KR102113588B1 - Mass Production System of Oligopeptide with His-Pro Repeats - Google Patents

Mass Production System of Oligopeptide with His-Pro Repeats Download PDF

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KR102113588B1
KR102113588B1 KR1020180034502A KR20180034502A KR102113588B1 KR 102113588 B1 KR102113588 B1 KR 102113588B1 KR 1020180034502 A KR1020180034502 A KR 1020180034502A KR 20180034502 A KR20180034502 A KR 20180034502A KR 102113588 B1 KR102113588 B1 KR 102113588B1
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histidine
mhp
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peptide
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최장원
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(주)터틀바이오
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]

Abstract

본 발명은 히스티딘-프롤린 반복서열을 갖는 펩타이드의 대량생산 시스템에 관한 것으로서, 보다 상세하게는 히스티딘-프롤린 반복서열을 갖는 펩타이드가 도입된 재조합 벡터로 형질전환된 고초균에서 히스티딘-프롤린 펩타이드의 발현 및 분비를 최적화하기 위한 방법을 포함하는 히스티딘-프롤린 반복서열을 갖는 펩타이드의 대량생산 시스템에 관한 것이다. The present invention relates to a system for mass production of a peptide having a histidine-proline repeat sequence, and more specifically, expression and secretion of a histidine-proline peptide in archaea transformed with a recombinant vector into which a peptide having a histidine-proline repeat sequence is introduced. It relates to a mass production system of a peptide having a histidine-proline repeat sequence comprising a method for optimizing.

Description

히스티딘-프롤린 반복서열을 갖는 올리고펩타이드의 대량생산 시스템{Mass Production System of Oligopeptide with His-Pro Repeats}Mass Production System of Oligopeptide with His-Pro Repeats

본 발명은 히스티딘-프롤린 반복서열을 갖는 펩타이드의 대량생산 시스템에 관한 것으로서, 보다 상세하게는 히스티딘-프롤린 반복서열을 갖는 펩타이드가 도입된 재조합 벡터로 형질전환된 고초균에서 히스티딘-프롤린 펩타이드의 발현 및 분비를 최적화하기 위한 방법을 포함하는 히스티딘-프롤린 반복서열을 갖는 펩타이드의 대량생산 시스템에 관한 것이다. The present invention relates to a system for mass production of a peptide having a histidine-proline repeat sequence, and more specifically, expression and secretion of a histidine-proline peptide in archaea transformed with a recombinant vector into which a peptide having a histidine-proline repeat sequence is introduced. It relates to a mass production system of a peptide having a histidine-proline repeat sequence comprising a method for optimizing.

Cyclo(His-Pro)(CHP)는 갑상선 자극호르몬 방출호르몬 (Thyrotropin-Releasing Hormone, TRH)의 대사 산물인 Histidine-Proline으로 구성된 자연적으로 발생하는 원형 dipeptide 또는 TRH 대사과정과 de novo로 체내에서 합성되기도 하는 bioactive dipeptide 로써 뇌 전반과 척수 및 위장관 등에 널리 분포하며, 간과 부신에 CHP receptor가 존재함이 보고되고 있다. Cyclo (His-Pro) (CHP) is a naturally occurring circular dipeptide or TRH metabolism composed of Histidine-Proline, a metabolite of Thyrotropin-Releasing Hormone (TRH), and is synthesized in the body through de novo As a bioactive dipeptide, it is widely distributed in the entire brain, spinal cord, and gastrointestinal tract, and it has been reported that CHP receptors exist in the liver and adrenal glands.

또한 CHP는 사람의 소화기관, 혈액, 뇌척수액, 척수, 정액 등에서 발견되며 뇌하수체로부터 prolactin의 분비를 저해하며, 알콜에 의한 부작용을 완화하고, 또한 식사욕구를 완화하여 비만을 예방할 수 있는 효과를 지니고 있다.In addition, CHP is found in the human digestive system, blood, cerebrospinal fluid, spinal cord, semen, etc., and inhibits the secretion of prolactin from the pituitary gland, relieves side effects caused by alcohol, and also has the effect of preventing obesity by alleviating the desire to eat. .

동물과 사람의 당뇨 유발 시 혈청 내의 TRH 수준이 감소되어지는 것을 확인하였으며, TRH는 인슐린 분비를 촉진하며, 조직 내에서는 뇌와 전립선에서 가장 높은 함량을 보이고 있다. When inducing diabetes in animals and humans, it was confirmed that the level of TRH in the serum decreased, and TRH promotes insulin secretion, and the highest content in the brain and prostate in tissues.

당뇨 환자의 혈당을 조절할 수 있는 인슐린 기능 개선에 대한 후보물질로 포도당 주입 후 혈중 CHP가 증가하는 것으로 보아 CHP가 혈당과 관련된 작용이 있다는 것이 제안되었고, CHP 수치가 당뇨병에 걸리지 않은 대조군 그룹에 비하여 상당히 낮은 것으로 보아 CHP가 당뇨병과 관련이 있다는 가능성도 대두되었다. As a candidate for improving insulin function that can control blood sugar in diabetic patients, it has been suggested that CHP increases blood glucose-related action after glucose infusion, and it has been suggested that CHP has a blood sugar-related action, and the CHP level is significantly higher than that of the control group without diabetes. Considering that it is low, the possibility that CHP is related to diabetes has also emerged.

그리고 아연과 함께 투여 시 당뇨 모델 쥐에서 인슐린 민감성 (insulin sensitivity) 및 포도당 소비율 (glucose clearance)을 향상시키며, 또한 CHP는 중추신경에서 도파민 기전과 관련되어 있고 렙틴과 유사한 작용을 한다고 보고되었다. In addition, when administered with zinc, it improves insulin sensitivity and glucose clearance in diabetic model rats, and CHP is also associated with dopamine mechanism in the central nervous system and has been reported to act similar to leptin.

이러한 보고들로부터 CHP는 당뇨병과 관련하여 인슐린과 렙틴의 작용에 있어 중요한 역할을 하는 것으로 생각되고 있다.From these reports, CHP is thought to play an important role in the action of insulin and leptin in relation to diabetes.

현재 CHP가 함유된 소재로는 소의 전립선 분말 (약 1% 함유)이 있으며 그 외에 새우, 젓갈, 참치, 흰 빵, 국수 등에서 CHP 함량이 보고되고 있으나 그 양이 매우 적어 ppm 수준으로 보고되고 있다. 식품에 존재하는 CHP를 활용할 경우 인체에 부작용을 야기하지 않을 것으로 생각되고 있으며, 실제로 현재 미국에서는 CHP가 함유된 카제인 또는 대두를 활용한 제품들이 개발되어 판매되고 있다. Currently, the material containing CHP is cow's prostate powder (containing about 1%). In addition, the CHP content is reported in shrimp, salted fish, tuna, white bread, noodles, etc., but it is reported in ppm level due to its very small amount. When using CHP present in food, it is thought that it will not cause side effects to the human body. In fact, products using casein or soybean containing CHP are developed and sold in the United States.

이와 관련하여, 한국등록특허 제10-1418968호에서는 대두에 효소처리를 하여 CHP 를 높은 함량으로 얻기 위한 제조방법에 대하여 개시하고 있다.In this regard, Korean Patent Registration No. 10-1418968 discloses a manufacturing method for obtaining CHP at a high content by enzymatic treatment on soybeans.

고초균 (Bacillus subtilis)은 대표적인 그람 양성균으로 성장이 빠르고 유전적 체계가 잘 확립되어 있어 유전자 조작이 용이하고, 넓은 범위의 codon 사용빈도를 가지며 또한 안전성이 입증된 GRAS (generally regarded as safe) 균주로 알려져 있다. Bacillus subtilis ( Bacillus subtilis ) is a representative Gram-positive bacterium. It is known as a GRAS (generally regarded as safe) strain that is easy to genetically manipulate, has a wide range of codon usage, and has been proven to be safe because of its rapid growth and well-established genetic system. have.

전통적인 발효과정에서 보듯이 고초균은 자체적으로 효율적인 단백질 분비시스템을 가지고 있어 많은 양의 활성적인 유용 단백질을 발효액으로 분비하는 이점이 있으며, 또한 단백질 분리 및 회수가 용이하므로 이종 단백질 생산의 주요 숙주세포로 많이 사용되고 있다.As seen in the traditional fermentation process, archaea bacteria have an efficient protein secretion system, which has the advantage of secreting a large amount of active useful protein into the fermentation broth, and also facilitates protein separation and recovery, making it a major host cell for producing heterologous proteins. Is being used.

본 발명자는 과거 고비용으로 ppm 수준의 소량 CHP 를 추출하던 문제점을 개선하기 위한 연구 개발의 일환으로 고초균에서 분비 시스템을 통하여 히스티딘-프롤린 펩타이드를 대량생산 가능함을 확인하였으며, 생산된 히스티딘-프롤린 펩타이드로부터 당뇨병 예방 및 치료 효과를 확인하여 본 발명에 이르게 되었다.The present inventors confirmed that it is possible to mass-produce histidine-proline peptides through the secretory system in archaea as part of research and development to improve the problem of extracting a small amount of CHP at a high cost in the past, and diabetes from the produced histidine-proline peptide It has led to the present invention by confirming the prophylactic and therapeutic effects.

한국등록특허 제10-1418968호(ＣＨＰ(ｃｙｃｌｏ(Ｈｉｓ-Ｐｒｏ))를 고농도로 함유한 대두 가수분해물을 포함하는 혈당 조절용 조성물)Korean Registered Patent No. 10-1418968 (Composition for regulating blood sugar containing soybean hydrolyzate containing high concentration of CYP

Prasad, C., C. W. Hilton, F. Svec, E. S. Onaivi, and P. Vo (1991) Could dietary proteins serve as cyclo(His-Pro) precursors? Neuropeptides 19: 17-21.Prasad, C., C. W. Hilton, F. Svec, E. S. Onaivi, and P. Vo (1991) Could dietary proteins serve as cyclo (His-Pro) precursors? Neuropeptides 19: 17-21.

상기와 같은 문제점을 해결하기 위한 본 발명의 목적은 히스티딘-프롤린 반복서열을 갖는 펩타이드를 대량생산 하기 위한 재조합 벡터, 상기 재조합 벡터로 형질전환된 고초균, 상기 고초균에서 히스티딘-프롤린 펩타이드의 발현 및 분비를 최적화하기 위한 방법을 제공하는 것이다.An object of the present invention for solving the above problems is to express and secrete a histidine-proline peptide from the recombinant vector, a recombinant bacterium transformed with the recombinant vector, and the histidine-proline peptide in a mass production of a peptide having a histidine-proline repeat sequence. It provides a way to optimize.

상기 과제를 해결하기 위한 히스티딘-프롤린 반복서열을 갖는 펩타이드의 대량생산 시스템은 His-Pro 반복서열로 구성된 36개 아미노산과 6개의 his tag를 포함하는 서열번호 1의 유전자가 도입된 재조합 벡터 pRBAS-mHP 를 제공한다.In order to solve the above problem, a mass production system of a peptide having a histidine-proline repeat sequence is a recombinant vector pRBAS-mHP in which the gene of SEQ ID NO: 1 containing 36 amino acids and six his tags composed of His-Pro repeat sequence is introduced. Provides

상기 재조합 벡터 pRBAS-mHP 는 하기와 같이 제조된다.The recombinant vector pRBAS-mHP is prepared as follows.

제한효소 EcoRI이 포함된 서열번호 4의 염기서열을 갖는 forward 프라이머(mHP primer F1)와 His tag, termination codon 및 Hind III가 포함된 서열번호 5의 염기서열을 갖는 reverse 프라이머(mHP primer R1) 를 이용하여 PCR를 수행하여 mHP peptide 유전자를 증폭하여 PCR 산물을 수득하고, 상기 PCR 산물을 클로닝 벡터 pGEM-T-Easy 에 클로닝하고 대장균에 도입하여 형질전환체를 선별하고, 상기 형질전환체로부터 확인된 유전자를 pT-mHP 로 명명하였다. The forward primer (mHP primer F1) having the nucleotide sequence of SEQ ID NO: 4 containing the restriction enzyme EcoRI and the reverse primer (mHP primer R1) having the nucleotide sequence of SEQ ID NO: 5 including His tag, termination codon and Hind III are used. PCR is performed to amplify the mHP peptide gene to obtain a PCR product, the PCR product is cloned into the cloning vector pGEM-T-Easy and introduced into E. coli to select transformants, and genes identified from the transformants Was named pT-mHP.

이후, 상기 pT-mHP 벡터를 주형으로 하여, 상기 서열번호 4와 서열번호 5의 프라이머 세트를 이용하여 PCR 증폭을 하고, 제한효소 EcoRI, Hind III로 자르고, 동일한 제한효소로 자른 pRBAS 벡터에 ligation 하여 최종적으로 pRBAS-mHP 벡터를 제조하였다(서열번호 2, 서열번호 3).Then, using the pT-mHP vector as a template, PCR amplification was performed using the primer sets of SEQ ID NO: 4 and SEQ ID NO: 5, cut with restriction enzymes EcoRI, Hind III, and ligated to the pRBAS vector cut with the same restriction enzyme. Finally, a pRBAS-mHP vector was prepared (SEQ ID NO: 2, SEQ ID NO: 3).

도 2는 서열번호 2의 염기서열과 이에 대응하는 서열번호 3의 아미노산 서열을 보여준다.Figure 2 shows the nucleotide sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 3 corresponding thereto.

또한, 본 발명은 서열번호 1의 유전자가 도입된 재조합 벡터 pRBAS-mHP 로 형질전환된 고초균을 제공한다. In addition, the present invention provides an archaebacteria transformed with the recombinant vector pRBAS-mHP in which the gene of SEQ ID NO: 1 is introduced.

또한, 본 발명은 서열번호 1의 유전자가 도입된 재조합 벡터로 형질전환된 고초균으로부터 수득된 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드를 포함하는 항당뇨 조성물을 제공한다. In addition, the present invention provides an anti-diabetic composition comprising an oligopeptide having a histidine-proline repeat sequence obtained from archaea transformed with a recombinant vector into which the gene of SEQ ID NO: 1 is introduced.

이때, 상기 항당뇨 조성물은 손상된 췌장 β-세포주 활성의 복구, nitric oxide (NO) 생성의 감소, DNA fragmentation 의 완화, CaM kinase II에 포함된 인슐린 분비량 증가 및 이들의 조합 중 어느 하나의 기전을 통하여 당뇨 및 당뇨 합병증 치료 및 예방효과를 갖는 것을 특징으로 한다. At this time, the anti-diabetic composition through the repair of any one of the mechanisms of restoration of damaged pancreatic β-cell line activity, reduction of nitric oxide (NO) production, alleviation of DNA fragmentation, increased insulin secretion contained in CaM kinase II, and combinations thereof It has characteristics of treating and preventing diabetes and diabetes complications.

또한, 서열번호 1의 유전자가 도입된 재조합 벡터로 형질전환된 고초균으로부터 수득된 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드는 당뇨 및 당뇨 합병증 치료 및 예방을 위한 조성물에만 한정하여 사용하는 것이 아닌, 손상된 췌장 β-세포주 활성의 복구, nitric oxide (NO) 생성의 감소, DNA fragmentation 의 완화, CaM kinase II에 포함된 인슐린 분비량 증가 및 이들의 조합 중 어느 하나의 기전을 통해 치료 및 예방 가능한 질병에 적용될 수 있다. In addition, the oligopeptide having a histidine-proline repeat sequence obtained from an archaebacteria transformed with a recombinant vector into which the gene of SEQ ID NO: 1 is introduced is not limited to a composition for the treatment and prevention of diabetes and diabetes complications, and is damaged pancreas. Restoration of β-cell line activity, reduction of nitric oxide (NO) production, alleviation of DNA fragmentation, increase of insulin secretion contained in CaM kinase II, and combinations thereof can be applied to treatable and preventable diseases .

본 발명의 항당뇨 조성물은 약학 조성물 또는 식품 조성물일 수 있으나, 구체적인 조성물의 형태가 이에 제한되는 것은 아니며, 항당뇨 효과를 획득하기 위해 적용될 수 있는 어떠한 섭취 형태의 항당뇨 조성물로 제조될 수 있다.The anti-diabetic composition of the present invention may be a pharmaceutical composition or a food composition, but the form of a specific composition is not limited thereto, and may be made of an anti-diabetic composition of any intake form that can be applied to obtain an anti-diabetic effect.

상기와 같은 약학 조성물은 서열번호 1의 유전자가 도입된 재조합 벡터로 형질전환된 고초균으로부터 수득된 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드를 약제학적으로 유효한 양으로 포함하는 것으로, 택일적으로 이들의 약제학적으로 허용가능한 염을 포함할 수 있으며, 이와 같은 약학 조성물을 환자에게 투여함으로써 당뇨병의 억제, 예방 및 치료 효과를 획득할 수 있다.The pharmaceutical composition as described above is to include an oligopeptide having a histidine-proline repeat sequence obtained from archaea transformed with a recombinant vector into which the gene of SEQ ID NO: 1 is introduced, in a pharmaceutically effective amount, or alternatively, these pharmaceutical compositions It may contain a scientifically acceptable salt, it is possible to obtain an inhibitory, preventive and therapeutic effect of diabetes by administering such a pharmaceutical composition to a patient.

나아가, 본 발명의 상기 조성물이 약학 조성물인 경우 상기 약학 조성물은 사용 목적에 맞게 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁제, 에멀젼, 시럽, 에어로졸 등의 경구 제형, 멸균 주사 용액의 형태 등 다양한 형태로 제형화하여 사용할 수 있으며, 경구 투여하거나 정맥 내, 복강 내, 피하, 직장, 국소 투여 등을 포함한 다양한 경로를 통해 투여될 수 있다.Furthermore, when the composition of the present invention is a pharmaceutical composition, the pharmaceutical composition is an oral formulation such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, sterile injection solution according to a conventional method according to the purpose of use. It can be formulated and used in various forms such as the form of orally, or it can be administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal and topical administration.

이러한 약학 조성물에 포함될 수 있는 적합한 담체, 부형제 및 희석제의 예로는 락토오스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 비정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 들 수 있다.Examples of suitable carriers, excipients and diluents that may be included in such pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

또한, 상기 당뇨병 예방 또는 치료용 약학 조성물은 충전제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.In addition, the pharmaceutical composition for preventing or treating diabetes may further include fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, and the like.

경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 당뇨병 예방 또는 치료용 약학 조성물에 적어도 하나 이상의 부형제, 예를 들면 전분, 탄산칼슘, 수크로스, 락토오스, 젤라틴 등을 섞어 제형화 할 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크와 같은 윤활제들도 사용될 수 있다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the pharmaceutical composition for preventing or treating diabetes, for example starch, calcium carbonate, sucrose , Can be formulated by mixing lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

경구용 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 액체 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, may be included.

비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성 용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름과 같은 주사 가능한 에스테르 등이 사용될 수 있다. 주사제의 기제로는 용해제, 등장화제, 현탁화제, 유화제, 안정화제 및 방부제와 같은 종래의 첨가제를 포함할 수 있다.Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents, suspensions may be used, such as propylene glycol, polyethylene glycol, injectable esters such as vegetable oils such as olive oil. Injectable bases may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

또한, 본 발명은 서열번호 1의 유전자가 도입된 재조합 벡터로 형질전환된 고초균을 이용하여 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드를 대량생산하기 위한 방법을 제공한다.In addition, the present invention provides a method for mass-producing an oligopeptide having a histidine-proline repeat sequence using archaea transformed with a recombinant vector into which the gene of SEQ ID NO: 1 is introduced.

상기 대량생산 방법은 히스티딘-프롤린 반복서열로 구성된 펩타이드의 PCR 산물을 클로닝 벡터에 도입한 후, 상기 PCR 산물이 도입된 클로닝 벡터를 대장균에 삽입 및 배양시켜 히스티딘-프롤린 유전자를 수득하는 단계(S100)와; 상기 히스티딘-프롤린 유전자를 대장균-고초균 셔틀 벡터에 도입하는 단계(S200);와 히스티딘-프롤린 유전자가 도입된 벡터를 고초균에 도입하여 형질전환된 고초균을 선별하고, 선별된 고초균을 배양하는 단계(S300)를 포함한다.In the mass production method, a PCR product of a peptide composed of a histidine-proline repeat sequence is introduced into a cloning vector, and then the cloning vector into which the PCR product is introduced is inserted into and cultured in E. coli to obtain a histidine-proline gene (S100). Wow; Introducing the histidine-proline gene into the E. coli-E. Coli shuttle vector (S200); and introducing the vector into which the histidine-proline gene is introduced into the E. coli bacteria to select the transformed E. coli and culturing the selected E. coli (S300) ).

이때, 상기 PCR 산물은 서열번호 4의 염기서열을 갖는 forward 프라이머와 서열번호 5의 염기서열을 갖는 reverse 프라이머 세트를 이용하여 수득되는 것을 특징으로 한다.In this case, the PCR product is characterized in that it is obtained by using a forward primer set having a nucleotide sequence of SEQ ID NO: 4 and a reverse primer set having a nucleotide sequence of SEQ ID NO: 5.

상기 고초균 배양단계(S300)는 배지 100 부피부에 대하여 선별된 형질전환 고초균을 0.5 내지 3 중량부 첨가하여 30 내지 37℃에서 12 내지 72시간 배양하는 것을 특징으로 한다.In the culture step of S. aureus (S300), 0.5 to 3 parts by weight of the transformed S. aureus selected for 100 parts by volume of the medium is added and cultured at 30 to 37 ° C for 12 to 72 hours.

상기 고초균 배양단계(S300)는 배지 100 부피부에 대하여, 탄소원 1 내지 5 중량부 및 질소원 1 내지 5 중량부를 더 첨가하는 것을 특징으로 한다. In the culture step of S. aureus (S300), 1 to 5 parts by weight of a carbon source and 1 to 5 parts by weight of a nitrogen source are further added to 100 parts by volume of the medium.

또한, 상기 탄소원은 barley 와 edible glycerol을 0.5 내지 1 : 1의 중량비로 혼합한 혼합 탄소원인 것을 특징으로 한다. In addition, the carbon source is characterized by being a mixed carbon source in which barley and edible glycerol are mixed in a weight ratio of 0.5 to 1: 1.

상술한 바와 같이, 본 발명에 따른 히스티딘-프롤린 반복서열을 갖는 펩타이드의 대량생산 시스템에 의하면, 과거 고비용으로 미량 수득되어 온 히스티딘-프롤린을 고효율적으로 대량생산할 수 있는 효과가 있다.As described above, according to the mass production system of a peptide having a histidine-proline repeat sequence according to the present invention, there is an effect capable of mass-producing histidine-proline, which has been obtained in trace amounts at a high cost in the past, with high efficiency.

본 발명에 따른 히스티딘-프롤린 반복서열을 갖는 펩타이드의 대량생산 시스템에 의해 생산된 히스티딘-프롤린은 당뇨 및 당뇨 합병증 치료 및 예방효과가 있다.The histidine-proline produced by the mass production system of a peptide having a histidine-proline repeat sequence according to the present invention has a treatment and prevention effect for diabetes and diabetes complications.

도 1은 본 발명에 따른 pRBAS-mHP 벡터를 제조하는 방법을 보여주는 개념도 및 개열지도.
도 2는 본 발명에 따른 pRBAS-mHP 벡터의 DNA sequence.
도 3은 pRBAS-mHP가 도입된 B. subtilis LSK의 배양 온도 및 질소원에 따른 mHP 펩타이드의 분비 효율을 보여주는 실험데이터.
도 4는 pRBAS-mHP가 도입된 B. subtilis LKS에 탄소원(barley, glycerol) 및 농도에 따른 mHP peptide의 분비 효율을 보여주는 실험데이터.
도 5는 pRBAS-mHP가 도입된 B. subtilis LKS 의 배양조건에 따른 mHP peptide의 분비량을 보여주는 실험데이터.
도 6은 STZ 처리된 RINm5F 세포주에서 mHP peptide 첨가에 따른 세포 생존능과 질소 생성을 보여주는 그래프.
도 7은 STZ 처리된 RINm5F 세포주에서 mHP peptide의 회복효과를 보여주는 실험데이터.1 is a conceptual diagram and a cleavage map showing a method of manufacturing a pRBAS-mHP vector according to the present invention.
2 is a DNA sequence of the pRBAS-mHP vector according to the present invention.
3 is an experimental data showing the secretion efficiency of mHP peptide according to the culture temperature and nitrogen source of B. subtilis LSK introduced pRBAS-mHP.
Figure 4 is the experimental data showing the secretion efficiency of mHP peptide according to the carbon source (barley, glycerol) and concentration in B. subtilis LKS introduced pRBAS-mHP.
5 is an experimental data showing the secretion amount of mHP peptide according to the culture conditions of B. subtilis LKS introduced pRBAS-mHP.
6 is a graph showing cell viability and nitrogen production according to the addition of mHP peptide in STZ-treated RINm5F cell line.
7 is experimental data showing the recovery effect of mHP peptide in STZ-treated RINm5F cell line.

본 발명의 구체적 특징 및 이점들은 이하에서 첨부도면을 참조하여 상세히 설명한다. 이에 앞서 본 발명에 관련된 기능 및 그 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 구체적인 설명을 생략하기로 한다.Specific features and advantages of the present invention will be described in detail below with reference to the accompanying drawings. Prior to this, when it is determined that the detailed description of the functions and configurations related to the present invention may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

이하, 본 발명을 바람직한 일 실시예를 참조하여 다음에서 구체적으로 상세하게 설명한다. 단, 다음의 실시예는 본 발명을 구체적으로 예시하기 위한 것이며, 이것만으로 한정하는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to one preferred embodiment. However, the following examples are intended to specifically illustrate the present invention, but are not limited thereto.

1. 재료와 방법1. Materials and Methods

1.1. 재료 및 시약1.1. Materials and reagents

Cyclo(His-Pro)는 Sigma (Darmstadt, Germany)사 및 Bachem(Bubendorf, Switzerland)사에서 구입하였으며, MTT (3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide)는 Generay(Shanghai, China)사, Griess reagent는 Sigma (Darmstadt, Germany)사에서 구입하였다. Cyclo (His-Pro) was purchased from Sigma (Darmstadt, Germany) and Bachem (Bubendorf, Switzerland), and MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) Generay (Shanghai, China) and Griess reagent were purchased from Sigma (Darmstadt, Germany).

또한, 제한효소는 KOSCHEM(Sungnam, Korea)사 및 NEB (Beverly, MA, USA)사로부터, T4 DNA ligase와 Taq polymerase는 TAKARA (Otsu, Shiga, Japan)사로부터, 올리고머는 Genoteck (Daegeon, Korea)사로부터 합성하여 사용하였다. In addition, restriction enzymes are from KOSCHEM (Sungnam, Korea) and NEB (Beverly, MA, USA), T4 DNA ligase and Taq polymerase are from TAKARA (Otsu, Shiga, Japan), and oligomers are Genoteck (Daegeon, Korea). It was synthesized from the company and used.

Immobilon-P PVDF membrane은 Millipore (Bedford, MA, USA)사, QIAEX agarose gel extraction kit는 Qiagen (Hilden, Germany)사, ECL western blotting system은 Amersham Pharmacia Biotech (Piscataway, NJ, USA)사로부터 구입하여 사용하였다. Immobilon-P PVDF membrane is purchased from Millipore (Bedford, MA, USA), QIAEX agarose gel extraction kit is from Qiagen (Hilden, Germany), ECL western blotting system is purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA) Did.

1차 및 2차 항체는 Santa Cruz Biotechnology (Santa Cruz, CA USA)사 및 Genscript (New Jersey, USA)사로부터, 나머지 시약들은 분석용 수준급을 사용하였다.The primary and secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA USA) and Genscript (New Jersey, USA), and the rest of the reagents were used for analysis.

1.2. Bacterial strains, 배지 및 1.2. Bacterial strains, media and plasmidplasmid

E. coli XL1-Blue MRF (F‘, proAB, lacIqZ△M15, thi, recA, gyrA, relA, supE, Tn10) (Stratagene, La Jolla, CA)는 형질전환 및 플라스미드 증폭을 위한 기본 숙주로 사용하였고, pGEM-T Easy vector (Promega, Madison, USA)는 PCR 산물을 클로닝 벡터로, Bacillus subtilis LKS (nprR2, nprE18, aprA3, amyE)균주 및 Bacillus subtilis 168 (wild type, GRAS and genome sequenced strain)은 외래단백질 발현 및 분비를 위한 숙주로 사용하였다. E. coli XL1-Blue MRF (F ', proAB, lacIqZΔM15, thi, recA, gyrA, relA, supE, Tn10) (Stratagene, La Jolla, CA) was used as the primary host for transformation and plasmid amplification. , pGEM-T Easy vector (Promega, Madison, USA) is a PCR product as a cloning vector, Bacillus subtilis LKS (nprR2, nprE18, aprA3, amyE) strains and Bacillus subtilis 168 (wild type, GRAS and genome sequenced strain) are foreign It was used as a host for protein expression and secretion.

유전자원으로는 pGEM-T Easy 벡터에 도입된 oligopeptide with His-Pro repeats (mHP peptide) 유전자를 사용하였고, 유전자의 발현 및 분비를 위하여 Bacillus 유래의 알칼리성 단백질분해효소 유전자의 프로모터와 신호서열을 함유하는 pRBAS벡터를 사용하여, mHP peptide 유전자가 도입된 재조합 pRBAS-mHP 벡터를 본 연구에서 구성하였다. As a gene source, the oligopeptide with His-Pro repeats (mHP peptide) gene introduced into the pGEM-T Easy vector was used, and contains the promoter and signal sequence of the alkaline protease gene derived from Bacillus for expression and secretion of the gene. Using the pRBAS vector, a recombinant pRBAS-mHP vector into which the mHP peptide gene was introduced was constructed in this study.

플라스미드가 도입된 대장균은 37℃에서 항생제 ampicillin (50 μg/mL)이 첨가된 Luria-Bertani (LB) 배지에서 키웠고, 재조합벡터가 도입된 Bacillus 균은 30℃ 또는 37℃에서 항생제 kanamycin (50 μg/mL)이 첨가된 LB, PY, MYP 배지에서 배양하였다. E. coli introduced with plasmid was grown in Luria-Bertani (LB) medium with antibiotic ampicillin (50 μg / mL) added at 37 ° C. Bacillus bacteria introduced with recombinant vector were antibiotic kanamycin (50 μg / 50 ° C) at 30 ° C or 37 ° C. mL) was added and cultured in LB, PY, MYP medium.

항당뇨 효과를 측정하기 위하여 insulinoma 세포인 RINm5F 세포를 사용하여 분석하였다.In order to measure the anti-diabetic effect, insulinoma cells were analyzed using RINm5F cells.

1.3. 1.3. PCR을PCR 이용한  Used mHPmHP peptide 유전자 증폭 및  peptide gene amplification and 클로닝Cloning

PCR (polymerase chain reaction)을 위하여 His-Pro의 반복서열로 구성된 mHP peptide 유전자에 대한 primer set를 제작하였으며, 해당하는 각 primer의 염기서열은 하기와 같다. For PCR (polymerase chain reaction), a primer set for mHP peptide gene composed of His-Pro repeat sequence was prepared, and the base sequence of each primer is as follows.

5' GGGGAATTCCATCCGCATCCT 3' (서열번호 4)5 'GGGGAATTCCATCCGCATCCT 3' (SEQ ID NO: 4)

5' GGGAAGCTTTTAATGGTGATGGTGATGGTGAGGATGAGGATG 3'(서열번호 5)5 'GGGAAGCTTTTAATGGTGATGGTGATGGTGAGGATGAGGATG 3' (SEQ ID NO: 5)

제한효소 EcoRI이 포함된 mHP primer F1 (서열번호 4)과 항원 항체반응을 통하여 발현된 mHP peptide를 정량하기 위하여 His tag, termination codon, 그리고 Hind III가 포함된 mHP primer R1 (서열번호 5)을 primer set로 Taq DNA 중합효소를 이용하여 PCR을 수행하였다. In order to quantify mHP primer F1 (SEQ ID NO: 4) containing the restriction enzyme EcoRI and mHP peptide expressed through an antigen-antibody reaction, primers were used with mHP primer R1 (SEQ ID NO: 5) containing His tag, termination codon, and Hind III. PCR was performed using Taq DNA polymerase as a set.

PCR 산물 (약 126 bp)은 1.5% agarose gel 전기영동을 통하여 확인하였고 pGEM-T Easy 벡터에 클로닝하여 E. coli XL1-Blue MRF에 도입하여 Sambrook 등의 방법에 따라 올바른 형질전환체를 선별하였다. The PCR product (about 126 bp) was confirmed through 1.5% agarose gel electrophoresis, cloned into a pGEM-T Easy vector, and introduced into E. coli XL1-Blue MRF to select the correct transformant according to Sambrook et al.

형질전환체는 ampicillin (50 μg/mL)이 첨가된 LB 액체배지에 접종하였고, 배양액으로부터 plasmid DNA를 추출하여 클로닝된 부위의 제한효소인 EcoRI과 HindIII로 절단한 다음, mHP 유전자가 도입되었는지를 전기영동에 의해 확인 후, 염기서열을 분석하여 pT-mHP로 명명하였다.The transformant was inoculated into LB liquid medium added with ampicillin (50 μg / mL), extracted with plasmid DNA from the culture medium, digested with the restriction enzymes EcoRI and HindIII at the cloned site, and then the electricity was introduced whether mHP gene was introduced. After confirming by Youngdong, the base sequence was analyzed and named as pT-mHP.

1.4. 1.4. mHPmHP peptide 유전자 포함 분비벡터 구축 및 형질전환 Construction and transformation of secretion vector containing peptide gene

Peptide mHP의 유전자 (126 bp)가 도입된 pRBAS 분비벡터를 구축하기 위하여, pT-mHP 벡터를 주형으로 하여 mHP primer F1과 R1을 사용하여 PCR증폭을 하여 EcoRI과 HindIII로 잘라 1.5% agarose gel에서 확인하여 elution 하였다. To construct the pRBAS secretion vector into which the peptide mHP gene (126 bp) was introduced, PCR amplification was performed using mHP primers F1 and R1 using the pT-mHP vector as a template, cut with EcoRI and HindIII, and confirmed on a 1.5% agarose gel. Was elution.

같은 제한효소로 자른 pRBAS 벡터와 ligation한 후, E.coli XL-1 Blue MRF로 형질전환하여 Sambrook 등의 방법에 따라 올바른 형질전환체를 선별하였고, 최종적으로 염기서열 분석에 의해 도입된 유전자 및 염기서열 frame을 확인하였다.After ligation with the pRBAS vector cut with the same restriction enzyme, it was transformed with E.coli XL-1 Blue MRF to select the correct transformant according to the method of Sambrook, etc. Finally, genes and bases introduced by sequencing The sequence frame was confirmed.

1.5. 1.5. Bacillus Bacillus subtilissubtilis 형질전환 및 단백질 확인Transformation and protein identification

고초균으로 pRBAS 및 pRBAS-mHP벡터 도입은 SPMM-I(Spizizen's minimal medium)과 SPMM-II를 사용하는 Sadaie and Kada 방법에 의해 행하였고 kanamycin (50 μg/mL)이 포함된 고체 LB 배지에서 형질전환체를 선별하였다. The introduction of pRBAS and pRBAS-mHP vector as an archaebacteria was performed by the Sadaie and Kada method using SPMM-I (Spizizen's minimal medium) and SPMM-II and transformants in solid LB medium containing kanamycin (50 μg / mL). Was selected.

얻어진 형질전환체에 도입된 재조합 벡터를 최종적으로 확인한 후, 온도, 배양시간, 균주, 배지, 등의 조건을 달리하여 발현 및 분비를 시도하였다. After finally confirming the recombinant vector introduced into the obtained transformant, expression and secretion were attempted by varying conditions such as temperature, culture time, strain, medium, and the like.

여러 형질전환체 중에 가장 발현 및 분비도가 높았던 균주를 사용하여 배양 조건을 확립한 다음, 5 L jar fermenter에서 대량생산을 시도하여 최적화된 분비시스템을 확립하였다.Culture conditions were established using the strains with the highest expression and secretion among the various transformants, and then an optimized secretion system was established by attempting mass production in a 5 L jar fermenter.

단백질 확인을 위하여 pRBAS 벡터 (control) 및 pRBASmHP벡터가 도입된 고초균 발효액 (250 μL)을 수거하여ethanol로 침전한 후, 멸균수 20 μL를 넣고 재현탁한 다음, 2x loading buffer (20 μL)를 넣고 100℃에서 가열 후 준비한 샘플들은 15% SDS-PAGE 에서 분석하였으며, 1차 항체 (His-tag in rabbit) 및 2차 항체 (Donkey anti-rabbit)를 사용하여 western blot을 통해 분비된 peptide 단백질을 최종 확인하였다.To identify the protein, the pRBAS vector (control) and pRBASmHP vector were introduced and the precipitated ethanol was precipitated with ethanol, precipitated with ethanol, added with 20 μL of sterile water, resuspended, and then added with 2x loading buffer (20 μL) 100 Samples prepared after heating at ℃ were analyzed on 15% SDS-PAGE, and finally confirmed peptide protein secreted through western blot using primary antibody (His-tag in rabbit) and secondary antibody (Donkey anti-rabbit) Did.

단백질 농도는 bovine serum albumin을 표준 단백질로 하여 재조합 mHP peptide 단백질이 포함된 발효액을 Bradford protein assay kit (Bio-Rad, USA)를 이용하여 측정하였다.The protein concentration was measured using a Bradford protein assay kit (Bio-Rad, USA) using a bovine serum albumin as a standard protein and a fermentation broth containing a recombinant mHP peptide protein.

1.6. 동물세포 배양 및 1.6. Animal cell culture and MTTMTT 분석 analysis

RINm5F세포는 쥐 췌장 세포종에서 유래된 insulinoma 세포주로서 American type culture collection (ATCC, USA)으로부터 구입하였고 세포배지는 fetal calf serum 10%, penicillin 100 unit/mL, streptomycin 100 μg/mL를 첨가한 RPMI1640을 사용하여 96 well plate 및 petri dish에서 배양하였다. RINm5F cells are insulinoma cell lines derived from rat pancreatic cell tumor, purchased from American type culture collection (ATCC, USA), and the cell medium is RPMI1640 with 10% fetal calf serum, 100 unit / mL penicillin, and 100 μg / mL streptomycin. And cultured in 96 well plates and petri dishes.

Trypsin EDTA를 넣고 37℃에서 20분간 배양하여 세포를 떼어내고, 3,000 rpm에서 원심분리한 후 상등액을 제거한 후, 신선한 배지를 넣고 현탁하여 hemocytometer(Thermo fisher scientific, Inc.)를 이용하여 세포수를 측정한 다음 실험에 사용하였다.Put Trypsin EDTA and incubate for 20 minutes at 37 ° C to separate cells, centrifuge at 3,000 rpm, remove the supernatant, add fresh medium and suspend it to measure the cell number using a hemocytometer (Thermo fisher scientific, Inc.). It was then used in the experiment.

Streptozotocin (STZ)을 처리하여 type I 당뇨를 유발시키고, 대조군과 함께 재조합 mHP peptide (0.01, 0.05, 0.1, 0.5, 1 mg/mL)를 포함하는 RPMI1640에서 세포증식 및 viability 측정에 의한 항당뇨 효과는 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT)가 formazan로 환원되는 것을 이용한 MTT분석에 의해 microplate reader (Molecular devices, Spectra MAX 250)를 사용하여 570 nm에서 흡광도를 측정하여 수행하였고, 모든 실험은 3회 반복하여 분석하였다.Anti-diabetic effect by measuring cell proliferation and viability in RPMI1640 containing Streptozotocin (STZ) treatment to induce type I diabetes and recombinant mHP peptide (0.01, 0.05, 0.1, 0.5, 1 mg / mL) with control At 570 nm using a microplate reader (Molecular devices, Spectra MAX 250) by MTT analysis using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) reduced to formazan Absorbance was measured and all experiments were repeated three times.

1.7. Nitric oxide 분석1.7. Nitric oxide analysis

세포 배양물에서 산화질소 (NO) 농도는 마이크로분석 방법으로 측정하였다. 즉, 세포를 96 well 플레이트 (200 μL volume, 2×104 cells/well)에 접종하고 STZ (2 mM) 처리 후에 37℃에서 30분 배양 후 배지를 제거하였다. The nitrogen oxide (NO) concentration in the cell culture was measured by a microanalysis method. That is, the cells were inoculated in a 96 well plate (200 μL volume, 2 × 10 4 cells / well) and cultured at 37 ° C. for 30 minutes after STZ (2 mM) treatment, and then the medium was removed.

대조군과 함께 mHP peptide (0.01, 0.05, 0.1, 0.5, 1 mg/mL)를 첨가하고 24시간 동안 37℃에서 배양한 후, 배양액 100 μL 취하여 Griess 시약 100 μL (1% sulfanilamide/0.1% N-(1-naphtyl)-ethylenediamine dihydrochloride/2.5% H3PO4)를 넣고 실온에서 10분 동안 방치하였다. 그 후, 540 nm에서 microplate reader (Molecular devices, Spectra MAX 250)를 사용하여 측정하였고, 산화질소 함량은 아질산 나트륨을 표준으로 비교하여 측정하였다.After adding mHP peptide (0.01, 0.05, 0.1, 0.5, 1 mg / mL) together with the control group, and incubating at 37 ° C for 24 hours, take 100 μL of the culture solution and 100 μL of Griess reagent (1% sulfanilamide / 0.1% N- ( 1-naphtyl) -ethylenediamine dihydrochloride / 2.5% H 3 PO 4 ) was added and left at room temperature for 10 minutes. Then, it was measured using a microplate reader (Molecular devices, Spectra MAX 250) at 540 nm, and the nitric oxide content was measured by comparing sodium nitrite as a standard.

1.8. DNA의 fragmentation 분석1.8. DNA fragmentation analysis

RINm5F (2×104 cells/mL)에 STZ 및 mHP peptide (0.1, 0.3, 0.5 mg/mL)를 처리하여 배양 후 수거한 세포 pellet 을 용해 완충액 500 μL (0.1 M NaCl, 0.01 M EDTA, 0.3 M Tris-HCl, pH 7.5, 0.2 M sucrose)에 재현탁하여 실온에서 배양 (15분)한 다음, 10% SDS (25 μL)를 첨가하여 65℃에서 30분 동안 더 배양하였다.RINm5F (2 × 10 4 cells / mL) was treated with STZ and mHP peptide (0.1, 0.3, 0.5 mg / mL) to dissolve the collected cell pellet and incubate 500 μL of lysis buffer (0.1 M NaCl, 0.01 M EDTA, 0.3 M Tris-HCl, pH 7.5, 0.2 M sucrose) was resuspended and incubated at room temperature (15 min), followed by further incubation at 65 ° C. for 30 min by adding 10% SDS (25 μL).

그 후, 5 M 아세트산 칼륨 (120 μL)을 첨가하여 얼음 하에서 1시간 배양하였고, 세포 용해물을 4℃에서 원심분리 (16,000×g)한 다음, DNA를 포함하는 상등액을 페놀/클로로포름 (1:1)으로 추출하고, 에탄올로 침전시켜 TE-RNase(10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 μg/mL RNase) 용액에 현탁하여 2% agarose gel 전기영동에 의해 분석하였다.Then, 5 M potassium acetate (120 μL) was added and incubated for 1 hour under ice, and the cell lysate was centrifuged (16,000 × g) at 4 ° C., and then the supernatant containing DNA was phenol / chloroform (1: Extracted with 1), precipitated with ethanol, suspended in a solution of TE-RNase (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 μg / mL RNase) and analyzed by 2% agarose gel electrophoresis.

1.9. 인슐린이 포함된 1.9. Containing insulin CaMCaM kinasekinase II 분비량 분석 II secretion analysis

Rat insulinoma cell line (RINm5F)은 NEDH rat islet cell tumor로부터 유도된 insulinoma 세포주로서 인슐린을 분비하는 세포이다. Rat insulinoma cell line (RINm5F) is an insulinoma cell line derived from NEDH rat islet cell tumor and is a cell that secretes insulin.

펩타이드 mHP가 STZ를 처리한 세포의 인슐린 분비에 영향을 주는지를 실험하기 위하여, RINm5F 세포에 STZ (2 mM)를 30분간 처리한 후 배지를 제거하고, 가장 항당뇨 효과가 좋았던 0.1 mg/mL 농도의 mHP peptide를 포함한 배지에서 24시간 동안 배양하였다. In order to test whether the peptide mHP affects insulin secretion of STZ-treated cells, the medium was removed after treatment with STZ (2 mM) for 30 minutes in RINm5F cells, and the concentration of 0.1 mg / mL, which had the best anti-diabetic effect Incubated for 24 hours in a medium containing mHP peptide.

인슐린이 분비된 세포의 배양액을 ethanol로 침전을 시킨 후, 3차 증류수 20μL (250μL 배양액에 해당하는 양)에 현탁하고, Laemmli 의 방법에 의해 15% SDS-PAGE에 로딩하여 약 55 kDa 크기에서 확인하였고, 1차 항체 (Insulin in rabbit), 2차 항체 (Donkey anti-rabbit)를 사용하여 western blot을 통해 인슐린 분비 정도를 측정하였다.After precipitating the culture medium of the cells secreted with insulin with ethanol, it was suspended in 20 μL of tertiary distilled water (amount equivalent to 250 μL culture medium), loaded on 15% SDS-PAGE by the Laemmli method, and confirmed at a size of about 55 kDa. In addition, the degree of insulin secretion was measured through western blot using a primary antibody (Insulin in rabbit) and a secondary antibody (Donkey anti-rabbit).

1.10. 통계적 분석1.10. Statistical analysis

결과는 통계적 유의성에 대한 사회 과학 (SPSS) 프로그램의 통계 패키지를 이용하여 분산 (ANOVA) 시험의 한 경로로 분석하였다. 모든 데이터는 평균±표준 편차 값으로 표현된다. 모든 분석에서 0.05보다 작은 P값은 통계적으로 유의한 것으로 간주하였다.The results were analyzed using a statistical package from the Social Sciences (SPSS) program for statistical significance as a route to the ANOVA test. All data are expressed as mean ± standard deviation values. P values less than 0.05 were considered statistically significant in all analyzes.

2. 결과 2. Results

2.1. 2.1. OligopeptideOligopeptide with His-Pro repeats를 포함하는 분비 시스템 구축 Build a secretion system that includes with His-Pro repeats

도 1은 본 발명에 따른 pRBAS-mHP 벡터를 제조하는 방법을 보여주는 개념도 및 개열지도를 보여준다. 1 shows a conceptual diagram and a cleavage map showing a method of manufacturing a pRBAS-mHP vector according to the present invention.

Oligopeptide with His-Pro repeats (mHP peptide)를 포함하는 분비시스템을 구축하기 위하여, 고초균 분비벡터인 alkaline protease promoter (0.45 kb), 리보솜결합서열 (GGAGAGGG)과 신호서열 (87 bp)을 가지고 있는 6.43 kb의 pRBAS벡터를 사용하였다. To build a secretion system that includes Oligopeptide with His-Pro repeats (mHP peptide), 6.43 kb with an alkaline protease promoter (0.45 kb), a ribosome binding sequence (GGAGAGGG) and a signal sequence (87 bp), an archaebacteria secretion vector PRBAS vector was used.

반복적인 (His-Pro) 유전자가 도입된 클론을 제작하기 위하여, pRBAS 벡터의 분비서열과 frame이 맞도록 디자인한 mHP primer F1 (5' GGGGAATTCCATCCGCATCCT 3')과 항원 항체 반응을 통하여 발현된 mHP peptide를 정량하기 위한 mHP primer R1 (5' GGGAAGCTTTTAATGGTGATGGTGATGGTGAGGATGAGGATG 3')을 사용하여 mHP peptide 유전자 3’ 말단에 His tag 유전자를 도입하였다.To produce a clone in which a repetitive (His-Pro) gene was introduced, mHP primer F1 (5 'GGGGAATTCCATCCGCATCCT 3') designed to match the secretion sequence and frame of the pRBAS vector and mHP peptide expressed through antigen antibody reaction His tag gene was introduced at the 3 'end of the mHP peptide gene using mHP primer R1 (5' GGGAAGCTTTTAATGGTGATGGTGATGGTGAGGATGAGGATG 3 ') for quantification.

PCR 에 의해 증폭하여 클로닝 한 pT-mHP 벡터에서 약 126 bp 크기의 DNA 단편을 확보하고 EcoRI 및 Hind III로 잘라 전기영동에 의해 DNA를 elution 하였다. 확보한 분비벡터와 mHP peptide의 유전자를 농도 측정 후 ligation 하여 대장균에 형질 전환하였고, 항생제 내성을 가지는 형질전환체를 분석하여 insert DNA가 들어간 형질전환체를 선별하였다. 최종적으로 plasmid DNA를 추출하여 염기서열 분석에 의해 mHP peptide 유전자가 도입된 것을 확인하였고 pRBAS-mHP (6.56 kb)로 명명하였다. A DNA fragment of about 126 bp in size was obtained from the pT-mHP vector amplified by PCR and cloned, and it was cut with EcoRI and Hind III to elution the DNA by electrophoresis. The obtained secretion vector and mHP peptide genes were measured for concentration, and then transformed into E. coli by ligation, and transformants with antibiotic resistance were analyzed to select transformants containing insert DNA. Finally, the plasmid DNA was extracted, and it was confirmed that the mHP peptide gene was introduced by sequencing and designated as pRBAS-mHP (6.56 kb).

2.2. 2.2. mHPmHP peptide 발현 및 분비시스템의 최적화 Optimization of peptide expression and secretion system

고초균의 형질전환은 SPMM-I (Spizizen's minimal medium)과 SPMM-II 배지를 사용하는 Sadaie and Kada 방법에 의해 pRBAS-mHP 벡터를 고초균 (Bacillus subtilis)으로 도입하였고, kanamycin (50 μg/mL)을 포함하는 LB 고체배지에서 형질전환체를 선별한 다음, plasmid 를 분리하여 DNA 염기서열 분석을 통하여 mHP peptide 유전자의 도입 여부를 확인하였다. Transformation of Bacillus subtilis was introduced into pRBAS-mHP vector by Bacillus subtilis by Sadaie and Kada method using SPMM-I (Spizizen's minimal medium) and SPMM-II medium, and contains kanamycin (50 μg / mL). After selecting the transformant from the LB solid medium, the plasmid was isolated to confirm whether the mHP peptide gene was introduced through DNA sequencing.

도입된 mHP peptide 유전자는 42개의 아미노산에 대한 정보(His-Pro로 구성된 36개 아미노산 및 6개 his tag)를 가지고 있고, 신호서열의 연결부위에 EcoRI 제한효소 부위에서 유래된 Glu (GAA)과 Phe (TTC) 2개의 아미노산이 첨가되어 44개의 아미노산으로 구성되어 약 4.84 kDa의 mHP peptide가 세포 외로 분비되는 것으로 확인하였다.The introduced mHP peptide gene has information on 42 amino acids (36 amino acids and 6 his tags composed of His-Pro), and Glu (GAA) and Phe derived from the EcoRI restriction enzyme site at the junction of the signal sequence. (TTC) It was confirmed that mHP peptide of about 4.84 kDa was secreted extracellularly, consisting of 44 amino acids by addition of 2 amino acids.

도 2는 mHP peptide가 도입된 재조합 벡터의 DNA sequence를 보여준다. Figure 2 shows the DNA sequence of the recombinant vector mHP peptide is introduced.

상기 재조합 벡터는 프로모터 영역(AprP, alkaline protease promoter, -35 & -10 region), +1 (전사시작부위), 신호서열(29 amino acids), GTG (개시코돈), TAA (종결코돈), His-tag (HHHHHH), processing site(peptidase cleavage site)을 갖는다.The recombinant vector is a promoter region ( Apr P, alkaline protease promoter, -35 & -10 region), +1 (transcription start site), signal sequence (29 amino acids), GTG (start codon), TAA (termination codon), His-tag (HHHHHH), processing site (peptidase cleavage site).

벡터 pRBAS-mHP를 함유하는 고초균 형질전환체 (Bacillus subtilis LKS/pRBAS-mHP)에서 mHP peptide 분비의 최적 배양조건을 확립하기 위하여, 우선적으로 seed culture에 의한 균주 활성화 후, 500 mL baffled flask 내에 100 mL 액상배지를 사용하여 균주 (B. subtilis LKS 및 168), 온도 (30℃ 및 37℃), 배지 (LB, PY, MYP) 등과 같은 조건하에서 일정 배양시간 (24 h 및 48 h)에 따른 mHP peptide의 분비효율을 조사하였다.In order to establish optimal culture conditions of mHP peptide secretion in the Bacillus subtilis LKS / pRBAS-mHP containing vector pRBAS-mHP, after first activating the strain by seed culture, 100 mL in 500 mL baffled flask MHP peptide according to the constant culture time (24 h and 48 h) under conditions such as strains (B. subtilis LKS and 168), temperature (30 ° C and 37 ° C), medium (LB, PY, MYP) using liquid medium The secretion efficiency of was investigated.

도 3은 pRBAS-mHP가 도입된 B. subtilis LSK의 배양 온도 및 질소원에 따른 mHP 펩타이드의 분비 효율을 보여준다.Figure 3 shows the secretion efficiency of mHP peptide according to the culture temperature and nitrogen source of B. subtilis LSK introduced pRBAS-mHP.

도 3(A)는 배양온도(30℃, 37℃)에 따른 분비효과를 보여주는 것으로서, 이때, Lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4-9, transformant 1-6 with pRBAS-mHP 이다. Figure 3 (A) shows the secretion effect according to the culture temperature (30 ℃, 37 ℃), at this time, Lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4-9, transformant 1-6 with pRBAS-mHP.

도 3(B)는 질소원으로서 soy peptone의 농도에 따른 분비효과를 보여주는 것으로서, 이때, Lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4-7, transformant with pRBAS-mHP (각각 1~4% soy peptone) 이다. 화살표는 mHP peptide의 위치를 나타낸다.3 (B) shows the secretion effect according to the concentration of soy peptone as a nitrogen source, and at this time, Lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4-7, transformant with pRBAS-mHP (1-4% soy peptone each). Arrows indicate the location of mHP peptide.

그 결과, 균주는 B. subtilis LKS, 온도는 30℃, 배지는 PY 배지가 mHP peptide 분비효율이 좋은 것으로 조사되었다.As a result, the strain was B. subtilis LKS, the temperature is 30 ℃, it was investigated that the medium is PY medium has good mHP peptide secretion efficiency.

따라서 100 mL 배양에서 얻어진 조건을 참고로 하여 3 L baffled flask (1 L PY 배지)에서 배양시간, 질소원 및 탄소원 등의 변화에 따라 mHP peptide의 최적화 분비 조건들을 확립하였고, 정량적 분석을 위한 대조군 (positive control)은 다른 시료들 (100 mL배양액)보다 2배 많은 0.5 mL 발효액을 농축하여 사용하였다.Therefore, with reference to the conditions obtained in 100 mL culture, optimized secretion conditions of mHP peptide were established according to changes in culture time, nitrogen source, and carbon source in a 3 L baffled flask (1 L PY medium), and a control for quantitative analysis (positive control) was used by concentrating 0.5 mL fermentation broth twice as much as other samples (100 mL culture).

즉, mHP 유전자를 함유하는 pRBAS-mHP분비벡터가 도입된 B. subtilis LKS를 PY 배지 1 L에서 48시간 동안 온도별 (30℃ 및 37℃)로 배양하였을 때, 37℃에서는 거의 분비되지 않았지만, 30℃에서는 mHP peptide가 30~40 μg/mL 수준으로 분비되므로 37℃보다 30℃의 분비 효율이 더 효과적으로 나타났다.That is, when the B. subtilis LKS containing the pRBAS-mHP secretion vector containing the mHP gene was cultured at 1 L of PY medium for 48 hours (30 ° C and 37 ° C) for 48 hours, it was hardly secreted at 37 ° C. At 30 ° C, mHP peptide was secreted at a level of 30-40 μg / mL, so the efficiency of secretion at 30 ° C was more effective than at 37 ° C.

따라서 이후의 배양은 30℃에서 수행하였고, 이는 Bacillus subtilis LKS 균주가 2종류의 단백질 분해효소, 즉 neutral protease 및 alkaline protease가 결여되어 있다고 해도 나머지 단백질 분해효소들의 활성이 30℃보다는 37℃에서 활성적이므로 mHP peptide가 분비되었다고 하더라도 단백질분해효소에 의해 분해되거나 다른 전사조절인자 등에 의해 영향을 받을 수 있을 것이라 추정된다. Therefore, the subsequent cultivation was performed at 30 ° C. Since Bacillus subtilis LKS strain lacks two types of proteolytic enzymes, that is, neutral protease and alkaline protease, the activity of the remaining proteases is active at 37 ° C. rather than 30 ° C. Even if mHP peptide is secreted, it is estimated that it may be degraded by proteolytic enzymes or affected by other transcription regulators.

분비 효율이 좋았던 30℃를 배양 온도로 하여 여러 가지 질소원 (soy peptone, peptone, potassium nitrate, corn steep liquor, defatted soybean meal, isolated soy protein) 중 가장 분비효율이 좋았던 soy peptone의 농도를 1~4%로 변화를 주어 배양하였을 때, 도 3B에서 보는 바와 같이, soy peptone 1% (48.3 μg/mL), 2% (48.3 μg/mL), 3% (48.3 μg/mL), 4% (49.8 μg/mL)로 농도가 증가하면서 mHP peptide 가 4% soy peptone의 경우 약간은 증가하였지만, soy peptone농도에 따라서 mHP peptide의 분비율에 큰 차이는 보이질 않았다.The concentration of soy peptone, which was the most secretory efficiency among the various nitrogen sources (soy peptone, peptone, potassium nitrate, corn steep liquor, defatted soybean meal, isolated soy protein), was 1 ~ 4% with 30 ° C, which had good secretion efficiency, as the culture temperature. When cultured with a change to, as shown in Figure 3B, soy peptone 1% (48.3 μg / mL), 2% (48.3 μg / mL), 3% (48.3 μg / mL), 4% (49.8 μg / mL), the mHP peptide increased slightly with 4% soy peptone as the concentration increased, but there was no significant difference in the secretion rate of mHP peptide depending on the soy peptone concentration.

도 4는 pRBAS-mHP가 도입된 B. subtilis LKS에 탄소원(barley, glycerol) 의 mHP peptide의 분비 효율을 보여준다. 여기서, 화살표는 mHP peptide의 위치를 나타낸다.Figure 4 shows the secretion efficiency of mHP peptide of the carbon source (barley, glycerol) in B. subtilis LKS introduced with pRBAS-mHP. Here, the arrow indicates the position of the mHP peptide.

도 4(A)는 barley 농도에 따른 분비효과를 보여주는 것으로서, 이때, Lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4-7, transformant with pRBAS-mHP (각각 순서대로 1, 2, 3, 4% barley)이다.Figure 4 (A) shows the secretion effect according to the barley concentration, at this time, Lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4-7, transformant with pRBAS-mHP (1, 2, 3, 4% barley in each order).

도 4(B)는 edible glycerol의 농도에 따른 분비효과를 보여주는 것으로서, 이때, Lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4-7, transformant with pRBAS-mHP (각각 순서대로 1-4% edible glycerol)이다. Figure 4 (B) shows the secretion effect according to the concentration of edible glycerol, at this time, Lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4-7, transformant with pRBAS-mHP (1-4% edible glycerol in each order).

도 4(C)는 barley와 edible glycerol 혼합 탄소원의 분비효과를 보여주는 것으로서, 이때, lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4, transformant with pRBAS-mHP (barley : edible glycerol = 3% : 2%); lane 5, (3% : 3%); lane 6, (3% : 4%); lane 7, (4% : 2%); lane 8, (4% : 3%); lane 9, (4% : 4%)이다. Figure 4 (C) is a barley and edible glycerol showing the secretion effect of the mixed carbon source, at this time, lane 1, MW marker; lane 2, pRBAS (control); lane 3, pRBAS-mHP (control); lane 4, transformant with pRBAS-mHP (barley: edible glycerol = 3%: 2%); lane 5, (3%: 3%); lane 6, (3%: 4%); lane 7, (4%: 2%); lane 8, (4%: 3%); lane 9, (4%: 4%).

탄소원의 경우 soy peptone 농도를 1(w/v)%로 고정하고, 여러 가지 탄소원(monodex, sucrose, rice bran, wheat flour, barley, edible glycerol) 중에서 적절한 분비율을 보이는 barley 및 edible glycerol을 각각 1~4(w/v)%로 함량을 변화시켜 배양하였을 때, barley의 경우 1, 2(w/v)%에서 거의 분비되지 않았지만, 3%에서는 41.4 μg/mL, 4%에서는 45.7 μg/mL로 분비되어 barley 농도가 증가하면서 peptide 분비가 증가되는 것으로 나타났으며, 그 중 4% barley에서 가장 높게 mHP peptide가 분비되었다(도 4A에 도시).In the case of carbon sources, the soy peptone concentration is fixed at 1 (w / v)%, and barley and edible glycerol each showing appropriate secretion rates among various carbon sources (monodex, sucrose, rice bran, wheat flour, barley, edible glycerol) 1 When cultured by changing the content to ~ 4 (w / v)%, barley hardly secreted at 1 and 2 (w / v)%, but at 4% at 41.4 μg / mL and at 4% at 45.7 μg / mL It was found that peptide secretion increased as the concentration of barley increased, and among them, mHP peptide was secreted the highest in 4% barley (shown in FIG. 4A).

또한, edible glycerol를 탄소원으로 사용한 경우 1(w/v)%에서 31.2 μg/mL, 2%는 31.5 μg/mL, 3%에서는 33.3 μg/mL, 4%에서는 35.6 μg/mL 로 barley와 마찬가지로 4%에서 가장 높게 mHP peptide가 분비되는 것으로 나타났다(도 4B에 도시).In addition, when edible glycerol was used as a carbon source, 1 (w / v)% to 31.2 μg / mL, 2% to 31.5 μg / mL, 3% to 33.3 μg / mL, and 4% to 35.6 μg / mL, similar to barley 4 % Showed the highest secretion of mHP peptide (shown in Figure 4B).

탄소원 함량변화 중 높은 발현 분비를 보였던 barley 3~4%와 edible glycerol 2~4%를 혼합하여 함량 변화에 따라 배양하였을 때, barley와 edible glycerol비율을 각각 (3:2)%, (3:3)%, (4:2)%, (4:3)%로 혼합한 배지에서는 평균적으로 21~27 μg/mL의 mHP peptide가 분비되었으며, (3:4)%에서는 59.6 μg/mL, (4:4)%일 때는 55.6 μg/mL의 분비를 보였다. 따라서 PY 배지에 3% barley와 4% edible glycerol이 첨가되었을 때 가장 높은 mHP peptide가 분비되었다(도 4C에 도시).Barley and edible glycerol ratios were (3: 2)%, (3: 3) when cultured according to the content change by mixing 3-4% of barley and 2-4% of edible glycerol, which showed high expression secretion during carbon source content change. In medium mixed with)%, (4: 2)%, (4: 3)%, mHP peptide of 21-27 μg / mL was secreted on average, and 59.6 μg / mL, (4) at (3: 4)% : 4)% showed 55.6 μg / mL secretion. Thus, when 3% barley and 4% edible glycerol were added to the PY medium, the highest mHP peptide was secreted (shown in FIG. 4C).

Batch culture에서 확립된 배양 조건이 대량생산을 위한 fermenter 조건과 정확하게 같지는 않지만 적정온도 30℃, 배지조성 (PY + 1% soy peptone + 3% barley + 4% edible glycerol) 및 배양시간 (24~48 h) 등을 고려하여 5 L jar fermenter(working vol. 3 L)에서 대량생산을 위한 조건을 확립하였다.The culture conditions established in batch culture are not exactly the same as the fermenter conditions for mass production, but the appropriate temperature is 30 ℃, medium composition (PY + 1% soy peptone + 3% barley + 4% edible glycerol) and culture time (24 ~ 48 h ) And established conditions for mass production in a 5 L jar fermenter (working vol. 3 L).

도 5는 pRBAS-mHP가 도입된 B. subtilis LKS 의 배양조건에 따른 mHP peptide의 분비량을 보여준다.Figure 5 shows the secretion amount of mHP peptide according to the culture conditions of B. subtilis LKS introduced pRBAS-mHP.

도 5(A)는 배양 시간에 따른 mHP peptide의 분비효율을 보여준다(Lane 1, MW marker; lane 2, pRBAS (negative control); lane 3, pRBAS-mHP (positive control); lane 4, pRBAS-mHP (12 h); lane 5, 24 h; lane 6, 48 h; lane 7, 72 h). 화살표는 mHP peptide의 위치를 나타낸다.Figure 5 (A) shows the secretion efficiency of mHP peptide according to the culture time (Lane 1, MW marker; lane 2, pRBAS (negative control); lane 3, pRBAS-mHP (positive control); lane 4, pRBAS-mHP (12 h); lane 5, 24 h; lane 6, 48 h; lane 7, 72 h). Arrows indicate the location of mHP peptide.

도 5(B)는 세포성장과 pH에 따른 mHP peptide의 분비량을 비교한 것이다. Figure 5 (B) is a comparison of the secretion of mHP peptide according to cell growth and pH.

분비벡터 pRBAS-mHP가 도입된 고초균 (B. subtilis LKS)을 PY 배지 3 L에 최종적으로 1(w/v)%가 되도록 접종하고, 300 rpm 에서 1 vvm의 공기 주입속도로 배양하여 시간에 따라 배양액을 수집하여 단백질을 분석한 결과, 48~72시간 배양했을 때 가장 좋은 수율의 최대 생산량 (43.7~46.8 μg of mHP peptide /mL)을 얻었고, optical density (OD)값에 따른 세포밀도가 증가할수록, pH값이 감소할수록 높은 양의 mHP peptide가 생산되는 것으로 확인되었다. Bacillus bacillus (B. subtilis LKS) into which the secretion vector pRBAS-mHP was introduced was inoculated to 3 L of PY medium to be finally 1 (w / v)%, and incubated at an air injection rate of 1 vvm at 300 rpm. As a result of analyzing the protein by collecting the culture solution, the maximum yield (43.7 ~ 46.8 μg of mHP peptide / mL) with the best yield was obtained when cultured for 48-72 hours, and the cell density according to the optical density (OD) value increased. , It was confirmed that a higher amount of mHP peptide was produced as the pH value decreased.

따라서 대량 생산의 경우 경제성 및 생산성을 고려해야 하므로 질소원 soy peptone은 1%로 사용하고, 탄소원 barley와 edible glycerol의 경우는 각각 3% 및 4%로 사용하여 48시간까지 배양을 시도하였다.Therefore, in the case of mass production, economic and productivity needs to be considered, so nitrogen source soy peptone was used as 1%, and carbon source barley and edible glycerol were used as 3% and 4%, respectively, and incubation was attempted up to 48 hours.

2.3. 2.3. RINm5FRINm5F 세포를 이용한 proliferation 및 viability 분석 Analysis of proliferation and viability using cells

STZ (streptozotocin)로 당뇨 (type I-like)를 유발시킨 RINm5F 세포들을 이용하여 mHP peptide 처리에 의한 항당뇨 효과를 MTT 분석을 통해 확인하였다. The anti-diabetic effect of mHP peptide treatment was confirmed by MTT analysis using RINm5F cells induced diabetes (type I-like) with STZ (streptozotocin).

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 분석은 탈수소효소작용에 의하여 노란색 수용성 기질인 MTT tetrazolium 이 세포 안으로 침투하여 미토콘드리아 내에서 그 안에 존재하는 reductase에 의해 환원되어 formazan (보라색) 결정체를 형성하는 빠른 색깔 변화로 세포 증식 또는 살아있는 세포를 측정할 수 있는 분석이므로, RINm5F 세포에서 mHP peptide가 독성 및 항당뇨 효과 여부를 알아보기 위하여 mHP peptide 처리에 따른 proliferation 및 viability 실험을 수행하였다. MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) analysis shows that the yellow water-soluble substrate, MTT tetrazolium, penetrates into cells by dehydrogenase action, and reductase is present in mitochondria. As an analysis that can measure cell proliferation or living cells by rapid color change that is reduced by tomazan (purple) crystals, proliferation according to mHP peptide treatment to determine whether mHP peptide is toxic and anti-diabetic in RINm5F cells And viability experiments.

도 6은 STZ 처리된 RINm5F cells에서 mHP peptide 첨가에 따른 세포 생존능과 질소 생성을 보여주는 그래프로서, (A)는 세포생존능, (B)는 질소생성 변화량을 보여준다. 측정은 3회 수행되었으며, 평균 ± 표준편차로 나타내었다. 6 is a graph showing cell viability and nitrogen production according to mHP peptide addition in STZ-treated RINm5F cells, (A) shows cell viability, and (B) shows nitrogen production change. Measurements were performed 3 times and are expressed as mean ± standard deviation.

대조군 RINm5F 세포의 정상적인 증식을 100% 기준으로 잡았을 때, mHP peptide를 농도별 (0.01, 0.05, 0.1, 0.5, 1mg/mL)로 투여하여 실험한 결과 세포증식은 거의 정상세포와 같은 수준을 유지하는 것으로 볼 때, mHP peptide의 실험적 투여 농도하에서 독성이 없는 것으로 나타났고, STZ (2 mM)를 처리한 세포는 약 49% 살아남았고, STZ 처리 후 mHP peptide를 0.01, 0.05, 0.1, 0.5, 1mg/mL로 처리한 세포들에서 각각 54%, 58%, 59%, 57%, 56% 정도로 세포 활성이 복구되는 것으로 나타났다.When the normal proliferation of the control RINm5F cells was set at 100%, the mHP peptide was administered by concentration (0.01, 0.05, 0.1, 0.5, 1 mg / mL), and as a result, cell proliferation maintained almost the same level as normal cells. In view of this, it was found that there was no toxicity under the experimental dose concentration of mHP peptide, and cells treated with STZ (2 mM) survived about 49%, and mHP peptide was 0.01, 0.05, 0.1, 0.5, 1 mg / after STZ treatment. It was found that the cell activity was restored to about 54%, 58%, 59%, 57%, and 56% in cells treated with mL, respectively.

이 중 STZ를 처리한 다음 (STZ only: cell viability) 세포활성을 가장 높게 회복시킨 mHP peptide 적정처리 농도는 0.1 mg/mL로 나타났으며, 약 10% 정도 세포활성을 복구시켰다(도 6A에 도시).Among them, after treating STZ (STZ only: cell viability), the mHP peptide titration concentration that restored the highest cell activity was found to be 0.1 mg / mL, and restored cell activity by about 10% (shown in FIG. 6A). ).

2.4. Nitric oxide 분석2.4. Nitric oxide analysis

Nitric oxide (NO)는 췌장 베타세포에서 미토콘드리아의 오작동과 염색체 DNA의 손상을 초래하며, STZ로 인한 당뇨병의 요인들은 알킬화 (alkylation)와는 관련이 없지만 NO donor로 잠재적인 역할을 한다고 알려져있다. Nitric oxide (NO) causes mitochondrial malfunction and damage to chromosomal DNA in pancreatic beta cells, and the factors of diabetes caused by STZ are not related to alkylation, but are known to play a potential role as NO donors.

따라서, mHP peptide가 만약 STZ로 인해 손상된 RINm5F 세포에서 생성되는 NO 가스를 감소시킬 수 있다면, mHP peptide의 회복효과가 증명되므로 세포 배양물에서 산화질소 농도를 Griess 시약(in MATERIALS AND METHODS)을 이용하여 마이크로분석 방법으로 540 nm에서 microplate reader로 측정하였다.Therefore, if mHP peptide can reduce the NO gas generated in RINm5F cells damaged by STZ, the recovery effect of mHP peptide is proved, so the concentration of nitric oxide in cell culture can be determined using Griess reagent (in MATERIALS AND METHODS). It was measured with a microplate reader at 540 nm using a microanalysis method.

산화질소 함량은 아질산 나트륨 0, 10, 20, 30, 40, 50, 100 μM 농도에 따른 각각의 OD값, 즉 0.037, 0.099, 0.247, 0.379, 0.533, 0.611, 0.887 수치를 standard curve화 하여 실험군에 대하여 정량하였다. The nitric oxide content was standardized to the experimental group by standardizing the respective OD values according to the concentrations of sodium nitrite 0, 10, 20, 30, 40, 50, and 100 μM, that is, 0.037, 0.099, 0.247, 0.379, 0.533, 0.611, and 0.887. Was quantified.

Nitric oxide (NO)의 발생은 2 mM STZ를 처리하였을 때 NO는 5.76 μM까지 증가하였지만, mHP peptide 농도를 0.01~1 mg/mL로 변화하여 처리하였을 때, mHP peptide 처리량에 따라 NO 발생이 조금씩 감소하다가 0.1 mg/mL 에서 3.75 μM로 급격히 감소하였다 (약 35%). The generation of Nitric oxide (NO) increased to 5.76 μM when treated with 2 mM STZ, but when mHP peptide concentration was changed to 0.01 to 1 mg / mL, NO generation decreased slightly depending on the mHP peptide throughput. Then, it decreased rapidly from 0.1 mg / mL to 3.75 μM (about 35%).

그러나 0.5 mg/mL에서는 3.91 μM, 1 mg/mL에서 4.06 μM 로 약간 증가하였으나 STZ만 처리하였을 때보다는 NO 발생은 감소하는 것으로 나타났다. However, although it was slightly increased from 0.5 mg / mL to 3.91 μM and from 1 mg / mL to 4.06 μM, NO generation was decreased compared to STZ alone.

여러 투여 농도 중에서 mHP peptide 농도를 0.1mg/mL로 처리한 경우에 NO 발생이 가장 낮은 것으로 나타났으며(도 6B에 도시), 이와 같은 결과는 mHP peptide를 0.1 mg/mL로 처리하였을 때 세포활성이 가장 높게 (10%) 복구되는 농도와 일치하며, 이와 같은 감소 현상은 당뇨병으로 인해 손상된 세포를 mHP peptide가 회복시킬 수 있다는 것을 보여주며, 항당뇨 효과까지 가지고 있다는 가능성을 뒷받침해주고 있다.Among the various administration concentrations, when mHP peptide concentration was treated with 0.1 mg / mL, NO was found to be the lowest (shown in FIG. 6B), and these results showed that cell activity when mHP peptide was treated with 0.1 mg / mL. This is consistent with the highest (10%) recovered concentration, and this reduction shows that mHP peptide can recover cells damaged by diabetes and supports the possibility of having an anti-diabetic effect.

2.5. DNA fragmentation 분석2.5. DNA fragmentation analysis

DNA ladder 현상은 베타세포를 포함한 다양한 세포들의 세포사멸 (programmed cell death)과 관련되어 있으며, STZ 처리로 인한 RINm5F 세포에서의 세포사멸 (apoptosis 현상)은 DNA fragmentation을 야기시키는 것으로 보고되어 있다. The DNA ladder phenomenon is related to the programmed cell death of various cells, including beta cells, and it has been reported that apoptosis in RINm5F cells due to STZ treatment causes DNA fragmentation.

우선적으로 STZ 처리의 적정농도를 측정하기 위하여, STZ 농도별 (1, 2, 3, 4, 5 mM)로 실험을 진행하였고 그 중 효과적으로 DNA fragmentation 현상을 보이는 농도 4 mM로 대조군 및 실험군 세포를 STZ 으로 처리하여 실험을 진행하였다. First of all, in order to measure the appropriate concentration of STZ treatment, experiments were conducted according to the concentration of STZ (1, 2, 3, 4, 5 mM), and among them, the control and experimental group cells with effective concentration of DNA fragmentation at 4 mM were subjected to STZ. The experiment was conducted by treatment with.

도 7은 STZ(4 mM)처리된 RINm5F 세포에서 mHP peptide의 회복효과를 보여주는 것으로서, 도 7(A)는 DNA fragmentation 테스트 결과를 보여준다.Figure 7 shows the recovery effect of mHP peptide in STZ (4 mM) treated RINm5F cells, Figure 7 (A) shows the DNA fragmentation test results.

여기서, Lane 1, 1 kb; lane 2, no STZ; lane 3, STZ; lane 4, STZ + pRBAS broth (0.5 mg/mL); lane 5, STZ + CHP (Sigma, 0.5 mg/mL); lane 6, STZ + CHP (Bachem, 0.5 mg/mL); lane 7, STZ + mHP (0.1 mg/mL); lane 8, STZ + mHP (0.3 mg/mL); lane 9, STZ + mHP (0.5 mg/mL) 이다.Here, Lane 1, 1 kb; lane 2, no STZ; lane 3, STZ; lane 4, STZ + pRBAS broth (0.5 mg / mL); lane 5, STZ + CHP (Sigma, 0.5 mg / mL); lane 6, STZ + CHP (Bachem, 0.5 mg / mL); lane 7, STZ + mHP (0.1 mg / mL); lane 8, STZ + mHP (0.3 mg / mL); lane 9, STZ + mHP (0.5 mg / mL).

STZ 처리로 인해 DNA fragmentation이 일어난 손상된 세포에 mHP peptide를 점진적으로 농도를 증가시켜서 (0.1, 0.3, 0.5 mg/mL)처리하였을 때, DNA fragmentation에 의한 ladder 현상이 현저하게 완화되었다. When the mHP peptide was gradually increased in concentration (0.1, 0.3, 0.5 mg / mL) to damaged cells where DNA fragmentation occurred due to STZ treatment, ladder phenomenon due to DNA fragmentation was significantly alleviated.

즉, 항당뇨 효과가 이미 입증된 positive control로 사용한 cyclo(His-Pro) (Sigma & Bachem:Fig. 6A lane 5 & 6)의 경우 DNA ladder 현상은 거의 복구되어지는 것으로 나타났고, mHP peptide의 경우에도 처리 농도에 따라 약간의 차이는 있지만 STZ만 처리한 세포에서 DNA ladder 현상과 비교해 볼 때 agarose gel상에서 거의 90% 이상 ladder 현상이 감소하는 것으로 나타났다. That is, in the case of cyclo (His-Pro) (Sigma & Bachem: Fig. 6A lane 5 & 6), which was used as a positive control with anti-diabetic effect, the DNA ladder phenomenon was found to be almost restored, and in the case of mHP peptide Although there are some differences depending on the concentration of Edo treatment, compared to the DNA ladder phenomenon in cells treated with STZ only, it was found that the ladder phenomenon was reduced by almost 90% or more on the agarose gel.

이와 같은 결과는 mHP peptide 처리에 따른 세포활성의 복구 및 NO 발생 억제 현상과도 일치하는 결과로 보인다.These results seem to be consistent with the restoration of cellular activity and inhibition of NO generation according to mHP peptide treatment.

2.6. 인슐린이 포함된 2.6. Containing insulin CaMCaM kinasekinase II 분비량 분석 II secretion analysis

RIN-m rat islet 세포주에서 유래된 RINm5F 세포는 인슐린을 분비하는 세포이므로, STZ를 처리한 세포에 mHP peptide를 처리하였을 때 인슐린 분비에 미치는 영향을 조사하기 위하여 Gray and Flatt 방법에 따라 실험을 수행하였다.Since RINm5F cells derived from the RIN-m rat islet cell line are insulin-secreting cells, experiments were performed according to the Gray and Flatt method to investigate the effect on insulin secretion when mHP peptide was treated with STZ-treated cells. .

즉, RINm5F 세포에 STZ (2 mM)를 30분간 처리한 후, 상기 실험 결과에서 가장 효과가 좋았던 0.1 mg/mL 농도로 mHP peptide가 포함된 배지에서 24시간 동안 배양하였다. 인슐린 (insulin secretion vesicle)을 함유한 calmodulin (CaM)-dependent protein kinase II 는 SDS-PAGE 상에서 분자량이 55 kDa 위치에 나타나며, 인슐린 항체로 western blot을 하였을 때 그 위치에서 검출되는 것으로 보고되었다. That is, after treating STZ (2 mM) in RINm5F cells for 30 minutes, the cells were cultured for 24 hours in a medium containing mHP peptide at a concentration of 0.1 mg / mL, which was the most effective in the experiment results. It has been reported that calmodulin (CaM) -dependent protein kinase II containing insulin (insulin secretion vesicle) has a molecular weight of 55 kDa on SDS-PAGE and is detected at western blot with insulin antibody.

도 7(B)는 인슐린이 포함된 CaM kinase II의 분석결과를 보여주는 것으로서 여기서, Lane 1, MW marker; lane 2, RPMI1640 배지; lane 3, glucose를 포함하지 않는 세포 상등액; lane 4, 11.1 mM glucose를 포함하는 세포 상등액; lane 5, STZ 로 처리된 세포 상등액; lane 6, STZ 과 pRBAS broth로 처리된 세포 상등액 ; lane7, STZ 과 mHP peptide에 의해 처리된 세포 상등액이다. 화살표는 인슐린이 포함된 CaM kinase II의 위치를 보여준다.Figure 7 (B) shows the analysis results of CaM kinase II containing insulin, where Lane 1, MW marker; lane 2, RPMI1640 medium; lane 3, cell supernatant without glucose; cell supernatant containing lane 4, 11.1 mM glucose; lane 5, cell supernatant treated with STZ; cell supernatant treated with lane 6, STZ and pRBAS broth; Cell supernatant treated with lane7, STZ and mHP peptide. The arrow shows the location of the CaM kinase II containing insulin.

하기의 표 1은 mHP peptide 처리에 의한 insulin secretion vesicle 포함CaM kinase II의 함량 변화를 보여준다.Table 1 below shows the change in the content of CaM kinase II including insulin secretion vesicle by mHP peptide treatment.

Figure 112018029857701-pat00001
Figure 112018029857701-pat00001

도 7(B)에서 보듯이 RINm5F 세포를 접종하지 않은 RPMI1640 배지에서는 인슐린 분비가 나타나지 않았고(lane 2), RINm5F 세포를 접종한 glucose를 포함하지 않은 배지에서는 21.20 μg/mL(lane 3), 11.1 mM glucose를 포함한 배지에서는 24.70 μg/mL(lane 4)로 glucose를 포함시킨 배지에서는 인슐린이 조금 더 분비되었다. As shown in FIG. 7 (B), insulin secretion was not observed in RPMI1640 medium not inoculated with RINm5F cells (lane 2), and 21.20 μg / mL (lane 3), 11.1 mM in medium not containing glucose inoculated with RINm5F cells. In the medium containing glucose, insulin was secreted a little more in the medium containing glucose at 24.70 μg / mL (lane 4).

Glucose (11.1 mM)가 포함된 배지에 RINm5F 세포에 STZ만 처리한 경우 세포 배양물에서 인슐린이 포함된 CaM kinase II 분비는 14.40 μg/mL (lane 5), STZ처리 후 pRBAS vector 함유 고초균 발효액을 처리한 세포 배양물에서는 14.50μg/mL (lane 6), 또한 STZ처리 후 0.1 mg/mL 농도로 mHP peptide를 처리한 세포 배양물에서는 16.82 μg/mL (lane 7)로 분비되었다. When only STZ is treated on RINm5F cells in a medium containing Glucose (11.1 mM), CaM kinase II secretion containing insulin in the cell culture is 14.40 μg / mL (lane 5), and after treatment with STZ, a pRBAS vector containing archaea fermentation broth is treated. It was secreted at 14.50 μg / mL (lane 6) in one cell culture and 16.82 μg / mL (lane 7) in a cell culture treated with mHP peptide at a concentration of 0.1 mg / mL after STZ treatment.

따라서 RINm5F 세포에 STZ를 처리한 경우보다mHP peptide를 처리한 세포 배양물에서 약 17% 정도 인슐린을 포함한 CaM kinase II가 더 분비되는 것으로 나타났다. Therefore, it was found that CaM kinase II containing insulin was secreted more than about 17% in cell culture treated with mHP peptide than STZ treatment with RINm5F cells.

상기 표 1에서 보는 바와 같이 대조군으로 사용한 RINm5F 세포에 11.1 mM glucose를 첨가한 배양물의 인슐린 분비량 기준을 100%로 본다면, STZ 만 처리한 경우 인슐린 분비량은 58%였고, 단지 pRBAS 벡터만 도입된 고초균 발효액을 처리하였을 때 인슐린 분비량에는 거의 변화가 없었고, STZ에 mHP peptide를 처리한 경우 인슐린 분비량은 68%로 나타났다. As shown in Table 1, if the standard for insulin secretion of a culture in which 11.1 mM glucose was added to RINm5F cells used as a control was 100%, the insulin secretion was 58% when only STZ was treated, and only the pRBAS vector was introduced. There was almost no change in insulin secretion when treated with and when mHP peptide was treated with STZ, insulin secretion was 68%.

따라서 RINm5F 세포에 STZ를 처리하여 type I과 유사한 당뇨를 유발하였을 때는 인슐린 분비 정도는 감소하였지만 (약 42% 정도), STZ처리 후 RINm5F세포에 mHP peptide를 처리하자 인슐린 분비는 STZ만 처리한 세포에서보다 약 10% 정도 증가하는 것으로 나타났다.Therefore, when STZ was treated on RINm5F cells to induce diabetes similar to type I, the insulin secretion level was reduced (about 42%), but after treatment with SHP, mHP peptide was treated on RINm5F cells, and insulin secretion was on cells treated with STZ only. It was found to increase by about 10%.

Cyclo(His-Pro)는 insulinoma cells, type I rat 및 type II ob/ob mouse에서 항당뇨 효과가 입증되었으나, 식품 소재에는 그 양이 매우 적은 수준으로 함유되어 있어 CHP 원료를 확보하기가 쉽지 않으므로, 본 발명에서는 His-Pro dipeptide가 multimer로 구성된 peptide (oligopeptide with His-Pro repeats, mHP peptide)를 충분히 확보하기 위하여 우선적으로 His-Pro의 dipeptide가 18 repeats로 반복되는 정보를 갖는 oligonucleotide를 제작하였고, 그 유전자를 pRBAS 분비 벡터에 클로닝하여 고초균에서 분비 및 최적화를 시도하였다. Cyclo (His-Pro) has been proven to have anti-diabetic effects in insulinoma cells, type I rats and type II ob / ob mice, but it is not easy to secure CHP raw materials because it contains very low levels of food ingredients. In the present invention, in order to sufficiently secure a peptide (oligopeptide with His-Pro repeats, mHP peptide) composed of multimers of His-Pro dipeptide, an oligonucleotide having information repeating His-Pro dipeptide with 18 repeats was prepared. The gene was cloned into a pRBAS secretion vector, and secretion and optimization were attempted by Bacillus subtilis.

그 결과, mHP peptide는 30℃, PY medium, 질소원 (1% soy peptone), 및 탄소원 (barley and glycerol)에서 가장 높은 수준 (59.6 μg/mL)으로 분비되었다. As a result, mHP peptide was secreted at the highest level (59.6 μg / mL) at 30 ° C., PY medium, nitrogen source (1% soy peptone), and carbon source (barley and glycerol).

Oligopeptide with His-Pro repeats (mHP peptide) 단백질 분비 최적화 및 항당뇨 효과를 확인하기 위하여, alkaline protease promoter (0.45 kb) 및 신호서열 (87 bp)을 포함하는 pRBAS 벡터에 mHP peptide 유전자를 도입하여 pRBAS-mHP (6.56 kb) 분비시스템을 구축하였고, GRAS 균주인 고초균 Bacillus subtilis LKS 로 도입하여 단백질 분비 최적화 실험을 시도하였다. Oligopeptide with His-Pro repeats (mHP peptide) In order to optimize protein secretion and to confirm anti-diabetic effect, pRBAS- was introduced by introducing mHP peptide gene into pRBAS vector containing alkaline protease promoter (0.45 kb) and signal sequence (87 bp). An mHP (6.56 kb) secretion system was constructed, and the protein secretion optimization experiment was attempted by introducing into the GRAS strain Bacillus subtilis LKS.

그 결과, 42 아미노산으로 구성된 mHP peptide가 높은 수율로 배양액에 분비되는 것이 확인되었고, 분비 단백질을 산업적으로 생산하기 위한 전 단계로서 5 L jar fermenter를 이용하여 대량생산을 위한 최적 배양조건을 확립하였다. As a result, it was confirmed that mHP peptide composed of 42 amino acids was secreted into the culture solution with a high yield, and optimum culture conditions for mass production were established using 5 L jar fermenter as a preliminary step for industrially producing secreted proteins.

또한, STZ를 처리한 RINm5F 세포에서 mHP peptide 처리에 의해 손상된 RINm5F 세포활성을 약 10% 정도 회복시킬 수 있었고, nitric oxide (NO)와 DNA fragmentation 현상을 감소시키고 인슐린 분비량도 약 17% 정도 복구되는 것으로 확인하였다. In addition, it was found that RINm5F cell activity damaged by mHP peptide treatment in STZ-treated RINm5F cells could be restored by about 10%, reduced nitric oxide (NO) and DNA fragmentation, and recovered insulin secretion by about 17%. Confirmed.

따라서, 과거 고비용으로 ppm 수준의 소량 CHP 를 추출하던 단점을 개선한 본 연구는 고초균에서 분비 시스템을 통하여 mHP peptide의 대량생산이 가능하므로 당뇨병 예방 및 치료제로써 경쟁력을 가질 수 있을 것으로 전망되며, 의료산업 외에 건강식품산업 등의 분야에도 활용될 수 있을 것이라고 예상된다. Therefore, this study, which improved the disadvantage of extracting a small amount of CHP at a high cost in the past, is expected to be competitive as a preventive and therapeutic agent for diabetes because mass production of mHP peptide is possible through the secretory system from archaea. In addition, it is expected to be used in fields such as the health food industry.

이상과 같이 본 발명은 첨부된 도면을 참조하여 바람직한 실시예를 중심으로 설명하였지만 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 본 발명의 특허청구범위에 기재된 기술적 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 또는 변형하여 실시할 수 있다. 따라서 본 발명의 범주는 이러한 많은 변형의 예들을 포함하도록 기술된 청구범위에 의해서 해석되어야 한다.As described above, the present invention has been mainly described with reference to the accompanying drawings, but a person having ordinary knowledge in the technical field to which the present invention pertains does not depart from the technical spirit and scope described in the claims of the present invention. In the present invention can be implemented by various modifications or variations. Therefore, the scope of the invention should be construed by the claims set forth to cover many examples of such modifications.

<110> Turtlebio Co., Ltd <120> Mass Production System of Oligopeptide with His-Pro Repeats <130> PX17-095 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 His Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro 1 5 10 15 His Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro 20 25 30 His Pro His Pro His His His His His His 35 40 <210> 2 <211> 803 <212> DNA <213> Artificial Sequence <220> <223> pRBAS-mHP <400> 2 tgcttggcga atgttcatct tatttcttcc tccctctcaa taattttttt attctatccc 60 ttttctgtaa agtttatttt tcagaatact tttatcatca tgctttgaaa aaatatcacg 120 ataatatcca ttgttctcac ggaagcatat acaggtcatt tgaacgaatt ttttcgacag 180 gaatttgcag ggactcagga gcatttaacc taaaaaagca tgacatttca gcataatgaa 240 catttactca tgtctatttt cgttcttttc tgtatgaaaa tagttatttc gagtctctac 300 ggaaatagcg agagatgata tacctaaata gagataaaat catctcaaaa aaatgggtct 360 actaaaatat tattccatct attacaataa attcacagaa tagtctttta agtaagtcta 420 ctctgaattt ttttaaaagg agagggtaaa gagtgagaag caaaaaattg tggatcagct 480 tgttgtttgc gttaacgtta atctttacga tggcgttcag caacacgtct gcgcaggctg 540 aattccatcc gcatcctcac cctcacccgc atcctcaccc tcaccctcat ccgcatccac 600 acccgcaccc tcatcctcat ccgcatcctc atccgcaccc tcaccctcat ccgcaccatc 660 accatcacca ttaaaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac 720 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg 780 cggtggtccc acctgacccc atg 803 <210> 3 <211> 73 <212> PRT <213> Artificial Sequence <220> <223> pRBAS-mHP <400> 3 Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Leu Ser Asn Met Ser Ala Gln Ala Glu Phe His 20 25 30 Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro His 35 40 45 Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro His 50 55 60 Pro His Pro His His His His His His 65 70 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 ggggaattcc atccgcatcc t 21 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 5 gggaagcttt taatggtgat ggtgatggtg aggatgagga tg 42 <210> 6 <211> 395 <212> DNA <213> Artificial Sequence <220> <223> Promoter sequence <400> 6 tgcttggcga atgttcatct tatttcttcc tccctctcaa taattttttt attctatccc 60 ttttctgtaa agtttatttt tcagaatact tttatcatca tgctttgaaa aaatatcacg 120 ataatatcca ttgttctcac ggaagcatat acaggtcatt tgaacgaatt ttttcgacag 180 gaatttgcag ggactcagga gcatttaacc taaaaaagca tgacatttca gcataatgaa 240 catttactca tgtctatttt cgttcttttc tgtatgaaaa tagttatttc gagtctctac 300 ggaaatagcg agagatgata tacctaaata gagataaaat catctcaaaa aaatgggtct 360 actaaaatat tattccatct attacaataa attca 395 <110> Turtlebio Co., Ltd <120> Mass Production System of Oligopeptide with His-Pro Repeats <130> PX17-095 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 His Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro   1 5 10 15 His Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro              20 25 30 His Pro His Pro His His His His His His          35 40 <210> 2 <211> 803 <212> DNA <213> Artificial Sequence <220> <223> pRBAS-mHP <400> 2 tgcttggcga atgttcatct tatttcttcc tccctctcaa taattttttt attctatccc 60 ttttctgtaa agtttatttt tcagaatact tttatcatca tgctttgaaa aaatatcacg 120 ataatatcca ttgttctcac ggaagcatat acaggtcatt tgaacgaatt ttttcgacag 180 gaatttgcag ggactcagga gcatttaacc taaaaaagca tgacatttca gcataatgaa 240 catttactca tgtctatttt cgttcttttc tgtatgaaaa tagttatttc gagtctctac 300 ggaaatagcg agagatgata tacctaaata gagataaaat catctcaaaa aaatgggtct 360 actaaaatat tattccatct attacaataa attcacagaa tagtctttta agtaagtcta 420 ctctgaattt ttttaaaagg agagggtaaa gagtgagaag caaaaaattg tggatcagct 480 tgttgtttgc gttaacgtta atctttacga tggcgttcag caacacgtct gcgcaggctg 540 aattccatcc gcatcctcac cctcacccgc atcctcaccc tcaccctcat ccgcatccac 600 acccgcaccc tcatcctcat ccgcatcctc atccgcaccc tcaccctcat ccgcaccatc 660 accatcacca ttaaaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac 720 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg 780 cggtggtccc acctgacccc atg 803 <210> 3 <211> 73 <212> PRT <213> Artificial Sequence <220> <223> pRBAS-mHP <400> 3 Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu   1 5 10 15 Ile Phe Thr Met Ala Leu Ser Asn Met Ser Ala Gln Ala Glu Phe His              20 25 30 Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro His          35 40 45 Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro His Pro His      50 55 60 Pro His Pro His His His His His His  65 70 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 ggggaattcc atccgcatcc t 21 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 5 gggaagcttt taatggtgat ggtgatggtg aggatgagga tg 42 <210> 6 <211> 395 <212> DNA <213> Artificial Sequence <220> <223> Promoter sequence <400> 6 tgcttggcga atgttcatct tatttcttcc tccctctcaa taattttttt attctatccc 60 ttttctgtaa agtttatttt tcagaatact tttatcatca tgctttgaaa aaatatcacg 120 ataatatcca ttgttctcac ggaagcatat acaggtcatt tgaacgaatt ttttcgacag 180 gaatttgcag ggactcagga gcatttaacc taaaaaagca tgacatttca gcataatgaa 240 catttactca tgtctatttt cgttcttttc tgtatgaaaa tagttatttc gagtctctac 300 ggaaatagcg agagatgata tacctaaata gagataaaat catctcaaaa aaatgggtct 360 actaaaatat tattccatct attacaataa attca 395

Claims (9)

히스티딘-프롤린 반복서열로 구성된 아미노산과 상기 반복서열의 말단에 his tag 로 이루어진 서열번호 1의 아미노산 서열을 코딩하는 유전자를 포함하는 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드 대량생산용 재조합 벡터에 있어서,
상기 재조합 벡터는
서열번호 6의 염기서열을 갖는 고초균 유래의 알칼리성 단백질분해효소 유전자의 프로모터와 상기 서열번호 1의 아미노산 서열을 코딩하는 유전자가 작동 가능하게 연결된 것이며,
상기 서열번호 1의 아미노산 서열을 코딩하는 유전자는
서열번호 4의 염기서열을 갖는 정방향 프라이머와 서열번호 5의 염기서열을 갖는 역방향 프라이머 세트에 의해 증폭되는 것을 특징으로 하는
히스티딘-프롤린 반복서열을 갖는 올리고펩타이드 대량생산용 재조합 벡터.
In the recombinant vector for mass production of oligopeptides having a histidine-proline repeat sequence comprising a gene consisting of an amino acid sequence consisting of histidine-proline repeat sequence and the amino acid sequence of SEQ ID NO: 1 consisting of his tag at the end of the repeat sequence,
The recombinant vector
The promoter of the alkaline protease gene derived from archaea having the nucleotide sequence of SEQ ID NO: 6 and the gene encoding the amino acid sequence of SEQ ID NO: 1 are operably linked,
The gene encoding the amino acid sequence of SEQ ID NO: 1
Characterized in that it is amplified by a set of forward primers having the nucleotide sequence of SEQ ID NO: 4 and reverse primers having the nucleotide sequence of SEQ ID NO: 5
A recombinant vector for mass production of oligopeptides having a histidine-proline repeat sequence.
제 1항의 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드 대량생산용 재조합 벡터에 의해 형질전환된 고초균.
An archaebacteria transformed by a recombinant vector for mass production of oligopeptides having the histidine-proline repeat sequence of claim 1.
제 2항의 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드 대량생산용 재조합 벡터에 의해 형질전환된 고초균으로부터 수득된 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드를 포함하는 항당뇨 조성물에 있어서,
상기 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드는
배지 100 부피부에 대하여 상기 형질전환된 고초균을 0.5 내지 3 중량부;와 보리와 식용글리세롤을 0.5 내지 1 : 1의 중량비로 혼합한 혼합 탄소원 1 내지 5 중량부;와 질소원 1 내지 5 중량부를 포함하며, 30 내지 37℃ 에서 12 내지 72시간 배양하여 수득되는 것을 특징으로 하는
히스티딘-프롤린 반복서열을 갖는 올리고펩타이드를 포함하는 항당뇨 조성물.
An anti-diabetic composition comprising an oligopeptide having a histidine-proline repeat sequence obtained from an archaea transformed by a recombinant vector for mass production of an oligopeptide having the histidine-proline repeat sequence of claim 2,
The oligopeptide having the histidine-proline repeat sequence is
0.5 to 3 parts by weight of the transformed archaea against 100 parts by volume of the medium; and 1 to 5 parts by weight of a mixed carbon source in which barley and edible glycerol are mixed in a weight ratio of 0.5 to 1: 1; and 1 to 5 parts by weight of nitrogen. And, characterized in that obtained by incubation for 12 to 72 hours at 30 to 37 ℃
Anti-diabetic composition comprising an oligopeptide having a histidine-proline repeat sequence.
제 3항에 있어서,
손상된 췌장 β-세포주 활성의 복구, nitric oxide (NO) 생성의 감소, DNA fragmentation 의 완화, CaM kinase II에 포함된 인슐린 분비량 증가 및 이들의 조합 중 어느 하나의 기전을 통하여 당뇨 및 당뇨 합병증 치료 및 예방효과를 갖는 것을 특징으로 하는
히스티딘-프롤린 반복서열을 갖는 올리고펩타이드를 포함하는 항당뇨 조성물.
According to claim 3,
Treatment and prevention of diabetes and diabetes complications through the mechanism of any one of the repair of damaged pancreatic β-cell line activity, reduction of nitric oxide (NO) production, alleviation of DNA fragmentation, increased insulin secretion in CaM kinase II, and combinations thereof Characterized by having an effect
Anti-diabetic composition comprising an oligopeptide having a histidine-proline repeat sequence.
히스티딘-프롤린 반복서열로 구성된 아미노산과 상기 반복서열의 말단에 his tag 로 이루어진 서열번호 1의 아미노산 서열을 코딩하는 유전자를 포함하는 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드를 대량생산하기 위한 방법에 있어서,
히스티딘-프롤린 반복서열로 구성된 아미노산과 상기 반복서열의 말단에 his tag 로 이루어진 서열번호 1의 아미노산 서열을 코딩하는 유전자를 포함하는 히스티딘-프롤린 반복서열을 갖는 올리고펩타이드 대량생산용 재조합 벡터를 제조하는 단계;와
상기 재조합 벡터를 고초균에 도입하여 형질전환된 고초균을 선별하고, 선별된 고초균을 배양하는 단계를 포함하며,
상기 재조합 벡터를 제조하는 단계는
서열번호 6의 염기서열을 갖는 고초균 유래의 알칼리성 단백질분해효소 유전자의 프로모터와 상기 서열번호 1의 아미노산 서열을 코딩하는 유전자가 작동 가능하게 연결되도록 하며,
상기 서열번호 1의 아미노산 서열을 코딩하는 유전자는
서열번호 4의 염기서열을 갖는 정방향 프라이머와 서열번호 5의 염기서열을 갖는 역방향 프라이머 세트에 의해 증폭되며,
상기 고초균을 배양하는 단계는
배지 100 부피부에 대하여 선별된 형질전환 고초균을 0.5 내지 3 중량부;와 보리와 식용글리세롤을 0.5 내지 1 : 1의 중량비로 혼합한 혼합 탄소원 1 내지 5 중량부;와 질소원 1 내지 5 중량부를 포함하며, 30 내지 37℃ 에서 12 내지 72시간 배양하는 것을 특징으로 하는
히스티딘-프롤린 반복서열을 갖는 올리고펩타이드의 대량생산 방법.
In the method for mass production of an oligopeptide having a histidine-proline repeat sequence comprising a gene encoding an amino acid sequence of SEQ ID NO: 1 consisting of an amino acid composed of a histidine-proline repeat sequence and a his tag at the end of the repeat sequence,
Preparing a recombinant vector for mass production of an oligopeptide having a histidine-proline repeat sequence comprising a gene encoding an amino acid sequence of SEQ ID NO: 1 consisting of an amino acid composed of a histidine-proline repeat sequence and a his tag at the end of the repeat sequence. ;Wow
Including the step of introducing the recombinant vector to the archaebacteria to transform the transformed archaebacteria, and culturing the selected archaebacteria,
The step of preparing the recombinant vector is
The promoter of the alkaline protease gene derived from archaea having the nucleotide sequence of SEQ ID NO: 6 and the gene encoding the amino acid sequence of SEQ ID NO: 1 are operably linked,
The gene encoding the amino acid sequence of SEQ ID NO: 1
Amplified by a set of forward primers having the nucleotide sequence of SEQ ID NO: 4 and reverse primers having the nucleotide sequence of SEQ ID NO: 5,
The step of culturing the archaea bacteria
0.5 to 3 parts by weight of the transformed archaebacteria selected for 100 parts by volume of the medium; and 1 to 5 parts by weight of a mixed carbon source in which barley and edible glycerol are mixed in a weight ratio of 0.5 to 1: 1; and 1 to 5 parts by weight of nitrogen. And, characterized in that the culture for 12 to 72 hours at 30 to 37 ℃
Method for mass production of oligopeptide having histidine-proline repeat sequence.
삭제delete 삭제delete 삭제delete 삭제delete
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KR101418968B1 (en) 2011-07-08 2014-07-14 대구대학교 산학협력단 Composition for blood sugar regulation comprising soybean hydrololysate with high chp(cyclo(his-pro))
CA3179537A1 (en) * 2012-02-27 2013-09-06 Amunix Pharmaceuticals, Inc. Xten conjugate compositions and methods of making same

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