KR20010076648A - Hexameric peptide promoting production of superoxide in human monocyte - Google Patents
Hexameric peptide promoting production of superoxide in human monocyte Download PDFInfo
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- KR20010076648A KR20010076648A KR1020000003916A KR20000003916A KR20010076648A KR 20010076648 A KR20010076648 A KR 20010076648A KR 1020000003916 A KR1020000003916 A KR 1020000003916A KR 20000003916 A KR20000003916 A KR 20000003916A KR 20010076648 A KR20010076648 A KR 20010076648A
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- amino acid
- hexapeptide
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- Engineering & Computer Science (AREA)
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Abstract
Description
본 발명은 사람 단핵구(monocyte)에서 수퍼옥사이드(superoxide)의 발생을촉진시키는 헥사펩티드(hexameric peptide)에 관한 것이다.The present invention relates to hexameric peptides that promote the generation of superoxide in human monocytes.
단핵구는 생체 면역 시스템의 초기 단계에서 중요한 역할을 한다. 외부에서 박테리아와 같은 미생물이 조직에 침입했을 때 생체 면역 시스템은 대식세포(macrophage), 중성구(neutrophil)와 같은 거식세포들이 직접 박테리아를 거식작용(phagocytosis)을 통해 세포 안으로 받아들여 파고좀(phagosome)에서 여러 가지 분해효소를 통해 초기단계에서 제거한다. 또한 단핵구는 미생물의 침입에 대응하여 조직에서 여러 가지 염증반응 결과 생성되는 물질을 거식작용을 통해 제거하는 중요한 역할을 한다. 이 때 파고좀과 세포막을 통해 막단백질인 NADPH 옥시데이즈에 의해 수퍼옥사이드가 생성되어 박테리아를 죽이는 등의 역할을 수행하게 된다.Monocytes play an important role in the early stages of the biological immune system. When microorganisms, such as bacteria, invade tissue, the biological immune system takes macrophages and macrophages, such as neutrophils, directly into the cells through phagocytosis, causing them to become pagosomes. Is removed at an early stage through various enzymes. In addition, monocytes play an important role in removing the substances produced by various inflammatory reactions in tissues through anorexic reaction in response to the invasion of microorganisms. At this time, the superoxide is produced by the membrane protein NADPH oxidases through the pagosome and the cell membrane to kill bacteria.
거식세포의 수용체에 특정작용 물질이 결합함으로써 거식세포에서 수퍼옥사이드의 발생이 촉진된다. 이러한 물질들에는 여러 가지 시토킨(cytokine)들, Fcγ, 리포폴리사카라이드(lipopolysaccharide, LPS), fMLP 그리고 최근에 밝혀진 헥사펩티드인 WKYMVm(여기에서, W는 트립토판, K는 리신, Y는 티로신, M은 메티오닌, V는 발린을 나타낸다) 등이 있다(Bae, et al,J. Leukoc. Biol.,65, 241-248(1999)). 또한 WKYMVm 외에도 수용체에 결합하는 다른 작용물질(agonist)의 결합부위와 유사하여 같은 효과를 나타낼 수 있는 활성 펩티드들이 존재할 가능성이 높다.The binding of specific agents to macrophage receptors promotes the development of superoxide in macrophages. These substances include various cytokines, Fcγ, lipopolysaccharide (LPS), fMLP and the recently discovered hexapeptide WKYMVm (where W is tryptophan, K is lysine, Y is tyrosine, M represents methionine, V represents valine), and others (Bae, et al, J. Leukoc. Biol. , 65 , 241-248 (1999)). In addition, in addition to WKYMVm, there is a high possibility that there are active peptides similar to the binding sites of other agonists that bind to the receptor.
이에 본 발명자들은 면역세포의 초기반응에서 중요한 역할을 하는 거식세포를 활성화하는 펩티드를 찾고자 계속 연구를 진행한 결과, 거식세포가 활성화된 직접적인 증거가 될 수 있는 수퍼옥사이드의 발생을 촉진시키는 헥사펩티드를 합성 펩티드 라이브러리 시스템을 이용하여 제조함으로써 본 발명을 완성하게 되었다.Therefore, the present inventors continued to search for a peptide that activates macrophages, which play an important role in the initial reaction of immune cells. As a result, the present inventors have found hexapeptides that promote the generation of superoxide, which may be direct evidence of macrophage activation. The present invention has been completed by preparing using a synthetic peptide library system.
본 발명의 목적은 사람 단핵구에서 수퍼옥사이드의 생성을 촉진하는 활성을 갖는 신규 헥사펩티드를 제공하는 것이다.It is an object of the present invention to provide novel hexapeptides having the activity of promoting the production of superoxide in human monocytes.
도 1a 내지 1f는 각각 N 말단으로부터 첫 번째 내지 여섯 번째 아미노산이 고정된 헥사펩티드 라이브러리로부터 사람 단핵구에서 수퍼옥사이드(superoxide)의 생성을 촉진시키는 활성을 갖는 펩티드를 동정한 결과를 보여주는 그래프이다.1A to 1F are graphs showing the results of identifying peptides having activity to promote the production of superoxide in human monocytes from hexapeptide libraries in which the first to sixth amino acids from the N terminus are immobilized.
도 2는 부분적 라이브러리로부터 사람 단핵구에서 수퍼옥사이드의 생성을 촉진시키는 활성을 갖는 펩티드를 동정한 결과를 보여주는 그래프이다.FIG. 2 is a graph showing the results of identifying peptides having activity that promotes the production of superoxide in human monocytes from partial libraries.
도 3은 서열번호: 1 내지 4의 단일 헥사펩티드가 사람 단핵구에서 농도의존적으로 수퍼옥사이드의 생성을 촉진시키는 것을 보여주는 그래프이다.3 is a graph showing that a single hexapeptide of SEQ ID NOs: 1 to 4 promotes the production of superoxides in a concentration dependent manner in human monocytes.
도 4는 서열번호: 1 및 2가 면역세포 특이적으로 세포내 칼슘을 증가시키는 것을 나타내는 그래프이다.4 is a graph showing that SEQ ID NOs: 1 and 2 increase immune cell specific intracellular calcium.
상기 목적에 따라, 본 발명에서는 서열번호: 1 내지 4의 아미노산 서열을 갖는 헥사펩티드로 이루어진 군으로부터 선택된, 수퍼옥사이드의 생성을 촉진하는 헥사펩티드를 제공한다.In accordance with the above object, the present invention provides a hexapeptide for promoting the production of superoxide, selected from the group consisting of hexapeptides having the amino acid sequence of SEQ ID NO: 1 to 4.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 헥사펩티드는 수퍼옥사이드의 생성을 촉진하는 활성을 갖는 서열번호: 1 내지 4의 아미노산 서열의 헥사펩티드들이다. 본 발명의 헥사펩티드는 통상적인 화학 합성법에 따라 합성하여 얻을 수 있다.The hexapeptides of the present invention are hexapeptides of the amino acid sequence of SEQ ID NOs: 1 to 4 having the activity of promoting the production of superoxide. The hexapeptide of the present invention can be obtained by synthesis according to a conventional chemical synthesis method.
본 발명의 헥사펩티드는 문헌(Houghtem, N.K.,Clin. Exp. Immunol.,7, 477 (1991); Pinilla, C. et al.,Biotechniques,901, 162 (1992); 및 대한민국 특허 공보 제 140262 호)의 방법을 변형하여 헥사펩티드 라이브러리로부터 수퍼옥사이드 생성을 촉진하는 활성을 측정하여 다음과 같이 선별할 수 있다. 구체적인 선별 과정은 첫 번째 내지 여섯 번째 아미노산 잔기 위치가 각각 특정 아미노산으로 고정된 헥사펩티드들을 포함하는 6개 세트의 헥사펩티드 라이브러리로부터 아미노산 잔기 위치별로 수퍼옥사이드 생성 촉진활성을 갖는 아미노산 잔기를 선별하는 1 차 선별 단계와, 1 차 선별 단계에서 선별된 아미노산 잔기로 이루어진 헥사펩티드들을 포함하는 부분적 라이브러리로부터 수퍼옥사이드 생성 촉진활성을 갖는 헥사펩티드를 선별하는 2 차 선별 단계로 이루어진다.Hexapeptides of the invention are described in Houghtem, NK, Clin. Exp. Immunol. , 7 , 477 (1991); Pinilla, C. et al., Biotechniques , 901 , 162 (1992); and Korean Patent Publication No. 140262 The method of) can be modified to determine the activity of promoting superoxide production from the hexapeptide library and selected as follows. A specific screening process is a primary step of selecting amino acid residues having superoxide-promoting activity by amino acid residue position from six sets of hexapeptide libraries including hexapeptides each of which has fixed hexapeptide positions of the first to sixth amino acid residue positions. And a second screening step of selecting a hexapeptide having superoxide-promoting activity from a partial library including hexapeptides consisting of amino acid residues selected in the first screening step.
상기 1 차 선별 단계에 사용되는 헥사펩티드 라이브러리는, (i) N 말단으로부터 첫 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 두 번째 내지 여섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (ii) N 말단으로부터 두 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 첫 번째, 세 번째 내지 여섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (iii) N 말단으로부터 세 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 첫 번째, 두 번째 및 네 번째 내지 여섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (iv) N 말단으로부터 네 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 첫 번째 내지 세 번째, 다섯 번째 및 여섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (v) N 말단으로부터 다섯 번째 아미노산 잔기가 시스테인을 제외한 19개 아미노산 중 어느 하나로 고정되고, 첫 번째 내지 네 번째 및 여섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (vi) N 말단으로부터 여섯 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 첫 번째 내지 다섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트로 이루어진다. 상기 라이브러리의 각 세트는 고정된 위치에 시스테인을 제외한 19 개 아미노산 중의 어느 하나가 특정되어 있고 나머지 고정되지 않은 위치에 시스테인을 제외한 임의의 아미노산 잔기가 무작위로 합성되어 있는 헥사펩티드를, 고정된 위치에 동일한 아미노산 잔기를 갖는 헥사펩티드들의 혼합물로서 19 종류씩 포함하고 있다. 상기 라이브러리를 구성하는 헥사펩티드들은 상기 문헌의 방법에 따라 6 회 합성 반응을 통해 C 말단으로부터 N 말단으로 아미노산 잔기 하나씩 차례로 합성할 수 있다.The hexapeptide library used in the primary selection step is (i) the first amino acid residue from the N terminus is fixed to any one of 19 amino acids except cysteine, and the second to sixth amino acid residues are any amino acid except cysteine. A set of phosphorus hexapeptides, (ii) a set of hexapeptides wherein the second amino acid residue from the N terminus is fixed to any of 19 amino acids except cysteine, and the first, third to sixth amino acid residues are any amino acids except cysteine, (iii) a set of hexapeptides wherein the third amino acid residue from the N terminus is anchored to any one of 19 amino acids except cysteine and the first, second and fourth to sixth amino acid residues are any amino acids except cysteine, (iv ) The fourth amino acid residue from the N terminus is cis A set of hexapeptides wherein the first to third, fifth and sixth amino acid residues are any amino acid except cysteine, and (v) the fifth amino acid residue from the N terminus is A set of hexapeptides wherein the first to fourth and sixth amino acid residues are any amino acid except cysteine, and (vi) the sixth amino acid residue from the N terminus is selected from nineteen amino acids except cysteine, Fixed to either, the first to fifth amino acid residues consist of a set of hexapeptides, which are any amino acid except cysteine. Each set of the library contains hexapeptides in which any one of 19 amino acids except cysteine is fixed at a fixed position and any amino acid residues except cysteine are randomly synthesized at a fixed position. Nineteen kinds are included as a mixture of hexapeptides having the same amino acid residue. The hexapeptides constituting the library can be synthesized one by one in amino acid residues from the C terminus to the N terminus through six synthesis reactions according to the method of the document.
상기 1 차 선별 단계에서는 헥사펩티드 라이브러리를 구성하는 각 세트의 19 종류의 헥사펩티드 혼합물에 대해 수퍼옥사이드 생성 촉진활성을 측정하여, 고정된 아미노산 위치별로 수퍼옥사이드 생성 촉진활성을 보이는 아미노산 잔기를 결정한다. 수퍼옥사이드 생성 촉진활성의 측정은 수퍼옥사이드에 의해 환원되는 시토크롬(cytochrome) c를 사용하는데, 구체적으로 사람 단핵구에 헥사펩티드와 시토크롬 c를 처리한 후 엘리자(ELISA) 탐색기를 통해 수퍼옥사이드에 의해 환원된 시토크롬 c의 550nm에서의 흡광도를 측정한다. 이 때 흡광도는 5분이 지나면 포화되어 더 이상 증가하지 않으므로 5분에서의 흡광도와 0분에서의 흡광도의 차이로 수퍼옥사이드의 발생정도를 측정할 수 있다.In the primary screening step, the superoxide production promoting activity is measured for each of the 19 types of hexapeptide mixtures constituting the hexapeptide library to determine amino acid residues showing the superoxide production promoting activity at fixed amino acid positions. Determination of superoxide production promoting activity is carried out using cytochrome c which is reduced by superoxide, specifically, the mononuclear cells treated with hexapeptide and cytochrome c and then reduced by superoxide through ELISA searcher. The absorbance at 550 nm of cytochrome c is measured. At this time, since the absorbance is saturated after 5 minutes and does not increase any more, the degree of occurrence of superoxide can be measured by the difference between the absorbance at 5 minutes and the absorbance at 0 minutes.
1 차 선별 단계에서 헥사펩티드의 N 말단으로부터 첫 번째 위치에 히스티딘 또는 메티오닌이, 두 번째 위치에 페닐알라닌이, 세 번째 위치에 타이로신이, 네번째 위치에 류신이, 다섯 번째 위치에 프폴린 또는 발린, 여섯 번째 위치에는 카복실기에 -NH2치환기를 선택적으로 갖는 아스파르트산, 글리신 또는 메티오닌이 존재하는 헥사펩티드 혼합물들이 수퍼옥사이드 생성을 촉진하는 활성을 갖는 것으로 확인되었다.In the first screening step, histidine or methionine in the first position, phenylalanine in the second position, tyrosine in the third position, leucine in the fourth position, propolin or valine in the fifth position, six from the N terminus of the hexapeptide In the first position, it was confirmed that hexapeptide mixtures containing aspartic acid, glycine or methionine optionally having a -NH 2 substituent on the carboxyl group have activity to promote superoxide production.
상기 2 차 선별 과정에 사용되는 부분적 헥사펩티드 라이브러리는, 1 차 선별 과정에서 얻어진 수퍼옥사이드 생성 촉진활성을 갖는 아미노산 자료에 근거하여 고안된 하기 화학식 1의 헥사펩티드 혼합물로 이루어져 있다.The partial hexapeptide library used in the secondary selection process is composed of a hexapeptide mixture of the following formula 1 designed based on amino acid data having superoxide generation promoting activity obtained in the primary selection process.
상기 식에서 Xaa1는 히스티딘 또는 메티오닌이고, Xaa2는 프롤린 또는 발린이며, Xaa3은 카복실기에 -NH2치환기를 선택적으로 갖는 아스파르트산, 글리신 또는 메티오닌이다.Wherein Xaa 1 is histidine or methionine, Xaa 2 is proline or valine, and Xaa 3 is aspartic acid, glycine or methionine optionally having a -NH 2 substituent on the carboxyl group.
부분적 라이브러리를 구성하는 헥사펩티드들은 상기 문헌의 방법에 따라 혼합물로 합성할 수 있다. 합성된 헥사펩티드 혼합물들은 프로테인-펩티드 C18칼럼 등을 이용한 고속 액체 크로마토그래피를 수행하여 각 피크의 분획별로 정제할 수 있으며, 각 피크의 헥사펩티드에 대해 수퍼옥사이드 생성 촉진활성을 측정하여 수퍼옥사이드 생성 촉진활성을 보이는 피크를 결정하고 이 피크의 헥사펩티드의 아미노산 조성을 분석하여 수퍼옥사이드 생성 촉진활성을 보이는 헥사펩티드의 아미노산 서열을 결정한다.The hexapeptides that make up the partial library can be synthesized in a mixture according to the methods described above. Synthesized hexapeptide mixtures can be purified by fractionation of each peak by high-performance liquid chromatography using a protein-peptide C 18 column, or the like. The peak showing the promoting activity is determined and the amino acid composition of the hexapeptide of the peak is analyzed to determine the amino acid sequence of the hexapeptide showing the superoxide generating promoting activity.
2 차 선별 단계에서 서열번호: 1 내지 4의 아미노산 서열을 갖는 헥사펩티드들이 수퍼옥사이드 생성 촉진활성을 갖는 것으로 확인되었다.It was confirmed that hexapeptides having an amino acid sequence of SEQ ID NOs: 1 to 4 in the secondary selection step had superoxide production promoting activity.
서열번호: 1 내지 4의 아미노산 서열을 갖는 단일 헥사펩티드를 합성하여 수퍼옥사이드 생성 촉진활성을 확인할 수 있으며, 이들은 200 nM의 농도에서도 수퍼옥사이드 생성 촉진활성을 나타낸다.A single hexapeptide having the amino acid sequence of SEQ ID NO: 1 to 4 can be synthesized to confirm superoxide production promoting activity, and they show superoxide production promoting activity even at a concentration of 200 nM.
특히 수퍼옥사이드 생성 촉진활성이 우수한 서열번호: 1 및 2의 헥사펩티드에 대하여 세포내 칼슘 증가 촉진여부를 측정하여 세포 특이성을 조사한 결과, 두 펩티드는 거식세포인 단핵구와 중성구를 활성화시켜 세포내 칼슘이온의 증가를 유도하였다. 또한, T 임파구 세포주인 Jurkat 세포는 서열번호: 1의 헥사펩티드에 의해서만 활성화되며, 상피 세포주인 3T3-L1 세포는 서열번호: 1 및 2의 두 헥사펩티드 모두에 의해 활성화되지 않아 본 발명의 헥사펩티드는 면역세포만을 특이적으로 활성화한다는 것을 알 수 있다.In particular, the cell specificity of the hexapeptides of SEQ ID NOs: 1 and 2 with excellent superoxide-promoting activity was measured and the cell specificity was examined. Both peptides activated intracellular calcium ions by activating monocytes and neutrophils, macrophages. Increased. In addition, J lymphat T cell line is activated only by the hexapeptide of SEQ ID NO: 1, epithelial cell line 3T3-L1 cells are not activated by both hexapeptides of SEQ ID NO: 1 and 2 hexapeptide of the present invention It can be seen that specifically activates only immune cells.
이하 본 발명을 하기 참조예 및 실시예에 의하여 더욱 상세하게 설명하고자 한다. 단, 하기 참조예 및 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples and Examples. However, the following reference examples and examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.
참조예 1: 사람 단핵구의 분리Reference Example 1: Isolation of Human Monocytes
울산 혈액원으로부터 백혈구 농축액을 수득하여 히스토파크(histopaque)1077 위에 넣은 후 상온에서 1500rpm으로 45분간 원심분리하였다. 이 중 백혈구 층만 취하여 Ca2+또는 Mg2+가 없는 항스 균일 염 용액(Hanks' balanced salt solution(HBSS), Gibco BRL, U.S.A.)에 현탁한 다음 3번 세척하였다. 이렇게 얻은 백혈구를 소혈청(bovine calf serum)이 10%(v/v)로 포함된 RPMI 배지(Gibco BRL, U.S.A.)에 현탁하여 배양용 접시에 분주하고 CO2배양기에서 37℃로 50분간 배양하였다. 50분 후에 혈청이 없는 37℃ RPMI로 2번 세척하고, 4℃의 혈청없는 RPMI로 배양용 접시에 붙어 있는 단핵구를 떼어내어 수확하였다.The leukocyte concentrate was obtained from Ulsan blood source, placed on Histopaque 1077, and centrifuged at 1500 rpm for 45 minutes at room temperature. Only the leukocyte layer was taken and suspended in Hans' balanced salt solution (HBSS), Gibco BRL, USA without Ca 2+ or Mg 2+ and washed three times. The leukocytes thus obtained were suspended in RPMI medium (Gibco BRL, USA) containing 10% (v / v) bovine calf serum, aliquoted in a culture dish and incubated at 37 ° C. for 50 minutes in a CO 2 incubator. . After 50 minutes, washed twice with 37 ° C. RPMI without serum, and harvested by removing the monocytes attached to the culture dish at 4 ° C. without serumI RPMI.
참조예 2: 사람 중성구의 분리 및 Jurkat 및 3T3-L1 세포 배양Reference Example 2: Isolation of Human Neutrophils and Culture of Jurkat and 3T3-L1 Cells
울산 혈액원으로부터 백혈구 농축액을 수득하여 히스토파크(histopaque) 1077 위에 넣은 후 상온에서 1500rpm으로 45분간 원심분리하였다. 이중 중성구층만을 취하여 PBS(8mM Na2HPO4, 1.5mM KH2PO4, 136mM NaCl, 2.6mM KCl, 0.5mM MgCl2, 0.6mM CaCl2, pH 7.4)에 현탁하였다. 1200rpm에서 7 분간 원심분리하여 다시 PBS에 재현탁하였다. 증류수로 적혈구를 용혈시킨 후 4% NaCl 용액으로 농도를 조절하여 원심분리하였다. 이 과정을 한번 더 반복하고 중성구를 PBS에 현탁하였다. 또한 T 임파구 세포주인 Jurkat 세포는 10% 소태아 혈청이 들어 있는 RPMI에서 적절한 수로 키운 다음, 혈청이 없는 RPMI로 수회 세척 후 수확하였으며, 상피 세포주인 3T3-L1은 10% 소태아 혈청이 들어있는 DMEM(GibcoBRL., U.S.A.)에 적절한 수로 키운 다음, 혈청이 없는 DMEM으로 수회 세척한 후 수확하였다.A leukocyte concentrate was obtained from Ulsan blood source, placed on a histopaque 1077, and centrifuged at 1500 rpm for 45 minutes at room temperature. Only a double neutrophil layer was taken and suspended in PBS (8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 136 mM NaCl, 2.6 mM KCl, 0.5 mM MgCl 2 , 0.6 mM CaCl 2 , pH 7.4). Centrifugation at 1200 rpm for 7 minutes and resuspended in PBS. Erythrocytes were hemolyzed with distilled water and centrifuged by adjusting the concentration with 4% NaCl solution. This process was repeated once more and the neutrophils were suspended in PBS. In addition, J lymphat cells, T lymphocyte cell lines, were grown in RPMI containing 10% fetal bovine serum and then washed several times with RPMI without serum, and epithelial cell line 3T3-L1 was loaded with DMEM containing 10% fetal bovine serum. (GibcoBRL., USA) was grown to the appropriate number, then washed several times with serum-free DMEM and harvested.
참조예 3: 세포내로 유리된 칼슘이온의 농도 측정Reference Example 3: Measurement of the concentration of free calcium ions into cells
면역세포 활성화 초기 신호를 측정하기 위하여, 세포내로 유리된 칼슘 이온의 농도를 칼슘에 결합력이 강한 염색물질인 Fura-2/AM을 이용하여 측정하였다. 즉, 참조예 1 및 2와 같은 방법으로 수확한 여러 세포를 1 x 107세포/㎖이 되게 RPMI 배지 또는 DMEM 배지에 재현탁하였다. 이어서 최종 3μM 농도의 Fura-2/AM(입수처: Molecular Probes, U.S.A.)을 첨가한 후 배양기에서 45분 동안 계속 교반하면서 방치하였다. 45분 후 세포를 수확하여 다시 RPMI 배지로 수회 세척하고 이를 250μM 농도의 설핀 피라죤(sulfin pyrazon)이 첨가된 적정량의 RPMI 배지에 현탁하여 세포내로 들어간 Fura-2가 세포외부로 유실되는 것을 방지하였다. 약 2 x 106세포를 매번 취하여 급속 원심분리로 수확하고 이를 칼슘이온이 첨가되지 않고 EGTA(ethylene glycol bis(2-amino-ethyl ether)-N,N,N',N'-tetra acetic acid) 0.2mM이 첨가된 Locke 용액(158.4mM NaCl, 5.6mM KCl, 1.2mM MgCl2, 5mM HEPES, pH 7.3, 10mM 글루코스, 0.2mM EGTA) 1㎖에 재현탁 후 스펙트로포토미터 상에서 340nm와 380nm의 두 파장에서의 흡광도의 비를 모니터링하면서 약 1분 후에 펩티드를 처리하고 두 파장에서의 흡광도 비의 차이를 조사하였고, 그린키윅스(Grynkiewicz)의 방법(Grynkiewicz, G., et al.,J. Biol. Chem.,260, 3440-3450(1985))에 따라 세포내로 유리된 칼슘이온의 농도로 환산하였다.In order to measure the initial signal of immune cell activation, the concentration of free calcium ions into the cells was measured using Fura-2 / AM, a staining material with strong binding to calcium. That is, several cells harvested in the same manner as in Reference Examples 1 and 2 were resuspended in RPMI medium or DMEM medium to 1 × 10 7 cells / ml. The final 3 μM concentration of Fura-2 / AM (Molecular Probes, USA) was then added and left under stirring for 45 minutes in the incubator. After 45 minutes, the cells were harvested and washed again several times with RPMI medium, which was suspended in an appropriate amount of RPMI medium added with 250 μM of sulfin pyrazon to prevent the loss of Fura-2 into the cells. . Approximately 2 x 10 6 cells are taken each time and harvested by rapid centrifugation, which is obtained by adding ethylene glycol bis (2-amino-ethyl ether) -N, N, N ', N'-tetra acetic acid (EGTA) without adding calcium ions. Resuspend in 1 ml of 0.2 mM added Locke solution (158.4 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl 2 , 5 mM HEPES, pH 7.3, 10 mM glucose, 0.2 mM EGTA), followed by two wavelengths of 340 nm and 380 nm on a spectrophotometer. The peptide was treated after about 1 minute while monitoring the ratio of absorbance at, and the difference in absorbance ratio at the two wavelengths was investigated and the method of Grynkiewicz (Grynkiewicz, G., et al., J. Biol. Chem. , 260 , 3440-3450 (1985)) was converted into the concentration of free calcium ions intracellularly.
참조예 4: 헥사펩티드의 아미노산 조성 분석 및 정량Reference Example 4: Analysis and Quantification of Amino Acid Composition of Hexapeptide
헥사펩티드에 6 N 염산을 가한 후 피코-텍 워크 스테이션(Pico-Tag work station, Waters)을 이용하여 진공과 질소 가스를 교대로 5회 주입하고 150 ℃에서 1 시간 동안 가열하여 펩티드를 가수분해시켰다. 이어서 반응물을 속도-진공 농축기(speed-vac concentrator, Savant)를 이용하여 완전히 건조시킨 다음, 에탄올/증류수/트리에틸아민(TEA)(2:2:1, v/v) 혼합액 20 ㎕를 가하고 속도-진공 농축기를 이용하여 다시 건조시켰다. 건조된 반응물에 메탄올/증류수/TEA/페닐이소티오시아네이트(7:1:1:1, v/v) 혼합액 20 ㎕를 가하고 실온에서 20 분간 반응시킨 후 다시 완전 건조시켜 유리 아미노산을 얻었다.6N hydrochloric acid was added to the hexapeptide, and then 5 times of vacuum and nitrogen gas were alternately injected using a Pico-Tag work station (Waters), and the peptide was hydrolyzed by heating at 150 ° C for 1 hour. . The reaction was then completely dried using a speed-vac concentrator (Savant), then 20 μl of ethanol / distilled water / triethylamine (TEA) (2: 2: 1, v / v) mixture was added and Dry again using a vacuum concentrator. 20 μl of a mixture of methanol / distilled water / TEA / phenylisothiocyanate (7: 1: 1: 1, v / v) was added to the dried reaction product, reacted at room temperature for 20 minutes, and then completely dried to obtain free amino acid.
얻어진 유리 아미노산을 희석액(인산 수소 이나트륨 710 ㎎, 0.5 % 아세토니트릴, 물 1 ℓ, pH 7.4) 적당량에 용해시킨 후 이를 피코-텍 고속 액체 크로마토그래피 컬럼(Pico-Tag column, Waters, U.S.A)에 적용하고 용매 A(아세트산나트륨 20 g, TEA 0.6 ㎖, 아세토니트릴 0.06 %, 물 ℓ, pH 6.4)와 용매 B(60 % 아세토니트릴)를 사용하여 20 분간 0 % 내지 46 %의 아세토니트릴 농도구배로 용출시켰다. 시료의 체류시간 및 면적을 비교 분석하여 각 아미노산의 조성과 양을 측정하였다.The obtained free amino acid was dissolved in an appropriate amount in a diluent (710 mg of sodium hydrogen phosphate, 0.5% acetonitrile, 1 L of water, pH 7.4), and then, the obtained free amino acid was dissolved in a Pico-Tag column, Waters, USA. And acetonitrile concentration gradient of 0% to 46% for 20 minutes using solvent A (20 g sodium acetate, 0.6 mL TEA, 0.06% acetonitrile, 1 L water, pH 6.4) and solvent B (60% acetonitrile). Eluted. The retention time and area of the sample were compared and analyzed to determine the composition and amount of each amino acid.
실 시 예 1 : 수퍼옥사이드의 생성을 촉진하는 헥사펩티드의 1차 선별Example 1 Primary Screening of Hexapeptides Promoting the Production of Superoxides
(단계 1) 헥사펩티드 라이브러리의 합성(Step 1) Synthesis of Hexapeptide Library
헥사펩티드 라이브러리는 (i) N 말단으로부터 첫 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 두 번째 내지 여섯 번째아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (ii) N 말단으로부터 두 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 첫 번째, 세 번째 내지 여섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (iii) N 말단으로부터 세 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 첫 번째, 두 번째 및 네 번째 내지 여섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (iv) N 말단으로부터 네 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 첫 번째 내지 세 번째, 다섯 번째 및 여섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (v) N 말단으로부터 다섯 번째 아미노산 잔기가 시스테인을 제외한 19개 아미노산 중 어느 하나로 고정되고, 첫 번째 내지 네 번째 및 여섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트, (vi) N 말단으로부터 여섯 번째 아미노산 잔기가 시스테인을 제외한 19 개 아미노산 중 어느 하나로 고정되고, 첫 번째 내지 다섯 번째 아미노산 잔기가 시스테인을 제외한 임의의 아미노산인 헥사펩티드 세트로 나누어 합성하였다.The hexapeptide library consists of (i) a set of hexapeptides wherein the first amino acid residue from the N terminus is fixed to any of 19 amino acids except cysteine, and the second to sixth amino acid residues are any amino acid except cysteine, (ii) N A set of hexapeptides wherein the second amino acid residue from the terminus is anchored to any of 19 amino acids except cysteine, and the first, third to sixth amino acid residues are any amino acid except cysteine, (iii) the third amino acid from the N terminus A set of hexapeptides wherein the residue is immobilized to any one of 19 amino acids except cysteine, the first, second and fourth to sixth amino acid residues are any amino acids except cysteine, (iv) a fourth amino acid residue from the N terminus Of 19 amino acids except cysteine Hexapeptide set, wherein the first to third, fifth, and sixth amino acid residues are any amino acid except cysteine, (v) the fifth amino acid residue from the N terminus is any of 19 amino acids except cysteine A set of hexapeptides wherein the first to fourth and sixth amino acid residues are any amino acids except cysteine, (vi) the sixth amino acid residue from the N terminus is fixed to any of 19 amino acids except cysteine, and the first The fifth to fifth amino acid residues were synthesized by dividing them into hexapeptide sets, which are any amino acids except cysteine.
각 세트의 헥사펩티드의 합성은 문헌(Houghten, N.K.,Clin. Exp. Immunol.,7, 477 (1991); Pinilla, C. et al.,Biotechniques,901, 162 (1992); 및 대한민국 특허공보 제 140262 호)에 기재된 방법에 따라 19 개의 반응관에서 6 회 합성 반응을 통해 C 말단부터 N 말단으로 아미노산 잔기 하나씩 차례로 합성하였다. 고정된 아미노산 잔기의 합성 반응에는 19개 반응관에 각각 서로 다른 특정 아미노산을 사용하였으며 나머지 고정되지 않은 아미노산 잔기의 합성 반응에는 모든 반응관에 19개 아미노산의 혼합물을 사용하였다. 예를 들어, N 말단으로부터 첫 번째 아미노산 잔기가 고정된 헥사펩티드 세트에서, C 말단으로부터 아미노산 잔기 5개를 합성하기 위한 첫 5 회의 합성 반응에는 19개 아미노산의 혼합물을 사용하고 N 말단 아미노산 잔기를 합성하기 위한 6 회 합성 반응에는 고정된 아미노산을 사용하였다.The synthesis of each set of hexapeptides is described in Houghten, NK, Clin. Exp. Immunol. , 7 , 477 (1991); Pinilla, C. et al., Biotechniques , 901 , 162 (1992); and Korean Patent Publication According to the method described in (140262), amino acid residues were synthesized one by one from the C to N terminus through six synthesis reactions in 19 reaction tubes. For the reaction of the immobilized amino acid residues, a specific specific amino acid was used for 19 reaction tubes, and the mixture of 19 amino acids was used for all reaction tubes for the synthesis of the remaining non-immobilized amino acid residues. For example, in a set of hexapeptides in which the first amino acid residue is immobilized from the N terminus, the first five synthetic reactions for synthesizing five amino acid residues from the C terminus use a mixture of 19 amino acids and synthesize the N terminal amino acid residue. Fixed amino acids were used in the six synthesis reactions.
헥사펩티드를 합성하기 위해, 19 개의 폴리프로필렌 반응관에 50μmol 링크 수지(Rink resin, 0.7 meq/g, 100-200 메쉬, Dupont Co., U.S.A.) 71 ㎎을 가하고, 자동 합성시스템(Auto peptide synthesis system, ACT369, Advanced Chemtech, U.S.A.)에 자치한 후, 25% 피페리딘/디메틸포름아미드 혼합액(1:1, v/v) 1㎖로 1회 흔들어 준 후 다시 동일 용액으로 9 분간 반응시켰다. 이어서 수지를 디메틸포름아미드 1.5 ㎖로 2회, 메탄올 1.5 ㎖로 2회, 디메틸포름아미드 1.5 ㎖로 2회 차례로 세척하고, NSC-아미노산과 하이드록시벤즈트리아졸의 혼합액 0.5 ㎖ 씩을 가하고 상온에서 1 시간 반응시켜 C-말단 아미노산 잔기를 합성하였다. 상기 세척 및 반응 과정을 반복하여 나머지 아미노산 잔기를 합성하였다. 얻어진 헥사펩티드를 25 % 피페리딘/디메틸포름아미드 혼합액(1:1, v/v)과 9 분간 반응시켜 최종 NSC기를 제거하고, 이어서 삼불화초산/에텐디티올/증류수/티오아니솔(trifluoroacetic acid/ethendithiol/distilled water/thioanisole) 혼합액(90:5:4:1, v/v)을 가하고 16 시간 동안 반응시켜 아미노산 곁사슬 보호기를 제거하였다. 이 반응액을 질소 가스 처리하여 삼불화초산을 증발시킨 다음, 남은 반응액을 다시 증류수 적당량에녹이고 디에틸에테르 10 ㎖로 5 회 추출하였다. 이 용액을 질소 가스 처리하여 잔류하는 디에틸에테르를 증발시켜 헥사펩티드 라이브러리를 얻었다. 얻어진 헥사펩티드 라이브러리를 적당하게 분주하고 -20 ℃에서 보관하였다.To synthesize hexapeptide, 71 μl of 50 μmol Link resin (Rink resin, 0.7 meq / g, 100-200 mesh, Dupont Co., USA) was added to 19 polypropylene reaction tubes, and an auto peptide synthesis system was added. , ACT369, Advanced Chemtech, USA), and then shaken once with 1 ml of a 25% piperidine / dimethylformamide mixture (1: 1, v / v) and reacted with the same solution for 9 minutes. Subsequently, the resin was washed twice with 1.5 ml of dimethylformamide, twice with 1.5 ml of methanol and twice with 1.5 ml of dimethylformamide, and 0.5 ml of a mixture of NSC-amino acid and hydroxybenztriazole was added thereto for 1 hour at room temperature. Reactions synthesized C-terminal amino acid residues. The wash and reaction process was repeated to synthesize the remaining amino acid residues. The resulting hexapeptide was reacted with a 25% piperidine / dimethylformamide mixture (1: 1, v / v) for 9 minutes to remove the final NSC group, followed by trifluoroacetic acid / ethenedithiol / distilled water / thioanisole (trifluoroacetic). acid / ethendithiol / distilled water / thioanisole) mixture (90: 5: 4: 1, v / v) was added and reacted for 16 hours to remove the amino acid side chain protecting group. The reaction solution was subjected to nitrogen gas treatment to evaporate trifluoroacetic acid, and the remaining reaction solution was again dissolved in an appropriate amount of distilled water and extracted five times with 10 ml of diethyl ether. This solution was subjected to nitrogen gas treatment to evaporate the remaining diethyl ether to obtain a hexapeptide library. The resulting hexapeptide library was properly dispensed and stored at -20 ° C.
(단계 2) 수퍼옥사이드를 생성하는 헥사펩티드의 1차 선별(Step 2) Primary Screening of Hexapeptide to Produce Superoxide
96웰 플레이트에 50μM의 시토크롬 c와 상기 단계 1에서 합성된 6개의 아미노산으로 이루어진 펩티드 라이브러리 0.5nM을 처리하고 참조예 1과 같은 방법으로 제조된 1.5 X 107개의 사람 단핵구를 37℃에서 1분간 배양하여 최종부피가 100㎕가 되도록 플레이트의 각 웰에 넣었다. 단핵구를 넣자마자 바로 엘리자 탐색기(ELISA reader)로 550nm에서의 흡광도를 1분 간격으로 10분 동안 측정하였다. 그 결과 시간이 지남에 따라 흡광도가 증가하여 5분 경과후 포화되는 것을 알 수 있었다. 5분 동안의 흡광도 증가량을 단핵구에서 발생한 수퍼옥사이드의 양으로 판단할 수 있다. 그 결과는 도 1에서 보듯이, N 말단에서부터 첫 번째 위치에 히스티딘 또는 메티오닌, 두 번째 위치에 페닐알라닌, 세 번째 위치에 타이로신, 네 번째 위치에 류신, 다섯 번째 위치에 프롤린 또는 발린, 여섯 번째 위치에 아스파르트산, 글리신 또는 메티오닌이 존재하는 헥사펩티드들이 비교적 높은 효과를 나타내었다.A 96-well plate was treated with 0.5 nM of peptide library consisting of 50 μM of cytochrome c and the six amino acids synthesized in Step 1, and incubated for 1.5 min at 37 ° C. for 1.5 X 10 7 human monocytes prepared in the same manner as in Reference Example 1. The final volume was added to each well of the plate so that the final volume was 100 μl. As soon as monocytes were added, the absorbance at 550 nm was measured for 10 minutes at 1 minute intervals with an ELISA reader. As a result, the absorbance increases with time, and it can be seen that after 5 minutes, it is saturated. The increase in absorbance for 5 minutes can be determined by the amount of superoxide generated in monocytes. The result is as shown in Figure 1, from the N-terminal histidine or methionine in the first position, phenylalanine in the second position, tyrosine in the third position, leucine in the fourth position, proline or valine in the fifth position, in the sixth position Hexapeptides with aspartic acid, glycine or methionine showed relatively high effects.
실 시 예 2 : 수퍼옥사이드의 생성을 촉진하는 헥사펩티드의 2차 선별Example 2 Secondary Screening of Hexapeptides Promoting the Production of Superoxides
실시예 1의 단계 2에서 얻은 수퍼옥사이드의 생성을 촉진하는 것으로 확인된 아미노산 잔기에 근거하여, 실시예 1의 단계 1과 유사한 방법으로 화학식 1의 부분적 헥사펩티드 라이브러리를 합성하였다.Based on the amino acid residue found to promote the production of the superoxide obtained in step 2 of Example 1, a partial hexapeptide library of Formula 1 was synthesized in a similar manner to Step 1 of Example 1.
화학식 1Formula 1
Xaa1-Phe-Tyr-Leu-Xaa2-Xaa3 Xaa 1 -Phe-Tyr-Leu-Xaa 2 -Xaa 3
상기 식에서 Xaa1는 히스티딘 또는 메티오닌이고, Xaa2는 프롤린 또는 발린이며, Xaa3은 카복실기에 -NH2치환기를 갖는 아스파르트산, 글리신 또는 메티오닌이다.Wherein Xaa 1 is histidine or methionine, Xaa 2 is proline or valine, and Xaa 3 is aspartic acid, glycine or methionine having a -NH 2 substituent on the carboxyl group.
합성된 부분적 라이브러리의 헥사펩티드를 이용하여 실시예 1과 동일한 방법으로 수퍼옥사이드 발생 촉진정도를 비교하였으나 뚜렷한 차이를 보이지 않아 각각의 펩티드 혼합물을 고속 액체 크로마토그래피(Hewlett Packard, U. S. A.)를 사용하여 순수분리하여 그 효과를 비교하였다.The hexapeptide of the synthesized partial library was compared in the same manner as in Example 1 to promote superoxide generation, but there was no significant difference. Each peptide mixture was purified by high-performance liquid chromatography (Hewlett Packard, USA). The effect was compared.
즉, 부분적 헥사펩티드 라이브러리를 프로테인-펩티드 C18칼럼(22 ㎝ x 25 ㎝, Vydac)에 적용하고 용매 A(증류수/0.1 % 삼불화초산)과 용매 B(아세토니트릴/0.1% 삼불화초산)를 사용하여 80 분 동안 0 % 내지 35 %의 아세토니트릴 농도 구배로 용출하였다. 위와 같은 방법으로 용출된 각 피크의 분획을 각각 모아 상온건조하여 증류수로 펩티드를 녹인 다음 정확한 농도를 결정하기 위해 참조예 4의 방법에 따라 아미노산 조성 분석 및 정량을 실시하였다. 그 결과 서열번호: 1 내지 8과 같이 헥사펩티드의 여섯 번째 위치에서 아스파르트산과 글리신은 분리가 안된 하나의 피크로 나타났고 메티오닌만이 분리되었다.That is, a partial hexapeptide library is applied to a protein-peptide C 18 column (22 cm x 25 cm, Vydac) and solvent A (distilled water / 0.1% trifluoroacetic acid) and solvent B (acetonitrile / 0.1% trifluoroacetic acid) Eluted with an acetonitrile concentration gradient of 0% to 35% for 80 minutes. The fractions of each of the peaks eluted in the above manner were collected and dried at room temperature to dissolve the peptides in distilled water, followed by amino acid composition analysis and quantification according to the method of Reference Example 4 to determine the correct concentration. As a result, aspartic acid and glycine in the sixth position of the hexapeptide, as shown in SEQ ID NO: 1 to 8 appeared as one unseparated peak and only methionine was isolated.
서열번호: 1 내지 8의 헥사펩티드를 실시예 1의 방법으로 수퍼옥사이드 발생 촉진 정도를 측정하여 그 결과를 도 2에 나타내었으며, 여기에서 보듯이 헥사펩티드의 여섯 번째 위치에 메티오닌이 있는 것이 아스파르트산과 글리신의 혼합물보다 수퍼옥사이드 생성 촉진활성이 월등히 높은 것을 알 수 있다.The hexapeptides of SEQ ID NOs: 1 to 8 were measured by the method of Example 1 to measure the degree of superoxide development, and the results are shown in FIG. 2, and as shown here, the presence of methionine at the sixth position of the hexapeptide was aspartic acid and It can be seen that the superoxide generation promoting activity is much higher than the mixture of glycine.
실 시 예 3 : 수퍼옥사이드 생성 촉진활성을 갖는 헥사펩티드의 3차 선별Example 3 Tertiary Screening of Hexapeptide with Superoxide Promoting Activity
상기 실시예 2에서 수퍼옥사이드 생성 촉진활성이 우수한 헥사펩티드로서 확인된 서열번호: 1 내지 4의 단일 헥사펩티드를 각각 0.2μM, 0.5μM, 1μM 및 10μM의 농도로 사용하여 실시예 1의 방법에 따라 550nm에서의 흡광도를 측정하여 그 결과를 도 3에 나타내었다. 여기에서 보듯이, 4개의 헥사펩티드가 200nM 농도에서도 수퍼옥사이드의 생성을 촉진하는 것을 알 수 있었으며, 특히 서열번호: 1 및 2가 가장 높은 활성을 보임을 알 수 있다.According to the method of Example 1 using a single hexapeptide of SEQ ID NO: 1 to 4 identified as hexapeptides excellent in superoxide production promoting activity in Example 2 at concentrations of 0.2 μM, 0.5 μM, 1 μM and 10 μM, respectively The absorbance at 550 nm was measured and the result is shown in FIG. 3. As shown here, it was found that four hexapeptides promote the production of superoxide even at 200 nM concentration, and in particular, SEQ ID NOs: 1 and 2 show the highest activity.
실 시 예 4 : 헥사펩티드의 세포 특이성 조사Example 4 Investigation of Cell Specificity of Hexapeptide
상기 실시예 3에서 수퍼옥사이드의 생성 촉진활성이 우수한 헥사펩티드로서 확인된 서열번호: 1 및 2의 단일 헥사펩티드의 사람 단핵구, 중성구, T 임파구인 Jurkat 세포 및 상피 세포주인 3T3-L1 세포에서 세포내 칼슘 증가활성을 Fura-2/AM(입수처 : Molecular Probes, U.S.A.)를 이용하여 참조예 3과 같은 방법으로 조사하였으며 그 결과를 도 4에 나타내었다. 여기에서 보듯이, 두 펩티드는 거식세포인 단핵구와 중성구를 활성화시켜 세포내 칼슘이온의 증가를 유도하였다. 또한, T 임파구 세포주인 Jurkat 세포는 서열번호: 2의 이루어진 헥사펩티드에 의해서만 활성화되며, 상피 세포주인 3T3-L1 세포는 두 헥사펩티드 모두에 의해 활성화되지 않는 것으로 나타나, 상기 펩티드들이 면역세포만을 특이적으로 활성화한다는 것을 알 수 있었다.In Example 3, human mononuclear cells, neutrophils, T lymphocytes of Jurkat cells and epithelial cell lines of single hexapeptides of SEQ ID NOs: 1 and 2, which were identified as hexapeptides having excellent superoxide-producing activity, were intracellular. Calcium increasing activity was investigated in the same manner as in Reference Example 3 using Fura-2 / AM (obtained from Molecular Probes, USA), and the results are shown in FIG. 4. As shown here, the two peptides activate macrophages, monocytes and neutrophils, leading to an increase in intracellular calcium ions. In addition, J lymphat T cell line is activated only by the hexapeptide consisting of SEQ ID NO: 2, epithelial cell line 3T3-L1 cells are not shown to be activated by both hexapeptide, so that the peptides specific for immune cells only You can see that it activates.
본 발명에 따른 서열번호: 1 내지 4의 헥사펩티드들은 수퍼옥사이드 생성을 촉진함으로써, 새로운 면역증강제로서 또는 면역세포의 활성화에 수반되는 신호전달 과정을 밝히는데 도구로서 사용될 수 있다.The hexapeptides of SEQ ID NOS: 1 to 4 according to the invention can be used as new immunopotentiators or tools as a tool for identifying signaling processes involved in the activation of immune cells by promoting superoxide production.
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