KR20230008466A - Vacuoles expressing histamine binding protein and composition for inhibiting histamine comprising thereof - Google Patents
Vacuoles expressing histamine binding protein and composition for inhibiting histamine comprising thereof Download PDFInfo
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- KR20230008466A KR20230008466A KR1020210089138A KR20210089138A KR20230008466A KR 20230008466 A KR20230008466 A KR 20230008466A KR 1020210089138 A KR1020210089138 A KR 1020210089138A KR 20210089138 A KR20210089138 A KR 20210089138A KR 20230008466 A KR20230008466 A KR 20230008466A
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- histamine
- binding protein
- vector
- yeast
- vacuole
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Abstract
Description
본 발명은 외막에 히스타민 결합 단백질을 발현하는 진핵 생물의 액포(vacuoles)의 제조 방법 및 이 액포의 히스타민 억제용 용도에 관한 것이다. The present invention relates to a method for producing eukaryotic vacuoles expressing a histamine-binding protein in their outer membrane and to the use of these vacuoles for histamine inhibition.
히스타민은 생체 내에 존재하는 바이오 아민으로, 외부 물질이 침입하면 면역반응으로 즉각적으로 많은 양이 분비되게 되고 빠르게 염증반응을 일으켜 외부 물질의 침입을 막을 수 있게 도와주는 면역 매개체이다. 히스타민은 정상적으로 조절되는 경우는 문제가 되지 않지만, 과다 분비되거나 히스타민을 분해하는 효소가 부족해지면 자가 면역질환을 일으키는 부작용을 나타낼 수 있다. 특히, 아토피 및 알레르기 반응에서 혈관 확장 및 가려움증을 유발한다.Histamine is a bio amine that exists in the body. When an external substance invades, a large amount is immediately secreted in an immune response, and it is an immune mediator that helps to prevent the invasion of external substances by rapidly causing an inflammatory response. When histamine is normally regulated, it is not a problem, but excessive secretion or lack of an enzyme that decomposes histamine can cause side effects that cause autoimmune diseases. In particular, it induces vasodilation and itching in atopic and allergic reactions.
항히스타민제는 히스타민 수용체와 결합하여 히스타민의 행동을 완화시키는데 도움이 되기 때문에, 알레르기 반응에 많이 쓰이는 약물이다. 하지만 항히스타민제는 히스타민 수용체 외에도 체내의 다른 수용체와 결합하여 졸음, 피로, 기억 상실 및 주의력 결핍과 같은 중추 신경계 부작용과 변비, 설사, 구역 및 구토와 같은 소화 장애를 유발하기 때문에 새로운 히스타민 억제제의 개발이 필요하다.Antihistamines are often used for allergic reactions because they bind to histamine receptors and help to alleviate the action of histamine. However, antihistamines bind to other receptors in the body in addition to histamine receptors, causing central nervous system side effects such as drowsiness, fatigue, memory loss and attention deficit, and digestive disorders such as constipation, diarrhea, nausea and vomiting. necessary.
본 발명은 히스타민 수용체에 작용하는 것이 아니라 히스타민을 직접적으로 억제하는 히스타민 억제제 및 이를 효율적으로 생산하는 방법을 제공하는 것을 해결과제로 한다. An object of the present invention is to provide a histamine inhibitor that directly inhibits histamine rather than acting on histamine receptors and a method for efficiently producing the same.
그러나 본 발명이 해결하고자 하는 과제는 이상에서 언급한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the problem to be solved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
상기와 같은 과제를 해결하기 위하여 본 발명은 일 측면에서, (a) 효모 액포 표면 발현 신호 서열의 유전자, 변형된 효모 액포 표면 발현 단백질 pVMA11의 유전자가 포함된 벡터를 제조하고 증폭시키는 단계; 및 (b) 단계 (a)에서 제조된 벡터에 히스타민 결합 단백질의 유전자를 삽입하고 증폭시키는 단계를 포함하는 히스타민 결합 단백질을 발현시키기 위한 벡터를 제조하는 방법을 제공한다. In order to solve the above problems, the present invention, in one aspect, (a) preparing and amplifying a vector containing the yeast vacuole surface expression signal sequence gene and the modified yeast vacuole surface expression protein pVMA11 gene; and (b) inserting and amplifying the histamine-binding protein gene into the vector prepared in step (a).
본 발명은 일 측면에서 상기 방법으로 제조된 재조합 벡터를 제공한다.In one aspect, the present invention provides a recombinant vector prepared by the above method.
본 발명은 일 측면에서 상기 재조합 벡터로 형질전환된 효모를 제공한다.In one aspect, the present invention provides yeast transformed with the recombinant vector.
또한, 본 발명은 일 측면에서, (a) 효모 액포 표면 발현 신호 서열의 유전자, 변형된 효모 액포 표면 발현 단백질 pVMA11의 유전자가 포함된 벡터를 제조하고 증폭시키는 단계; (b) 단계 (a)에서 제조된 벡터에 히스타민 결합 단백질의 유전자를 삽입하고 증폭시키는 단계; (c) 단계 (b)에서 제조된 벡터로 효모를 형질전환 시키는 단계; 및 (d) 단계 (c)에서 제조된 형질전환 효모로부터 액포를 분리하는 단계를 포함하는 히스타민 결합 단백질이 발현된 액포를 제조하는 방법을 제공한다. In one aspect, the present invention provides (a) preparing and amplifying a vector containing the yeast vacuole surface expression signal sequence gene and the modified yeast vacuole surface expression protein pVMA11 gene; (b) inserting and amplifying the histamine binding protein gene into the vector prepared in step (a); (c) transforming yeast with the vector prepared in step (b); and (d) isolating vacuoles from the transformed yeast prepared in step (c).
본 발명은 일 측면에서, 상기 방법에 의하여 제조된 히스타민 결합 단백질이 발현된 액포를 제공한다.In one aspect, the present invention provides a vacuole expressing the histamine-binding protein prepared by the above method.
본 발명은 일 측면에서, 히스타민 결합 단백질이 발현된 액포를 유효성분으로 포함하는 조성물의 히스타민 억제용 용도를 제공한다. In one aspect, the present invention provides a use of a composition comprising, as an active ingredient, a vacuole expressing a histamine-binding protein for inhibiting histamine.
본 발명에 의하면 히스타민 억제 및 면역 증진 소재 개발 방법이 제공된다.According to the present invention, a method for developing a material for suppressing histamine and enhancing immunity is provided.
본 발명에서 제공하는 히스타민 결합 단백질이 발현된 액포를 제조하는 방법은 히스타민 결합 단백질이 발현된 액포의 효율적으로 대량생산이 쉽고 개발에 큰 어려움이 없다는 장점을 가지고 있다. The method for preparing a histamine-binding protein-expressing vacuole provided by the present invention has the advantage of being able to efficiently mass-produce histamine-binding protein-expressing vacuoles without much difficulty in development.
본 발명을 통해 개발된 외막 상에 히스타민 결합 단백질이 발현된 액포는 기존의 액포의 장점과 함께 히스타민을 억제 활성을 가진다. 진핵 미생물의 액포는 세포 내로 외부 유해 물질이 침입했을 때 미생물, 곰팡이, 유해활성산소 뿐만 아니라 바이러스에 대한 처리 효율이 검증된 바 있다. The vacuoles in which the histamine-binding protein is expressed on the outer membrane developed according to the present invention have the advantages of conventional vacuoles and histamine inhibitory activity. Eukaryotic vacuoles have been proven to be effective in treating viruses as well as microorganisms, fungi, and harmful reactive oxygen species when external harmful substances invade the cells.
본 발명의 히스타민 결합 단백질이 표면에 발현된 액포는 히스타민의 행동을 억제하고, 과도한 염증반응으로 약해진 면역을 증진시키는 면역 증진제로서 사용될 수 있으며, 알레르기, 아토피, 천식의 예방 또는 치료를 위한 약학적 조성물로 사용될 수 있다 또한, 본 발명의 히스타민 결합 단백질이 표면에 발현된 액포는 화장료 조성물로 사용될 수 있으며, 히스타민을 억제하는 것이 필요한 시험관 내 실험을 위하여 사용될 수 있다. The vacuoles in which the histamine-binding protein of the present invention is expressed on the surface suppress the action of histamine, can be used as an immune enhancer that enhances immunity weakened by excessive inflammatory reactions, and is a pharmaceutical composition for preventing or treating allergy, atopy, and asthma. In addition, the vacuoles in which the histamine-binding protein of the present invention is expressed on the surface can be used as cosmetic compositions and for in vitro experiments that require inhibition of histamine.
본 발명의 효과는 이런 문언적 기재에만 한정되지 않고, 통상의 기술자가 본 발명을 통해 유추할 수 있는 것까지 모두 포함한다.The effects of the present invention are not limited to these literal descriptions, but also include everything that a person skilled in the art can infer through the present invention.
도 1은 히스타민 결합 단백질을 효모 세포 소기관인 액포의 외막에 발현시키기 위해 설계된 벡터의 도면이다.
도 2는 히스타민 결합 단백질을 효모 내의 액포의 외막에 발현시킨 모식도이다.
도 3은 재조합 단백질의 발현을 확인하기 위해 형광 현미경을 통해 GFP 발현을 확인한 결과를 나타낸다. 재조합 효모는 SD 배지로 24 시간동안 배양 후, 갈락토즈를 첨가하여 단백질 발현을 유도시켰다.
도 4는 액포의 외막에 발현된 히스타민 결합 단백질을 확인하기 위하여 Anti-His tag을 이용한 웨스턴 블롯을 실시한 결과를 나타낸다.
도 5는 HBP vacuoles의 RAW 264.7 세포 대한 적정 처리 농도를 측정하기 위한 MTT assay 결과를 나타낸다.
도 6은 LPS를 처리해 히스타민 방출을 유도한 RAW 264.7 세포에 대한 HBP vacuoles의 히스타민 효과를 나타낸다.
도 7은 HBP vacuoles에 대한 면역 반응을 확인해보기 위해, RAW 264.7 세포에 HBP vacuoles을 처리한 후 실시한 식균작용 분석 (phagocytosis assay) 결과를 나타낸다. 1 is a diagram of a vector designed to express histamine-binding proteins in the outer membrane of vacuoles, which are yeast cell organelles.
Fig. 2 is a schematic diagram showing the expression of histamine-binding protein on the outer membrane of vacuoles in yeast.
Figure 3 shows the result of confirming GFP expression through a fluorescence microscope to confirm the expression of the recombinant protein. After culturing the recombinant yeast in SD medium for 24 hours, protein expression was induced by adding galactose.
Figure 4 shows the results of Western blotting using the Anti-His tag to confirm the histamine binding protein expressed on the outer membrane of the vacuole.
Figure 5 shows the MTT assay results for measuring the appropriate treatment concentration of HBP vacuoles for RAW 264.7 cells.
Figure 6 shows the histamine effect of HBP vacuoles on RAW 264.7 cells treated with LPS to induce histamine release.
7 shows the result of phagocytosis assay performed after treating RAW 264.7 cells with HBP vacuoles to confirm the immune response to HBP vacuoles.
본 발명은 히스타민 결합 단백질을 표면에 발현하는 액포(vacuoles)를 제조하는 방법 및 상기 액포의 용도에 관한 것이다. The present invention relates to a method for preparing vacuoles that express a histamine binding protein on their surface and to the use of such vacuoles.
이하에서는 본 발명을 실시하기 위한 내용을 구체적 실시예를 첨부한 도면을 참조하여 자세히 설명한다. 종래기술과 다르지 않은 부분으로서 본 발명의 기술적 사상을 이해하는데 필요하지 않은 사항은 설명에서 제외한다. Hereinafter, details for carrying out the present invention will be described in detail with reference to the drawings accompanying specific embodiments. Matters that are not different from the prior art and are not necessary for understanding the technical idea of the present invention are excluded from the description.
본 발명의 발명자들은 부작용이 많이 보고된 히스타민 수용체에 작용하는 항히스타민제를 대체하기 위한 방법을 연구한 결과, 진드기가 생산하는 히스타민 결합 단백질을 표면에 발현시킨 액포를 사용하면, 히스타민 결합 단백질이 히스타민의 활성 자체를 억제하고 면역세포의 식균 작용을 증진시킨다는 것을 발견하고 본 발명을 완성하였다.The inventors of the present invention studied a method for replacing antihistamines that act on histamine receptors, which have been reported to have many side effects. The present invention was completed by discovering that it inhibits the activity itself and enhances the phagocytosis of immune cells.
이와 같은 발명자들의 연구결과를 바탕으로 본 발명은 다음을 제공한다.Based on the findings of the inventors, the present invention provides the following.
본 발명은 일 측면에서, (a) 효모 액포 표면 발현 신호 서열의 유전자, 변형된 효모 액포 표면 발현 단백질 pVMA11의 유전자가 포함된 벡터를 제조하고 증폭시키는 단계; 및 (b) 단계 (a)에서 제조된 벡터에 히스타민 결합 단백질의 유전자를 삽입하고 증폭시키는 단계를 포함하는 히스타민 결합 단백질을 발현시키기 위한 벡터를 제조하는 방법을 제공한다. In one aspect, the present invention comprises the steps of (a) preparing and amplifying a vector containing the yeast vacuole surface expression signal sequence gene and the modified yeast vacuole surface expression protein pVMA11 gene; and (b) inserting and amplifying the histamine-binding protein gene into the vector prepared in step (a).
본 발명은 일 측면에서 상기 방법으로 제조된 재조합 벡터로 효모를 형질전환시켜 히스타민 결합 단백질을 발현하는 액포를 함유하는 형질전환된 효모를 제조하는 방법을 제공한다.In one aspect, the present invention provides a method for producing transformed yeast containing vacuoles expressing a histamine-binding protein by transforming yeast with the recombinant vector prepared by the above method.
또한, 본 발명은 일 측면에서, (a) 효모 액포 표면 발현 신호 서열의 유전자, 변형된 효모 액포 표면 발현 단백질 pVMA11의 유전자가 포함된 벡터를 제조하고 증폭시키는 단계; (b) 단계 (a)에서 제조된 벡터에 히스타민 결합 단백질의 유전자를 삽입하고 증폭시키는 단계; (c) 단계 (b)에서 제조된 벡터로 효모를 형질전환 시키는 단계; 및 (d) 단계 (c)에서 제조된 형질전환 효모로부터 액포를 분리하는 단계를 포함하는 히스타민 결합 단백질이 발현된 액포를 제조하는 방법을 제공한다. In one aspect, the present invention provides (a) preparing and amplifying a vector containing the yeast vacuole surface expression signal sequence gene and the modified yeast vacuole surface expression protein pVMA11 gene; (b) inserting and amplifying the histamine binding protein gene into the vector prepared in step (a); (c) transforming yeast with the vector prepared in step (b); and (d) isolating vacuoles from the transformed yeast prepared in step (c).
상기 제조방법은 히스타민 결합 단백질의 발현을 확인하기 위해 GFP 유전자를 벡터에 추가로 포함시킬 수 있다. 또한 상기 제조방법은 벡터에 His tag를 추가로 포함시킬 수 있다.In the above production method, a GFP gene may be further included in the vector to confirm the expression of the histamine binding protein. In addition, the above production method may further include a His tag in the vector.
상기 히스타민 결합 단백질은 진드기 (Rhipicephalus appendiculatus)가 생산하는 히스타민 결합 단백질 2 (Histamine binding protein 2)일 수 있다. 상기 변형된 효모 액포 막 단백질(pVMA11)은 N-말단과 C-말단이 모두 세포질로 노출되도록 변형된 것일 수 있다. The histamine binding protein may be
상기 효모 액포 표면 발현 신호 서열의 유전자, 변형된 효모 액포 표면 발현 단백질 pVMA11의 유전자 및 His tag의 유전자 및 GFP 유전자가 포함된 벡터는 pYES2, pYES3 등을 사용하여 제조할 수 있으나 이로 제한되는 것은 아니다.A vector containing the yeast vacuolar surface expression signal sequence gene, the modified yeast vacuole surface expression protein pVMA11 gene, His tag gene, and GFP gene may be prepared using pYES2, pYES3, etc., but is not limited thereto.
상기 벡터의 증폭은 대장균 등에서 실시할 수 있으며, 이로 제한되는 것은 아니다. Amplification of the vector can be performed in E. coli, etc., but is not limited thereto.
본 발명은 일 측면에서 상기 방법으로 제조된 재조합 벡터를 제공한다.In one aspect, the present invention provides a recombinant vector prepared by the above method.
본 발명은 일 측면에서 상기 재조합 벡터로 형질전환된 효모를 제공한다.In one aspect, the present invention provides yeast transformed with the recombinant vector.
본 발명은 일 측면에서, 상기 방법에 의하여 제조된 히스타민 결합 단백질이 발현된 액포를 제공한다.In one aspect, the present invention provides a vacuole expressing the histamine-binding protein prepared by the above method.
본 발명은 일 측면에서, 히스타민 결합 단백질이 발현된 액포를 유효성분으로 포함하는 조성물의 히스타민 억제용 용도를 제공한다. In one aspect, the present invention provides a use of a composition comprising, as an active ingredient, a vacuole expressing a histamine-binding protein for inhibiting histamine.
본 발명은 일 측면에서 히스타민 결합 단백질이 발현된 액포를 유효성분으로 포함하는 히스타민 억제용 조성물을 히스타민의 활성을 억제할 필요가 있는 개체에게 투여하는 것을 포함하는 히스타민 과다로 인한 질병의 예방 또는 치료 방법을 제공한다.In one aspect, the present invention provides a method for preventing or treating a disease caused by excessive histamine, comprising administering a composition for inhibiting histamine containing, as an active ingredient, a vacuole in which a histamine binding protein is expressed to a subject in need of inhibiting histamine activity. provides
상기 개체는 인간 또는 인간 이외의 생물, 예를 들면 소, 원숭이, 새, 고양이, 마우스, 렛트, 햄스터, 돼지, 개, 토끼, 양, 말 등의 비인간 포유동물을 의미하는 것으로, 창상 또는 피부 조직이 손상된 개체에 이용할 수 있다. 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The subject refers to a human or non-human organism, for example, a non-human mammal such as a cow, monkey, bird, cat, mouse, rat, hamster, pig, dog, rabbit, sheep, horse, wound or skin tissue available for this damaged object. The dosage varies depending on the condition and weight of the patient, the severity of the disease, the type of drug, the route of administration and time, but can be appropriately selected by those skilled in the art.
본 발명은 일 측면에서 히스타민 결합 단백질이 발현된 액포를 유효성분으로 포함하는 히스타민 억제용 조성물을 제공한다. In one aspect, the present invention provides a composition for inhibiting histamine comprising, as an active ingredient, a vacuole in which a histamine binding protein is expressed.
상기 조성물은 과도한 염증반응, 알레르기, 아토피, 천식의 예방 또는 치료를 위한 약학적 조성물일 수 있다.The composition may be a pharmaceutical composition for preventing or treating excessive inflammatory reaction, allergy, atopy, or asthma.
또한, 상기 조성물은 면역 증진 효과를 추가로 나타내는 약학적 조성물일 수 있다.In addition, the composition may be a pharmaceutical composition that further exhibits an immune enhancing effect.
상기 조성물은 시험관 내에서 히스타민을 억제하기 위한 실험에 사용될 수 있는 조성물일 수 있다.The composition may be a composition that can be used in an experiment for inhibiting histamine in vitro.
본 발명은 일 측면에서 히스타민 결합 단백질이 발현된 액포를 유효성분으로 포함하는 히스타민 억제용 조성물을 포함하는 키트를 제공한다.In one aspect, the present invention provides a kit comprising a composition for inhibiting histamine containing, as an active ingredient, a vacuole in which a histamine binding protein is expressed.
본 발명의, "치료"는 본 발명에 따른 약학적 조성물의 투여에 의해 질환에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.In the present invention, "treatment" refers to all activities in which symptoms of a disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.
본 발명의, "개체"는 질환의 치료를 필요로 하는 대상을 의미하며, 보다 구체적으로 인간 또는 비-인간인 영장류, 생쥐(mouse), 개, 고양이, 말 및 소 등의 포유류를 의미한다.In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically means a mammal such as a human or non-human primate, mouse, dog, cat, horse, and cow.
본 발명의 "개선"이란 치료되는 상태와 관련된 파라미터, 예를 들면, 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다."Improvement" in the present invention means any action that at least reduces the parameters associated with the condition being treated, eg, the severity of symptoms.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 당업자에 의해 적절하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, It can be appropriately determined by those skilled in the art according to sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including drugs used concurrently, and other factors well known in the medical field.
일 예로, 본 발명의 히스타민 결합 단백질이 발현된 액포는 1일 0.0001 내지 100mg/kg으로, 바람직하게는 0.001 내지 10mg/kg으로 투여하는 것이 좋다. 투여는 하루에 한 번 투여할 수도 있고, 수 회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. For example, vacuoles expressing the histamine binding protein of the present invention may be administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 10 mg/kg per day. Administration may be administered once a day or divided into several times. The dosage is not intended to limit the scope of the present invention in any way.
본 발명에 따른 약학적 조성물은 개별 치료제로 투여하거나 다른 항히스타민제와 병용하여 투여될 수 있고, 또는 상기한 유효성분과는 별도로 항염증, 면역증진과 관련이 있는 기능을 갖는 다른 통상적인 약제를 더 포함할 수 있는데, 이러한 통상적인 약제를 포함시키는 것은 본 발명이 속하는 기술적 분야에서 통상의 지식을 가진 자라면 용이하게 실시할 수 있을 것이다. 본 발명의 약학적 조성물과 상기 다른 약제는 동시에, 별도로, 또는 순차적으로 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other antihistamines, or may further include other conventional drugs having functions related to anti-inflammatory and immune enhancement apart from the above active ingredients. It can be done, but the inclusion of such conventional drugs will be easily carried out by those skilled in the art to which the present invention belongs. The pharmaceutical composition of the present invention and the other agents may be administered simultaneously, separately, or sequentially, and may be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, which can be easily determined by those skilled in the art.
또한, 본 발명에 의한 약학적 조성물은 화장품, 세척용 로션 또는 자외선 차단제 등에 부가하여 사용할 수도 있다.In addition, the pharmaceutical composition according to the present invention may be used in addition to cosmetics, washing lotion or sunscreen.
본 발명의 조성물은 상기 조성물의 투여량, 투여경로, 투여횟수, 적응증 및 부작용 중 하나 이상을 개시한 지시서와 함께 히스타민 과다로 인한 질병의 치료용 키트로 제공될 수 있음이 자명하다.It is obvious that the composition of the present invention can be provided as a kit for treating diseases caused by excessive histamine together with instructions disclosing at least one of the dosage, administration route, frequency of administration, indications and side effects of the composition.
본 발명의 히스타민 결합 단백질이 발현된 액포는 콜로이드 현탁액, 분말, 식염수, 지질, 리포좀, 미소구체( microspheres), 또는 나노 구형입자와 같은 약학적으로 허용될 수 있는 담체에 운반될 수 있다. 이들은 운반 수단과 복합체를 형성하거나 관련될 수 있고, 지질, 리포좀, 미세입자, 금, 나노입자, 폴리머, 축합 반응제, 다당류, 폴리아미노산, 덴드리머, 사포닌, 흡착 증진 물질 또는 지방산과 같은 당업계에 공지된 운반 시스템을 사용하여 생체 내 운반될 수 있다.The vacuole expressing the histamine binding protein of the present invention can be transported in a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline solution, lipid, liposome, microspheres, or nano-spherical particles. They may be complexed with or associated with the delivery vehicle and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.
이 외에도, 약학적으로 허용되는 담체는 제제시 통상적으로 이용되는 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아, 고무, 인산칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세 결정성 셀룰로스, 폴리비닐 피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필 히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함할 수 있으나, 이에 한정되는 것은 아니다. 또한, 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.In addition, pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like may be further included.
본 발명은 히스타민 결합 단백질이 발현된 액포를 유효성분으로 함유하는 가려움증, 알레르기 증상 치료 또는 개선을 위한 피부 외용제를 제공한다.The present invention provides an external skin preparation for treating or improving symptoms of itching and allergy, which contains, as an active ingredient, vacuoles in which histamine binding protein is expressed.
본 발명의 피부 외용제는 화장품학 또는 피부과학적으로 허용 가능한 매질 또는 기제를 함유하여 피부 국소 도포용으로 제형화될 수 있다. 이는 국소적용에 적합한 모든 제형으로서, 예를 들면, 용액, 겔, 고체, 반죽무수생성물, 수상에 유상을 분산시켜 얻은 에멀젼, 현탁액, 마이크로에멀젼, 마이크로캡슐, 미세과립구 또는 이온형(리포좀) 및 비이온형의 소낭 분산제의 형태로, 또는 크림, 스킨, 로션, 파우더, 연고, 스프레이 또는 콘실 스틱의 형태로 제공될 수 있으며, 이로 제한되는 것은 아니다. 또한, 폼(foam)의 형태로 또는 압축된 추진제를 더 함유한 에어로졸 조성물의 형태로도 사용될 수 있다. 이들 조성물은 당해 분야의 통상적인 방법에 따라 제조될 수 있다.The skin external preparation of the present invention may be formulated for topical skin application by containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, e.g. solutions, gels, solids, anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and ratios. It may be provided in the form of an ionic follicular dispersant, or in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick, but is not limited thereto. It can also be used in the form of a foam or an aerosol composition further containing a compressed propellant. These compositions can be prepared according to conventional methods in the art.
또한, 본 발명의 피부 외용제는 지방 물질, 유기용매, 용해제, 농축제, 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속이온봉쇄제, 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 또는 친유성 활성제, 지질 소낭 또는 화장품에 통상적으로 사용되는 임의의 다른 성분과 같은 화장품학 또는 피부과학 분야에서 통상적으로 사용되는 보조제를 함유할 수 있다. 상기 보조제는 화장품학 또는 피부과학 분야에서 일반적으로 사용되는 양으로 도입된다.In addition, the skin external preparation of the present invention may contain a fatty substance, an organic solvent, a solubilizing agent, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, ionic or non-ionic. ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredient commonly used in cosmetics; It may contain adjuvants commonly used in the field of cosmetology or dermatology. The adjuvant is introduced in an amount generally used in the field of cosmetology or dermatology.
본 발명의 또 다른 일 구현예에 있어서, 본 발명의 히스타민 결합 단백질이 발현된 액포를 포함하는 피부 외용 약제 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 비경구적 사용을 위한 조성물들은 단위량 투약 형태(예를 들어 일회 분량의 앰플)나 몇 회분을 포함하는 작은 병 그리고 적절한 부형제가 첨가된 형태로 제공되기도 한다. 조성물은 용액, 현탁액, 유탁액, 주입 장치 또는 삽입을 위한 운반 장치의 형태로 있을 수도 있고, 사용 전에 물이나 또 다른 적합한 용액에 타서 원래대로 되게 하도록 건조분말 형태로 존재하기도 한다. 본 발명의 조성물은 비경구적으로 적합한 운반체들 그리고/또는 첨가제들을 포함하기도 한다. In another embodiment of the present invention, the pharmaceutical composition for external application to the skin comprising the vacuole expressing the histamine binding protein of the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. there is. Compositions for parenteral use may be presented in unit-dose dosage form (e.g., in single-dose ampoules) or in vials containing several doses, with appropriate excipients added. The composition may be in the form of a solution, suspension, emulsion, injection device or delivery device for insertion, or may be in the form of a dry powder to be infused with water or another suitable solution prior to use. Compositions of the present invention may also contain parenterally suitable carriers and/or additives.
또한, 본 발명은 히스타민 결합 단백질을 발현하는 액포를 유효성분으로 함유하는 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition containing, as an active ingredient, vacuoles expressing histamine binding proteins.
본 발명은 일 측면에서 히스타민 결합 단백질이 발현된 액포를 유효성분으로 포함하는 화장료 조성물을 제공한다.In one aspect, the present invention provides a cosmetic composition comprising, as an active ingredient, vacuoles in which histamine binding protein is expressed.
본 발명에 따른 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화 될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 본 발명의 조성물이 화장료 조성물로 사용될 때에는 화장수, 에멀젼, 크림, 에센스, 젤, 팩 및 클렌징크림으로부터 선택되는 기초 화장료; 화운데이션 등의 메이크업 화장료로 구성되는 군으로 부터 선택되는 제형으로 제조될 수 있다.The cosmetic composition according to the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing It may be formulated as a cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, when the composition of the present invention is used as a cosmetic composition, a basic cosmetic selected from lotion, emulsion, cream, essence, gel, pack and cleansing cream; It can be prepared in a formulation selected from the group consisting of makeup cosmetics such as foundation.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소 결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Star cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.
본 발명의 제형이 계면활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether Sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.
또한 본 발명의 화장료 조성물은 각각의 제형에 상기 기재한 성분들 이외에 일반 피부화장료에 배합되는 유분, 물, 계면활성제, 보습제, 저급알코올, 증점제, 킬레이트제, 색소, 방부제, 또는 향료 등을 필요에 따라 적절히 배합하여 사용할 수 있다.In addition, the cosmetic composition of the present invention contains oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, colorant, preservative, or flavoring agent, etc., which are formulated in general skin cosmetics, in addition to the components described above in each formulation, if necessary. It can be mixed and used appropriately.
예컨대, 유연화장수는 본 발명에 따른 액포 이외에 다가알콜류 (프로필렌글리콜, 글리세린 등) 1∼10 중량% 및 계면활성제 (폴리에틸렌올레일에테르, 폴리옥시에틸렌 경화피마자유 등) 0.05∼2 중량%를 함유하도록 제조할 수 있다. 또한, 영양화장수 및 영양크림은 본 발명에 따른 액포 이외에 오일류(스쿠알란, 바셀린, 옥틸도데칸올 등) 5∼20 중량% 및 왁스성분(세탄올, 스테아릴알콜, 밀납 등) 3∼15 중량%를 함유하도록 제조할 수 있으며, 에센스는 본 발명에 따른 액포 이외에 글리세린, 프로필렌글리콜 등의 다가알콜류 5∼30 중량%를 함유하여 제조할 수 있다. 마사지 크림은 본 발명에 따른 액포 이외에 유동파라핀, 바셀린, 이소노닐이소노나노에이트 등의 오일 30∼70 중량%를 함유하여 제조되며, 팩은 본 발명에 따른 액포 이외에 폴리비닐알콜 5∼20 중량%를 함유하는 필 오프(peel off) 팩 또는 일반유화형 화장료에 본 발명에 따른 펩타이드 이외에 카올린, 탈크, 산화아연, 이산화티탄 등의 안료가 5∼30 중량% 함유된 워시오프(wash off) 팩으로 제조할 수 있다.For example, the softening lotion contains 1 to 10% by weight of polyhydric alcohols (propylene glycol, glycerin, etc.) and 0.05 to 2% by weight of surfactants (polyethylene oleyl ether, polyoxyethylene hydrogenated castor oil, etc.) in addition to the vacuole according to the present invention. can be manufactured In addition, the nutrient lotion and nutrient cream according to the present invention contain 5 to 20% by weight of oils (squalane, vaseline, octyldodecanol, etc.) and 3 to 15% by weight of wax components (cetanol, stearyl alcohol, beeswax, etc.) in addition to the vacuoles according to the present invention. In addition to the vacuole according to the present invention, the essence may be prepared by containing 5 to 30% by weight of polyhydric alcohols such as glycerin and propylene glycol. Massage cream is prepared by containing 30 to 70% by weight of oil such as liquid paraffin, vaseline, isononyl isononanoate, etc. As a wash off pack containing 5 to 30% by weight of pigments such as kaolin, talc, zinc oxide, titanium dioxide, etc. in addition to the peptide according to the present invention in a peel off pack or general emulsion type cosmetic containing can be manufactured
본 발명의 화장료 조성물에서, 본 발명에 따른 액포는 상기 조성물의 총 중량을 기준으로 0.01 내지 90 중량% 함유되어 있을 수 있고, 바람직하게는 3 내지 15 중량% 함유되어 있을 수 있으나, 바람직한 효과를 제공할 수 있는 유효량을 포함한다면 이에 제한되지 않는다. In the cosmetic composition of the present invention, the vacuole according to the present invention may contain 0.01 to 90% by weight, preferably 3 to 15% by weight, based on the total weight of the composition, but provides desirable effects. It is not limited thereto as long as it includes an effective amount that can be used.
아래에서는 발명의 구체적인 구현예에 따른 외막에 히스타민 결합 단백질을 발현하는 진핵 생물의 액포를 제조하는 방법 및 상기 액포의 히스타민 억제, 면역 증진 효과에 대해 보다 상세히 설명하기로 한다. 다만, 이는 발명의 하나의 예시로서 제시되는 것으로, 이에 의해 발명의 권리범위가 한정되는 것은 아니며, 발명의 권리범위 내에서 구현예에 대한 다양한 변형이 가능함은 당업자에게 자명하다.Hereinafter, a method for preparing a eukaryotic vacuole expressing a histamine-binding protein in the outer membrane and the histamine-suppressing and immune-enhancing effects of the vacuole will be described in detail according to a specific embodiment of the present invention. However, this is presented as an example of the invention, whereby the scope of the invention is not limited, and it is obvious to those skilled in the art that various modifications to the embodiments are possible within the scope of the invention.
<실시예 1.> 히스타민 결합 단백질을 액포 표면에 발현시키기 위한 벡터의 제작<Example 1.> Construction of a vector for expressing histamine-binding protein on the surface of the vacuole
진드기 (Rhipicephalus appendiculatus)가 생산하는 히스타민 결합 단백질 2 (Histamine binding protein 2)를 효모의 액포의 외막에 발현시키기 위해, 변형된 효모 액포 막 단백질(pVMA11)과 이의 신호 서열 (서열번호 2)을 사용했다.A modified yeast vacuole membrane protein (pVMA11) and its signal sequence (SEQ ID NO: 2) were used to express
액포의 막단백질인 VMA11 단백질이 액포의 표면에 위치하며, N 말단과 C 말단이 세포질 쪽으로 노출되도록 변형시켜 만든 pVMA11의 유전자 서열 (서열번호 3)이 합성되어진 pYES2 벡터를 제조하였다.The pYES2 vector was prepared by synthesizing the gene sequence (SEQ ID NO: 3) of pVMA11, which was modified so that the VMA11 protein, a membrane protein of the vacuole, was located on the surface of the vacuole, and the N-terminus and C-terminus were exposed to the cytoplasm.
상기 벡터의 VMA11 신호 서열과 pVMA11의 서열 사이에 GFP 유전자를 삽입하기 위해 MCS (Multi Cloning Site)에 있는 제한효소 자리의 BamHI과 XhoI 부분을 잘라내고, 그 사이에 GFP 서열을 T4 ligase를 통해 이어 붙어 벡터를 완성시켰다. 완성된 벡터는 대장균에 열충격법을 통해 형질전환시켰고, 대장균을 배양시켜 유전자를 증폭시켰다.In order to insert the GFP gene between the VMA11 signal sequence and the pVMA11 sequence of the vector, the BamHI and XhoI parts of the restriction enzyme site in the MCS (Multi Cloning Site) were cut out, and the GFP sequence was connected through T4 ligase between them. vector is complete. The completed vector was transformed into E. coli by heat shock method, and the gene was amplified by culturing E. coli.
상기 증폭시킨 벡터를 추출하여 히스타민 결합 단백질 유전자를 삽입한 다음 대장균에 형질전환시켜 벡터를 완성시켰다 (도면 1). 상기 증폭시킨 벡터는 Plasmid isolation kit (GeneAll Exprep Plamid SV)을 사용하여 추출되었으며, MCS에 있는 제한효소 자리의 KpnI과 BamHI 부분을 잘라내고, 히스타민 결합 단백질의 유전자 서열을 이어 붙였다. 상기 히스타민 결합 단백질은 신호서열과 정지코돈을 배제한 히스타민 결합 단백질만의 서열이다 (서열번호 1). 완성된 벡터는 대장균에 열충격법을 통해 형질전환시켰고, 대장균을 배양시켜 유전자를 증폭시켰다.The amplified vector was extracted, the histamine binding protein gene was inserted, and then transformed into E. coli to complete the vector (Fig. 1). The amplified vector was extracted using a Plasmid isolation kit (GeneAll Exprep Plamid SV), the KpnI and BamHI portions of the restriction enzyme site in the MCS were cut out, and the histamine-binding protein gene sequences were spliced together. The histamine-binding protein is a sequence of only histamine-binding protein excluding signal sequences and stop codons (SEQ ID NO: 1). The completed vector was transformed into E. coli by heat shock method, and the gene was amplified by culturing E. coli.
완성된 벡터를 다음 서열의 프라이머를 사용하여 염기 서열 분석을 수행하여 확인하였다.The completed vector was confirmed by performing nucleotide sequence analysis using primers of the following sequence.
상기 히스타민 결합 단백질 서열을 확인하기 위해 5'- ACG GTA CCA ATC AGC CAG ATT GGG CCG A-3' (HBP F primer), 5'-ACT GGA TCC CTC TAG GCA AGC ACT TGT G-3' (HBP R primer)을 사용하였다. GFP 서열을 확인하기 위해서는 5'-AAT GGA TCC GTG AGC AAG GGC GAG GAG-3' (GFP F primer), 5'- GAG CTC GAG CTT GTA CAG CTC GTC CAT G-3' (GFP R primer)을 사용했다.To confirm the histamine binding protein sequence, 5'-ACG GTA CCA ATC AGC CAG ATT GGG CCG A-3' (HBP F primer), 5'-ACT GGA TCC CTC TAG GCA AGC ACT TGT G-3' (HBP R primer) was used. To confirm the GFP sequence, use 5'-AAT GGA TCC GTG AGC AAG GGC GAG GAG-3' (GFP F primer), 5'-GAG CTC GAG CTT GTA CAG CTC GTC CAT G-3' (GFP R primer) did.
상기 벡터의 제작에 사용된 유전자의 염기서열은 표 1과 같다.Table 1 shows the nucleotide sequence of the gene used in the construction of the vector.
< 표 1 > 벡터 제작에 사용된 유전자의 염기 서열<Table 1> Base sequence of genes used for vector construction
<실시예 2.> 히스타민 결합 단백질의 액포 표면에의 발현<Example 2.> Expression of histamine-binding protein on the vacuole surface
실시예 1의 방법으로 제작된 벡터로 효모 (S. cerevisiae 2805)을 형질전환하여 히스타민 결합 단백질의 발현을 유도하였다. Yeast ( S. cerevisiae 2805) was transformed with the vector constructed by the method of Example 1 to induce the expression of histamine binding protein.
실시예 1의 방법으로 제작된 벡터를 리튬 아세테이트를 사용하여 알카리성 양이온이 효모의 세포막에 작용하여 DNA 유입이 용이하도록 한 다음, PEG와 상기 완성된 벡터를 혼합한 후 열 충격을 주어 효모 균주 안으로 주입하였으며, 이를 영양 선택적 배지 (SD 배지 (0.67% Yeast nitrogen base without amino acid, 0.5% Casamino acid, 2% Agar를 통해 선별했다. The vector prepared by the method of Example 1 was subjected to alkaline cations using lithium acetate to act on the cell membrane of yeast to facilitate DNA incorporation, and then mixed with PEG and the completed vector and subjected to heat shock to inject into the yeast strain. and selected through a nutrient selective medium (SD medium (0.67% Yeast nitrogen base without amino acid, 0.5% Casamino acid, 2% Agar).
이후 본 배양 과정 (SD 배지, 30 ?, 180 rpm, 24 시간동안 배양을 거쳐 벡터 내 갈락토스 프로모터를 이용하여 갈락토즈를 첨가하여 재조합 단백질 발현을 유도했다 (도 2). After the main culture process (SD medium, 30 ?, 180 rpm, 24 hours of culture, galactose was added using the galactose promoter in the vector to induce recombinant protein expression (FIG. 2).
히스타민 결합 단백질의 발현을 확인하기 위해 형광 현미경을 이용하여 GFP 발현을 확인했다 (도 3). 또한 발현된 히스타민 결합 단백질은 His tag를 지니고 있으므로, 이 tag를 이용하여 Western blot을 이용하여 발현을 확인했다 (도 4). 재조합 단백질의 크기는 61.3 kDa이었고, 대조군으로 사용된 Mock vacuoles은 pYES2 벡터로만 형질전환된 재조합 효모의 액포이다. HBP vacuoles은 히스타민 결합 단백질이 발현된 재조합 효모 액포를 나타낸다.To confirm the expression of histamine binding protein, GFP expression was confirmed using a fluorescence microscope (Fig. 3). In addition, since the expressed histamine-binding protein has a His tag, expression was confirmed by Western blot using this tag (FIG. 4). The size of the recombinant protein was 61.3 kDa, and mock vacuoles used as a control were recombinant yeast vacuoles transformed only with the pYES2 vector. HBP vacuoles represent recombinant yeast vacuoles in which histamine-binding proteins are expressed.
<실시예 3.> 상기 히스타민 결합 단백질이 발현된 액포의 분리<Example 3.> Separation of vacuoles expressing the histamine binding protein
재조합된 효모는 24시간 동안 2% 포도당이 함유된 SD 배지에서 배양되었고, 이후 2% 갈락토스가 함유된 SG 배지에서 20시간 동안 단백질 발현이 유도되었다. 단백질 발현이 끝난 효모는 원심분리를 통해 수거되었고, Tris-SO₄ buffer (0.1 M Tris-SO₄ (pH 9.4), 10 mM DTT)를 세포 중량(g)당 5 ml를 첨가 후 30℃ 인큐베이터에서 15분 동안 반응시켰다. The recombinant yeast was cultured in SD medium containing 2% glucose for 24 hours, and then protein expression was induced in SG medium containing 2% galactose for 20 hours. The yeast with protein expression was collected by centrifugation, and after adding 5 ml of Tris-SO₄ buffer (0.1 M Tris-SO₄ (pH 9.4), 10 mM DTT) per cell weight (g), they were incubated in a 30°C incubator for 15 minutes. reacted during
그 다음 원심분리를 한 후 상층액을 제거하고, 세포와 동일 중량의 glass bead를 첨가 후 파쇄 완충액 (breaking buffer) (20 mM Tris-HCl (pH 7.4), 0.6 M Sorbitol)를 세포 중량(g)당 4 ml를 첨가 후, 1 분 vortexing 후 1 분 얼음 위에 놓는 과정을 총 10번 반복해 세포 파쇄를 진행했다. After centrifugation, the supernatant was removed, glass beads of the same weight as the cells were added, and breaking buffer (20 mM Tris-HCl (pH 7.4), 0.6 M Sorbitol) was added to the cell weight (g). After adding 4 ml of sugar, vortexing for 1 minute and placing on ice for 1 minute was repeated a total of 10 times to disrupt the cells.
그 후, 원심분리 (500 g, 5분)를 통해 세포소기관만 포함된 상층액을 분리했고, 다시 원심분리 (20,000 g, 30분)을 통해 효모 액포가 포함된 추출물을 수득하였다.Thereafter, the supernatant containing only organelles was separated by centrifugation (500 g, 5 minutes), and again centrifuged (20,000 g, 30 minutes) to obtain an extract containing yeast vacuoles.
<실시예 4.> 히스타민 결합 단백질이 발현된 액포의 RAW 264.7 세포에 대한 적정 처리 농도 확인<Example 4.> Confirmation of appropriate treatment concentration for RAW 264.7 cells of histamine-binding protein-expressed vacuoles
히스타민 결합 단백질이 발현된 효모 액포에 대한 면역세포인 RAW 264.7 세포의 세포 생존률로 적정 처리 농도를 확인했다. RAW 264.7 세포는 510⁴/ml로 24-well에 seed했고, 24시간동안 배양 후 12시간동안 기아상태로 유지했다. 그 후 다양한 농도의 효모 액포를 6시간동안 처리한 다음, MTT 어세이를 진행하여 생포 생존률을 측정했다. 그 결과 0.01 μg/ml의 HBP vacuoles이 80% 이상의 세포 생존율을 가진 가장 높은 농도로서, 적정 처리 농도로 확인되었다. 모든 실험은 세 번씩 반복되었으며 데이터는 평균표준오차로 표현되었다 (도 5).The appropriate treatment concentration was confirmed by the cell viability of RAW 264.7 cells, which are immune cells for yeast vacuoles expressing histamine-binding protein. RAW 264.7 cells were seeded in 24-well at 510⁴/ml, incubated for 24 hours and starved for 12 hours. Thereafter, yeast vacuoles of various concentrations were treated for 6 hours, and then the MTT assay was performed to measure the viability of the yeast vacuoles. As a result, 0.01 μg/ml of HBP vacuoles was the highest concentration with a cell viability of 80% or more, which was confirmed as an appropriate treatment concentration. All experiments were repeated three times and data were expressed as standard error of the mean (FIG. 5).
<실시예 5> 히스타민 결합 단백질이 발현된 액포에 의한 히스타민 농도 감소<Example 5> Reduction of histamine concentration by vacuoles expressing histamine binding protein
LPS 자극으로 인해 RAW 264.7 세포에서 방출된 히스타민이 액포 표면에 발현된 히스타민 결합 단백질과 결합되어 억제되는지를 확인했다. It was confirmed whether histamine released from RAW 264.7 cells due to LPS stimulation was bound to and inhibited by histamine-binding protein expressed on the vacuole surface.
RAW 264.7 세포는 실시예 4에서와 같이 510⁴/ml로 24-well에 seed하고, 24시간동안 배양 후 12시간 동안 기아상태로 유지한 다음, free serum DMEM에 희석한 효모 액포를 상층액에 6시간 동안 처리하였다. RAW 264.7 cells were seeded in 24-well at 510⁴/ml as in Example 4, cultured for 24 hours, starved for 12 hours, and yeast vacuoles diluted in free serum DMEM were added to the supernatant for 6 hours. treated during.
그 후 세포 상층액을 원심분리 (20,000g, 30분) 후, Histamine ELISA를 이용하여 남아있는 히스타민을 정량하였다. 최종 히스타민 농도는 Histamine ELISA를 통해 측정되었다. 대조군으로 효모 액포 막 단백질인 pVMA11만 발현된 pVMA11 vacuoles을 이용했다. Thereafter, the cell supernatant was centrifuged (20,000 g, 30 minutes), and remaining histamine was quantified using Histamine ELISA. Final histamine concentrations were measured via Histamine ELISA. As a control, pVMA11 vacuoles expressing only the yeast vacuolar membrane protein pVMA11 were used.
그 결과, HBP vacuoles (막 표면에 히스타민 결합 단백질이 발현된 액포)의 처리 농도에 따라 히스타민 감소율이 증가하였고, 이로써 발현된 단백질이 히스타민을 억제한다는 사실을 알 수 있었다. 대조군으로 처리한 pVMA11 vacuoles은 HBP vacuoles 만큼 히스타민을 억제하지 못했다. 모든 실험은 세 번씩 반복되었으며 데이터는 평균표준오차로 표현되었다 (도 6).As a result, the histamine reduction rate increased according to the treatment concentration of HBP vacuoles (vacuoles in which histamine-binding protein was expressed on the membrane surface), and it was found that the expressed protein inhibits histamine. Control-treated pVMA11 vacuoles did not inhibit histamine as well as HBP vacuoles. All experiments were repeated three times and data were expressed as standard error of the mean (FIG. 6).
<실시예 6.> RAW 264.7 세포의 식균 작용에 대한 히스타민 결합 단백질이 발현된 액포의 영향<Example 6.> Effect of vacuoles expressing histamine-binding protein on phagocytosis of RAW 264.7 cells
RAW 264.7세포의 식균작용 활성에 대한 HBP vacuoles의 효과를 확인했다.The effect of HBP vacuoles on the phagocytotic activity of RAW 264.7 cells was confirmed.
RAW 264.7 세포는 실시예 4에서와 같이 510⁴/ml로 24-well에 seed하고, 24시간동안 배양 후 12시간동안 기아상태로 유지한 다음, free serum DMEM에 희석한 효모 액포를 상층액에 6시간동안 처리하였다. RAW 264.7 cells were seeded in 24-well at 510⁴/ml as in Example 4, cultured for 24 hours, starved for 12 hours, and yeast vacuoles diluted in free serum DMEM were added to the supernatant for 6 hours. treated during.
그 후 RAW 264.7 세포에 RAW 264.7 세포에 히스타민 결합 단백질이 발현된 액포 (HBP vacuoles)을 6시간 처리한 후, zymosan (Cell Biolabs)을 처리하여 식균작용을 유도하고 phagocytosis 분석 키트 (Cell Biolabs)를 이용하여 식균작용 활성을 측정했다. 대조군으로 식균작용 억제제 (Cytochalasin D)와 pVMA11 vacuoles을 처리하였다. 모든 실험은 세 번씩 반복되었으며 데이터는 평균표준오차로 표현되었다 (도 7). 그 결과, HBP vacuoles의 처리 농도에 따라 식균작용 활성이 증가함을 확인하였다.After that, RAW 264.7 cells were treated with vacuoles (HBP vacuoles) in which histamine-binding protein was expressed in RAW 264.7 cells for 6 hours, and then treated with zymosan (Cell Biolabs) to induce phagocytosis using a phagocytosis assay kit (Cell Biolabs) phagocytosis activity was measured. As a control, phagocytosis inhibitor (Cytochalasin D) and pVMA11 vacuoles were treated. All experiments were repeated three times and data were expressed as standard error of the mean (FIG. 7). As a result, it was confirmed that the phagocytosis activity increased according to the treatment concentration of HBP vacuoles.
이상에서는 본 발명을 설명하였으나, 본 발명은 개시된 실시예 및 첨부된 도면에 의하여 한정되지 않으며 본 발명의 기술적 사상을 벗어나지 않는 범위 이내에서 통상의 기술자에 의하여 다양하게 변형될 수 있다. 또한 본 발명의 실시예에서 설명한 기술적 사상은, 각각 독립적으로 실시될 수도 있고, 둘 이상이 서로 조합되어 실시될 수도 있다.Although the present invention has been described above, the present invention is not limited by the disclosed embodiments and the accompanying drawings, and may be variously modified by a person skilled in the art without departing from the technical spirit of the present invention. In addition, the technical ideas described in the embodiments of the present invention may be implemented independently, or two or more may be combined with each other.
<110> INDUSTRIAL COOPERATION FOUNDATION JEONBUK NATIONAL UNIVERSITY <120> Vacuoles expressing histamine binding protein and composition for inhibiting histamine comprising thereof <130> JB21033 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 513 <212> DNA <213> Artificial Sequence <220> <223> Histamine binding protein 2 <400> 1 aatcagccag attgggccga tgaagcggca aatggtgcac accaagacgc ctggaagagt 60 ctgaaagcgg acgttgaaaa cgtttactac atggtgaagg ccacctataa gaatgaccca 120 gtgtggggca atgacttcac ttgcgtgggt gttatggcaa atgatgtcaa cgaggatgag 180 aagagcattc aagcagagtt tttgtttatg aataatgctg acacaaacat gcaattcgcc 240 actgaaaagg tgactgctgt taaaatgtat ggttacaata gggaaaacgc cttcagatac 300 gagacggagg atggccaagt tttcacagac gtcattgcat actctgatga caactgcgat 360 gtcatctacg ttcctggcac agacggaaat gaggaaggtt acgaactatg gactacggat 420 tacgacaaca ttccagccaa ttgtttaaat aagtttaatg agtacgctgt aggtagggag 480 acaagggatg tattcacaag tgcttgccta gag 513 <210> 2 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> signal sequence of VMA11 <400> 2 atgtcaacgc aactcgcaag taacatatat gctccattgt acgctccctt tttcgggttc 60 gcaggttgtg cagctgccat ggtgctttcc tgtttgggag ctgccattgg tacagct 117 <210> 3 <211> 397 <212> DNA <213> Artificial Sequence <220> <223> modified VMA11 <400> 3 gtcggtgacg ttggtgttag aaagtatatg caccaaccaa ggctttttgt cggtatcgtt 60 ttgattctaa ttttctctga agttttaggg ttatatggta tgattgtagc tttgattttg 120 aacactagag gctctgaatg aagcatgccc accaagacca aagtcaggta ttggtatcgc 180 cggtataggt actttcaagc cggaattgat catgaagtct ttgattcctg tggttatgag 240 tggtatctta gccatttatg ggcttgttgt ggccgtttta attgcaggta atttatctcc 300 taccgaagac tatactctct tcaatgggtt catgcacttg agttgtgggc tctgtgtggg 360 atttgcctgt ttgagtagtg gctacgccat tggtatg 397 <210> 4 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> Green fluorescence protein <400> 4 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caag 714 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> His tag <400> 5 caccaccacc accaccactg a 21 <110> INDUSTRIAL COOPERATION FOUNDATION JEONBUK NATIONAL UNIVERSITY <120> Vacuoles expressing histamine binding protein and composition for inhibiting histamine including <130> JB21033 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 513 <212> DNA <213> artificial sequence <220> <223> Histamine binding protein 2 <400> 1 aatcagccag attgggccga tgaagcggca aatggtgcac accaagacgc ctggaagagt 60 ctgaaagcgg acgttgaaaa cgtttactac atggtgaagg ccacctataa gaatgaccca 120 gtgtggggca atgacttcac ttgcgtgggt gttatggcaa atgatgtcaa cgaggatgag 180 aagagcattc aagcagagtt tttgtttatg aataatgctg acacaaacat gcaattcgcc 240 actgaaaagg tgactgctgt taaaatgtat ggttacaata gggaaaacgc cttcagatac 300 gagacggagg atggccaagt tttcacagac gtcattgcat actctgatga caactgcgat 360 gtcatctacg ttcctggcac agacggaaat gaggaaggtt acgaactatg gactacggat 420 tacgacaaca ttccagccaa ttgtttaaat aagtttaatg agtacgctgt aggtagggag 480 acaagggatg tattcacaag tgcttgccta gag 513 <210> 2 <211> 117 <212> DNA <213> artificial sequence <220> <223> signal sequence of VMA11 <400> 2 atgtcaacgc aactcgcaag taacatatat gctccattgt acgctccctt tttcgggttc 60 gcaggttgtg cagctgccat ggtgctttcc tgtttgggag ctgccattgg tacagct 117 <210> 3 <211> 397 <212> DNA <213> artificial sequence <220> <223> modified VMA11 <400> 3 gtcggtgacg ttggtgttag aaagtatatg caccaaccaa ggctttttgt cggtatcgtt 60 ttgattctaa ttttctctga agttttaggg ttatatggta tgattgtagc tttgattttg 120 aacactagag gctctgaatg aagcatgccc accaagacca aagtcaggta ttggtatcgc 180 cggtataggt actttcaagc cggaattgat catgaagtct ttgattcctg tggttatgag 240 tggtatctta gccatttatg ggcttgttgt ggccgtttta attgcaggta atttatctcc 300 taccgaagac tatactctct tcaatgggtt catgcacttg agttgtgggc tctgtggggg 360 atttgcctgt ttgagtagtg gctacgccat tggtatg 397 <210> 4 <211> 714 <212> DNA <213> artificial sequence <220> <223> Green fluorescence protein <400> 4 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caag 714 <210> 5 <211> 21 <212> DNA <213> artificial sequence <220> <223> His tag <400> 5 caccaccacc accaccactg a 21
Claims (11)
(b) 단계 (a)에서 제조된 벡터에 히스타민 결합 단백질의 유전자를 삽입하고 증폭시키는 단계를 포함하는 히스타민 결합 단백질을 발현시키기 위한 벡터를 제조하는 방법.
(a) preparing and amplifying a vector containing the yeast vacuole surface expression signal sequence gene and the modified yeast vacuole surface expression protein pVMA11 gene; and
(b) a method for preparing a vector for expressing a histamine-binding protein comprising the step of inserting and amplifying a histamine-binding protein gene into the vector prepared in step (a).
The method of claim 1, wherein the step of amplifying the vector comprises transforming and culturing E. coli with the prepared vector.
A recombinant vector prepared by the method of claim 1 or 2.
Yeast transformed with the recombinant vector of claim 3.
(b) 단계 (a)에서 제조된 벡터에 히스타민 결합 단백질의 유전자를 삽입하고 증폭시키는 단계;
(c) 단계 (b)에서 제조된 벡터로 효모를 형질전환 시키는 단계; 및
(d) 단계 (c)에서 제조된 형질전환 효모로부터 액포를 분리하는 단계를 포함하는 히스타민 결합 단백질이 발현된 액포를 제조하는 방법.
(a) preparing and amplifying a vector containing the yeast vacuole surface expression signal sequence gene and the modified yeast vacuole surface expression protein pVMA11 gene;
(b) inserting and amplifying the histamine binding protein gene into the vector prepared in step (a);
(c) transforming yeast with the vector prepared in step (b); and
(d) a method for producing a histamine-binding protein-expressed vacuole comprising the step of isolating vacuoles from the transformed yeast prepared in step (c).
A vacuole expressing the histamine-binding protein prepared by the method of claim 5.
A composition for inhibiting histamine comprising, as an active ingredient, a vacuole in which a histamine binding protein is expressed.
According to claim 7, excessive inflammatory reaction, allergy, atopy, a pharmaceutical composition for the prevention or treatment of asthma.
The pharmaceutical composition according to claim 8, which further exhibits an immune enhancing effect.
8. The composition of claim 7 for inhibiting histamine in vitro.
The cosmetic composition according to claim 7, which inhibits histamine.
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