KR101899209B1 - Proteasome Sensitive NIS-MODC Fusion Protein - Google Patents
Proteasome Sensitive NIS-MODC Fusion Protein Download PDFInfo
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- KR101899209B1 KR101899209B1 KR1020160086812A KR20160086812A KR101899209B1 KR 101899209 B1 KR101899209 B1 KR 101899209B1 KR 1020160086812 A KR1020160086812 A KR 1020160086812A KR 20160086812 A KR20160086812 A KR 20160086812A KR 101899209 B1 KR101899209 B1 KR 101899209B1
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- South Korea
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- leu
- nis
- cells
- cell
- modc
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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Abstract
본 발명은 NIS(sodium/iodide symporter)와 mODC(mouse ornithine decarboxylase degradation domain)이 결합된 재조합 융합단백질, 이를 암호화하는 재조합 폴리뉴클레오타이드, 이를 포함하는 발현벡터, 및 이를 이용한 세포 영상화 또는 치료 방법에 관한 것이다. 본 발명에 의하면, 생체내 암줄기세포와 프로테아좀 활성이 낮은 세포, 프로테아좀 억제제로 치료를 받은 세포를 실시간으로 검출하고 추적할 수 있는 새로운 분자영상기술을 제공할 뿐만 아니라, 치료용 방사성 요오드를 이용함으로써 표적 세포를 제거하는 치료기술로도 활용할 수 있다.The present invention relates to a recombinant fusion protein comprising a sodium / iodide symporter (NIS) and a mouse ornithine decarboxylase degradation domain (mODC), a recombinant polynucleotide encoding the recombinant fusion protein, an expression vector containing the same, and a cell imaging or therapeutic method using the same . According to the present invention, not only new molecular imaging technology capable of detecting and tracing in vivo cytoskeletal cells and cells treated with proteasome inhibitor, and cells treated with proteasome inhibitor can be provided, and therapeutic radioactive iodine Can be utilized as a therapeutic technique for removing target cells.
Description
본 발명은 NIS(sodium/iodide symporter)와 mODC(mouse ornithine decarboxylase degradation domain)이 결합된 재조합 융합단백질, 이를 암호화하는 재조합 폴리뉴클레오타이드, 이를 포함하는 발현벡터, 및 이를 이용한 세포 영상화방법 및 치료방법에 관한 것이다.The present invention relates to a recombinant fusion protein comprising a sodium / iodide symporter (NIS) and a mouse ornithine decarboxylase degradation domain (mODC), a recombinant polynucleotide encoding the recombinant fusion protein, an expression vector containing the same, and a cell imaging method and a therapeutic method using the same will be.
살아 있는 세포는 세포질 안의 단백질 pool을 끊임없이 변화시키고 있으며, 이와 관련된 시스템 중에서 프로테아좀은 수명이 짧은 조절 단백질과 손상된 단백질의 제거를 담당하는 세포의 일차적 단백질 붕괴 시스템에 해당한다. 프로테아좀을 통한 제거 신호는 대상 단백질의 lysine기에 결합되어 있는 ubiquitin chain이 담당하고 있다. Living cells are constantly changing the protein pool in the cytoplasm, and among these systems, proteasome is the primary protein degradation system in cells responsible for the removal of short-lived regulatory proteins and damaged proteins. The signal removed by the proteasome is carried by the ubiquitin chain bound to the lysine of the target protein.
최근에 질병 치료를 위해 세포의 프로테아좀 시스템 기능에 대해 관심이 높아지고 있다. 그 예로 bortezomib(VELCADETM)이라는 약물은 최초로 개발된 프로테아좀 억제제로서 다발성 골수종이라는 암의 치료제로 미국 FDA 허가를 획득하였다. 이 허가의 기반연구를 통해 프로테아좀 기능이 암세포의 증식과 생존에 종요하며 살아있는 몸속에서 이 기능을 안전하게 억제하는 것이 가능함이 확인되었다. 반대로 신경퇴행성 질환 등에서는 프로테아좀 기능이 부족하여 독성 단백질이 축적될 수 있다. 이와 같이, 질병의 극복을 위해 프로테아좀 기능을 억제하는 약제와 기능을 촉진하는 약제 모두 개발대상이 되고 있으며, 따라서 항진된 프로테아좀 기능과 저하된 프로테아좀 기능 모두 질병진단이나 치료효과 평가를 위한 영상기술의 표적이 될 수 있다. Recently, attention has been paid to the function of cellular proteasome systems to treat diseases. For example, bortezomib (VELCADE TM ) is the first developed proteasome inhibitor and has been approved by the US FDA for the treatment of multiple myeloma. Based on this grant, it has been shown that proteasome function is essential for the proliferation and survival of cancer cells and that it is possible to safely suppress this function in the living body. Conversely, in neurodegenerative diseases and the like, toxic proteins may accumulate due to lack of proteasome function. Thus, in order to overcome disease, both proteasome inhibiting agents and agents promoting function are being developed. Therefore, both enhanced proteasome function and decreased proteasome function are diagnosed or treated Can be a target of imaging technology for.
한편, 최근에 항암제에 저항성을 보이는 암줄기세포가 항암치료 내성에 관여한다고 여겨지고 있다. 암줄기세포(cancer stem cell)는 무제한 재생능력을 가진 암세포로서 동물모델에 주입하면 원발 종양의 고유 세포 다양성이 재현된 암을 쉽게 만든다. 이러한 세포 다양성은 일반 줄기세포의 분화 다양성과 유사하며 암세포의 항암제 내성에 중요한 역할을 한다. 따라서, 암줄기세포의 형질 특성은 항암제와 방사선에 대한 민감도가 저하되는 암치료 내성에 기여하며, 이들 암세포를 표적하는 새로운 암치료 전략의 필요성이 대두되고 있다.On the other hand, cancer stem cells, which have recently been shown to be resistant to anticancer drugs, are thought to be involved in chemoresistance. Cancer stem cells are cancer cells with an unlimited regenerative capacity and when injected into an animal model, native cell diversity of the primary tumor is easily reproduced. Such cell diversity is similar to the differentiation diversity of general stem cells and plays an important role in cancer cell resistance to anticancer drugs. Therefore, the trait characteristics of cancer stem cells contribute to the resistance to cancer therapy, which is less susceptible to anticancer drugs and radiation, and a new cancer treatment strategy targeting these cancer cells is required.
암정복에 성공하려면 암의 항암치료 내성 메카니즘을 밝히고 이를 억제하는 전략 개발이 중요함에도 불구하고 지금까지는 이에 대한 연구가 제한적이고 암줄기세포의 원천과 그 유지 기작에 대해서는 확실하게 밝혀진 바가 없다. 따라서, 정상 세포에는 손상을 주지 않으면서 암줄기세포만을 표적하는 치료를 효율적으로 수행하기 위해서는 암줄기세포의 형질 유지와 조절에 중요한 분자생물학적인 조절 기작에 대한 지식과 이해를 토대로 살아있는 세포에서도 이를 평가하고 모니터할 수 있는 새로운 영상기술의 개발이 중요하다.To succeed in conquering cancer, it is important to develop a strategy to inhibit the cancer-resistant resistance mechanism of cancer. However, the research on cancer chemosis has been limited so far and the origin and maintenance mechanism of cancer stem cells has not been clarified. Therefore, in order to efficiently perform treatment targeting only cancer stem cells without damaging normal cells, it is necessary to evaluate and monitor the survival cells based on knowledge and understanding of molecular biologic control mechanisms important for the maintenance and regulation of cancer stem cells It is important to develop a new imaging technology that can do this.
암줄기세포를 체내에서 영상화하는 기술은 항암 신약개발에서 동물 전임상시험에 적용하면 치료효과를 조기에 평가하여 신약 개발기간을 크게 단축시킬 수 있을 것이다. 항암제에 대한 종양의 일시적인 크기 감소는 증식속도가 빠른 일반 암세포에 대한 효과를 반영할 뿐 암줄기세포에 대한 효과를 반영하지 못하기 때문에, 치료과정 중에 항암제 내성을 평가하기 위해서는 종양 안에 소수로 존재하는 암줄기세포를 검출하고 평가할 수 있는 새로운 영상기술이 필요하다. The technique of imaging cancer stem cells in the body can be applied to animal pre-clinical tests in the development of anti-cancer drugs, and early evaluation of treatment effects will greatly shorten the development period of new drugs. Since the temporal size reduction of the tumor to the anticancer drug reflects the effect on the normal cancer cell with a rapid growth rate, it does not reflect the effect on the cancer stem cell. Therefore, in order to evaluate the anticancer drug resistance during the treatment, New imaging techniques are needed to detect and evaluate the cells.
암줄기세포의 형질을 검출하기 위한 종래기술은 조직을 침습적으로 채취하여 절편을 만들고 CD133이나 CD44 등 세표표면 마커를 면역화학염색을 통해 현미경으로 확인하는 수준에 머물러 있는데, 이러한 기술로는 살아있는 세포에서 실시간 모니터링이 어렵고 몸속에서 영상하기 불가능하다. 따라서, 암줄기세포에 대한 치료를 시작할 때는 약물의 반응을 가늠할 수 있는 지금까지의 약물반응 척도와는 다른 새로운 영상검사 등 비관혈적인 평가방법이 고안되어야 한다. Conventional techniques for detecting the traits of cancer stem cells are invasive sampling of tissues to make slices and microscopic examination of the surface markers such as CD133 and CD44 by immunochemical staining. It is difficult to monitor and impossible to image in the body. Therefore, when starting treatment for cancer stem cells, non-invasive evaluation methods such as new imaging tests, which are different from conventional drug response scales that can measure the response of drugs, should be devised.
암줄기세포의 세포학적 특성 중에는 프로테아좀(proteasome) 활성이 감소하는 성질이 있는데, 프로테아좀은 세포내에서 단백질을 분해하는 거대 분자로 세포 안의 여러 단백질을 파괴하는 역할을 한다. 즉, 세포내 단백질들은 유비퀴틴과 결합하고, 폴리유비퀴틴 인지 도메인을 갖고 있는 프로테아좀의 19S regulator 부분과 결합한 후, deubiquitinating enzyme에 의해 유비퀴틴은 단백질에서 떨어져 나간다. 프로테아좀에 붙어 있는 단백질은 defolding되어 프로테아좀의 프로테아제(protease) 활성을 갖는 20S core 부분으로 들어가 단백질 분해가 일어난다.The cytosolic characteristics of cancer stem cells have a property of decreasing proteasome activity. Proteasome is a macromolecule that breaks down proteins in cells and serves to destroy various proteins in cells. That is, intracellular proteins bind to ubiquitin and bind to the 19S regulator part of proteasome, which has a polyubiquitin cognate domain, and then the ubiquitin is released from the protein by deubiquitinating enzyme. The protein attached to the proteasome is defolded and enters the 20S core region with the protease activity of proteasome, resulting in protein degradation.
이상과 관련하여, mODC(mouse ornithine decarboxylase degradation domain)은 프로테아좀에 민감하여 프로테아좀 활성이 있는 세포에서는 빠르게 분해되나, 프로테아좀 활성이 저하된 세포에서는 축적되기 때문에, 이러한 원리를 이용하여 리포터 영상기법(reporter imaging)에 응용할 수 있다. 이 기술은 암줄기세포를 포함하여 프로테아좀 활성이 저하된 각종 세포의 검출과 프로테아좀 활성을 억제시키는 치료약물의 효과 평가에 활용할 수 있다.In relation to the above, mODC (mouse ornithine decarboxylase degradation domain) is sensitive to proteasome and rapidly degrades in cells with proteasome activity, but accumulates in cells with reduced proteasome activity. Therefore, It can be applied to reporter imaging. This technique can be used for the detection of various cells with reduced proteasome activity, including cancer stem cells, and for evaluating the efficacy of therapeutic drugs that inhibit proteasome activity.
한편, NIS(sodium/iodide symporter)는 갑상선 세포에 존재하는 요오드 운반체로서, 요오드의 선택적인 세포 섭취를 매개한다. NIS는 13개의 도메인을 갖는 단백질로서 sodium-potassium ATPase에 의하여 생기는 나트륨 이온의 세포막 안팎의 농도 차에 의하여 운반능이 발생하고 유지된다. On the other hand, NIS (sodium / iodide symporter) is an iodine carrier present in thyroid cells and mediates selective cell uptake of iodine. NIS is a protein with 13 domains. The sodium ion produced by sodium-potassium ATPase is transported and maintained by the concentration difference between the inside and outside of the cell membrane.
이에, 본 발명자들은 암줄기세포를 포함하여 프로테아좀 활성이 저하된 세포와 프로테아좀 억제 치료제의 치료효과를 생체내에서 실시간 모니터하고 선택적으로 방사성옥소로 표적하여 치료하는 새로운 기술을 개발하고자 한다. 이를 위해 프로테아좀 활성에 민감한 mODC에 NIS를 결합시킨 융합 단백질(fusion protein)을 개발하여, 프로테아좀 활성이 저하된 세포에서 방사성 옥소(radio-iodide)의 섭취를 증가시켜 영상화하거나 치료할 수 있는 새로운 기법을 제공하고자 한다.Accordingly, the present inventors intend to develop a new technique for monitoring the therapeutic effect of the proteasome inhibiting therapeutic agent and the cell with reduced proteasome activity including the cancer stem cells in vivo and selectively targeting with radioactive iodine. To this end, we have developed a fusion protein with NIS-binding mODC, which is sensitive to proteasome activity, and is capable of imaging or treating by increasing the uptake of radio-iodide in cells with reduced proteasome activity We want to provide a new technique.
따라서, 본 발명의 목적은, NIS(sodium/iodide symporter)와 mODC(mouse ornithine decarboxylase degradation domain)이 결합된 재조합 융합단백질, 재조합 폴리뉴클레오타이드, 발현벡터 등을 제공하는 데 있다.Accordingly, an object of the present invention is to provide a recombinant fusion protein, a recombinant polynucleotide, an expression vector and the like, wherein the NIS (sodium / iodide symporter) and mODC (mouse ornithine decarboxylase degradation domain) are combined.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.
상기와 같은 과제를 해결하기 위하여, 본 발명은 NIS(sodium/iodide symporter)와 mODC(mouse ornithine decarboxylase degradation domain)이 결합된, 재조합 융합단백질을 제공한다.In order to solve the above problems, the present invention provides a recombinant fusion protein comprising a sodium / iodide symporter (NIS) and a mouse ornithine decarboxylase degradation domain (mODC).
본 발명의 일 구현예로, 상기 융합단백질은 서열번호 1의 아미노산 서열로 이루어지는 것을 특징으로 한다.In one embodiment of the present invention, the fusion protein is characterized by comprising the amino acid sequence of SEQ ID NO: 1.
본 발명의 다른 구현예로, 상기 NIS 단백질은 인간, 마우스, 래트, 개 또는 돼지 유래인 것을 특징으로 한다.In another embodiment of the present invention, the NIS protein is derived from a human, mouse, rat, dog or pig.
또한, 본 발명은 상기 융합단백질을 암호화하는 재조합 폴리뉴클레오타이드를 제공한다.In addition, the present invention provides a recombinant polynucleotide encoding said fusion protein.
본 발명의 일 구현예로, 상기 폴리뉴클레오타이드는 서열번호 2의 염기서열로 이루어지는 것을 특징으로 한다.In one embodiment of the present invention, the polynucleotide is characterized by comprising the nucleotide sequence of SEQ ID NO: 2.
또한, 본 발명은 상기 폴리뉴클레오타이드를 포함하는 재조합 발현벡터를 제공한다.The present invention also provides a recombinant expression vector comprising the polynucleotide.
또한, 본 발명은 상기 발현벡터를 포함하는, 프로테아좀 활성이 저하된 세포의 모니터링용 조성물을 제공한다.In addition, the present invention provides a composition for monitoring a cell with reduced proteasome activity comprising the expression vector.
본 발명의 일 구현예로, 상기 프로테아좀 활성이 저하된 세포는 암줄기세포 또는 프로테아좀 억제제로 치료를 받은 세포인 것을 특징으로 한다.In one embodiment of the present invention, the cell with reduced proteasome activity is a cell treated with a cancer stem cell or proteasome inhibitor.
본 발명의 다른 구현예로, 상기 조성물은 방사성동위원소 섭취 모니터링용인 것을 특징으로 한다.In another embodiment of the present invention, the composition is for monitoring radioisotope uptake.
본 발명의 또 다른 구현예로, 상기 방사성동위원소는 123I, 124I, 또는 125I인 것을 특징으로 한다.In another embodiment of the present invention, the radioactive isotope is 123 I, 124 I, or 125 I.
또한, 본 발명은 상기 발현벡터를 포함하는, 프로테아좀 활성이 저하된 세포의 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the treatment of cells with reduced proteasome activity, comprising the above expression vector.
본 발명의 일 구현예로, 상기 프로테아좀 활성이 저하된 세포는 암줄기세포인 것을 특징으로 한다.In one embodiment of the present invention, the cell with reduced proteasome activity is characterized by being a cancer stem cell.
본 발명의 다른 구현예로, 상기 조성물은 방사성동위원소 섭취에 의한 치료용인 것을 특징으로 한다.In another embodiment of the present invention, the composition is for treatment by radioisotope ingestion.
본 발명의 또 다른 구현예로, 상기 방사성동위원소는 131I인 것을 특징으로 한다.In another embodiment of the present invention, the radioactive isotope is 131 I.
또한, 본 발명은, (a) 인비트로에서(in vitro) 상기 조성물을 프로테아좀 활성이 저하된 세포에 처리하는 단계; (b) 방사성동위원소를 상기 세포에 처리하는 단계; 및 (c) 세포에서 방사성동위원소를 측정하는 단계를 포함하는 세포의 영상화 방법을 제공한다.The present invention also relates to a method for the treatment of cancer, comprising the steps of: (a) treating said composition in vitro with cells having reduced proteasome activity; (b) treating the cell with a radioactive isotope; And (c) measuring the radioisotope in the cell.
본 발명의 일 구현예로, 상기 프로테아좀 활성이 저하된 세포는 암줄기세포 또는 프로테아좀 억제제로 치료를 받은 세포인 것을 특징으로 한다.In one embodiment of the present invention, the cell with reduced proteasome activity is a cell treated with a cancer stem cell or proteasome inhibitor.
본 발명의 다른 구현예로, 상기 방사성동위원소는 123I, 124I, 또는 125I인 것을 특징으로 한다.In another embodiment of the present invention, the radioactive isotope is 123 I, 124 I, or 125 I.
본 발명에 의하면, 생체내 암줄기세포를 실시간으로 검출하고 추적할 수 있는 새로운 분자영상기술을 제공함으로써, 종양 형성과 전이 과정에서 암줄기세포의 역할을 밝힐 수 있다. According to the present invention, it is possible to reveal the role of cancer stem cells in tumorigenesis and metastasis by providing a new molecular imaging technique capable of detecting and tracking cancer stem cells in vivo in real time.
또한, 본 발명에 의하면, 암줄기세포 외에도 프로테아좀 활성이 저하된 각종 질환 조직을 이미징하는 데 활용될 수 있다. Further, according to the present invention, in addition to the cancer stem cells, it can be utilized for imaging various disease tissues in which the proteasome activity is decreased.
또한, 본 발명에 의하면, 프로테아좀을 표적하는 치료에 대한 효과를 모니터링할 수 있다. Further, according to the present invention, the effect on treatment for targeting proteasome can be monitored.
또한, 본 발명에 의하면, 방사성 요오드 치료를 통하여 암줄기세포 등 프로테아좀 활성이 저하된 질병세포를 선택적으로 치료하는 기술로 활용될 수 있다.In addition, according to the present invention, it is possible to utilize radioactive iodine as a technique for selectively treating diseased cells having reduced proteasome activity such as cancer stem cells.
도 1은, 프로테아좀 민감형 NIS-mODC 융합 단백질을 이용하여 방사성 동위원소(방사성 옥소) 섭취에 의한 영상 또는 치료 기법의 개념을 도식화한 것이다.
도 2는, 재조합 발현벡터 pQCXIN-huNIS-mODC의 제작을 위한 pZsProSensor-1 벡터와 pQCXIN retroviral 벡터의 map을 나타낸 것이다.
도 3은, 프로테아좀 민감형 NIS-mODC 융합 단백질을 암호화하는 DNA 염기서열을 나타낸 것이다.
도 4는, MCF7 유방암 세포주에 NIS-mODC 융합 단백질을 암호화하는 DNA를 트랜스펙션시킨 후, 프로테아좀 억제제인 MG132 처리군(MG132+)과 비처리군(MG132-)에 대하여 항-NIS 항체로 각각 웨스턴 블랏팅을 수행한 결과이다.
도 5는, CT26 대장암 세포주에 NIS-mODC 융합 단백질을 암호화하는 DNA를 트랜스펙션시킨 후, 프로테아좀 억제제인 PS341 처리군(PS341+)과 비처리군(PS341-)에 대하여 방사성 옥소 섭취율(radioiodide uptake)을 측정하여 정량화한 그래프이다.
도 6은, CT26 대장암 세포주에 NIS-mODC 융합 단백질을 암호화하는 DNA를 트랜스펙션시켜 제조된 huNIS-mODC가 안정적으로 발현된 CT26 cell에 프로테아좀 억제제인 PS341 처리군(PS341 +)과 비처리군(PS341 -)에 대하여 방사성 옥소 섭취율(radioiodide uptake)을 측정하여 정량화한 그래프이다.
도 7은, huNIS-mODC가 안정적으로 발현된 CT26 대장암 세포주(CT26 cell)와 CT26 세포를 암줄기세포에 대한 선택적 배지에 배양하여 얻은 암줄기세포(cancer stem cell; CT26 CSC)를 이용하여 프로테아좀 억제제인 PS341을 처리한 후 방사성 옥소 섭취율(radioiodide uptake)을 측정하여 정량화한 그래프이다.
도 8은, NIS-mODC가 트랜스펙션되지 않은 CT26 대장암 세포주(CT26 cell) 와 huNIS-mODC가 안정적으로 발현된 CT26 cell(stable cell)에 프로테아좀 억제제인 PS341을 처리한 후 항-NIS 항체로 각각 웨스턴 블랏팅을 수행한 결과이다.
도 9는, huNIS-mODC가 안정적으로 발현된 CT26 대장암 세포를 8주령 wild type Balb/C 마우스의 어깨에 피하 주사하여 종양을 형성시킨 다음, PS341을 처리한 군과 처리하지 않은 군에 (군당 6마리) I-124을 정맥주사하고 1 시간 뒤 양전자방출단층촬영을 하여 얻은 동물영상 관상단면(coronal section) 소견, 그리고 종양조직을 떼어낸 후 근육대비 섭취율을 측정하여 비교한 도면이다((1):PET 영상소견, (2): I-124의 종양 대 근육 섭취비).
도 10은 huNIS-mODC가 안정적으로 발현된 CT26 대장암 세포를 8주령 wild type Balb/C 마우스의 어깨에 피하 주사하여 종양을 형성시킨 다음, PS341을 처리한 군과 처리하지 않은 군에 대하여 종양조직을 떼어 내여 절편을 얻고 NIS에 대한 면역조직화학염색을 수행한 결과를 나타낸 도면이다.Figure 1 is a schematic representation of the concept of imaging or therapeutic techniques by the ingestion of radioisotopes (radioactive iodine) using a proteasome-sensitive NIS-mODC fusion protein.
Fig. 2 shows a map of the pZsProSensor-1 vector and the pQCXIN retroviral vector for the production of the recombinant expression vector pQCXIN-huNIS-mODC.
Fig. 3 shows the DNA base sequence encoding the proteasome-sensitive NIS-mODC fusion protein.
FIG. 4 shows the results of the transfection of DNA encoding the NIS-mODC fusion protein in MCF7 breast cancer cell lines and the anti-NIS antibody against the proteasome inhibitor MG132 (MG132 +) and the non-treatment group (MG132-) The results are obtained by performing Western blotting, respectively.
FIG. 5 shows the results of transfection of DNA encoding the NIS-mODC fusion protein into the CT26 colon cancer cell line, and then, with respect to the proteasome inhibitor PS341 + (PS341 +) and the non-treatment group (PS341-) radioiodide uptake) was measured and quantified.
Fig. 6 is a graph showing the relationship between the PS341 + treatment group (PS341 +) and the proteasome inhibitor (PS341 +) in the CT26 cell stably expressing huNIS-mODC prepared by transfecting the CT26 colon cancer cell line with the DNA encoding the NIS- (Radioactive iodine uptake) of the treated group (PS341 -).
FIG. 7 is a graph showing the results obtained by using CT26 CSC, which is obtained by culturing CT26 colon cancer cell line (CT26 cell) and CT26 cell stably expressing huNIS-mODC in a selective medium for cancer stem cells, (Radioiodide uptake) after PS341, which is an inhibitor, was measured and quantified.
FIG. 8 is a graph showing the results of treatment of a CT26 colon cancer cell line (CT26 cell) in which NIS-mODC was not transfected and PS341, which is a proteasome inhibitor, in CT26 cell (stable cell) stably expressing huNIS-mODC, And Western blotting, respectively.
FIG. 9 is a graph showing the results of tumor growth of CT26 colon cancer cells stably expressing huNIS-mODC on the shoulder of 8-week-old wild-type Balb / C mice by subcutaneous injection, 6 corneal sections obtained by intravenous injection of I-124 and
Fig. 10 shows the results of tumor-forming CT26 colon cancer cells stably expressing huNIS-mODC on the shoulder of 8-week-old wild-type Balb / C mice, And the sections were obtained and immunohistochemical staining for NIS was performed.
본 발명은 NIS(sodium/iodide symporter)와 mODC(mouse ornithine decarboxylase degradation domain)이 결합된 재조합 융합단백질, 이를 암호화하는 재조합 폴리뉴클레오타이드, 및 이를 포함하는 발현벡터를 제공한다.The present invention provides a recombinant fusion protein comprising a sodium / iodide symporter (NIS) and a mouse ornithine decarboxylase degradation domain (mODC), a recombinant polynucleotide encoding the recombinant fusion protein, and an expression vector comprising the same.
본 발명의 융합단백질은 통상의 발현벡터를 이용하여 제조할 수 있으며, 보다 상세하게는 NIS를 코딩하는 유전자, 및 mODC의 전장 아미노산 서열 중 41 내지 422번째 아미노산 서열(PETS 서열)을 코딩하는 유전자, 및 이 두 유전자가 작동 가능하게 연결된 하나의 프로모터를 포함하는, 재조합 발현벡터일 수 있다. 후술하는 본 발명의 실시예에서는 이러한 발현벡터 중 대표적인 하나의 발현벡터를 예로 들어 도 2에 도시하였다.The fusion protein of the present invention can be prepared using a conventional expression vector, and more specifically, a gene encoding NIS and a gene encoding the amino acid sequence 41 to 422 of the full amino acid sequence of mODC (PETS sequence) And one promoter in which these two genes are operably linked. In an embodiment of the present invention described later, a typical expression vector among these expression vectors is shown in Fig. 2 as an example.
본 발명에 있어서, "발현벡터"란 유전공학에 이용되는 벡터로서, 플라스미드 벡터가 바람직하나 이에 한정되지 않고, 예를 들면 바이러스 벡터, 코스미드 벡터, 세균 인공 염색체(BAC), 효모 인공 염색체(YAC) 및 다른 비플라스미드 벡터도 이용될 수 있다.In the present invention, "expression vector" is a vector used in genetic engineering and is preferably a plasmid vector, but is not limited thereto. Examples thereof include virus vectors, cosmid vectors, bacterial artificial chromosomes (BAC), yeast artificial chromosomes ) And other non-plasmid vectors may also be used.
상기 "프로모터"란 특정한 숙주 세포에서 작동 가능하게 연결된 핵산 서열의 발현을 조절하는 DNA 서열을 의미하며, 발현용으로 사용되는 것이라면 특별히 한정되지 않는다. 또한, 상기 "작동 가능하게 연결(operably linked)"이란 하나의 핵산 단편이 다른 핵산 단편과 결합되어 그의 기능 또는 발현이 다른 핵산 단편에 의해 영향을 받는 것을 말한다. As used herein, the term "promoter " means a DNA sequence that controls the expression of a nucleic acid sequence operatively linked to a specific host cell, and is not particularly limited as long as it is used for expression. The term "operably linked " also means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments.
아울러, 본 발명의 발현벡터에는 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열 및 전사 및 해독의 종결을 조절하는 서열을 추가로 포함할 수 있다.In addition, the expression vector of the present invention may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.
상기 발현벡터에 도입된 NIS와 mODC의 코딩 유전자는 각각 프로모터의 활성에 의한 상기 각 유전자의 발현을 효과적으로 유도할 수 있도록 발현벡터 상에 존재하는 프로모터의 전사 방향과 일치하도록 배열하는 것일 수 있고, 이때 상기 NIS유전자는 상기 mODC의 유전자의 아미노 말단 또는 카르복시 말단에 위치하도록 배열하는 것일 수 있으나, 바람직하게는 융합단백질을 효과적으로 발현할 수 있도록 카르복시 말단에 위치하도록 배열하는 것일 수 있다.The NIS and mODC coding genes introduced into the expression vector may be arranged so as to coincide with the transcription direction of the promoter present on the expression vector so as to effectively induce the expression of each gene by the activity of the promoter, The NIS gene may be arranged to be located at the amino terminal or carboxy terminal of the mODC gene, but may be arranged to be positioned at the carboxy terminal so as to effectively express the fusion protein.
또한, 상기 융합단백질은 서열번호 1의 아미노산 서열로 이루어질 수 있고, 상기 폴리뉴클레오타이드는 서열번호 2의 염기서열로 이루어질 수 있으나 이에 한정되지 않는다. 즉, 서열번호 1의 아미노산 서열과 적어도 약 50%, 60%, 70% 또는 75%, 바람직하게는 적어도 약 80-90%, 더욱 바람직하게는 적어도 약 92-94%, 및 가장 바람직하게는 적어도 약 95%, 98%, 99% 또는 그 이상의 동일성을 공유하는 폴리펩티드일 수 있고, 서열번호 2의 염기서열과 적어도 약 약 50%, 60%, 70% 또는 75%, 바람직하게는 적어도 약 80-90%, 더욱 바람직하게는 적어도 약 92-94%, 및 가장 바람직하게는 적어도 약 95%, 98%, 99% 또는 그 이상의 동일성을 공유하는 폴리뉴클레오타이드일 수 있다.In addition, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 1, and the polynucleotide may comprise the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto. At least about 50%, 60%, 70% or 75%, preferably at least about 80-90%, more preferably at least about 92-94%, and most preferably at least about 50% 2, at least about 50%, 60%, 70% or 75%, preferably at least about 80-fold, at least about 80%, at least about 80% 90%, more preferably at least about 92-94%, and most preferably at least about 95%, 98%, 99% or more identity with the nucleotide sequence of the polynucleotide.
또한, 상기 NIS 단백질은 포유동물 유래이면 제한이 없으나, 인간, 마우스, 래트, 개 또는 돼지 유래인 것이 바람직하다.In addition, the NIS protein may be derived from mammals, but is preferably derived from human, mouse, rat, dog or pig.
본 발명은 상기 발현벡터를 포함하는, 프로테아좀 활성이 저하된 세포의 모니터링용 또는 치료용 조성물을 제공한다. 이때, 세포 모니터링이란 방사성 동위원소의 섭취를 모니터링할 수 있으며, 모니터링용 방사성 동위원소에는 제한이 없으나 감마선을 방출하는 방사성 요오드 123I, 124I, 또는 125I인 것이 바람직하다. 또한, 세포 치료란 방사성 피폭을 통해 해당 세포를 선택적으로 고사시켜 제거하는 것을 의미하며, 치료용 방사성 동위원소에는 제한이 없으나 베타선을 방출하는 방사성 요오드 131I인 것이 바람직하다. The present invention provides a composition for monitoring or treating cells with reduced proteasome activity comprising the expression vector. At this time, the cell monitoring can monitor the uptake of radioactive isotopes. The radioactive isotope for monitoring is not limited, but is preferably radioactive iodine 123 I, 124 I, or 125 I, which emits gamma rays. In addition, the term "cell therapy" refers to selective removal and elimination of the cells through radioactive exposure. It is preferable that the therapeutic radioactive isotope is radioactive iodine 131 I which releases beta rays.
즉, 감마선을 방출하는 종류의 방사성 요오드(I-123, I-124, I-125)를 살아있는 몸속에 투여하면 NIS가 발현된 세포에 섭취되어 감마카메라나 양전자방출단층촬영을 이용하여 비침습적으로 이미징할 수 있다. 이 원리를 이용하여 mODC가 부착된 NIS 단백질을 이용하면 프로테아좀 활성이 낮은 세포를 방사성 요오드와 영상장비로 검출할 수 있다. 나아가 감마선 대신 베타선을 방출하는 방사성 요오드(I-131)를 이용하면 프로테아좀 활성이 낮은 세포에 섭취되어 방사성 피폭을 통해 해당 세포를 선택적으로 고사시켜 제거시킴으로써 질병치료에도 활용할 수 있다.In other words, administration of gamma-emitting radioactive iodine (I-123, I-124, I-125) to the living body can be achieved by ingesting NIS-expressing cells and using a gamma camera or positron emission tomography Imaging is possible. Using this principle, NIS proteins with mODCs can be used to detect cells with low proteasome activity using radioactive iodine and imaging equipment. Furthermore, by using radioactive iodine (I-131) that releases beta rays instead of gamma rays, it is taken into cells having low proteasome activity and can be used for the treatment of diseases by selectively eliminating the cells through radioactive exposure.
또한, 프로테아좀 활성이 저하된 세포에는 제한이 없으나 암줄기세포인 것이 바람직하다.In addition, there is no limitation on the cell with reduced proteasome activity, but it is preferably a cancer stem cell.
OCD(Ornithine decarboxylase의 C-terminal degron)은 대표적인 프로테아좀 표적이므로, 원하는 단백질의 유전자 말단에 OCD의 염기서열을 연결하면 그 단백질은 다른 모든 세포에서는 파괴되지만 프로테아좀 활성이 낮은 세포에만 파괴되지 않고 축적이 된다. 이러한 원리를 이용하면 OCD가 퓨전된 리포터(reporter) 단백질이 암줄기세포 등 프로테아좀 활성이 낮은 세포에만 축적되도록 하여 체내 영상이 가능하다(도 1 참조). Since OCD (C-terminal degron of Ornithine decarboxylase) is a typical proteasome target, if the nucleotide sequence of OCD is connected to the gene end of the desired protein, the protein will be destroyed in all other cells but not in cells with low proteasome activity Accumulate. Using this principle, an in vivo image can be obtained by allowing the OCD-fused reporter protein to accumulate only in cells with low proteasome activity such as cancer stem cells (see FIG. 1).
본 발명에 의하면, OCD가 융합된 NIS 리포터로 베타선을 방출하는 종류의 방사성옥소를 섭취하게 하여, 암줄기세포를 검출하고 영상하는 기술 뿐만 아니라, 암줄기세포 등 프로테아좀 활성이 낮은 세포를 방사성옥소로 표적하는 치료기술에 응용할 수 있다.According to the present invention, it is possible to use a NIS reporter fused with OCD to take a kind of radioactive iodine that emits a betaine to detect and image cancer stem cells, as well as a technique for imaging cells having low proteasome activity, such as cancer stem cells, And can be applied to targeted therapeutic techniques.
즉, 본 발명은, (a) 상기 발현벡터를 포함하는 조성물을 프로테아좀 활성이 저하된 세포에 처리하는 단계; (b) 방사성 동위원소를 상기 세포에 처리하는 단계; 및 (c) 세포에서 방사성동위원소를 측정하는 단계를 포함하는, 세포의 영상 및 치료방법을 제공한다.That is, the present invention relates to a method for producing a protein comprising the steps of: (a) treating a composition comprising the expression vector to cells having reduced proteasome activity; (b) treating the cell with a radioactive isotope; And (c) measuring the radioisotope in the cell.
본 발명의 실시예에서는, pZsProSensor-1 벡터와 pQCXIN retroviral 벡터를 이용하여 재조합 발현벡터 pQCXIN-huNIS-mODC를 제작한 후(도 2 참조), 프로테아좀 활성을 차단제로 억제시킨 MCF-7 세포에서 NIS 단백질이 축적되는 것을 웨스턴 블랏팅으로 확인하고(도 4 참조), 프로테아좀 활성을 차단제로 억제시킨 CT26 세포에서 NIS 단백질 증가에 의한 방사성 옥소 섭취 항진을 확인하였다(도 5 참조). 즉, 세포에 프로테아좀 억제제(MG132 또는 PS341)를 처리하여 프로테아좀 활성을 저해시키면, 항-NIS 항체로 검출되는 NIS 단백질양이나 방사성 옥소 섭취율이 대조군에 비하여 증가함을 알 수 있었다. In the examples of the present invention, a recombinant expression vector pQCXIN-huNIS-mODC was prepared using pZsProSensor-1 vector and pQCXIN retroviral vector (see FIG. 2), and then MCF-7 cells inhibited proteasome activity as a blocking agent The accumulation of NIS protein was confirmed by Western blotting (see FIG. 4), and the increase in NIS protein-induced radioiodine uptake was confirmed in CT26 cells inhibiting proteasome activity as a blocker (see FIG. 5). In other words, when the proteasome inhibitor (MG132 or PS341) was treated to inhibit proteasome activity, the amount of NIS protein and the amount of radioiodine uptake detected by the anti-NIS antibody were increased compared to the control.
또한, huNIS-mODC가 안정적으로 발현된 CT26 안정세포주(stable cell line)에서 프로테아좀 억제제(PS341)을 처리한 경우에 NIS 단백질의 유의한 축적 증가와 (도 8) NIS 활성에 의한 방사성 옥소 섭취 항진을 (도 6) 확인하였다. 나아가. huNIS-mODC가 안정적으로 발현된 CT26 안정세포주(stable cell line)에서는 프로테아좀 억제제(PS341)을 처리한 경우에만 방사성 옥소 섭취 항진을 볼 수 있는데 반하여, CT26 안정세포주를 암줄기세포 선택적 배지에서 배양하여 확보한 CT26 유래 암줄기세포에서는 프로테아좀 억제제(PS341) 처리 여부와 관계없이 방사성 옥소 섭취 항진을 확인하였다(도 7). 이는 암줄기세포의 경우 프로테아좀 활성이 저하되어 있음이 알려져 있는 바와 일치하는 결과였다.In addition, when the proteasome inhibitor (PS341) was treated in a stably expressed CT26 stable cell line of huNIS-mODC, significant accumulation of NIS protein was increased and (Fig. 8) radioisotope uptake by NIS activity (Fig. 6). Furthermore. In stable CT26 stable cell line in which huNIS-mODC was stably expressed, the CT26 stable cell line was cultured in a cancer stem cell selective medium while the proteasome inhibitor (PS341) The obtained CT26-derived cancer stem cells were confirmed to have enhanced radioiodine uptake regardless of treatment with proteasome inhibitor (PS341) (FIG. 7). This is in agreement with what is known to be a decrease in proteasome activity in cancer stem cells.
아울러, CT26 안정세포주를 피하 이식한 마우스 종양모델 실험에서도 상기 동일한 수준의 결과를 얻을 수 있었다. 즉, 프로테아좀 억제제(PS341) 처리한 경우 살아있는 동물의 몸속 종양의 방사성 옥소 섭취 항진을 PET 영상으로도 확인하였고 종양을 꺼내어 방사능을 정량한 결과도 일치하였다.In addition, the CT26 stable cell line was subcutaneously transplanted into a mouse tumor model experiment to obtain the same level of results. In other words, when the proteasome inhibitor (PS341) was treated, the radioactive iodine uptake of the somatic tumor of living animals was also confirmed by PET image, and the result of quantifying radioactivity was also consistent with the tumor.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
실시예 1: 재조합 발현벡터(pQCXIN-huNIS-mODC) 제작Example 1 Production of Recombinant Expression Vector (pQCXIN-huNIS-mODC)
우선, pZsProSensor-1 벡터의 ZsGreen-mODC 5' end에 GAATTC(EcoRI), 3' end에 GGATCC(BamHI) mutant base를 추가한 후, pQCXIN retroviral vector MCS(Multiple Cloning Site) BamHI과 EcoRI제한효소 부위 사이에 상기 ZsGreen-mODC 유전자를 클로닝하였다.First, GAATTC (EcoRI) was added to the ZsGreen-mODC 5 'end of the pZsProSensor-1 vector and GGATCC (BamHI) mutant base was added to the 3' end. Then, pQCXIN retroviral vector MCS (Multiple Cloning Site) The ZsGreen-mODC gene was cloned.
또한, pcDNA vector inserted full length NIS (gbU66088.1) (delete stop codon)의 5' end에 ATGGAGGCCGTGG 서열을 추가하고 이를 포함한 NIS full length 양쪽 말단에 제한효소 부위 CCGCGG (SacII) TAATTA (PacI) mutant base를 추가하였다. 그 다음, pQCXIN-mODC의 SacII과 PacI 제한효소 부위 사이에 NIS 유전자를 클로닝하여, 최종적으로 pQCXIN-huNIS-mODC 발현벡터를 제작하였다.The ATGGAGGCCGTGG sequence was added to the 5 'end of the pcDNA vector inserted full length NIS (gbU66088.1) (delete stop codon) and the restriction enzyme site CCGCGG (SacII) TAATTA (PacI) mutant base was added at both ends of the NIS full length Respectively. Next, the NIS gene was cloned between SacII and PacI restriction enzyme sites of pQCXIN-mODC, and finally pQCXIN-huNIS-mODC expression vector was constructed.
이때, pZsProSensor-1와 pQCXIN 벡터의 map은 도 2에 나타낸 바와 같으며, 최종 클로닝된 huNIS-mODC 융합단백질을 암호화하는 염기서열은 도 3 및 서열번호 2에 나타내었다.In this case, the maps of pZsProSensor-1 and pQCXIN vectors are as shown in FIG. 2, and the nucleotide sequences encoding the final cloned huNIS-mODC fusion protein are shown in FIG. 3 and SEQ ID NO: 2.
실시예 2: 웨스턴 블랏팅Example 2: Western blotting
상기 실시예 1에서 제작한 pQCXIN-huNIS-mODC 발현벡터를 이용하여, NIS 단백질의 발현(축적) 여부를 웨스턴 블랏팅으로 확인하였으며, 구체적인 실험방법은 다음과 같다.Using the pQCXIN-huNIS-mODC expression vector prepared in Example 1, the expression (accumulation) of the NIS protein was confirmed by Western blotting. Specific experimental methods are as follows.
우선, MCF-7 유방암 세포주를 6-well 플레이트에 2×104 개/well로 씨딩하고, DMEM 배양배지(10% FBS)로 37℃, 5% CO2에서 24 시간 배양하였다. 이후, pQCXIN-huNIS-mODC 발현벡터와 대조군(Mock DNA) 각각을 10 μg/well의 양으로 리포펙타민 LTX 키트(Lipofectamin LTX kit; (주)invitrogen)를 이용하여, 상기 유방암 세포주에 트렌스펙션시켰다. First, MCF-7 breast cancer cell lines were seeded at 6 × 10 4 cells / well in a 6-well plate and cultured in DMEM culture medium (10% FBS) for 24 hours at 37 ° C. and 5% CO 2 . Then, each of the pQCXIN-huNIS-mODC expression vector and the control (Mock DNA) was transfected into the breast cancer cell line using a lipofectamine LTX kit (Lipofectamin LTX kit; Invitrogen) in an amount of 10 μg / .
또한, 트렌스펙션 후 약 48 시간 경과 했을 때, MG132(proteasome inhibitor)를 50 μM 농도로 처리하고, 24 시간 후에 세포를 수거, 파괴하여 단백질(25 μg)에 대해 전기영동한 후, 항-NIS 항체로 웨스턴 블랏팅을 실시하였다.After about 48 hours from the transfection, MG132 (proteasome inhibitor) was treated at a concentration of 50 μM. After 24 hours, the cells were harvested and disrupted, and the protein (25 μg) was electrophoresed. Western blotting.
그 결과, 도 4에 나타낸 바와 같이, 프로테아좀 억제제인 MG132를 처리한 군(MG132+)에서는 비처리군(MG132-)에 비하여 NIS-mODC 융합 단백질의 발현이 증가함을 알 수 있었다.As a result, as shown in FIG. 4, the expression of NIS-mODC fusion protein was increased in the MG132 + treated group (MG132 +), which is a proteasome inhibitor, compared to the untreated group (MG132-).
실시예 3: 대장암 세포주에서 방사성 옥소(Example 3: In a colon cancer cell line, radioiodine ( 125125 I) 섭취율 확인I) Confirm ingestion rate
3.1. 대장암 세포주에서 방사성 옥소(3.1. In colorectal cancer cell lines, 125125 I) 섭취율 확인I) Confirm ingestion rate
상기 실시예 1에서 제작한 pQCXIN-huNIS-mODC 발현벡터를 이용하여, NIS 단백질의 활성 여부를 방사성 옥소(I-125)의 uptake 정도로 확인하였으며, 구체적인 실험방법은 다음과 같다.Using the pQCXIN-huNIS-mODC expression vector prepared in Example 1, the activity of the NIS protein was confirmed to be about the uptake of radioiodine (I-125). Specific experimental methods are as follows.
우선, CT26 대장암 세포주를 24well 플레이트에 5×104 개/well로 씨딩하고, DMEM 배양배지(10% FBS)로 37℃, 5% CO2에서 24 시간 배양하였다. 이후, pQCXIN-huNIS-mODC 발현벡터와 대조군(Mock DNA) 각각을 2 μg/well의 양으로 리포펙타민 LTX 키트(Lipofectamin LTX kit; (주)invitrogen)를 이용하여, 상기 대장암 세포주에 트렌스펙션시켰다. First, the CT26 colon cancer cell line was seeded on a 24-well plate at 5 × 10 4 cells / well and cultured in DMEM culture medium (10% FBS) for 24 hours at 37 ° C. and 5% CO 2 . Then, each of the pQCXIN-huNIS-mODC expression vector and the control (Mock DNA) was transfected into the colon cancer cell line using a lipofectamine LTX kit (Invitrogen) in an amount of 2 μg / .
또한, 트렌스펙션 후 약 48 시간 경과 했을 때, PS341(proteasome inhibitor)를 4 μM 농도로 처리하고, 16 시간 후에 I-125 2 μCi/well을 처리한 후 1 시간 동안 incubator에서 세포에 uptake시켰다. 그 다음, 세포를 세정한 후 떼어내어 테스트 튜브에 옮기고 감마-카운터를 이용해 세포에 섭취된 I-125 활성을 측정하였다.In addition, PS341 (proteasome inhibitor) was treated at a concentration of 4 μM for about 48 hours after transfection, treated with 2 μCi / well of I-125 for 16 hours, and then uptake was carried out in an incubator for 1 hour. The cells were then washed, removed and transferred to test tubes and the I-125 activity in the cells was measured using a gamma-counter.
그 결과, 도 5에 나타낸 바와 같이, 프로테아좀 억제제인 PS341을 처리한 군(PS341+)에서는 비처리군(PS341-)에 비하여 I-125의 섭취가 증가함을 알 수 있었다.As a result, as shown in FIG. 5, it was found that I-125 was increased in the PS341-treated group (PS341 +), which is a proteasome inhibitor, compared to the non-treated group (PS341-).
3.2. 안정 세포주에서 방사성 옥소(3.2. In stable cell lines, 125125 I) 섭취율 확인I) Confirm ingestion rate
상기 실시예 3.2.와 유사하게, 실시예 1에서 제작한 pQCXIN-huNIS-mODC 발현벡터를 이용하여, NIS 단백질의 활성 여부를 방사성 옥소(I-125)의 uptake 정도로 확인하였으며, 구체적인 실험방법은 다음과 같다.Using the pQCXIN-huNIS-mODC expression vector prepared in Example 1, the activity of the NIS protein was confirmed to be about the uptake of radioiodine (I-125), similar to Example 3.2. Respectively.
CT26 마우스 대장암 세포에 huNIS-mODC retroviral vector를 transfection 하였고, 200 μg/ml geneticin 항생제로 selection 하면서 huNIS-mODC가 안정적으로 발현된 CT26 cell line (stable cell clone #1)을 제조하였다. geneticin 200 μg/ml를 넣은 배지로 유지하고 있는 stable cells 를 24-well plate에 60-70% confluence로 seeding 하고 24 시간 배양하였다.CT26 mouse colon cancer cells were transfected with a retroviral vector of huNIS-mODC and selected as 200 μg / ml geneticin antibiotics to produce CT26 cell line (stable cell clone # 1) stably expressing huNIS-mODC. Stable cells maintained in a medium containing 200 μg / ml of geneticin were seeded on a 24-well plate with 60-70% confluence and cultured for 24 hours.
이 후, 20 % fetal bovine serum (FBS)을 넣은 배지로 교체해주었고, 6시간 후에 PS341 (proteasome inhibitor) 처리군에 20 nM 농도로 처리하였다. 처리 후 48 시간에, I-125 2 μCi/well 을 처리하였고, 1 시간 동안 incubator에서 세포에 섭취시켰다. 그 다음, 세정하여준 후, 세포를 떼내어 실험 튜우브에 옮기고 감마카운터를 이용해 세포에 섭취된 I-125 활성을 측정하였다.Subsequently, the medium was replaced with medium supplemented with 20% fetal bovine serum (FBS), and treated with PS341 (proteasome inhibitor) at 20 nM after 6 hours. At 48 hours after treatment, I-125 2 μCi / well was treated and incubated in the incubator for 1 hour. Then, after washing, the cells were removed, transferred to an experimental tube, and the I-125 activity in the cells was measured using a gamma counter.
그 결과, 도 6에 나타낸 바와 같이, 프로테아좀 억제제인 PS341(20 nM)을 처리한 군(PS341+)에서는 비처리군(PS341-)에 비하여 I-125의 섭취가 유의하게 증가함을 알 수 있었다.As a result, as shown in FIG. 6, the intake of I-125 was significantly increased in the group treated with PS341 (20 nM) as a proteasome inhibitor (PS341 +) compared with the group not treated (PS341-) there was.
실시예Example 4. 4. 암줄기세포Cancer stem cell 배양 및 방사성 옥소( Culture and radioiodine ( 125125 I) 섭취율 확인I) Confirm ingestion rate
상기 실시예 3.2에서 제조한 stable cell clone #1 세포를 100 mm ultra-low attachment plate (corning,NY) 에 seeding 한 후, DMEM/F12 배지를 기본으로 하고 다양한 성분이 추가된 암줄기세포 선택 배지 (0.4 % bovine serum albumin, 5 mL/L B27 (Gibco, MA), 5 μg/ml bovine insulin, 4 μg/ml heparin, 20 ng/ml fibroblast growth factor 2, 20 ng/ml epidermal growth factor를 포함하는 DMEM/F12 base)로 2-3일에 한번씩 교체해주면서 7-10일간 유지하였다.The stable
NIS-mODC 발현 CT26 stable cell 및 상기 방법으로 배양한 CT26 유래 암줄기세포를 떼내어 matrigel을 코팅한 24-well plate 에 1x105 cells/well 로 seeding 해주었다. 24시간 후 20% FBS를 넣은 배지로 갈아주었고 6 시간 후에 PS341 처리군에 PS341을 200 nM 농도로 처리해주었다. 처리 후 36 시간에, I-125 2 μCi/well 을 처리했고 1 시간 동안 incubator에서 세포에 섭취시켰다.NIS-mODC-expressing CT26 stable cells and CT26-derived cancer stem cells cultured by the above method were removed and seeded in a 24-well plate coated with matrigel at 1 × 10 5 cells / well. After 24 hours, the medium was changed to 20% FBS medium. After 6 hours, the PS341-treated group was treated with PS341 at a concentration of 200 nM. At 36 hours after the treatment, I-125 was treated with 2 μCi / well and the cells were taken in the incubator for 1 hour.
세정시킨 후 세포를 떼내어 test tube에 옮기고 감마카운터를 이용해 세포에 섭취된 I-125 activity를 측정하였고, 그 결과를 도 7에 나타내었다.After washing, the cells were removed and transferred to a test tube, and the I-125 activity of the cells was measured using a gamma counter. The results are shown in FIG.
도 7에 나타낸 바와 같이, 암줄기세포주(CT26 CSCs)에서는 프로테아좀 억제제인 PS341(20 nM)의 처리와 관계없이 I-125의 섭취가 증가한다는 것을 확인하였다. 이를 통해 본 발명의 융합단백질을 암줄기세포에 적용할 경우 프로테아좀 활성이 이미 저하되어 있기 때문에 억제제를 사용하지 않아도 I-125 섭취가 증가됨을 알 수 있다.As shown in Fig. 7, in the cancer stem cell line (CT26 CSCs), it was confirmed that the intake of I-125 was increased regardless of the treatment with the proteasome inhibitor PS341 (20 nM). Thus, when the fusion protein of the present invention is applied to cancer stem cells, the proteasome activity is already lowered, so that the I-125 uptake is increased without using the inhibitor.
실시예 5. 안정 세포에서 hNIS의 웨스턴블랏팅Example 5. Western blotting of hNIS in stable cells
상기 실시예 3.2에서 제작한 NIS-mODC stably expressed CT26 세포(stable cell clone #1)와 NIS-mODC가 트랜스펙션 되지 않은 CT26 세포를 이용하여 NIS 단백질의 축적을 웨스턴 블랏팅으로 확인하였으며, 구체적인 실험방법은 다음과 같다.The accumulation of NIS protein was confirmed by Western blotting using NIS-mODC stably expressed CT26 cells (stable cell clone # 1) prepared in Example 3.2 and CT26 cells not transfected with NIS-mODC, The method is as follows.
NIS-mODC가 트랜스펙션 되지 않은 CT26 세포와 stable cell clone #1 세포를 6-well plate 에 60-70% confluence로 seeding 하고 24시간 동안 배양하였다. 20% FBS를 넣은 배지로 교체하였고 6 시간 후에 PS341 처리군에 200 nM 농도로 처리하였다. 처리 후 36 시간이 경과했을 때, 세포를 깨고 단백질을 뽑은 후 35 μg의 단백질을 전기영동 하고 hNIS 항체를 이용해 western blotting을 실시하였다.NIS-mODC-untransfected CT26 cells and stable
그 결과, 도 8에 나타낸 바와 같이, NIS-mODC가 트랜스펙션되지 않은 CT26 세포(CT26)에서는 프로테아좀 억제제인 PS341을 처리한 군(PS341+)과 처리하지 않은 실험군(PS341-) 모두에서 NIS-mODC 융합 단백질의 발현이 거의 보이지 않았다. 그러나, NIS-mODC stably expressed CT26 세포(stable cell clone #1)에서는 프로테아좀 억제제인 PS341을 처리한 군(PS341+)에서는 비처리군(PS341-)에 비하여 NIS-mODC 융합 단백질의 발현이 유의하게 증가한다는 것을 확인하였다.As a result, as shown in Fig. 8, in the CT26 cell (CT26) in which NIS-mODC was not transfected, the expression of NIS in both the PS341-treated group (PS341 +) and the untreated experimental group (PS341-) -mODC fusion protein. However, in NIS-mODC stably expressed CT26 cells (stable cell clone # 1), the expression of NIS-mODC fusion protein was significantly higher in the PS341-treated group (PS341 +) than the untreated group (PS341-) .
실시예 6. 동물 실험(In vivo study)Example 6. In vivo study
6.1. 영상 촬영 및 방사성 옥소(6.1. Imaging and radioiodine ( 124124 I)의 섭취 확인I) intake
상기 실시예 3.2에서 제작한 NIS-mODC stably expressed CT26 세포(stable cell clone #1)을 100 mm plate (80-90% confluence) 에서 배양하고, 25개 plate 분량의 세포를 떼내어 BALB/C 마우스 15 마리 (PS341 군; n = 8, 대조군; n = 7)에 일정하게 나누어 피하주사로 접종하였다.The NIS-mODC stably expressed CT26 cells (stable cell clone # 1) prepared in Example 3.2 were cultured on a 100 mm plate (80-90% confluence), and 25 plate-like cells were removed from BALB / C mouse 15 (PS341 group; n = 8, control group; n = 7).
2주 후에 암의 크기가 1-1.5 cm에 도달 하였을 때, 대조군 마우스에는 vehicle (DMSO) 을, PS341 군 마우스에는 PS341을 1 mg/kg 농도로 12 시간 간격으로 총 2 회 복강 주사하였다.Two weeks later, when the size of the cancer reached 1-1.5 cm, vehicle (DMSO) was injected into control mice and PS341 was injected intraperitoneally at a dose of 1 mg / kg in PS341 group mice every 12 hours.
두 번째 주사 후, 양전자방출단층영상(PET)을 위한 방사성동위원소인 Iodine-124를 115-119 μCi/mouse씩 꼬리정맥 주사하고, 1 시간 뒤 PET/CT를 20분 동안 촬영한 결과를 도 9(1)에 나타내었다. 그 결과 PS341을 처리한 군에서 더 뚜렷하게 I-124가 발견되어, NIS에 의한 I-124의 섭취가 증가했다는 것을 알 수 있다.After the second injection, Iodine-124, a radioisotope for positron emission tomography (PET), was injected into the tail vein by 115-119 μCi / mouse, and the PET / (1). As a result, I-124 was found to be more pronounced in the PS341-treated group, indicating that the intake of I-124 by NIS was increased.
영상 촬영이 끝난 후 암과 반대편 위치의 앞다리 muscle을 control로 적출하여 무게를 재고 감마카운트로 I-124의 activity를 측정한 결과를 도 9(2)에 나타내었다. PS341을 처리한 경우 더 높은 I-124의 섭취율을 보였다.Figure 9 (2) shows the result of measuring the activity of I-124 by counting the weight of the forelimb muscle at the opposite side of the cancer after the imaging was completed. PS341 treatment resulted in higher I-124 uptake.
6.2. 면역 조직 화학 염색법을 이용한 NIS 단백질의 확인6.2. Identification of NIS proteins by immunohistochemical staining
상기 실시예 6.1에서 영상 획득 후 적출하여 카운트를 측정한 종양 조직을 즉시 4% 포름알데히드 고정액에 72 시간 동안 고정하고 파라핀 블록을 제작하였다.After capturing the images in Example 6.1, tumor tissues counted and counted were immediately fixed in a 4% formaldehyde fixative for 72 hours to prepare paraffin blocks.
상기 파라핀 블록 조직을 serially microsection 한 슬라이드로 NIS에 대한 면역조직화학염색을 실시하였다. 먼저 rabbit unconjugated anti-NIS polyclonal antibody (sc-134545; Santa Cruz)를 1:100 농도로 희석하여 조기절편 슬라이드에 4°C에서 overnight 반응시켰다. 이어서 2차 항체로 peroxidase-conjugated polymer backbone goat anti-rabbit IgG 항체를 (DAKO) 1:200 농도로 희석하여 조기절편 슬라이드에 37°C에서 40 분간 반응시켰다. 표적에 부착한 항체를 DAB를 사용하여 EnVision™ Detection System으로 발색시켰다. 최종적으로 헤마톡실린-에오신으로 가볍게 대조 염색한 다음 슬라이드에 커버유리를 적용시킨 다음 현미경으로 판독하였다.Immunohistochemical staining of NIS was performed by serially microsecting the paraffin block tissue. First, rabbit unconjugated anti-NIS polyclonal antibody (sc-134545; Santa Cruz) was diluted 1: 100 and reacted overnight at 4 ° C on slides. Then, peroxidase-conjugated polymer backbone goat anti-rabbit IgG antibody (DAKO) was diluted 1: 200 with secondary antibody and reacted at 37 ° C for 40 minutes. Antibodies attached to the target were developed with EnVision Detection System using DAB. Finally, lightly contrasted with hematoxylin-eosin, stained with a cover glass followed by microscopic reading.
대조군(Vehicle-C3)과 PS341 처리군(PS341-PS3)의 종양 슬라이드에서의 NIS 염색 정도를 비교하였다. 그 결과, PS341 처리군에서 NIS 단백질의 발현이 더 증가한다는 것을 확인하였다.The degree of NIS staining in the tumor slides of the control (Vehicle-C3) and PS341-treated (PS341-PS3) groups was compared. As a result, it was confirmed that expression of NIS protein was further increased in PS341-treated group.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.
<110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Proteasome Sensitive NIS-MODC Fusion Protein <130> MP16-362 <150> KR 10-2015-0098074 <151> 2015-07-10 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 697 <212> PRT <213> Artificial Sequence <220> <223> huNIS-mODC recombinant protein <400> 1 Met Glu Ala Val Glu Thr Gly Glu Arg Pro Thr Phe Gly Ala Trp Asp 1 5 10 15 Tyr Gly Val Phe Ala Leu Met Leu Leu Val Ser Thr Gly Ile Gly Leu 20 25 30 Trp Val Gly Leu Ala Arg Gly Gly Gln Arg Ser Ala Glu Asp Phe Phe 35 40 45 Thr Gly Gly Arg Arg Leu Ala Ala Leu Pro Val Gly Leu Ser Leu Ser 50 55 60 Ala Ser Phe Met Ser Ala Val Gln Val Leu Gly Val Pro Ser Glu Ala 65 70 75 80 Tyr Arg Tyr Gly Leu Lys Phe Leu Trp Met Cys Leu Gly Gln Leu Leu 85 90 95 Asn Ser Val Leu Thr Ala Leu Leu Phe Met Pro Val Phe Tyr Arg Leu 100 105 110 Gly Leu Thr Ser Thr Tyr Glu Tyr Leu Glu Met Arg Phe Ser Arg Ala 115 120 125 Val Arg Leu Cys Gly Thr Leu Gln Tyr Ile Val Ala Thr Met Leu Tyr 130 135 140 Thr Gly Ile Val Ile Tyr Ala Pro Ala Leu Ile Leu Asn Gln Val Thr 145 150 155 160 Gly Leu Asp Ile Trp Ala Ser Leu Leu Ser Thr Gly Ile Ile Cys Thr 165 170 175 Phe Tyr Thr Ala Val Gly Gly Met Lys Ala Val Val Trp Thr Asp Val 180 185 190 Phe Gln Val Val Val Met Leu Ser Gly Phe Trp Val Val Leu Ala Arg 195 200 205 Gly Val Met Leu Val Gly Gly Pro Arg Gln Val Leu Thr Leu Ala Gln 210 215 220 Asn His Ser Arg Ile Asn Leu Met Asp Phe Asn Pro Asp Pro Arg Ser 225 230 235 240 Arg Tyr Thr Phe Trp Thr Phe Val Val Gly Gly Thr Leu Val Trp Leu 245 250 255 Ser Met Tyr Gly Val Asn Gln Ala Gln Val Gln Arg Tyr Val Ala Cys 260 265 270 Arg Thr Glu Lys Gln Ala Lys Leu Ala Leu Leu Ile Asn Gln Val Gly 275 280 285 Leu Phe Leu Ile Val Ser Ser Ala Ala Cys Cys Gly Ile Val Met Phe 290 295 300 Val Phe Tyr Thr Asp Cys Asp Pro Leu Leu Leu Gly Arg Ile Ser Ala 305 310 315 320 Pro Asp Gln Tyr Met Pro Leu Leu Val Leu Asp Ile Phe Glu Asp Leu 325 330 335 Pro Gly Val Pro Gly Leu Phe Leu Ala Cys Ala Tyr Ser Gly Thr Leu 340 345 350 Ser Thr Ala Ser Thr Ser Ile Asn Ala Met Ala Ala Val Thr Val Glu 355 360 365 Asp Leu Ile Lys Pro Arg Leu Arg Ser Leu Ala Pro Arg Lys Leu Val 370 375 380 Ile Ile Ser Lys Gly Leu Ser Leu Ile Tyr Gly Ser Ala Cys Leu Thr 385 390 395 400 Val Ala Ala Leu Ser Ser Leu Leu Gly Gly Gly Val Leu Gln Gly Ser 405 410 415 Phe Thr Val Met Gly Val Ile Ser Gly Pro Leu Leu Gly Ala Phe Ile 420 425 430 Leu Gly Met Phe Leu Pro Ala Cys Asn Thr Pro Gly Val Leu Ala Gly 435 440 445 Leu Gly Ala Gly Leu Ala Leu Ser Leu Trp Val Ala Leu Gly Ala Thr 450 455 460 Leu Tyr Pro Pro Ser Glu Gln Thr Met Arg Val Leu Pro Ser Ser Ala 465 470 475 480 Ala Arg Cys Val Ala Leu Ser Val Asn Ala Ser Gly Leu Leu Asp Pro 485 490 495 Ala Leu Leu Pro Ala Asn Asp Ser Ser Arg Ala Pro Ser Ser Gly Met 500 505 510 Asp Ala Ser Arg Pro Ala Leu Ala Asp Ser Phe Tyr Ala Ile Ser Tyr 515 520 525 Leu Tyr Tyr Gly Ala Leu Gly Thr Leu Thr Thr Val Leu Cys Gly Ala 530 535 540 Leu Ile Ser Cys Leu Thr Gly Pro Thr Lys Arg Ser Thr Leu Ala Pro 545 550 555 560 Gly Leu Leu Trp Trp Asp Leu Ala Arg Gln Thr Ala Ser Val Ala Pro 565 570 575 Lys Glu Glu Val Ala Ile Leu Asp Asp Asn Leu Val Lys Gly Pro Glu 580 585 590 Glu Leu Pro Thr Gly Asn Lys Lys Pro Pro Gly Phe Leu Pro Thr Asn 595 600 605 Glu Asp Arg Leu Phe Phe Leu Gly Gln Lys Glu Leu Glu Gly Ala Gly 610 615 620 Ser Trp Thr Pro Cys Val Gly His Asp Gly Gly Arg Asp Gln Gln Glu 625 630 635 640 Thr Asn Leu Pro Arg Ser Arg Pro Met Trp Gln Leu Met Lys Gln Ile 645 650 655 Gln Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr 660 665 670 Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala 675 680 685 Ala Cys Ala Ser Ala Arg Ile Asn Val 690 695 <210> 2 <211> 2094 <212> DNA <213> Artificial Sequence <220> <223> huNIS-mODC recombinant polynucleotide <400> 2 atggaggccg tggagaccgg ggaacggccc accttcggag cctgggacta cggggtcttt 60 gccctcatgc tcctggtgtc cactggcatc gggctgtggg tcgggctggc tcggggcggg 120 cagcgcagcg ctgaggactt cttcaccggg ggccggcgcc tggcggccct gcccgtgggc 180 ctgtcgctgt ctgccagctt catgtcggcc gtgcaggtgc tgggcgtgcc gtcggaggcc 240 tatcgctatg gcctcaagtt cctctggatg tgcctgggcc agcttctgaa ctcggtcctc 300 accgccctgc tcttcatgcc cgtcttctac cgcctgggcc tcaccagcac ctacgagtac 360 ctggagatgc gcttcagccg cgcagtgcgg ctctgcggga ctttgcagta cattgtagcc 420 acgatgctgt acaccggcat cgtaatctac gcaccggccc tcatcctgaa ccaagtgacc 480 gggctggaca tctgggcgtc gctcctgtcc accggaatta tctgcacctt ctacacggct 540 gtgggcggca tgaaggctgt ggtctggact gatgtgttcc aggtcgtggt gatgctaagt 600 ggcttctggg ttgtcctggc acgcggtgtc atgcttgtgg gcgggccccg ccaggtgctc 660 acgctggccc agaaccactc ccggatcaac ctcatggact ttaaccctga cccgaggagc 720 cgctatacat tctggacttt tgtggtgggt ggcacgttgg tgtggctctc catgtatggc 780 gtgaaccagg cgcaggtgca gcgctacgtg gcttgccgca cagagaagca ggccaagctg 840 gccctgctca tcaaccaggt cggcctgttc ctgatcgtgt ccagcgctgc ctgctgtggc 900 atcgtcatgt ttgtgttcta cactgactgc gaccctctcc tcctggggcg catctctgcc 960 ccagaccagt acatgcctct gctggtgctg gacatcttcg aagatctgcc tggagtcccc 1020 gggcttttcc tggcctgtgc ttacagtggc accctcagca cagcatccac cagcatcaat 1080 gctatggctg cagtcactgt agaagacctc atcaaacctc ggctgcggag cctggcaccc 1140 aggaaactcg tgattatctc caaggggctc tcactcatct acggatcggc ctgtctcacc 1200 gtggcagccc tgtcctcact gctcggagga ggtgtccttc agggctcctt caccgtcatg 1260 ggagtcatca gcggccccct gctgggagcc ttcatcttgg gaatgttcct gccggcctgc 1320 aacacaccgg gcgtcctcgc gggactaggc gcgggcttgg cgctgtcgct gtgggtggcc 1380 ttgggcgcca cgctgtaccc acccagcgag cagaccatga gggtcctgcc atcgtcggct 1440 gcccgctgcg tggctctctc agtcaacgcc tctggcctcc tggacccggc tctcctccct 1500 gctaacgact ccagcagggc ccccagctca ggaatggacg ccagccgacc cgccttagct 1560 gacagcttct atgccatctc ctatctctat tacggtgccc tgggcacgct gaccactgtg 1620 ctgtgcggag ccctcatcag ctgcctgaca ggccccacca agcgcagcac cctggccccg 1680 ggattgttgt ggtgggacct cgcacggcag acagcatcag tggcccccaa ggaagaagtg 1740 gccatcctgg atgacaactt ggtcaagggt cctgaagaac tccccactgg aaacaagaag 1800 ccccctggct tcctgcccac caatgaggat cgtctgtttt tcttggggca gaaggagctg 1860 gagggggctg gctcttggac cccctgtgtt ggacatgatg gtggtcgaga ccagcaggag 1920 acaaacctcc cgcggtcacg gccaatgtgg caactcatga aacagatcca gagccatggc 1980 ttcccgccgg aggtggagga gcaggatgat ggcacgctgc ccatgtcttg tgcccaggag 2040 agcgggatgg accgtcaccc tgcagcctgt gcttctgcta ggatcaatgt gtag 2094 <110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Proteasome Sensitive NIS-MODC Fusion Protein <130> MP16-362 <150> KR 10-2015-0098074 <151> 2015-07-10 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 697 <212> PRT <213> Artificial Sequence <220> <223> huNIS-mODC recombinant protein <400> 1 Met Glu Ala Val Glu Thr Gly Glu Arg Pro Thr Phe Gly Ala Trp Asp 1 5 10 15 Tyr Gly Val Phe Ala Leu Met Leu Leu Val Ser Thr Gly Ile Gly Leu 20 25 30 Trp Val Gly Leu Ala Arg Gly Gly Gln Arg Ser Ala Glu Asp Phe Phe 35 40 45 Thr Gly Arg Arg Leu Ala Leu Pro Val Gly Leu Ser Leu Ser 50 55 60 Ala Ser Phe Met Ser Ala Val Gln Val Leu Gly Val Ser Ser Glu Ala 65 70 75 80 Tyr Arg Tyr Gly Leu Lys Phe Leu Trp Met Cys Leu Gly Gln Leu Leu 85 90 95 Asn Ser Val Leu Thr Ala Leu Leu Phe Met Pro Val Phe Tyr Arg Leu 100 105 110 Gly Leu Thr Ser Thr Tyr Glu Tyr Leu Glu Met Arg Phe Ser Arg Ala 115 120 125 Val Arg Leu Cys Gly Thr Leu Gln Tyr Ile Val Ala Thr Met Leu Tyr 130 135 140 Thr Gly Ile Val Ile Tyr Ala Pro Ala Leu Ile Leu Asn Gln Val Thr 145 150 155 160 Gly Leu Asp Ile Trp Ala Ser Leu Leu Ser Thr Gly Ile Ile Cys Thr 165 170 175 Phe Tyr Thr Ala Val Gly Gly Met Lys Ala Val Val Trp Thr Asp Val 180 185 190 Phe Gln Val Val Met Leu Ser Gly Phe Trp Val Val Leu Ala Arg 195 200 205 Gly Val Met Leu Val Gly Gly Pro Arg Gln Val Leu Thr Leu Ala Gln 210 215 220 Asn His Ser Arg Ile Asn Leu Met Asp Phe Asn Pro Asp Pro Arg Ser 225 230 235 240 Arg Tyr Thr Phe Trp Thr Phe Val Gly Gly Thr Leu Val Trp Leu 245 250 255 Ser Met Tyr Gly Val Asn Gln Ala Gln Val Gln Arg Tyr Val Ala Cys 260 265 270 Arg Thr Glu Lys Gln Ala Lys Leu Ala Leu Leu Ile Asn Gln Val Gly 275 280 285 Leu Phe Leu Ile Val Ser Ser Ala Cys Cys Gly Ile Val Met Phe 290 295 300 Val Phe Tyr Thr Asp Cys Asp Pro Leu Leu Leu Gly Arg Ile Ser Ala 305 310 315 320 Pro Asp Gln Tyr Met Pro Leu Leu Val Leu Asp Ile Phe Glu Asp Leu 325 330 335 Pro Gly Val Pro Gly Leu Phe Leu Ala Cys Ala Tyr Ser Gly Thr Leu 340 345 350 Ser Thr Ala Ser Thr Ser Ile As Ala Ala Ala Val Thr Val Glu 355 360 365 Asp Leu Ile Lys Pro Arg Leu Arg Ser Leu Ala Pro Arg Lys Leu Val 370 375 380 Ile Ile Ser Lys Gly Leu Ser Leu Ile Tyr Gly Ser Ala Cys Leu Thr 385 390 395 400 Val Ala Leu Ser Ser Leu Leu Gly Gly Gly Val Leu Gln Gly Ser 405 410 415 Phe Thr Val Met Gly Val Ile Ser Gly Pro Leu Leu Gly Ala Phe Ile 420 425 430 Leu Gly Met Phe Leu Pro Ala Cys Asn Thr Pro Gly Val Leu Ala Gly 435 440 445 Leu Gly Ala Gly Leu Ala Leu Ser Leu Trp Val Ala Leu Gly Ala Thr 450 455 460 Leu Tyr Pro Pro Ser Glu Gln Thr Met Arg Val Leu Pro Ser Ser Ala 465 470 475 480 Ala Arg Cys Val Ala Leu Ser Val Asn Ala Ser Gly Leu Leu Asp Pro 485 490 495 Ala Leu Leu Pro Ala Asn As Ser Ser Ser Ala Pro Ser Ser Gly Met 500 505 510 Asp Ala Ser Arg Ala Leu Ala Asp Ser Phe Tyr Ala Ile Ser Tyr 515 520 525 Leu Tyr Tyr Gly Ala Leu Gly Thr Leu Thr Thr Val Leu Cys Gly Ala 530 535 540 Leu Ile Ser Cys Leu Thr Gly Pro Thr Lys Arg Ser Thr Leu Ala Pro 545 550 555 560 Gly Leu Leu Trp Trp Asp Leu Ala Arg Gln Thr Ala Ser Val Ala Pro 565 570 575 Lys Glu Glu Val Ala Ile Leu Asp Asp Asn Leu Val Lys Gly Pro Glu 580 585 590 Glu Leu Pro Thr Gly Asn Lys Lys Pro Pro Gly Phe Leu Pro Thr Asn 595 600 605 Glu Asp Arg Leu Phe Phe Leu Gly Gln Lys Glu Leu Glu Gly Ala Gly 610 615 620 Ser Trp Thr Pro Cys Val Gly His Asp Gly Gly Arg Asp Gln Gln Glu 625 630 635 640 Thr Asn Leu Pro Arg Ser Ser Pro Met Trp Gln Leu Met Lys Gln Ile 645 650 655 Gln Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr 660 665 670 Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala 675 680 685 Ala Cys Ala Ser Ala Arg Ile Asn Val 690 695 <210> 2 <211> 2094 <212> DNA <213> Artificial Sequence <220> <223> huNIS-mODC recombinant polynucleotide <400> 2 atggaggccg tggagaccgg ggaacggccc accttcggag cctgggacta cggggtcttt 60 gccctcatgc tcctggtgtc cactggcatc gggctgtggg tcgggctggc tcggggcggg 120 cagcgcagcg ctgaggactt cttcaccggg ggccggcgcc tggcggccct gcccgtgggc 180 ctgtcgctgt ctgccagctt catgtcggcc gtgcaggtgc tgggcgtgcc gtcggaggcc 240 tatcgctatg gcctcaagtt cctctggatg tgcctgggcc agcttctgaa ctcggtcctc 300 accgccctgc tcttcatgcc cgtcttctac cgcctgggcc tcaccagcac ctacgagtac 360 ctggagatgc gcttcagccg cgcagtgcgg ctctgcggga ctttgcagta cattgtagcc 420 acgatgctgt acaccggcat cgtaatctac gcaccggccc tcatcctgaa ccaagtgacc 480 gggctggaca tctgggcgtc gctcctgtcc accggaatta tctgcacctt ctacacggct 540 gtgggcggca tgaaggctgt ggtctggact gatgtgttcc aggtcgtggt gatgctaagt 600 ggcttctggg ttgtcctggc acgcggtgtc atgcttgtgg gcgggccccg ccaggtgctc 660 acgctggccc agaaccactc ccggatcaac ctcatggact ttaaccctga cccgaggagc 720 cgctatacat tctggacttt tgtggtgggt ggcacgttgg tgtggctctc catgtatggc 780 gtgaaccagg cgcaggtgca gcgctacgtg gcttgccgca cagagaagca ggccaagctg 840 gccctgctca tcaaccaggt cggcctgttc ctgatcgtgt ccagcgctgc ctgctgtggc 900 atcgtcatgt ttgtgttcta cactgactgc gaccctctcc tcctggggcg catctctgcc 960 ccagaccagt acatgcctct gctggtgctg gacatcttcg aagatctgcc tggagtcccc 1020 gggcttttcc tggcctgtgc ttacagtggc accctcagca cagcatccac cagcatcaat 1080 gctatggctg cagtcactgt agaagacctc atcaaacctc ggctgcggag cctggcaccc 1140 aggaaactcg tgattatctc caaggggctc tcactcatct acggatcggc ctgtctcacc 1200 gtggcagccc tgtcctcact gctcggagga ggtgtccttc agggctcctt caccgtcatg 1260 ggagtcatca gcggccccct gctgggagcc ttcatcttgg gaatgttcct gccggcctgc 1320 aacacaccgg gcgtcctcgc gggactaggc gcgggcttgg cgctgtcgct gtgggtggcc 1380 ttgggcgcca cgctgtaccc acccagcgag cagaccatga gggtcctgcc atcgtcggct 1440 gcccgctgcg tggctctctc agtcaacgcc tctggcctcc tggacccggc tctcctccct 1500 gctaacgact ccagcagggc ccccagctca ggaatggacg ccagccgacc cgccttagct 1560 gacagcttct atgccatctc ctatctctat tacggtgccc tgggcacgct gaccactgtg 1620 ctgtgcggag ccctcatcag ctgcctgaca ggccccacca agcgcagcac cctggccccg 1680 ggattgttgt ggtgggacct cgcacggcag acagcatcag tggcccccaa ggaagaagtg 1740 gccatcctgg atgacaactt ggtcaagggt cctgaagaac tccccactgg aaacaagaag 1800 ccccctggct tcctgcccac caatgaggat cgtctgtttt tcttggggca gaaggagctg 1860 gagggggctg gctcttggac cccctgtgtt ggacatgatg gtggtcgaga ccagcaggag 1920 acaaacctcc cgcggtcacg gccaatgtgg caactcatga aacagatcca gagccatggc 1980 ttcccgccgg aggtggagga gcaggatgat ggcacgctgc ccatgtcttg tgcccaggag 2040 agcgggatgg accgtcaccc tgcagcctgt gcttctgcta ggatcaatgt gtag 2094
Claims (17)
1. A recombinant fusion protein comprising the amino acid sequence of SEQ ID NO: 1, wherein NIS (sodium / iodide symporter) and mODC (mouse ornithine decarboxylase degradation domain) are combined.
A recombinant polynucleotide encoding the fusion protein of claim 1.
5. The recombinant polynucleotide of claim 4, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2.
A recombinant expression vector comprising the polynucleotide of claim 4.
(a) 인비트로(in vitro)에서 제6항의 재조합 발현벡터를 포함하는 조성물을 프로테아좀 활성이 저하된 세포에 처리하는 단계;
(b) 방사성동위원소를 상기 세포에 처리하는 단계; 및
(c) 상기 세포에서 상기 방사성동위원소를 측정하는 단계.
A method of imaging a cell, comprising the steps of:
(a) treating a composition comprising the recombinant expression vector of claim 6 in vitro in a cell with reduced proteasome activity;
(b) treating the cell with a radioactive isotope; And
(c) measuring the radioactive isotope in the cell.
16. The method of claim 15, wherein the cell with reduced proteasome activity is a cell that has been treated with a cancer stem cell or proteasome inhibitor.
16. The method of claim 15, wherein the radioisotope is 123 I, 124 I, or 125 I.
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Non-Patent Citations (2)
| Title |
|---|
| Oncology Report, Vol. 34, Pages 59-66(공개일: 2015.)* |
| pZsProSensor-1 Clontech Catalog No. 632425(공개일: 2002. 12. 19.)* |
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