kDa의 질량 변화는 당화(Glycosylation)에 의해서 나타난 것으로 볼 수 있다. 비슷하게 hACE2의 질량은 약 102kDa으로 측정되었고, dimer와 전하가 2인 peak도 함께 나타났다. 단백질 서열로부터 계산한 hACE2의 질량이 84.4kDa인 것을 고려하면, 약 17.5kDa의 질량 변화가 당화(Glycosylation)에 의해 나타난 것으로 볼 수 있다. The mass of intact protein was measured using MALDI-TOF. RBD was measured to be 30 kDa. In addition to 30 kDa, the peak appeared as a dimer, such as a 60 kDa peak, a monomer with a charge (z) of 2 (m/z = 15 kDa), and a trimer (m/z = 45 kDa) with a charge (z) of 2. Considering that the mass of RBD calculated from the protein sequence is 25.9 kDa, the mass change of 4.1 kDa can be seen as caused by glycosylation. Similarly, the mass of hACE2 was measured to be about 102 kDa, and a dimer and a peak with a charge of 2 appeared together. Considering that the mass of hACE2 calculated from the protein sequence is 84.4 kDa, it can be seen that a mass change of about 17.5 kDa is caused by glycosylation.
CTP-δ1의 분석 결과, 단백질 서열로부터 계산한 질량은 6.396kD이었는데, MALDI-TOF로 측정된 질량은 6.221kD으로, N-말단 'M'이 탈락된 서열로 확인되었다(도 3). As a result of analysis of CTP-δ1, the mass calculated from the protein sequence was 6.396 kD, but the mass measured by MALDI-TOF was 6.221 kD, confirming that the N-terminal 'M' was omitted (FIG. 3).
7. 단백질 서열 확인7. Protein Sequence Verification
RBD, hACE2, CTP 델타의 서열을 확인하기 위하여 LC-MS/MS를 분석을 수행하였다. 각각의 단백질은 단백질 분해효소인 Trypsin과 Glu-C를 사용하여 단백질 분석을 진행하였으며, RBD와 hACE2는 PNGase F 효소활성에 의해 Asn이 Asp로 바뀐 아미노산 잔기를 검색하여 N-linked 당화(Glycosylation) 된 Asn을 찾았다. LC-MS/MS analysis was performed to confirm the sequences of RBD, hACE2, and CTP delta. Each protein was analyzed using proteolytic enzymes Trypsin and Glu-C, and RBD and hACE2 searched for amino acid residues where Asn was changed to Asp by PNGase F enzymatic activity, resulting in N-linked glycosylation. Found Asn.
N, C-말단이 확인된 CTP 델타의 MS/MS 분석결과를 도 4에 나타냈다. N, C-말단의 서열 분석은 단백질의 상태를 확인하는 수단 중의 하나이며, 단백질의 말단이 확인되지 않는다면 단백질의 보존 상태에 문제가 있어 분해된 것으로 간주할 수 있다. RBD와 hACE2의 경우 단백질의 말단이 ID 되지 않는 경향을 보이므로, 서열의 부정확성이나 PTM의 효과가 아니라면, 이들은 잘 분해되는 단백질이니 보존에 있어 주의가 필요하다.The results of MS/MS analysis of CTP delta with confirmed N and C-terminus are shown in FIG. 4 . Sequence analysis of the N-terminus and C-terminus is one of means for confirming the state of the protein, and if the terminus of the protein is not confirmed, it can be regarded as degraded due to a problem with the preservation state of the protein. In the case of RBD and hACE2, since the end of the protein tends not to be ID, unless it is the inaccuracy of the sequence or the effect of PTM, these are proteins that are easily degraded, so care must be taken in preservation.
<< 실시예Example 5> 코로나바이러스 감염증 5> Coronavirus infection COVIDCOVID -19 치료제 단백질 -19 therapeutic protein CTPCTP 델타의 2차 구조 안정성 Secondary structural stability of delta 및 기능and function /유효성 검증//validate
1. One. 원형이색상편광circular dichroic polarization (Circular (Circular DichorismDichorism ; CD) 실험; CD) experiment
Far UV 원형이색상편광(Circular Dichroism)을 RBD(PBS buffer), hACE2(PBS buffer), CTP 델타에 대해 측정하였다. JASCO-1500 기계를 이용하여 20℃ 온도에서 단백질의 농도는 0.2mg/ml, 0.1cm의 석영(quartz) 셀을 사용하였다. 190-250nm의 far UV 파장에서 20nm/min의 측정 속도, 5nm의 bandwidth, 4초의 D.I.T(Digital Integration Time)을 이용하여 측정하였다. 단백질에 대해 5회 측정한 데이터를 버퍼(buffer)를 5회 측정한 데이터로 감산하여 그래프를 나타내었다.Far UV circular dichroism was measured for RBD (PBS buffer), hACE2 (PBS buffer), and CTP delta. Using a JASCO-1500 machine, the protein concentration was 0.2 mg/ml at 20 °C and a 0.1 cm quartz cell was used. Measurement was performed at a far UV wavelength of 190-250 nm using a measurement speed of 20 nm/min, a bandwidth of 5 nm, and a digital integration time (D.I.T) of 4 seconds. A graph was shown by subtracting the data measured for
그 결과, hACE2의 경우 α-helix를 2차 구조로 가진 단백질인 것을 알 수 있고, RBD는 beta sheet의 2차 구조를 가진 단백질임을 알 수 있다. CTP 델타는 helical 구조와 coil 구조가 함께 있는 것을 알 수 있다(도 5).As a result, it can be seen that hACE2 is a protein having an α-helix secondary structure, and RBD is a protein having a beta sheet secondary structure. It can be seen that the CTP delta has both a helical structure and a coil structure (FIG. 5).
2. 미세규모 2. Microscale 열영동thermophoresis (( MicroscaleMicroscale ThermophoresisThermophoresis ; MST) 실험 ; MST) experiment
8μM 100μL의 RBD(0.05% Tween-20 추가한 PBS buffer)에 Monolith Protein Labeling Kit RED-NHS 2nd Generation를 이용하여 형광을 부착하였다. Ligand(hACE2(PBS buffer), CTP 델타는 1:1 연속희석법(serial dilution)을 사용하여 16μM 부터 0.488nM 까지 16개 농도로 각각 10μL 준비하였다. 형광이 부착된 RBD를 30nM로 희석하여 준비된 16개 농도의 리간드에 각각 10μL 추가하여 15nM의 RBD와 8μM ~ 0.244nM의 리간드 혼합 용액을 준비하였다. Monolith NT.115 기계로 60% Excitation power와 Medium MST power를 이용하여 각 리간드에 대해서 MST를 5회 측정하였다. Monolith 회사에서 제공하는 MO affinity analysis 프로그램의 Kdmodel을 이용하여 Kd(dissociation constant) 값을 계산하였다. 5회의 실험 중 중앙값의 Kd 값을 가지는 실험결과를 그래프로 나타내었다.Fluorescence was attached to 100 μL of 8 μM RBD (PBS buffer with 0.05% Tween-20 added) using Monolith Protein Labeling Kit RED-NHS 2nd Generation. Ligand (hACE2 (PBS buffer), CTP delta) were prepared in 10 μL each at 16 concentrations from 16 μM to 0.488 nM using 1:1 serial dilution. 16 samples prepared by diluting fluorescent RBD to 30 nM A mixed solution of 15 nM RBD and 8 μM ~ 0.244 nM ligand was prepared by adding 10 μL of each ligand to each concentration.MST was measured 5 times for each ligand using a Monolith NT.115 machine at 60% excitation power and medium MST power. The Kd (dissociation constant) value was calculated using the Kdmodel of the MO affinity analysis program provided by Monolith, and the results of the experiment with the median Kd value among the five experiments were shown in a graph.
그 결과, 농도의 변화에 따른 형광의 세기의 변화가 관측되어 hACE2와 CTP 델타가 모두 RBD에 결합하는 것을 알 수 있다. 결합하는 세기를 나타내는 Kd 값은 CTP-δ1은 56.3nM, hACE2는 42.2nM 이다(도 6). RBD와 hACE2의 Kd 값은 Science 논문에서(Wrapp et al, Science 367, 1260-1263(2020)) BLI(biolayer interferometry)와 SPR(Surface Plasmon Resonance)을 이용하여 측정한 34.6nM의 Kd 값과 유사하다.As a result, a change in fluorescence intensity according to a change in concentration was observed, indicating that both hACE2 and CTP delta bind to RBD. The Kd values representing the binding strength were 56.3 nM for CTP-δ1 and 42.2 nM for hACE2 (FIG. 6). The Kd values of RBD and hACE2 are similar to the Kd value of 34.6 nM measured using BLI (biolayer interferometry) and SPR (Surface Plasmon Resonance) in the Science paper (Wrapp et al, Science 367, 1260-1263 (2020)). .
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시예일 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. Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> Daegu Gyeongbuk Institute of Science and Technology
KOREA BASIC SCIENCE INSTITUTE
<120> Therapeutic protein CTP delta for treating coronavirus disease
COVID-19 and use thereof
<130> ADP-2020-0556
<160> 1
<170> KopatentIn 2.0
<210> 1
<211> 46
<212> PRT
<213> Artificial Sequence
<220>
<223> CTP-Delta1
<400> 1
Met Gly Glu Phe Ala Asp Lys Leu Lys Asn Glu Leu Ala Lys Thr Glu
1 5 10 15
Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His Glu Ala Glu Asp
20 25 30
Leu Phe Tyr Gln Ser Ser Gly Leu Gly Lys Gly Asp Phe Arg
35 40 45
<110> Daegu Gyeongbuk Institute of Science and Technology
KOREA BASIC SCIENCE INSTITUTE
<120> Therapeutic protein CTP delta for treating coronavirus disease
COVID-19 and its use
<130> ADP-2020-0556
<160> 1
<170> KopatentIn 2.0
<210> 1
<211> 46
<212> PRT
<213> artificial sequence
<220>
<223> CTP-Delta1
<400> 1
Met Gly Glu Phe Ala Asp Lys Leu Lys Asn Glu Leu Ala
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