KR20160072451A - Production Methods of Human Growth Hormone Protein using a Vector for Transforming Pleurotus eryngii - Google Patents
Production Methods of Human Growth Hormone Protein using a Vector for Transforming Pleurotus eryngii Download PDFInfo
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- KR20160072451A KR20160072451A KR1020140180064A KR20140180064A KR20160072451A KR 20160072451 A KR20160072451 A KR 20160072451A KR 1020140180064 A KR1020140180064 A KR 1020140180064A KR 20140180064 A KR20140180064 A KR 20140180064A KR 20160072451 A KR20160072451 A KR 20160072451A
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- mushroom
- vector
- protein
- growth hormone
- recombinant
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- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
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Abstract
Description
본 발명은 새송이버섯 형질전환용 재조합 벡터를 이용한 재조합 인간 성장 호르몬 (human growth hormone) 단백질 제조방법에 관한 것이다.
The present invention relates to a method for producing a recombinant human growth hormone protein using a recombinant vector for transformation of mushroom mushroom.
유용 단백질 소재는 인체 치료용뿐만 아니라 산업용으로도 사용함으로써 그 활용 폭이 점점 확대되고 있어, 전 세계적으로 그 중요성이 부각되고 있다. 그러나 이러한 유용 단백질들은, 특히 동물 유래의 단백질의 경우 생체에서 미량으로 만들어지기 때문에 치료 목적으로 단백질을 사용하기 위해서는 대량 생산이 필요할 뿐만 아니라, 혈액에서 분리하는 단백질의 경우에는 바이러스 등의 감염 위험성, 잠재적인 암 유발인자의 잔류 가능성 등과 같은 심각한 문제점을 가지고 있다. 상기의 여러 가지 문제점을 해결하기 위하여 유용 단백질의 유전자를 미생물 등의 단백질 발현 숙주 세포에서 대량으로 생산하는 유전자 재조합 기술을 이용함으로써 해결할 수가 있다. 유전자 재조합 기술은 산업적, 의학적으로 중요한 기능을 갖는 단백질(효소, 호르몬, 성장인자 등)을 미생물, 효모 등의 세포에 해당 유전자를 도입하여 목적하는 단백질을 합성하도록 하는 기술로서 생체 유래 생리 활성 단백질의 대량생산이 가능하게 하는 기술이다. 재조합 단백질 제품으로 1982년 Genentech 사가 대장균에서 생산한 사람 인슐린이 FDA의 승인받아 최초로 판매된 이래로, 산업자원부의 산업기술개발 사업보고서에 의하면 단백질 제품은 2005년 기준 약 130여 종의 단백질 제품이 판매되고 있으며, 세계 시장 규모는 약 500억 달러에서, 2011년에는 1,100억 달러에 이를 것으로 예측되고 있는 고부가가치의 산업으로 전 세계적으로 100개 이상의 다국적기업이 참여하고 있는 것으로 알려져 있다. 따라서 유전자 재조합 기술을 이용한 단백질 생산은 단백질 시장에 매우 빠른 속도로 확장되고 있다. 재조합 단백질은 사용 목적, 단백질의 종류 및 생산 규모에 따라 여러 종류의 발현시스템을 사용하여 생산되고 있으며 대표적으로 많이 쓰이는 세포는 대장균이나 효모와 같은 미생물을 이용하거나 곤충세포, 동물세포, 식물세포를 이용하고 있다. 특히, 대장균을 이용한 단백질 발현은 유전자 조작이 쉽고, 균체의 성장 속도가 빠르며, 배양이 용이하고, 비교적 많은 양으로 생산하여 경제적인 방법으로 단백질 합성이 가능하다. 그러나 당화 단백질(glycoprotein)과 같은 진핵세포 유래의 단백질을 생산하고자 할 경우에는 대장균이 당화 단백질을 합성하지 못하고 많은 단백질의 경우 접힘(folding)이 제대로 이루어지지 않는 봉입체(inclusion body) 형태의 불용성 단백질로 생산되어 생물학적 활성을 갖게 하기 위해서는 재접힘(refolding) 공정이 필요하고, 분비 단백질의 생산이 어려움 등의 여러 가지 문제점을 가지고 있다. 초기의 재조합 단백질 의약품은 주로 대장균에서 생산하였으나 기술이 발전함에 따라 2000년대에는 대장균에서 생산한 재조합 단백질의 비율이 감소한 반면, 효모나 곤충, 식물 및 동물세포에서의 생산이 증가하고 있다. 동물세포나 곤충세포의 경우에는 인체 유래 단백질을 대부분 완전한 활성형의 단백질로 생산이 가능하다는 장점을 가지고 있으나 배양 시간이 길며, 배양 조건이 까다롭고, 배지의 가격이 비싸다는 문제점을 가지고 있다. 식물을 이용하여 생산한 재조합 단백질은 인체 유래 유용 단백질들의 생물학적 활성에 필수적인 당화, 인산화 등과 같은 화학적 수식 작용을 가지고 있다. 또한 생물학적 안전성을 갖는 단백질을 생산할 수 있다는 장점을 가지고 있으며, 당(sugar)과 염(salt)을 주 배지 성분으로 사용하여 경제적인 면에서도 저렴하게 생산이 가능하나 느린 성장속도, 낮은 수율 등의 단점들을 지니고 있다(Biotechnol. Bioeng. 1997, 56, 473-484). 상기의 재조합 단백질의 생산 시스템이 가지고 있는 여러 가지 문제점들로 인하여 현재 새로운 재조합 단백질의 생산 시스템의 필요성이 대두 되고 있다.Useful protein materials are used not only for the treatment of human body but also for industrial use, and their use is getting wider, and their importance is becoming increasingly important worldwide. However, these useful proteins, especially those derived from animals, are produced in a very small amount in the living body. Therefore, in order to use proteins for therapeutic purposes, mass production is necessary. In the case of proteins separated from blood, And the possibility of residual cancer inducing factors. In order to solve the above-mentioned problems, the present invention can be solved by using a gene recombination technique which mass-produces a gene of a useful protein in a protein expression host cell such as a microorganism. BACKGROUND ART [0002] Genetic recombination technology is a technology for synthesizing a desired protein by introducing a gene having an important function for industrial and medical functions (enzyme, hormone, growth factor, etc.) into a cell such as microorganism or yeast, It is a technology that enables mass production. Since recombinant protein products were first sold by Genentech in 1982 to E. coli, insulin was approved by the FDA. According to the Industrial Technology Development Project of the Ministry of Commerce, Industry and Energy, about 130 kinds of protein products were sold in 2005 It is a high-value-added industry with a global market size estimated at about $ 50 billion, and in 2011 it is expected to reach $ 110 billion, with more than 100 multinational corporations participating worldwide. Therefore, protein production using genetic recombination technology is rapidly expanding into the protein market. Recombinant proteins are produced using various expression systems depending on the purpose of use, the type of protein, and the production scale. Typically used cells are microorganisms such as Escherichia coli or yeast, or cells such as insect cells, animal cells, and plant cells . In particular, protein expression using E. coli is easy to genetically manipulate, has a high growth rate of cells, is easy to cultivate, can be produced in a relatively large amount, and can be synthesized in an economical manner. However, when it is intended to produce proteins derived from eukaryotic cells such as glycoprotein, E. coli can not synthesize glycosylated proteins, and many proteins are insoluble proteins in the form of inclusion bodies which do not fold properly In order to produce biologically active products, refolding process is required and production of secreted proteins is difficult. Early recombinant protein drugs were mainly produced in E. coli, but as technology developed, the proportion of recombinant proteins produced in E. coli decreased in the 2000s, while production in yeast, insects, plant and animal cells is increasing. In the case of animal cells or insect cells, most of the protein derived from human body can be produced as a fully active protein, but it has a problem that the culture time is long, the culture condition is difficult, and the cost of the culture medium is high. The recombinant proteins produced using plants have chemical modifying functions such as saccharification and phosphorylation, which are essential for the biological activity of useful proteins derived from human body. In addition, it has the advantage of producing biologically stable proteins. It can be produced economically inexpensively by using sugar and salt as main components. However, it has disadvantages such as slow growth rate and low yield ( Biotechnol. Bioeng., 1997, 56, 473-484). Due to various problems of the recombinant protein production system, there is a need for a new recombinant protein production system.
버섯은 분류학상으로 고등균류 중 진균류(Eumycetes)에 속하며, 대부분은 담자균류에 속한다. 새송이버섯은 남유럽 일대, 북아프리카, 동아시아, 남러시아 등지에 분포하고 있으며 식용으로 널리 사용되어 왔고, 학명은 Pleurotus eryngii이며, 분류학적으로 진정담자균강, 동담자균아강(모균아강), 주름버섯목, 느타리과에 속하는 담자균류(Basidiomycetes)의 일종이다. 일반명은 왕굴버섯(King Oyster Mushroom) 또는 초원버섯(Boletus of the Steppes)이며, 우리나라에서는 큰느타리버섯 또는 왕느타리버섯으로 알려져 왔다. 이 버섯은 죽은 나무의 그루터기 또는 줄기에 부착하여 자생하는 기생균으로서 몇몇 식물의 조직에서도 생장이 가능하며, 산형과, 분과, 부처꽃과 등 초본식물의 뿌리에 질병을 유발시키는 병원균으로 보고되어 있다.Mushrooms are taxonomically belonging to the fungi (Eumycetes) of higher fungi, most of which belong to the family Mycobacterium. The mushroom is distributed in southern Europe, North Africa, East Asia, South Russia and has been widely used for edible purposes. Its scientific name is Pleurotus eryngii. It is taxonomically classified into the genus Bacillus subtilis, (Basidiomycetes) belonging to the genus. The common name is King Oyster Mushroom or Boletus of the Steppes, and it has been known in Korea as large oyster mushroom or king oyster mushroom. This mushroom is a parasitic organism that attaches to the stump or stem of a dead tree and is able to grow in some plant tissues and has been reported as a pathogen causing illnesses in the roots of herbaceous plants such as mountain type,
한편, 그람 음성 박테리아인 아그로박테리움 투메파시언스(Agrobacterium tumefaciens)은 식물에 뿌리혹병(crown gall)을 일으키는 식물 병원성 균으로 알려져 있다. 아그로박테리움 투메파시언스는 식물 세포 염색체 DNA에 끼어들어갈 수 있는 Ti 플라스미드 DNA를 가지고 있어 목적하고자 하는 유전자를 식물로 도입하는데 많이 이용되고 있다. Ti 플라스미드에는 Ti DNA 운송, 식물세포 염색체 DNA에 삽입되는 과정에 필요한 여려 가지 vir gene 들을 가지고 있으며, 아세토시린곤(acetosyringone)과 같은 방향족 화합물(phenolic compounds)에 의하여 유도되어 Ti 플라스미드 DNA가 식물세포로 운송되어 단일사슬 DNA 형태로 숙주세포의 염색체 DNA에 삽입되는 과정을 거친다 (Plant Physiol, 2000, 124, 13631371). 아그로박테리움으로 매개하여 식물의 형질전환은 식물에 유전자를 도입하기 위하여 오래 동안 사용되어 왔으며, 식물에 돌연변이 도입에도 사용되어 왔으며, 백색부후균의 형질전환 방법으로도 이용되고 있다. 또한 아스퍼질러스(Aspergillus; 누룩곰팡이), 푸사리움(Fusarium; 곰팡이)와 트리코더마(Trichoderma; 곰팡이) 등과 같은 여러 가지 곰팡이 에도 성공적인 형질전환 방법으로 사용되어 왔다(Nature Biotechn., 1998, 16, 839-842; J. Appl. Microbiol, 2002, 92, 189-195; Symbiosis. J, 2006, 41, 71-79). 그러나 현재까지 버섯류에서 유용한 단백질을 생산하는 유전자를 도입하는 형질전환시스템은 시도된 연구가 매우 제한적이어서, 유용 재조합 단백질 생산에 관한 산업화 연구는 아직까지 많이 이루어지지 않고 있다. On the other hand, gram-negative bacterium, Agrobacterium-to-mepa sieon's (Agrobacterium tumefaciens) is known as a plant pathogenic fungus that causes clubroot (crown gall) to the plant. Agrobacterium tumefaciens has a Ti plasmid DNA that can interfere with plant cell chromosomal DNA and is widely used to introduce a desired gene into a plant. The Ti plasmid contains a number of vir genes necessary for the transport of Ti DNA and its insertion into plant cell chromosomal DNA and is induced by phenolic compounds such as acetosyringone, ( Plant Physiol, 2000, 124, 13631371). ≪ Desc /
유용 단백질 중 하나인 성장 호르몬(growth hormone)은 뇌하수체전엽에서 217개의 아미노산으로 구성된 단백질로 합성된 후, N-말단 부분의 리더서열(leader peptide)이 제거되어 191개(N-말단 알라닌) 또는 190개(N-말단 페닐알라닌) 길이의 분자량이 약 22,000 달톤에 달하는 단백질로 전환되어 생물학적 활성을 갖는다(Pro. Natl. Acad. Sci. 1977, 74, 2432-2436). 성장호르몬의 분비조절은 시상하부(hypothalamus)에서 분비되는 성장호르몬 방출 호르몬(growth hormone releasing hormone, GHRH)에 의해 자극받고, 소마토스타틴(somatostatin)에 의해 분비가 억제된다. 분비된 성장호르몬은 간에서 인슐린유사 성장인자-1(insulin-like growth factor-1, IGF-1)의 생산을 유도하여 단백질, 탄수화물, 지방 및 무기물 대사 조절에 관여하여 성장을 촉진시키는 기능을 하는 단백질로서(Nature, 1982, 300, 611-615), 성장호르몬이 결여된 왜소증 환자의 치료 및 축산업에서 동물의 성장을 촉진하는데 이용이 되고 있다. Growth hormone, one of the useful proteins, is synthesized as a protein consisting of 217 amino acids in the anterior pituitary gland, and then the leader peptide of the N-terminal part is removed, resulting in 191 (N-terminal alanine) or 190 (N-terminal phenylalanine) is converted into a protein having a molecular weight of about 22,000 daltons and has biological activity ( Pro. Natl. Acad. Sci. 1977, 74, 2432-2436). Growth hormone secretion regulation is stimulated by growth hormone releasing hormone (GHRH) secreted from the hypothalamus and secreted by somatostatin. The secreted growth hormone is involved in the regulation of the metabolism of proteins, carbohydrates, fats and minerals by inducing the production of insulin-like growth factor-1 (IGF-1) in the liver, As a protein ( Nature, 1982, 300, 611-615), it is being used to promote animal growth in the treatment and livestock industry of dwarfism patients lacking growth hormone.
본 발명은 유용한 재조합 단백질을 생산할 수 있는 형질전환 백색부후균을 얻기 위하여 아그로박테리움을 이용하여 새송이버섯의 형질전환 방법 및 이러한 방법에 의해 얻은 새송이버섯 형질전환체를 제공하며, 짧은 시간 내에 활성을 갖는 유용 재조합 단백질을 대량생산할 수 있음을 확인하고 본 발명을 완성하였다.The present invention provides a method for transformation of mushroom mushroom using Agrobacterium to obtain a transformed white rot fungus capable of producing a useful recombinant protein, and a mushroom transformant obtained by this method. And thus the present invention has been completed.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 새송이버섯 형질전환용 재조합 벡터 및 이를 이용한 재조합 인간 성장 호르몬 단백질 제조방법을 개발하고자 예의 연구 노력하였다. 그 결과, 새송이버섯 형질전환에 적합한 발현 컨스트럭트를 포함하는 벡터를 구축하였고, 이를 이용하여 새송이 버섯을 형질전환시켜 성공적으로 목적 단백질을 생산함으로써 본 발명을 완성하게 되었다
The present inventors have made extensive efforts to develop a recombinant vector for transformation of mushroom mushroom and a method for producing recombinant human growth hormone protein using the recombinant vector. As a result, a vector containing an expression construct suitable for transformation of mushroom mushroom was constructed, and the mushroom was transformed using this vector to successfully produce the desired protein, thereby completing the present invention
상기한 목적을 달성하기 위한 본 발명의 특징은, 새송이버섯 형질전환용 재조합 벡터를 제공하는데 있다. In order to accomplish the above object, a feature of the present invention is to provide a recombinant vector for transformation of mushroom mushroom.
그 결과, 새송이버섯 형질전환에 적합한 발현 컨스트럭트를 포함하는 벡터를 구축하였고, 이를 이용하여 버섯을 형질전환시켜 성공적으로 목적 단백질을 생산하였다. As a result, a vector containing an expression construct suitable for transformation of mushroom mushroom was constructed, and the target protein was successfully produced by transforming mushroom using the vector.
본 발명의 버섯 형질전환용 재조합 벡터에서 프로모터로서 CaMV 35S 프로모터(cauliflower mosaic virus 35S promoter)가 이용된다. 그리고 본 발명의 다른 특징은, 본 발명에서 사용한 비해석 부위는 CaMV 35S 유래의 비해석 부위이다.
The
본 발명은 버섯 형질전환에 적합한 발현 컨스트럭트를 포함하는 벡터 및 이를 이용한 형질전환 버섯으로부터 인간 성장 호르몬 단백질 등의 목적 단백질을 성공적으로 제조하는 방법을 제공한다. The present invention provides a method for successfully producing a target protein such as a human growth hormone protein from a vector containing an expression construct suitable for mushroom transformation and a transforming mushroom using the same.
이러한 결과를 바탕으로 재조합 발현 벡터는 새송이버섯을 형질전환 하는 데에 매우 적합하며, 다양한 단백질을 버섯, 특히 새송이버섯에서 발현 및 수득할 수 있도록 한다.Based on these results, recombinant expression vectors are well suited for transformation of mushrooms and allow the expression and acquisition of a variety of proteins in mushrooms, particularly mushrooms.
또한, 대량생산이 가능한 단백질 발현시스템 확립으로 산업적으로 활용이 가능하게 한다.
In addition, it can be used industrially by establishing protein expression system capable of mass production.
도 1은 본 발명에 따른 재조합 인간 성장 호르몬을 포함하는 벡터의 제작과정을 나타내는 도면,
도 2는 본 발명에 따라 재조합 인간 성장 호르몬을 포함하는 벡터의 DNA를 주형으로 하여 PCR로 분석한 hGH 유전자를 확인한 전기영동 사진,
도 3는 본 발명에 따른 재조합 인간 성장 호르몬을 포함하는 벡터로 형질전환 방법을 나타내는 도면,
도 4는 본 발명에 따라 재조합 인간 성장 호르몬을 포함하는 벡터로 형질전환된 새송이버섯 형질전환체의 염색체 DNA를 주형으로 하여 PCR로 분석한 hGH 유전자를 확인한 전기영동 사진.
도 5은 본 발명에 따라 재조합 인간 성장 호르몬을 포함하는 벡터로 형질전환된 새송이버섯 균사체를 배양하였을 때 발현된 hGH 단백질을 웨스턴블럿 방법으로 분석한 사진이다. Brief Description of Drawings Fig. 1 is a diagram showing a process for producing a vector containing recombinant human growth hormone according to the present invention,
FIG. 2 is an electrophoresis image showing the hGH gene analyzed by PCR using DNA of a vector containing a recombinant human growth hormone according to the present invention as a template,
FIG. 3 is a diagram showing a method of transforming a vector containing recombinant human growth hormone according to the present invention,
FIG. 4 is an electrophoresis image of a hGH gene analyzed by PCR using a chromosomal DNA of a transformed mushroom transformant transformed with a vector containing recombinant human growth hormone according to the present invention as a template.
FIG. 5 is a photograph showing Western blot analysis of expressed hGH protein when cultured Mycelium of mushroom cultured with a vector containing recombinant human growth hormone according to the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
실시예 Example
<실시예 1> 재조합 단백질을 생산하는 벡터 제조≪ Example 1 > Preparation of vector for producing recombinant protein
<1-1> 벡터의 제조<1-1> Preparation of vector
대장균 배양액으로부터 알칼리 분리 방법(alkaline lysis method) 에 따라 분리한 pPEV 벡터를 BamHI으로 37℃에서 2시간 동안 반응시키고 아가로스 겔 전기영동으로 약 10.6 kb 크기의 DNA 단편을 아가로스 겔로부터 추출하였다. The pPEV vector isolated from the Escherichia coli culture by alkaline lysis method was reacted with Bam HI at 37 ° C for 2 hours, and a DNA fragment of about 10.6 kb in size was extracted from the agarose gel by agarose gel electrophoresis.
<1-2> hGH 유전자의 제조≪ 1-2 > Production of hGH gene
인간 성장호르몬 유전자의 경우 hGH가 클로닝 되어있는 pGEMT-hGH 플라스미드로부터 BamHI로 37℃에서 2시간 동안 반응시키고 아가로스 겔 전기영동으로 약 0.57 kb 크기의 DNA 단편을 아가로스 겔로부터 추출하여 pPEV 벡터에 클로닝할 hGH 유전자 DNA로 사용하였다. In the case of the human growth hormone gene, a pGEMT-hGH plasmid in which hGH was cloned was reacted with Bam HI at 37 ° C for 2 hours, and a DNA fragment of about 0.57 kb in size was extracted from the agarose gel by agarose gel electrophoresis, And used as hGH gene DNA to be cloned.
<1-3> 재조합 유전자 벡터의 제조 ≪ 1-3 > Preparation of Recombinant Gene Vector
상기 실시예 <1-1>에서 제조한 접착 말단(cohesive end)을 갖는 pPEV 벡터와 상기 실시예 <1-2>에서 제조한 hGH 유전자를 Sam Brook 등의 방법에 따라 pPEV 벡터와 hGH 유전자 단편을 라이게이션(ligation)하고 라이게이션 반응액을 대장균(E. coli XL-1 Blue MRF')에 형질전환 하였다(Proc. Natl. Acad. Sci. U.S.A., 1972, 69, 2110-2114). LB 배지에 접종한 E. coli XL-1 Blue MRF'를 600 nm에서 흡광도가 약 0.5가 될 때까지 37℃에서 200 rpm으로 교반하면서 배양하고, 3500 rpm으로 10분간 원심분리하여 대장균을 회수하고 차가운 0.1 M CaCl2 용액으로 간단하게 세척하여 준 후, 얻어진 대장균을 1/10 부피의 차가운 0.1 M CaCl2 용액에 잘 현탁하여 얼음에서 30분 동안 방치하였다. 원심분리하여 CaCl2로 처리된 대장균을 회수하여 1/2 부피의 차가운 0.1 M CaCl2 용액에 잘 현탁하였다. 대장균 현탁액 200 ㎕에 상기의 방법으로 각각 제조한 라이게이션 반응액을 잘 혼합하여 주고, 얼음에서 30분 동안 방치하고 42℃에서 1분 동안 열 충격을 가한 후 얼음에서 2분 동안 방치하였다. 이 반응액에 0.5 ㎖의 LB 배지를 가하고 37℃에서 1시간 동안 방치하고, 이 용액으로부터 0.1 ㎖을 50 ㎍/㎖의 카나마이신(kanamycin)을 포함하는 LB 한천 배지에 도말하고 37℃에서 하루 동안 배양하여 콜로니(colony)가 생성되도록 하였다. 선별된 콜로니로부터 알카리 분해법으로 재조합 벡터를 분리하고, hGH 유전자가 클로닝되어 있는 클론은 PCR을 이용하여 약 0.57 kb 크기의 DNA 밴드를 을 확인하였으며 제조된 재조합 벡터를 pPEV-hGH로 명명하였다(도 1 및 도 2 참조).
The pPEV vector having the cohesive end prepared in Example <1-1> and the hGH gene prepared in Example <1-2> were transformed into pPEV vector and hGH gene fragment by the method of Sam Brook et al. Ligation and the ligation reaction solution was transformed into E. coli XL-1 Blue MRF '( Proc. Natl. Acad. Sci. USA, 1972, 69, 2110-2114). The E. coli XL-1 Blue MRF inoculated on LB medium was cultured at 600 nm with stirring at 200 rpm at 37 DEG C until the absorbance reached about 0.5, and E. coli was recovered by centrifugation at 3500 rpm for 10 minutes, After briefly washing with 0.1 M CaCl 2 solution, the obtained E. coli was suspended in 1/10 volume of cold 0.1 M CaCl 2 solution and left on ice for 30 minutes. The CaCl 2 -treated E. coli was recovered by centrifugation and suspended in 1/2 volume of cold 0.1 M CaCl 2 solution. The ligation reaction mixture prepared in the above manner was mixed well in 200 μl of Escherichia coli suspension, allowed to stand in ice for 30 minutes, thermally shocked at 42 ° C for 1 minute, and left on ice for 2 minutes. 0.5 ml of LB medium was added to the reaction solution, and the mixture was allowed to stand at 37 占 폚 for 1 hour. From this solution, 0.1 ml was plated on LB agar medium containing 50 占 퐂 / ml of kanamycin and cultured at 37 占 폚 for one day To produce a colony. The recombinant vector was isolated from the selected colonies by the alkaline digestion method. The clone in which the hGH gene was cloned was confirmed to have a DNA band of about 0.57 kb by PCR, and the recombinant vector thus constructed was named pPEV-hGH And Fig. 2).
<실시예 2> 형질전환≪ Example 2 > Transformation
<2-1> 아그로박테리움의 형질전환<2-1> Transformation of Agrobacterium
새송이버섯(Pleurotus eryngii)에 상기 실시예 1에서 제조한 pPEV-hGH를 도입시키기 위하여 아그로박테리움에 의한 형질전환 방법을 사용하였다(Plasmid, 1978, 1, 456-467). YEP 배지(1% Bacto tryptone, 1% yeast extract, 0.5% NaCl)에 접종한 아그로박테리움 투메파시언스(Agrobacterium tumefaciens) LBA4404를 600 nm에서 흡광도가 약 0.5 가 될 때까지 37에서 200 rpm으로 교반하면서 배양하고, 배양액을 얼음에서 10분간 방치하여 냉각시켰다. 3500 rpm으로 10분간 원심분리하여 균을 회수하고 1 ㎖의 차가운 20 mM CaCl2 용액에 잘 현탁하고, 아그로박테리움 현탁액 200 에 상기의 방법으로 각각 제조한 재조합 벡터 pPEV-hGH 용액을 잘 혼합하여 주고, 혼합액을 액체 질소에 방치하여 급속 냉동시킨 후, 얼려진 각 혼합액을 37℃에서 5분 동안 방치하여 녹여 주었다. 그런 후, 혼합액의 10배 부피의 YEP 배지를 가하여 28℃에서 2시간 동안 잘 저어 주면서 균을 배양하였다. 3500 rpm으로 10분간 원심분리하여 균을 회수하고, 균을 0.3 ㎖의 YEP 배지에 잘 현탁시키고, 이 중 0.1 ㎖을 취하여 50 ㎍/㎖의 카나마이신을 포함하는 YEP 한천 배지에 도말하고 37℃에서 하루 동안 배양하여 콜로니(colony)가 생성되도록 하였다. 선별된 콜로니로부터 알카리 분해법으로 각 재조합 벡터를 분리하고, 실시예 1-3에 기재한 방법으로 hGH 유전자가 클로닝되어 있는 클론을 확인하였으며(도 2 참조), DNA 시퀀싱에 의하여 재조합 벡터에 삽입된 유전자의 서열을 최종적으로 확인되었다. In order to introduce the pPEV-hGH prepared in Example 1 into Pleurotus eryngii , Agrobacterium-mediated transformation was used ( Plasmid , 1978, 1, 456-467). Agrobacterium tumefaciens LBA4404 inoculated on YEP medium (1% Bacto tryptone, 1% yeast extract, 0.5% NaCl) was stirred at 37 to 200 rpm until the absorbance reached about 0.5 at 600 nm , And the culture solution was allowed to stand in ice for 10 minutes and then cooled. The bacteria were recovered by centrifugation at 3500 rpm for 10 minutes, suspended well in 1 ml of cold 20 mM CaCl 2 solution, and the recombinant vector pPEV-hGH solution prepared in the above manner was mixed well in Agrobacterium suspension 200 , The mixed solution was left to stand in liquid nitrogen for rapid freezing, and each frozen mixed solution was left to stand at 37 ° C for 5 minutes to dissolve it. Then, 10 times as much volume of YEP medium as the mixed solution was added, and the bacteria were cultured while being stirred at 28 ° C for 2 hours. The bacteria were recovered by centrifugation at 3500 rpm for 10 minutes, the bacteria were suspended in 0.3 ml of YEP medium, 0.1 ml of the suspension was plated on a YEP agar medium containing 50 μg / ml of kanamycin, Lt; RTI ID = 0.0 > colony. ≪ / RTI > Each recombinant vector was isolated from the selected colonies by the alkaline digestion method, and the clone in which the hGH gene was cloned was confirmed by the method described in Example 1-3 (see Fig. 2). The gene inserted into the recombinant vector by DNA sequencing Was finally confirmed.
<2-2> 아그로박테리움 투메파시언스에 의한 새송송버섯의 형질전환≪ 2-2 > Transformation of new mushroom by Agrobacterium tumefaciens
아그로박테리움 투메파시언스에 의한 새송이버섯의 형질전환은 Michielse 등의 방법을 약간 수정하여 사용하였다(Nat. Protoc, 2008, 3, 16711678). 간단하게, 상기 실시예 1에서 제조한 pPEV-hGH로 형질전환된 아그로박테리움 LBA4404를 50 ㎍/㎖의 카나마이신을 포함하는 5 ㎖의 최소배지(0.2% 글루코오스(glucose), 0.205% 다이포타슘 포스페이트(dipotassium phosphate; K2HPO4), 0.145% 모노포타슘 포스페이트(monopotassium phosphate; KH2PO4), 0.015% 소듐클로라이드(sodium choloride; NaCl), 0.05% 마그네슘 설페이트(magnesium sulfate; MgSO47H2O), 0.01% 칼슘클로라이드 헥사하이드레이트(calcium chloride hexahydrate; CaCl26H2O), 0.00025% 페러스 설페이트(ferrous sulfate; FeSO47H2O), 0.05% 암모늄 설페이트(ammonium sulfate; (NH4)2SO4))접종하고 28℃에서 2일 동안 잘 저어주면서 배양하였다. 아그로박테리움 배양액을 600 nm에서 흡광도가 약 0.5가 되게 유도배지(induction medium) (10 mM 글루코오스, 0.205% 다이포타슘 포스페이트, 0.145% 모노포타슘 포스페이트, 0.015% 소듐클로라이드, 0.05% 마그네슘 설페이트, 0.01% 칼슘클로라이드 헥사하이드레이트, 0.00025% 페러스 설페이트, 0.05% 암모늄 설페이트, 0.5% 글라이세롤(glycerol), 200 uM 아세토시린곤(acetosyringone), 40 mM 모르폴리노에탄설폰산(2-morpholinoethanesulfonic acid; MES)으로 희석하고 같은 조건에서 3시간 동안 더 배양하여 준 후, 동일한 부피의 새송이버섯 분생자 현탁액(conidial suspension, 106 conidia/)과 혼합하여 주었다(Eur. Mol. Biol. Organ, 1995, 14, 3206-3214). 이 혼합액 200 ㎕를 3 MM filter paper(왓트만사, 미국)에 가하고, 200 uM의 아세토시린곤을 포함하는 유도배지 및 아세토시린곤을 포함하지 않는 유도배지에 각각 상기의 이 필터페이퍼를 담가두고 28℃에서 2일 동안 공동배양(co-cultivation)하였다. 각 공동배양 후에, 3 MM 필터페이퍼에서 자란 버섯균(새송이버섯; Pleurotus eryngii) 및 미생물균(아그로박테리움 투메파시언스)을 200 uM 세포탁심을 포함하는 PDA(potato dextrose agar) 배지로 옮겨 아그로박테리움을 죽게 하였다. PDA 배지 상에서 자란 각 형질전환된 새송이버섯의 분생자(conidia)를 멸균수에 현탁하고 200 uM 세포탁심을 포함하는 PDB 배지(0.4% 감자전분(Potato Starch), 2% 덱스트로즈(Dextrose))에 접종하고 28℃에서 100 rpm으로 진탕배양하였다. PDB 배지에서 자란 새송이버섯 형질전환체를 원심분리하여 회수하고, 여러 가지 분석을 위하여 사용하였다.
Transformation of the mushroom by Agrobacterium tumefaciens was slightly modified by Michielse et al . ( Nat. Protoc, 2008, 3, 16711678). Briefly, Agrobacterium LBA4404 transformed with pPEV-hGH prepared in Example 1 was inoculated into 5 ml of a minimal medium (0.2% glucose, 0.205% diapotassium phosphate (pH 7.0) containing 50 μg / ml of kanamycin dipotassium phosphate; K 2 HPO 4) , 0.145% mono-potassium phosphate (monopotassium phosphate; KH 2 PO 4 ), 0.015% sodium chloride (sodium choloride; NaCl), 0.05 % of magnesium sulfate (magnesium sulfate; MgSO 4 7H 2 O), 0.01% calcium chloride hexahydrate (CaCl 2 6H 2 O), 0.00025% ferrous sulfate (FeSO 4 7H 2 O), 0.05% ammonium sulfate (NH 4 ) 2 SO 4, ) And incubated at 28 째 C for 2 days while stirring well. Agrobacterium cultures were grown in an induction medium (10 mM glucose, 0.205% diphotassium phosphate, 0.145% monopotassium phosphate, 0.015% sodium chloride, 0.05% magnesium sulfate, 0.01% calcium (2-morpholinoethanesulfonic acid (MES)) in the presence of 0.00025% ferrous sulfate, 0.05% ammonium sulfate, 0.5% glycerol, 200 uM acetosyringone and 40 mM morpholinoethanesulfonic acid Biol. Organ, 1995, 14, 3206-8) . The cells were then incubated for 3 h at the same conditions and then mixed with the same volume of conidial suspension (10 6 conidia / 3214). 200 占 퐇 of this mixed solution was added to a 3 MM filter paper (Whatman, USA), and an induction medium containing 200 占 아세 of acetosyringone and an induction medium containing no acetocyrinone Were co-cultivated for 2 days at 28 ° C. After each co-cultivation, the filter paper of 3 mm filter paper ( Pleurotus eryngii ) and microorganisms (Agrobacterium tumefaciens ) Were transferred to a PDA (potato dextrose agar) medium containing 200 uM cell troclin to cause Agrobacterium to die. The conidia of each transformed mushroom grown on the PDA medium were suspended in sterilized water (0.4% Potato Starch, 2% Dextrose) containing 200 uM cell trophoblasts and shake-cultured at 28 rpm at 100 rpm. [0091] The mushroom transformant grown in the PDB medium Were recovered by centrifugation and used for various analyzes.
<실시예 3> 형질전환된 새송이버섯의 염색체 DNA 분석Example 3: Chromosomal DNA analysis of transformed mushroom
상기 실시예 2에 의하여 얻어진 새송이버섯 형질전환체를 200 uM 세포탁심을 포함하는 PDB 배지에서 배양하고, 회수한 균사체로부터 염색체 DNA를 변형한 헥사데실트리메틸 암모늄 브로마이드(hexadecyltrimethylammonium bromide; CTAB) 방법(FOCUS, 1990, 12, 13-15)으로 분리하였다. 간단하게, 상기 실시예 2에서 얻어진 새송이버섯 균사체 0.1g 을 0.5 의 DNA 추출 완충액(0.1 M Tris-Cl (pH 9), 20 mM 에틸렌다이아민테트라아세트산(ethylenediaminetetraacetic acid; EDTA), 1.4 M 염화나트륨(NaCl), 2% 헥사데실트리메틸 암모늄 브로마이드, 1% 폴리에텔렌 글리콜(polyethylene glycol), 0.2% 베타-머캡토에탄올(-mercaptoethanol))를 가하여 조직을 파쇄하고, 74℃에서 70분간 열처리 하였다. 상기의 용액을 200 ㎕의 클로로포름/이소아밀알콜(chloroform/isoamyl alcohol) (24:1)로 추출하고, 0℃에서 30분간 방치한 후, 15,000 rpm으로 5분간 원심분리하여 상층액을 회수하였으며, 용출 추출과정을 한 번 더 반복하였다. 상층액을 회수하고 0.55배 부피의 이소프로판올(isopropanol)을 가하고 상온에서 5분간 방치한 후, 15,000 rpm으로 10분간 원심분리하여 침전물을 회수하여, 염색체 DNA를 얻었다. DNA 침전물을 50 ㎕의 40 ㎍/㎖의 리보핵산가수분해효소 A(RNase A)를 포함하는 TE(Tris-EDTA) 용액에 녹여, 중합효소 연쇄 반응(polymerase chain reaction; PCR)의 주형 DNA로 사용하였다. 이 염색체 DNA를 주형으로 하여 hGH 유전자 분석은 PCR 하여 hGH 유전자의 도입을 확인하였으며, 약 0.57 kb 크기의 DNA 밴드를 확인하여(도 4), hGH 유전자로 형질전환 여부를 확인하였으며 최종적으로 각 중합효소 연쇄 반응 산물을 클로닝하여 DNA 서열을 분석하여 최종적으로 정확한 서열의 유전자로 형질전환 되었음을 확인하였다.
The transformed mushroom transformant obtained in Example 2 was cultivated in a PDB medium containing 200 uM cytotoxicity, and hexadecyltrimethylammonium bromide (CTAB) method ( FOCUS, 1990, 12, 13-15). Briefly, 0.1 g of the mushroom mycelia obtained in Example 2 was dissolved in 0.5 ml of DNA extraction buffer (0.1 M Tris-Cl (pH 9), 20 mM ethylenediaminetetraacetic acid (EDTA), 1.4 M sodium chloride ), 2% hexadecyltrimethylammonium bromide, 1% polyethylene glycol, and 0.2% beta-mercaptoethanol) was added to the tissue and the tissue was disrupted and heat-treated at 74 ° C for 70 minutes. The above solution was extracted with 200 μl of chloroform / isoamyl alcohol (24: 1), left at 0 ° C. for 30 minutes, centrifuged at 15,000 rpm for 5 minutes to recover supernatant, The elution extraction procedure was repeated one more time. The supernatant was recovered and 0.55 volume of isopropanol was added. The mixture was allowed to stand at room temperature for 5 minutes and then centrifuged at 15,000 rpm for 10 minutes to recover the precipitate to obtain chromosomal DNA. The DNA precipitate was dissolved in 50 μl of a TE (Tris-EDTA) solution containing 40 μg / ml of ribonucleic acid hydrolase A (RNase A) and used as template DNA for polymerase chain reaction Respectively. Using this chromosomal DNA as a template, the hGH gene analysis was confirmed by PCR to confirm the introduction of the hGH gene. A DNA band of about 0.57 kb was confirmed (FIG. 4), and the hGH gene was transfected. Finally, The sequence product was cloned and the DNA sequence was analyzed to confirm that it was finally transformed into the gene of the correct sequence.
<실시예 4> 형질전환된 새송이버섯 균사체에서 hGH gene 의 발현 확인 Example 4 Expression of hGH Gene in Mycelia of Transgenic Shrimp Mushroom
pPEV-hGH 벡터로 형질전환된 새송이버섯 균사체를 PDB 배지에서 접종하고 25℃에서 5일 동안 100 rpm으로 진탕배양으로 배양하였다. 이 균 배양액 1 ㎖을 50 ㎖의 Mushroom Complete 배지(0.2% 트립톤(Bacto tryptone), 0.2% 효모추출물(yeast extract), 2% 글루코오스, 0.05% 마그네슘 설페이트)를 포함하는 250 ㎖의 삼각플라스크에 접종하고 25℃에서 7일 동안 100 rpm으로 진탕배양 하였다. 각 시간대별로 균사체를 회수하고, 회수한 균사체를 균사체 1 g 당 3 ㎖의 초음파 파쇄 버퍼(buffer)(10 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM 베타-머캡토에탄올)에 현탁하고, 균질기(homogenizer) 및 초음파 파쇄기로 새송이버섯 균사체를 파쇄하였다. 균사체를 파쇄 후, 15,000 rpm으로 5분 동안 원심분리하여 상층액을 회수하여 단백질 발현 분석 시료로 사용하였다. 세포 추출액 0.5 ㎖에 동일한 부피의 99% 에탄올을 가하여 잘 섞어준 후, -20℃에서 30분간 방치하고 12,000 rpm으로 10분 동안 원심분리하여 단백질 침전물을 회수하고, 이 침전물은 공기 중에 약 30분 간 방치하여 건조시켰다. 건조한 단백질 추출물은 20 ㎕의 증류수에 잘 현탁시키고 20 ㎕의 2X SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis) 샘플 버퍼(0.1 M Tris-HCl (pH 6.8), 0.2 M 디티오트레이톨(Dithiothreitol; DTT), 4% SDS, 20% 글리세롤, 0.2% 브로모페놀블루(bromophenolblue))를 가하고, 100℃에서 5분간 가열하여 단백질 전기영동(SDS-PAGE)방법으로 확인하였다(Nature, 1970, 227, 680-685). 일정한 전기장에서 분리가 끝난 젤은 쿠마시 브릴리언트 블루 용액(Coomassie brilliant blue)에 넣어 1시간 정도 염색한 후 탈색용액(30% 메탄올, 10% 아세트산)에 옮겨 탈색하여 생산된 단백질을 확인하였다. 또한 SDS-PAGE를 전개한 단백질을 PVDF(Polyvinylidene fluoride) 막(membrane)으로 이동시킨 후 토끼에서 제조한 anti-hGH 항체를 이용하여 형질전환된 새송이버섯 균사체 배양액에서 발현된 hGH 단백질을 공지의 항체 블럿 방법으로 확인하였다. 그 결과, 본 발명의 pPEV-hGH로 형질전환된 새송이버섯 균사체 배양액은 배양 시간이 지남에 따라 분자량 약 21 kDa의 hGH 단백질의 발현됨을 확인하였다(도 5 참조).The mushroom mycelium transformed with the pPEV-hGH vector was inoculated in PDB medium and cultured at 25 DEG C for 5 days with shaking at 100 rpm. 1 ml of the bacterial culture was inoculated into a 250 ml Erlenmeyer flask containing 50 ml of Mushroom Complete medium (0.2% Bacto tryptone, 0.2% yeast extract, 2% glucose, 0.05% magnesium sulfate) Lt; RTI ID = 0.0 > 25 C < / RTI > for 7 days. Mycelia were collected at each time zone, and the recovered mycelium was suspended in 3 ml of an ultrasonic disruption buffer (10 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM beta-mercaptoethanol) per g of the mycelium , A homogenizer and an ultrasonic crusher. The mycelium was disrupted and centrifuged at 15,000 rpm for 5 minutes. The supernatant was recovered and used as a protein expression assay sample. The same volume of 99% ethanol was added to 0.5 ml of the cell extract, followed by mixing well. The mixture was allowed to stand at -20 ° C for 30 minutes and centrifuged at 12,000 rpm for 10 minutes to recover the protein precipitate. Left to dry. The dried protein extracts were suspended in 20 μl of distilled water and suspended in 20 μl of 2 × SDS-PAGE buffer (0.1 M Tris-HCl (pH 6.8), 0.2 M dithiothreitol (DTT ), 4% SDS, 20% glycerol, 0.2% bromophenol was added and the blue (bromophenolblue)), was heated at 100
Claims (5)
(a) CaMV 35S 프로모터(cauliflower mosaic virus 35S promoter);
(b) CaMV 35S 5-비해석부위(untranslated sequence, UTS);
(c) CaMV 35S 3-비해석부위;
(d) NOS 터미네이터(nopaline synthase terminator); 및
(e) 상기 (b)와 (c) 사이에 위치한 목적 단백질 코딩 뉴클레오타이드 서열.A recombinant vector for mushroom transformation comprising:
(a) CaMV 35S promoter (cauliflower mosaic virus 35S promoter);
(b) CaMV 35S 5-untranslated sequence (UTS);
(c) CaMV 35S 3-noninterpected region;
(d) a nopaline synthase terminator; And
(e) a nucleotide sequence encoding a target protein located between (b) and (c) above.
The transformed mushroom according to claim 3, wherein the mushroom is a fresh mushroom.
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