KR102543962B1 - Composition for Preventing or Treating Infectious Disease of Enterotoxigenic Escherichia Coli Comprising Peptide Specifically Binding to Enterotoxigenic Escherichia Coli Heat-Labile Enterotoxi-B - Google Patents
Composition for Preventing or Treating Infectious Disease of Enterotoxigenic Escherichia Coli Comprising Peptide Specifically Binding to Enterotoxigenic Escherichia Coli Heat-Labile Enterotoxi-B Download PDFInfo
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- KR102543962B1 KR102543962B1 KR1020220151129A KR20220151129A KR102543962B1 KR 102543962 B1 KR102543962 B1 KR 102543962B1 KR 1020220151129 A KR1020220151129 A KR 1020220151129A KR 20220151129 A KR20220151129 A KR 20220151129A KR 102543962 B1 KR102543962 B1 KR 102543962B1
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- Prior art keywords
- peptide
- escherichia coli
- gm1a
- enterotoxigenic escherichia
- present
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Abstract
Description
본 발명은 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 펩타이드, 이를 암호화하는 핵산 분자, 핵산 분자를 포함하는 재조합 발현 벡터, 재조합 발현 벡터로 형질 전환된 형질전환체, 상기 펩타이드를 포함하는 장독소성대장균 감염 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention provides a peptide that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT), a nucleic acid molecule encoding the same, a recombinant expression vector containing the nucleic acid molecule, a transformant transformed with the recombinant expression vector, It relates to a pharmaceutical composition for preventing or treating enterotoxigenic Escherichia coli infection comprising the peptide.
장독소원성 대장균(enterotoxigenic E. coli, ETEC)은 열에 불안정한 이열성 독소((heat labile enterotoxin, LT)의 생산을 담당한다. 장독소원성 대장균은 매년 50,000명 이상의 사망자와 수백만 건의 설사와 관련이 있다. 위생 시설의 발전에도 불구하고 질병의 사망률이 떨어지지 않지만 이환율도 떨어지지 않는다. 어린이가 장독소원성 대장균에 감염되면 종종 발달 장애 및 인지 능력 저하와 같은 장기적인 건강 문제가 발생하여 빈곤의 악순환이 시작될 수 있다. 또한, 발병 지역을 방문하는 여행자, 특히 의료 및 군사 전문가는 질병에 걸리기 쉽다. 또한 이 질병은 과민성 대장 증후군과 같은 만성 질환과 관련이 있다. 장독소에 의해 유발되는 묽은 설사는 감염에 의해 악화되는 대변-구강 경로를 통해 질병이 확산되는 것을 돕는다.Enterotoxigenic E. coli (ETEC) is responsible for the production of heat labile enterotoxin (LT). Enterotoxigenic E. coli is associated with more than 50,000 deaths and millions of cases of diarrhea each year. .Despite advances in sanitation, the mortality rate of the disease has not fallen, but neither has the morbidity.Infection with enterotoxigenic E. coli often causes long-term health problems, such as developmental disabilities and cognitive decline, which can set off a vicious cycle of poverty. In addition, travelers visiting endemic areas, particularly medical and military professionals, are susceptible to the disease, which is also associated with chronic conditions such as irritable bowel syndrome, watery diarrhea caused by enterotoxins, exacerbated by infection. It helps spread the disease through the fecal-oral route.
장독소성대장균은 이열성 독소(heat labile enterotoxin, LT)과 내열성 독소(heat stable enterotoxin, ST) 중 한 개 혹은 두 개의 독소를 동시에 생산한다. 이열성 독소는 항원구조나 작용기전이 콜레라 독소와 유사하며, 단백질 구조상 하나의 A subunit(분자량 25,000)과 5 개의 B subunit(분자량 11,500)로 구성되어 있고, 각각 18개와 21개의 signal 펩타이드와 함께 합성된다. 촉매 활성 A-소단위체는 상피 세포에 대한 결합을 담당하는 원환체 모양의 B-펜타머의 코어에 묶여 있다. 이 두 가지 구성 요소가 함께 단백질 복합체를 구성한다. 이열성 독소(LT)는 일반적으로 콜레라 독소(cholera toxin, CT)보다 더 불규칙적이고, 다양한 글리코스핑고리피드(glycosphingolipids)에 결합합니다. 여기에는 디시알로강글리오시드 GD2, GM1 및 락토-N-네오테트라오실세라마이드(LNnT-Cer; 파라글로보시드)와 같은 글리코스핑고지질과 장내 폴리글리코실세라마이드 및 시알산 함유 글리코스핑고가 포함된다. LT-B 소단위체는 동물 상피 세포의 원형질막 표면에서 발견되는 모노시알로강글리오시드인 GM1a에 결합하여 숙주 세포에 들어갈 수 있다. Enterotoxic Escherichia coli simultaneously produces one or both toxins, heat labile enterotoxin (LT) and heat stable enterotoxin (ST). Heterophilic toxin is similar to cholera toxin in its antigenic structure or mechanism of action. It consists of one A subunit (molecular weight: 25,000) and five B subunits (molecular weight: 11,500) in protein structure, and is synthesized with 18 and 21 signal peptides, respectively. do. The catalytically active A-subunit is bound to the torus-shaped core of the B-pentamer responsible for binding to epithelial cells. These two components together constitute a protein complex. Heterophilic toxin (LT) is generally more irregular than cholera toxin (CT) and binds to a variety of glycosphingolipids. These include glycosphingolipids such as disialogangliosides GD2 and GM1 and lacto-N-neotetraosylceramide (LNnT-Cer; paragloboside) and enteric polyglycosylceramides and glycosphingos containing sialic acid. included The LT-B subunit can enter host cells by binding to GM1a, a monosialoganglioside found on the surface of the plasma membrane of animal epithelial cells.
한편, 병원성 대장균을 포함한 세균에 의한 대부분의 감염질환과 질병치료를 위해 화학치료제에 의존할 수밖에 없으며, 대부분은 항생제로 치료하고 있다.On the other hand, for the treatment of most infectious diseases and diseases caused by bacteria, including pathogenic Escherichia coli, there is no choice but to rely on chemotherapeutic agents, and most of them are treated with antibiotics.
그러나 이러한 항생제의 무분별한 남용과 오용에 의하여 현재는 많은 종류의 세균들이 항생제에 대해 내성을 획득하였을 뿐만 아니라 내성균의 출연빈도도 점차 증가하고 있다.However, due to the indiscriminate abuse and misuse of these antibiotics, many types of bacteria have now acquired resistance to antibiotics, and the frequency of appearance of resistant bacteria is gradually increasing.
대장균에 의한 질환은 다른 세균성 질병과 다르게 지속적으로 발생한다는 특성이 있으며, 이를 예방하기 위해서는 철저한 소독, 위생적인 환경을 유지하여 병인체에 노출되는 것을 차단하는 것이 최우선이나 이는 현실적으로 불가능하다. Unlike other bacterial diseases, diseases caused by Escherichia coli have the characteristic of continuously occurring, and in order to prevent this, thorough disinfection and maintaining a hygienic environment to prevent exposure to pathogens is the top priority, but this is practically impossible.
이에 세포막 표면에서 GM1a에 이열성독소(LT)의 부착을 억제할 수 있도록 선택적으로 이열성독소(LT)의 B 서브유닛과 결합하는 물질은 이열성독소(LT)의 후속 생리학적 결과를 방지하여, 장독소대장균 이열성독소(LT) 감염에 대한 예방 또는 치료가 가능하여 새로운 치료제로 사용될 수 있다. Therefore, a substance that selectively binds to the B subunit of thermophilic toxin (LT) to inhibit the attachment of thermophilic toxin (LT) to GM1a on the cell membrane surface prevents the subsequent physiological consequences of thermophilic toxin (LT). , it is possible to prevent or treat enterotoxin E. coli heat-febrile toxin (LT) infection, so it can be used as a new therapeutic agent.
본 발명의 과제는 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 서열번호 1의 아미노산 서열을 포함하는 펩타이드를 제공하는 것이다.An object of the present invention is to provide a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT).
본 발명의 다른 과제는 상기 전술한 펩타이드의 아미노산 서열을 암호화하는 핵산 분자를 제공하는 것이다.Another object of the present invention is to provide a nucleic acid molecule encoding the amino acid sequence of the aforementioned peptide.
본 발명의 다른 과제는 상기 핵산 분자를 포함하는 재조합 발현벡터를 제공하는 것이다.Another object of the present invention is to provide a recombinant expression vector containing the nucleic acid molecule.
본 발명의 다른 과제는 발현 벡터로 형질전환된 형질전환체를 제공하는 것이다.Another object of the present invention is to provide a transformant transformed with an expression vector.
본 발명의 다른 과제는 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 서열번호 1의 아미노산 서열을 포함하는 펩타이드를 유효성분으로 포함하는 장독소성대장균 감염 예방 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is a pharmaceutical for preventing or treating enterotoxinic E. coli infection comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxin heat-labile toxin (LT) as an active ingredient to provide a composition.
본 발명의 또 다른 과제는 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 서열번호 1의 아미노산 서열을 포함하는 펩타이드를 포함하는 장독소성대장균 감염 예방 또는 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or improving enterotoxinic E. coli infection comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxin heat-labile toxin (LT) is to provide
상기한 과제를 달성하기 위해 본 발명은 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 서열번호 1의 아미노산 서열을 포함하는 펩타이드를 제공한다.In order to achieve the above object, the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT).
또한 본 발명은 상기 전술한 펩타이드의 아미노산 서열을 암호화하는 핵산 분자를 제공한다.In addition, the present invention provides a nucleic acid molecule encoding the amino acid sequence of the aforementioned peptide.
또한 본 발명은 상기 핵산 분자를 포함하는 재조합 발현벡터를 제공한다.In addition, the present invention provides a recombinant expression vector containing the nucleic acid molecule.
또한 본 발명은 발현 벡터로 형질전환된 형질전환체를 제공한다.In addition, the present invention provides a transformant transformed with the expression vector.
또한 본 발명은 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 서열번호 1의 아미노산 서열을 포함하는 펩타이드를 포함하는 장독소성대장균 감염 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating enterotoxinic E. coli infection comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxin heat-labile toxin (LT).
또한 본 발명은 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 서열번호 1의 아미노산 서열을 포함하는 펩타이드를 포함하는 장독소성대장균 감염 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving enterotoxinic E. coli infection comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxin heat-labile toxin (LT).
본 발명에 따른 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 펩타이드는 GM1a 유사 펩타이드로 GM1a에 이열성독소(LT)의 부착을 억제할 수 있도록 선택적으로 이열성독소(LT)의 B 서브유닛과 결합함으로써 이열성독소(LT)의 후속 생리학적 결과를 방지하여, 장독소대장균 이열성독소(LT) 감염에 대한 예방 또는 치료가 가능하여 새로운 치료제로 사용될 수 있다.The peptide that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT) according to the present invention is a GM1a-like peptide, and selectively heat-heat toxin (LT) to suppress the attachment of heat-heat toxin (LT) to GM1a By binding to the B subunit of LT), it prevents the subsequent physiological consequences of heat-labile toxin (LT), enabling prevention or treatment of enterotoxin E. coli heat-heat toxin (LT) infection, and thus can be used as a new therapeutic agent.
도 1은 LT-B에 대한 면역요법이 병원체에 대한 면역 반응에서 숙주 GM1a 글리칸 펩타이드의 관여를 결정함으로써 감염성 장애를 예방하고 치료하는 데 사용될 수 있다는 것을 보여주는 모식도이다.
도 2는 본 발명의 일 실시예에서 플라클 리프트 분석을 통해 GM1a 강글리오사이드와 관련된 LT-B 렉틴 인자를 밝혀낸 결과를 나타낸 것이다.
도 3은 본 발명의 일 실시예에서 GM1a 글리칸에 결합하는 렉틴 후보인 LT-B와의 도킹 시뮬레이션을 사용하여, 결합 친화도를 결정하고, 예상되는 결합 부위에서 발견된 3개의 리간드 결합 도메인(LBD)를 나타낸 모식도이다.
도 4의 A는 본 발명의 일 실시예에서 LT-B에 대한 GM1a-복제 펩타이드, P1, P2, P3의 결합 친화도를 나타낸 그래프, B는 GM1a-복제 펩타이드 P1의 농도에 따른 저해율을 나타낸 그래프이다(*P < 0.05 및 **P < 0.01)).
도 5는 본 발명의 일 실시예에서 장 상피 세포 HCT-8 공격 감염 후, P1의 영향을 검출하기 위해 ELISA를 사용하여 염증 유발 사이토카인 IL-1β, IL-6 및 TNF-α의 생산 수준을 비교한 결과를 나타낸 그래프이다. Figure 1 is a schematic showing that immunotherapy against LT-B can be used to prevent and treat infectious disorders by determining the involvement of host GM1a glycan peptides in the immune response to pathogens.
Figure 2 shows the results of revealing the LT-B lectin factor related to GM1a ganglioside through plaque lift analysis in one embodiment of the present invention.
Figure 3 is a docking simulation with LT-B, a lectin candidate that binds to GM1a glycan in one embodiment of the present invention, to determine the binding affinity, and three ligand binding domains (LBD) found at the expected binding site ) is a schematic diagram showing.
4A is a graph showing the binding affinity of the GM1a-replicating peptides P1, P2, and P3 to LT-B in an embodiment of the present invention, and B is a graph showing the inhibition rate according to the concentration of the GM1a-replicating peptide P1 (*P < 0.05 and **P < 0.01)).
Figure 5 shows the production levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α using ELISA to detect the effect of P1 after HCT-8 challenge infection of intestinal epithelial cells in one embodiment of the present invention. This is a graph showing the comparison results.
본 발명자들은 동물 상피 세포의 원형질막 표면에서 발견되는 모노시알로강글리오시드인 GM1a에 장독소성대장균 이열성독소(LT)의 부착을 억제하기 위한 목적으로 GM1a 유사 펩타이드를 개발하였다. 파지 디스플레이 라이브러리에서 GM1a와 구조적으로 유사한 LT-B 결합 펩타이드를 선택하기 위해 LT-B 소단위를 사용했다. Biopanning은 LT-B 인식을 기반으로 하는 3개의 GM1a 유사 펩타이드를 식별하는 데 사용되었다. GM1a 유사 펩타이드가 LT-B가 GM1a에 결합하는 것을 차단함으로써 GM1a을 모방할 수 있음을 입증했다. 엔벨로프 단백질에 표시되는 파지-보여 펩타이드 라이브러리 기술을 사용하여 LT-B가 GM1a에 결합하는 것을 성공적으로 억제하는 3개의 펩타이드를 얻을 수 있었다. 이들 GM1a 유사 펩타이드는 GM1a와 유사한 기능적 활성을 가져, GM1a가 LT-B에 결합하는 것을 억제하는 효과가 있었다. 또한 인간 장 상피 세포주인 HCT-8 세포를 사용하여 GM1a 유사 펩타이드의 결합 능력, 기능적 억제 활성 및 시험관 내 효과를 평가하였으며, 이를 통해 GM1a 유사 펩타이드가 상피 세포에 대한 LT-B 결합을 억제하고 장독소성대장균 이열성독소(LT)에 의한 후속 생리학적 결과를 방지하기 위한 새로운 치료제로 작용할 수 있다는 것을 확인하였다. The present inventors developed a GM1a-like peptide for the purpose of inhibiting the attachment of enterotoxin E. coli heat-labile toxin (LT) to GM1a, a monosialoganglioside found on the plasma membrane surface of animal epithelial cells. The LT-B subunit was used to select LT-B binding peptides structurally similar to GM1a from the phage display library. Biopanning was used to identify three GM1a-like peptides based on LT-B recognition. We demonstrated that a GM1a-like peptide can mimic GM1a by blocking the binding of LT-B to GM1a. Using the phage-visible peptide library technology displayed on the envelope protein, we were able to obtain three peptides that successfully inhibited the binding of LT-B to GM1a. These GM1a-like peptides had a functional activity similar to that of GM1a, and had the effect of inhibiting the binding of GM1a to LT-B. In addition, the binding ability, functional inhibitory activity, and in vitro effect of GM1a-like peptides were evaluated using HCT-8 cells, a human intestinal epithelial cell line. It was confirmed that it can act as a new therapeutic agent to prevent subsequent physiological consequences caused by E. coli heat-labile toxin (LT).
상술한 목적을 달성하기 위한 일 실시양태로서, 본 발명은 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 서열번호 1의 아미노산 서열을 포함하는 펩타이드를 제공한다. As one embodiment for achieving the above object, the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT).
상기 펩타이드는 당해 분야에 공지된 화학적 펩타이드 합성방법으로 제조하거나, 상기 펩타이드를 코딩하는 유전자를 PCR(polymerase chain reaction)에 의해 증폭하거나 공지된 방법으로 합성한 후 발현벡터에 클로닝하여 발현시켜서 제조할 수 있다.The peptide may be prepared by a chemical peptide synthesis method known in the art, or by amplifying a gene encoding the peptide by PCR (polymerase chain reaction) or synthesizing it by a known method, and then cloning it into an expression vector and expressing it. there is.
본 발명의 일 실시예에서, LT-B를 이용한 접착력 검사에서 서열번호 1 내지 서열번호 3에 해당하는 GM1a 유사 펩타이드는 모두 대조군과 비교할 때 LT-B에 결합하는 수준이 상당히 높았으며, 특히 서열번호 1의 아미노산 서열을 갖는 글리칸 모방 펩타이드 P1은 다른 GM1a 유사 펩타이드인 P2(서열번호 2), P3(서열번호 3)에 비해 가장 높은 결합력을 나타내는 것으로 나타났다. In one embodiment of the present invention, in the adhesion test using LT-B, all of the GM1a-like peptides corresponding to SEQ ID NO: 1 to SEQ ID NO: 3 showed significantly higher levels of binding to LT-B than the control group, especially SEQ ID NO: The glycan-mimicking peptide P1 having an amino acid sequence of 1 showed the highest binding affinity compared to other GM1a-like peptides, P2 (SEQ ID NO: 2) and P3 (SEQ ID NO: 3).
본 발명의 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 펩타이드 범위에 포함될 수 있는 생물학적 기능 균등물은 본 발명의 펩타이드와 균등한 생물학적 활성을 발휘하는 아미노산 서열의 변이를 포함하는데에 한정될 것이라는 것은 당업자에게 명확하다.Biological functional equivalents that can be included in the range of peptides that specifically bind to the B subunit of enterotoxin E. coli heat-labile toxin (LT) of the present invention are mutations in amino acid sequences that exhibit biological activity equivalent to those of the peptides of the present invention. It is clear to those skilled in the art that the inclusion will be limited to.
다른 하나의 양태로서, 본 발명은 상기 펩타이드를 코딩하는 핵산분자를 제공한다. 상기 핵산 분자는 하나 이상의 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있다. 뉴클레오티드 서열을 화학적으로 합성하여 제조하는 경우, 당업계에 널리 공지된 합성법, 예를 들어 문헌(Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988)에 기술된 방법을 이용할 수 있으며, 트리에스테르, 포스파이트, 포스포르아미다이트 및 H-포스페이트 방법, PCR 및 기타 오토프라이머 방법, 고체 지지체상의 올리고뉴클레오타이드 합성법 등을 이용하여 합성할 수 있다.As another aspect, the present invention provides a nucleic acid molecule encoding the peptide. The nucleic acid molecule may be mutated by substitution, deletion, insertion, or a combination of one or more bases thereof. When preparing a nucleotide sequence by chemical synthesis, a synthesis method well known in the art, for example, a method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988) can be used , triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other auto-primer methods, and oligonucleotide synthesis methods on solid supports.
또 다른 하나의 양태로서, 본 발명은 상기 핵산 분자를 포함하는 발현벡터, 상기 발현벡터를 포함하는 형질전환체를 제공한다.As another aspect, the present invention provides an expression vector containing the nucleic acid molecule and a transformant containing the expression vector.
본 발명의 용어 "발현벡터"란, 목적하는 숙주세포에서 목적 펩타이드를 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 의미한다. 상기 발현벡터는 포유류 세포(예를 들어, 사람, 원숭이, 토끼, 래트, 햄스터, 마우스 세포 등), 식물세포, 효모 세포, 곤충 세포 또는 박테리아 세포(예를 들어, 대장균 등)를 포함하는 진핵 또는 원핵세포에서 상기 핵산 분자를 복제 및/또는 발현할 수 있는 벡터가 될 수 있고, 바람직하게는 숙주세포에서 상기 핵산 분자가 발현될 수 있도록 적절한 프로모터에 작동가능하도록 연결되며, 적어도 하나의 선별마커를 포함하는 벡터가 될 수 있다. The term "expression vector" of the present invention refers to a recombinant vector capable of expressing a desired peptide in a desired host cell, and refers to a genetic construct containing essential regulatory elements operably linked to express a gene insert. The expression vector may be a mammalian cell (eg, human, monkey, rabbit, rat, hamster, mouse cell, etc.), a plant cell, a yeast cell, an insect cell, or a eukaryotic or bacterial cell (eg, E. coli, etc.) It can be a vector capable of replicating and/or expressing the nucleic acid molecule in a prokaryotic cell, preferably operably linked to an appropriate promoter so that the nucleic acid molecule can be expressed in a host cell, and comprising at least one selectable marker. It can be a vector that contains
본 발명에서 제공하는 상기 발현벡터가 도입될 수 있는 숙주세포는 상기 핵산 분자를 발현시켜서 상기 펩타이드를 생산할 수 있는 한 특별히 제한되지 않는데, 바람직하게는 대장균, 스트렙토미세스, 살모넬라 티피뮤리움 등의 박테리아 세포; 효모 세포; 피치아 파스토리스 등의 균류 세포; 드로조필라, 스포도프테라 Sf9 세포 등의 곤충 세포; CHO, COS, NSO, 293, 보우 멜라노마 세포 등의 동물 세포; 또는 식물 세포가 될 수 있고, 보다 바람직하게는 포유류 동물 세포가 될 수 있다.The host cell into which the expression vector provided in the present invention can be introduced is not particularly limited as long as it can produce the peptide by expressing the nucleic acid molecule, and is preferably a bacterial cell such as Escherichia coli, Streptomyces, or Salmonella typhimurium. ; yeast cells; fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; or a plant cell, more preferably a mammalian animal cell.
또 다른 하나의 양태로서, 본 발명은 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 서열번호 1의 아미노산 서열을 포함하는 펩타이드를 유효성분으로 포함하는 장독소성대장균 감염 예방 또는 치료용 약학 조성물을 제공한다. In another aspect, the present invention provides a peptide containing the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT) as an active ingredient. Or it provides a pharmaceutical composition for treatment.
본 발명의 약학 조성물에는 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 펩타이드 이외에 보조제(adjuvant)를 추가로 포함할 수 있다. 상기 보조제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있으나, 예를 들어 프로인드(Freund)의 완전 보조제 또는 불완전 보조제를 더 포함하여 그 효과를 증가시킬 수 있다.The pharmaceutical composition of the present invention may further include an adjuvant in addition to a peptide that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT). Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect.
본 발명에 따른 약학 조성물은 유효성분을 약학적으로 허용된 담체에 혼입시킨 형태로 제조될 수 있다. 여기서, 약학적으로 허용된 담체는 제약 분야에서 통상 사용되는 담체, 부형제 및 희석제를 포함한다. 본 발명의 약학 조성물에 이용할 수 있는 약학적으로 허용된 담체는 이들로 제한되는 것은 아니지만, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀전, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. .
제제화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 그러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘 카르보네이트, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 일반적으로 사용되는 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수용성용제, 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브유와 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used.
본 발명에 따른 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 모든 방식이 예상될 수 있는데, 예를 들면 경구, 정맥, 근육, 피하, 복강내 주사에 의해 투여될 수 있다.The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.
본 발명에 따른 약학 조성물의 투여량은 개체의 연령, 체중, 성별, 신체 상태 등을 고려하여 선택된다. 상기 약학 조성물 중 포함되는 상기 펩타이드의 농도는 대상에 따라 다양하게 선택할 수 있음은 자명하며, 바람직하게는 약학 조성물에 0.01 ~ 5,000 ㎍/ml의 농도로 포함되는 것이다. 그 농도가 0.01 ㎍/ml 미만일 경우에는 약학 활성이 나타나지 않을 수 있고, 5,000 ㎍/ml를 초과할 경우에는 인체에 독성을 나타낼 수 있다.The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the peptide included in the pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmacological activity may not appear, and if the concentration exceeds 5,000 μg/ml, toxicity to the human body may be exhibited.
본 발명에 따른 펩타이드는 장독소성 대장균 감염의 예방 및 치료 효과를 나타낼 수 있다. 상기 장독소성대장균에 의한 감염 질환은 장독소성대장균 감염에 따른 급성위장관염일 수 있으나, 이에 제한되는 것은 아니다. The peptide according to the present invention may exhibit preventive and therapeutic effects of enterotoxin E. coli infection. Infectious diseases caused by the enterotoxin E. coli may be acute gastroenteritis due to enterotoxin E. coli infection, but is not limited thereto.
또 다른 하나의 양태로서, 본 발명은 상기 장독소성대장균 이열성독소(LT)의 B 서브유닛에 특이적으로 결합하는 펩타이드를 유효성분으로 포함하는 장독소성대장균 감염 예방 또는 개선용 식품 조성물을 제공할 수 있다.In another aspect, the present invention provides a food composition for preventing or ameliorating enterotoxic E. coli infection comprising a peptide that specifically binds to the B subunit of the enterotoxin heat-labile toxin (LT) as an active ingredient. can
본 발명의 식품 조성물은 유효성분인 펩타이드를 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.The food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions, in addition to containing peptides as active ingredients.
상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 상술한 향미제는 천연 향미제(타우마틴), 스테비아 추출물 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 본 발명의 식품 조성물은 상기 약학적 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조 식품류 등이 있다.Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the above-mentioned flavors, natural flavors (thaumatin), stevia extracts, and synthetic flavors (saccharin, aspartame, etc.) can advantageously be used. The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. there is
또한 상기 식품 조성물은 유효성분인 추출물 외에 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition, the food composition, in addition to extracts as active ingredients, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. In addition, the food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages.
본 발명의 식품 조성물은 장독소성대장균 감염 예방 또는 개선 목적으로, 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공될 수 있다. 본 발명에서 '건강기능성 식품 조성물'이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다. 본 발명의 건강기능식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다. 상기 '식품 첨가물 공전'에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료 제제, 타르색소제제 등의 혼합제제류 등을 들 수 있다. The food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving enterotoxinic E. coli infection. In the present invention, 'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to Health Functional Food Act No. 6727, and the structure and function of the human body It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions. The health functional food of the present invention may contain ordinary food additives, and the suitability as a food additive is determined according to the general rules of the Food Additive Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.
이하 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited by the following examples.
실시예Example 1 One
T7 페이지 디스플레이T7 page display
바이오패닝 및 파지 증식을 포함한 모든 작업은 제조업체에서 제공한 지침에 따라 수행하였다. 제조사(TB178 T7Select®System; Novagen)에서 제공한 지침에 따라 다클론 항체를 사용하여 음성 및 양성 선택 모두로 차등 바이오 패닝을 수행했다. PBS(phosphate-buffered saline)로 헹구고 1% 농도의 소 혈청 알부민에서 1시간 동안 차단한 후, 25리터의 단백질 G+ 아가로스 비드를 처리(BSA)했다. 비드를 PBS로 3회 세척한 후 T7 파지 디스플레이 cDNA 라이브러리와 함께 추가로 2시간 동안 재인큐베이션하였다. 이 과정을 두 번 반복하였다. 면역 전 IgG와 반응하는 단백질을 제거하기 위해 이 감산 바이오패닝 전략을 사용할 필요가 있다. 그 후, T7 파지 cDNA 라이브러리의 상청액을 단백질 G+ 아가로스 비드에 고정화된 다클론 항체로 처리하고, 혼합물을 섭씨 4도에서 24시간 동안 인큐베이션하도록 두었다. 결합된 T7 파지 cDNA 라이브러리는 1 PBS로 3회 세척한 후 1% 나트륨 도데실 설페이트로 용리되었다. 바이오패닝의 후속 사이클 동안, 용리액은 E. coli 균주 EDL933을 사용하여 증폭되었다. 4번의 바이오패닝 과정을 거친 후, 선택된 파지 라이브러리는 면역 스크리닝을 거쳤다.All operations including biopanning and phage propagation were performed according to the instructions provided by the manufacturer. Differential biopanning was performed with both negative and positive selection using polyclonal antibodies according to the instructions provided by the manufacturer (TB178 T7Select®System; Novagen). After rinsing with PBS (phosphate-buffered saline) and blocking in 1% bovine serum albumin for 1 hour, 25 liters of protein G+ agarose beads were treated (BSA). The beads were washed three times with PBS and then re-incubated with the T7 phage display cDNA library for an additional 2 hours. This process was repeated twice. It is necessary to use this subtractive biopanning strategy to remove proteins that react with IgG prior to immunization. Then, the supernatant of the T7 phage cDNA library was treated with polyclonal antibody immobilized on protein G+ agarose beads, and the mixture was left to incubate at 4 degrees Celsius for 24 hours. The bound T7 phage cDNA library was washed three times with 1 PBS and then eluted with 1% sodium dodecyl sulfate. During subsequent cycles of biopanning, the eluate was amplified using E. coli strain EDL933. After four rounds of biopanning, the selected phage library was subjected to immunological screening.
플라크 리프트 분석plaque lift assay
한천 플레이트에서 약 1000개의 잘 분산된 개별 파지 클론을 플라크 리프트 기술을 사용하여 니트로셀룰로오스 막으로 옮겼다. 니트로셀룰로오스 멤브레인(직경 82mm HATF 필터, Millipore Corp.)을 한천 위에 씌우고 실온에서 10-15분 동안 배양했다. 한천과 플라크를 방해하지 않고 니트로셀룰로오스 막을 조심스럽게 들어 올렸다. 면역 염색 절차에는 1-2시간 동안 결합 글리칸(GM1a) LT-B와 함께 초기 배양이 포함되었다. 그런 다음 멤브레인을 헹구고 검출 시약을 적용했다. 검출 시약에는 양고추냉이 퍼옥시다제 결합 염소 항-마우스 IgG가 포함되며 3,3-디아미노벤지딘(DAB) 및 0.01% H2O2를 사용한 발색이 뒤따렀다.Approximately 1000 well-dispersed individual phage clones from agar plates were transferred to nitrocellulose membranes using the plaque lift technique. A nitrocellulose membrane (82 mm diameter HATF filter, Millipore Corp.) was placed over the agar and incubated for 10-15 minutes at room temperature. The nitrocellulose membrane was carefully lifted without disturbing the agar and plaque. The immunostaining procedure included an initial incubation with combined glycan (GM1a) LT-B for 1–2 h. The membrane was then rinsed and detection reagent applied. Detection reagents included horseradish peroxidase conjugated goat anti-mouse IgG followed by color development with 3,3-diaminobenzidine (DAB) and 0.01% H 2 O 2 .
도킹 시뮬레이션docking simulation
이 방법을 통해 병원체의 렉틴 구조를 해독하고 숙주의 수용체에 부착되는 부위를 식별할 수 있었다. 상동 모델 접근법인 SWISS-MODEL을 사용하여 GM1a(PDB ID: 6LF2) 3D 구조를 사용하여 LT(PDB ID: 1LTS)의 3차원(3D) 구조를 생성했다. 도킹 시뮬레이션은 GM1a의 3차원 구조를 이용하여 진행하였다. Chem-office 애플리케이션(http://www.cambridgesoft.com, 버전: 7.0)도 에너지 사용을 줄이기 위해 사용되었다. Autodock Vina 1.1.2는 도킹 시뮬레이션에 사용되었다. 도킹 시뮬레이션 결과를 기반으로 LigPlot에서 기본 설정으로 HBPLUS 및 비결합 접촉 매개변수를 사용하여 가능한 수소 결합 및 소수성 상호 작용을 식별했다.This method allowed them to decipher the pathogen's lectin structure and identify the site where it attaches to the host's receptor. A three-dimensional (3D) structure of LT (PDB ID: 1LTS) was generated using the homology model approach, SWISS-MODEL, using the GM1a (PDB ID: 6LF2) 3D structure. Docking simulation was performed using the 3D structure of GM1a. The Chem-office application (http://www.cambridgesoft.com, version: 7.0) was also used to reduce energy use. Autodock Vina 1.1.2 was used for docking simulation. Based on the docking simulation results, possible hydrogen bonding and hydrophobic interactions were identified using HBPLUS and nonbonding contact parameters with default settings in LigPlot.
고체상 펩타이드 합성Solid phase peptide synthesis
수지에서 펩타이드 합성의 초기 단계는 C-말단 아미노산을 수지에 연결하는 것이었다. 일시적인 보호 그룹은 알파 아미노 그룹과 반응성 측쇄를 중합으로부터 보호한다. 다음으로 수지를 여과하고 세척하여 부산물과 과잉 시약을 제거했다. 그런 다음 N-알파 보호기를 제거하고 수지를 다시 세척하여 부산물과 과잉 시약을 제거했다. 그 다음, 펩타이드 서열이 완성될 때까지 다음 아미노산을 첨가하였다. 그런 다음 보호 그룹을 제거하기 위해 헹구고 펩타이드를 수지에서 방출했다. 상기 과정과 마찬가지로 P1, P2, P3에 대해서도 동일한 프로토콜로 펩타이드 제조 합성을 진행하였다. GM1a 유사 펩타이드, P1(KILSYTESMAGKREMVIIT: 서열번호 1), P2(SYTESMAGKRE: 서열번호 2) 및 P3(TLRITYLTETK: 서열번호 3)도 유사하게 합성되었다.The initial step in peptide synthesis on resin was to link the C-terminal amino acid to the resin. Temporary protecting groups protect the alpha amino group and reactive side chains from polymerization. Next, the resin was filtered and washed to remove by-products and excess reagents. The N-alpha protecting group was then removed and the resin washed again to remove byproducts and excess reagent. Then, the next amino acid was added until the peptide sequence was complete. The peptide was then released from the resin after rinsing to remove the protecting group. As in the above process, peptide synthesis was performed with the same protocol for P1, P2, and P3. The GM1a-like peptides, P1 (KILSYTESMAGKREMVIIT: SEQ ID NO: 1), P2 (SYTESMAGKRE: SEQ ID NO: 2) and P3 (TLRITYLTETK: SEQ ID NO: 3) were similarly synthesized.
펩타이드 결합 및 억제 분석Peptide Binding and Inhibition Assay
P1, P2 및 P3 펩타이드를 플레이트의 각 웰에 웰당 25ng의 농도로 코팅하였다. 블로킹 후 각 웰에 LT-B를 상온에서 2시간 동안 채우고 1% BSA로 5회 세척하였다. 다음으로, 플레이트를 4℃에서 15-16시간 동안 인큐베이션했다. 또 다른 세척 후, 플레이트를 1:10,000 희석의 플루오레세인 이소티오시아네이트-접합 염색 시약으로 염색하였다. 실온에서 2시간 후 플레이트를 세척하고 마이크로 리더를 이용하여 형광을 검출하였다.P1, P2 and P3 peptides were coated on each well of the plate at a concentration of 25 ng per well. After blocking, each well was filled with LT-B for 2 hours at room temperature and washed 5 times with 1% BSA. Next, the plates were incubated at 4° C. for 15-16 hours. After another wash, the plates were stained with fluorescein isothiocyanate-conjugated staining reagent at a 1:10,000 dilution. After 2 hours at room temperature, the plate was washed and fluorescence was detected using a micro reader.
T7 파지 디스플레이를 이용한 using T7 phage display GM1aGM1a 강글리오사이드와with ganglioside 관련된 Related LTLT -B의 렉틴 수용체 식별 결과Results of lectin receptor identification of -B
mRNA는 T7 파지 디스플레이에 사용될 수 있도록 ETEC에서 분리되었다. mRNA를 추출한 후 cDNA를 생성하고 파지 라이브러리를 조립했다. 올리고(dT), 무작위 또는 일치하는 서브유닛 특이적 프라이머를 사용한 cDNA 합성은 동일한 양의 전사체를 생성했다. 면역 스크리닝 방법은 업계의 요구 사항과 일치하는 방식으로 수행되다. 플라크 리프트 테스트는 GM1a 글리칸과 연결된 렉틴 인자를 밝혀냈다. 두꺼운 패치가 선택 및 시퀀싱을 위해 선택되었고, 결과를 조사했다. 이에 직접적인 결과로 LT-B가 대부분 확인되었으며, LT-B를 이용하여 다음과 같은 테스트를 진행하였다(도 2 참조).mRNA was isolated from ETEC so that it could be used for T7 phage display. After mRNA was extracted, cDNA was generated and a phage library was assembled. cDNA synthesis using oligo(dT), random or matched subunit specific primers produced equal amounts of transcript. Immune screening methods are performed in a manner consistent with industry requirements. Plaque lift testing revealed lectin factors associated with GM1a glycans. Thick patches were selected for selection and sequencing, and the results investigated. As a direct result of this, LT-B was mostly confirmed, and the following tests were conducted using LT-B (see FIG. 2).
LTLT -B의 리간드 결합 도메인(The ligand binding domain of -B ( LBDLBD )의 결정) of determination
GM1a 강글리오시드 결합 친화도에 결합하는 LT-B의 리간드 결합 도메인(LBD)은 GM1a 강글리오시드에 결합하는 렉틴 후보인 LT-B와의 도킹 시뮬레이션을 사용하여 결정되었다. LT-B의 리간드 결합 도메인(LBD)을 찾기 위해 리간드와 상호작용하는 LT-B 잔기에 대한 수소결합, 이온결합, 친화도를 고려하여 조사했다. 이것은 모든 리간드에 대해 수행했다. 이것의 직접적인 결과로 예상되는 결합 부위에서 3개의 LBD가 발견되었다(도 3 참조).The ligand binding domain (LBD) of LT-B that binds to GM1a ganglioside binding affinity was determined using docking simulations with LT-B, a lectin candidate that binds to GM1a ganglioside. In order to find the ligand-binding domain (LBD) of LT-B, hydrogen bonding, ionic bonding, and affinity for the ligand-interacting LT-B residue were investigated. This was done for all ligands. As a direct result of this, three LBDs were found at the expected binding sites (see Fig. 3).
접착을 억제하는 to inhibit adhesion LTLT -B -B LBDLBD 펩타이드의 능력 비교 및 접착 친화도 비교 Comparison of peptide ability and adhesion affinity
조사가 완료된 후 3개의 리간드 결합 도메인(LBD)를 발견했다. 펩타이드 서열 KILSYTESMAGKREMVIIT(서열번호 1), SYTESMAGKRE(서열번호 2), 및 TLRITYLTETK(서열번호 3)는 모두 결합 부위의 잠재적 후보였고, 이 시퀀스를 각각 P2, P1 및 P3이라고 명명했다. LT-B를 이용한 접착력 검사를 했다. 그 결과는 도 4에 나타내었다. After the investigation was completed, they found three ligand-binding domains (LBDs). The peptide sequences KILSYTESMAGKREMVIIT (SEQ ID NO: 1), SYTESMAGKRE (SEQ ID NO: 2), and TLRITYLTETK (SEQ ID NO: 3) were all potential candidates for binding sites, and these sequences were designated P2, P1, and P3, respectively. Adhesion was tested using LT-B. The results are shown in FIG. 4 .
도 4의 A를 참조하면, 글리칸 모방 펩타이드인 P1은 다른 펩타이드에 비해 가장 높은 결합력을 나타내는 것으로 나타났고, P1, P2 및 P3은 모두 대조군과 비교할 때 LT-B에 결합하는 능력 수준이 상당히 높았다. 대조군(control)은 펩타이드가 없는 매트릭스의 결합 친화도를 의미한다. 대조군과 비교할 때, P1-P3 펩타이드는 통계적으로 유의한 변화를 나타냈다. Referring to A in Figure 4, the glycan-mimicking peptide P1 showed the highest binding affinity compared to other peptides, and all P1, P2 and P3 showed significantly higher binding ability to LT-B than the control group. . Control refers to the binding affinity of the matrix without peptide. Compared to the control group, P1-P3 peptides showed statistically significant changes.
도 4의 B를 참조하면, 0, 10, 100 및 1000 nM의 농도로 P1이 처리되었고, 펩타이드 P1을 사용한 시험관내 처리는 농도 의존적 방식으로 LT-B 활성을 억제하였다. Referring to FIG. 4B , P1 was treated at concentrations of 0, 10, 100, and 1000 nM, and in vitro treatment with peptide P1 inhibited LT-B activity in a concentration-dependent manner.
펩타이드 P1에 의한 HCT-8 세포에서 LT-B 사이토카인 생성억제Inhibition of LT-B cytokine production in HCT-8 cells by peptide P1
장 상피 세포 HCT-8에 의한 공격 감염 후, P1의 영향을 검출하기 위해 ELISA를 사용하여 사이토카인 수준을 측정하였다. 감염 후 주요 염증 인자인 TNF-a, IL-6, IL-1b의 수치를 측정한 결과, 이들 3가지 염증 마커가 모두 감소한 것으로 나타났다(도 5 참조). 이러한 발견은 P1이 세포 표면의 GM1a와 상호작용하는 LT-능력 B를 억제하고 배양 배지에 존재할 때 이를 중화할 수 있다는 결정적인 증거를 제공한다.After challenge infection with intestinal epithelial cells HCT-8, cytokine levels were measured using ELISA to detect the effect of P1. As a result of measuring the levels of TNF-a, IL-6, and IL-1b, which are major inflammatory factors after infection, it was found that all three inflammatory markers were reduced (see FIG. 5). These findings provide conclusive evidence that P1 can inhibit LT-capable B interacting with GM1a on the cell surface and neutralize it when present in the culture medium.
Claims (6)
A food composition for preventing or improving enterotoxigenic Escherichia coli infection comprising the peptide according to claim 1 as an active ingredient.
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US8877161B2 (en) * | 2011-10-19 | 2014-11-04 | Georgia Regents Research Institute, Inc. | GM1-like peptides and uses thereof |
KR20160089466A (en) * | 2013-11-26 | 2016-07-27 | 이데미쓰 고산 가부시키가이샤 | Vaccine against colibacillosis |
US9534020B2 (en) * | 2010-09-29 | 2017-01-03 | New York University | Immunogenic polypeptides comprising a modified loop peptide presenting the HIV-1 GP120 3074 mAb epitope and scaffold proteins containing said peptide |
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US9534020B2 (en) * | 2010-09-29 | 2017-01-03 | New York University | Immunogenic polypeptides comprising a modified loop peptide presenting the HIV-1 GP120 3074 mAb epitope and scaffold proteins containing said peptide |
US8877161B2 (en) * | 2011-10-19 | 2014-11-04 | Georgia Regents Research Institute, Inc. | GM1-like peptides and uses thereof |
KR20160089466A (en) * | 2013-11-26 | 2016-07-27 | 이데미쓰 고산 가부시키가이샤 | Vaccine against colibacillosis |
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