KR100427299B1 - Recombination plasmid pGHOPG(KCTC 1019BP) to produce human osteoprotegerin - Google Patents

Recombination plasmid pGHOPG(KCTC 1019BP) to produce human osteoprotegerin Download PDF

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KR100427299B1
KR100427299B1 KR10-2001-0048270A KR20010048270A KR100427299B1 KR 100427299 B1 KR100427299 B1 KR 100427299B1 KR 20010048270 A KR20010048270 A KR 20010048270A KR 100427299 B1 KR100427299 B1 KR 100427299B1
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hopg
pghopg
kctc
plasmid
milk
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KR20030013968A (en
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이철상
성윤영
오건봉
박정선
이경광
이기녕
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한국생명공학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C12N2015/8518—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles

Abstract

본 발명은 인체 골 재흡수 억제인자(human osteoprotegerin, 이하 hOPG)를 생산하는 재조합 플라스미드 pGHOPG(KCTC 1019BP)에 관한 것으로서, 더욱 상세하게는 흑염소 유래의 5.5 kb 크기의 베타-카세인 유전자의 프로모터 및 인체 성장 호르몬 유전자의 엑손 1, 2, 인트론 1과 전사종결요소, 그리고 인체 OPG cDNA를 포함하는 신규 플라스미드 pGHOPG(KCTC 1019BP) 및 이를 사용하여 포유동물을 형질 전환시켜 유선에서만 조직 특이적으로 생물학적 활성을 가진 고농도의 hOPG가 함유된 유즙을 안정적으로 생산하는 방법에 관한 것이다.The present invention relates to a recombinant plasmid pGHOPG (KCTC 1019BP) that produces human osteoprotegerin (hOPG), and more specifically, to promoter and human growth of a 5.5 kb beta-casein gene derived from black goat. Novel plasmid pGHOPG (KCTC 1019BP) containing exons 1, 2, intron 1 and transcription terminators of hormone genes, and human OPG cDNA and transformed mammals using them to produce tissue-specific biological activity only in the mammary gland It relates to a method for stably producing milk containing hOPG.

Description

인체 골 재흡수 억제인자（ｈＯＰＧ）를 생산하는 재조합 플라스미드 ｐＧＨＯＰＧ（ＫＣＴＣ １０１９ＢＰ）{Recombination plasmid pGHOPG(KCTC 1019BP) to produce human osteoprotegerin}Recombination plasmid pGHOPG (KCTC 1019BP) to produce human osteoprotegerin}

본 발명은 인체 골 재흡수 억제인자(human osteoprotegerin, 이하 hOPG)를 생산하는 재조합 플라스미드 pGHOPG(KCTC 1019BP)에 관한 것으로서, 더욱 상세하게는 흑염소 유래의 5.5 kb 크기의 베타-카세인 유전자의 프로모터 및 인체 성장 호르몬 유전자의 엑손 1, 2, 인트론 1과 전사종결요소, 그리고 인체 OPG cDNA를 포함하는 신규 플라스미드 pGHOPG(KCTC 1019BP) 및 이를 사용하여 포유동물을 형질 전환시켜 유선에서만 조직 특이적으로 생물학적 활성을 가진 고농도의 hOPG가 함유된 유즙을 안정적으로 생산하는 방법에 관한 것이다.The present invention relates to a recombinant plasmid pGHOPG (KCTC 1019BP) that produces human osteoprotegerin (hOPG), and more specifically, to promoter and human growth of a 5.5 kb beta-casein gene derived from black goat. Novel plasmid pGHOPG (KCTC 1019BP) containing exons 1, 2, intron 1 and transcription terminators of hormone genes, and human OPG cDNA and transformed mammals using them to produce tissue-specific biological activity only in the mammary gland It relates to a method for stably producing milk containing hOPG.

OPG(osteoprotegerin)는 TNF(tumor necrosis factor) 수용체 계열의 하나로써, 막통과 도메인(transmembrane domain)과 세포질 도메인(cytoplasmic domain)이 결여된 분비형 당단백질이다[Simonet et al., Cell., 89, 309 (1997)]. 분비된 인간의 OPG 단백질은 생물학적 활성을 유도하는데 필수적인 N-말단의 시스테인-리치 도메인(cystein-rich domain) 4개, DDH(death domain homologous region)1, DDH2, 그리고 1개의 C-말단 도메인으로 이루어진 401개의 아미노산으로 구성되며, 분자량은 약 55 kDa이고 호모다이머(homodimer)를 형성하게 되면 약 110 kDa을 갖게 된다[Simonet et al., Cell., 89, 309 (1997); Tsuda et al., Biochem. Biophys. Res. Commun., 139, 1329 (1997)]. 이러한 OPG는 동물 실험들을 통하여 파골세포(osteoclast)에 의한 골 재흡수를 억제함으로써 골다공증의 치료제로 이용될 수 있음이 확인되었으며[Simonet et al., Cell., 89, 309 (1997); Yasudaet al., Endocrinology., 139, 1329 (1998); Bucay et al., Genes Dev., 12, 1260 (1998)], 최근에는 Amgen 사에 의해 OPG의 최초 임상적인 시도가 이루어졌다[Bekker et al., J Bone Miner Res., 16, 348 (2001)]. 즉, OPG를 단독으로 폐경기 여성에게 피하 주입할 경우, 골 재흡수와 관련된 골 교체(turnover)의 감소를 보여줌으로써 골다공증을 비롯한 류마티스 관절염, 염증성 관절염 등, 골의 손실과 관련된 질병의 치료제로서의 가능성을 제시하였다.Osteoprotegerin (OPG) is a family of tumor necrosis factor (TNF) receptors and is a secreted glycoprotein that lacks a transmembrane domain and a cytoplasmic domain [Simonet et al., Cell., 89, 309 (1997). Secreted human OPG protein consists of four N-terminal cystein-rich domains, a death domain homologous region (DDH) 1, DDH2, and one C-terminal domain essential for inducing biological activity. Consists of 401 amino acids, has a molecular weight of about 55 kDa and forms a homodimer and has about 110 kDa [Simonet et al., Cell., 89, 309 (1997); Tsuda et al., Biochem. Biophys. Res. Commun., 139, 1329 (1997). Animal experiments have shown that OPG can be used as a therapeutic agent for osteoporosis by inhibiting bone resorption by osteoclasts [Simonet et al., Cell., 89, 309 (1997); Yasuda et al., Endocrinology., 139, 1329 (1998); Bucay et al., Genes Dev., 12, 1260 (1998), recently the first clinical trial of OPG by Amgen (Bekker et al., J Bone Miner Res., 16, 348 (2001)). ]. In other words, when subcutaneous injection of OPG alone into a postmenopausal woman shows a decrease in bone turnover associated with bone resorption, it can be used as a therapeutic agent for diseases related to bone loss, including osteoporosis, rheumatoid arthritis and inflammatory arthritis. Presented.

한편, 유용한 당단백질을 지속적으로 대량 생산하는 방법으로 생체기관인 포유동물의 유선을 이용하는 방법이 있는데, 이는 외래 유전자가 유선 특이적으로 발현될 수 있도록 포유동물을 형질 전환시킴으로써 비유기의 유즙을 통해 목적하는 유용 단백질을 대량 생산하는 방법이다[Gordon et al.,Bio/Technol.,5, 1183 (1987)]. 동물의 유선에서 유용 단백질을 대량 생산하기 위해서는 유선 특이적 발현 벡타를 제작하여 그 발현능력을 실험동물에서 검증하는 과정이 선행되어야 한다. 유선 특이적 발현 벡타는 유용 단백질 유전자의 프로모터와 생산하려는 유용단백질의 유전자로 구성되는데, 일반적으로 유용단백질의 유전자로는 목적으로 하는 단백질의 게놈 유전자가 cDNA 유전자에 비해 월등히 효율적인 것으로 알려져 있으나, 게놈 유전자는 cDNA 유전자에 비해 길이가 훨씬 길어 다루기 어려울 뿐만 아니라, 클로닝 되어 있지 않는 경우도 있는 등, 활용상의 어려움이 많다[L.-M. Houdebine, J Biotech., 34, 269 (1994); N.S. Rudolph, TIBTECH., 17, 367 (1999)].On the other hand, there is a method of continuously producing a large amount of useful glycoproteins using a mammal's mammary gland, which is a living organ, which transforms a mammal so that foreign genes can be specifically expressed in mammary gland. Is a method of mass production of useful proteins [Gordon et al., Bio / Technol. , 5 , 1183 (1987). In order to mass-produce useful proteins in the mammary gland of animals, a process of preparing a mammary gland specific expression vector and verifying its expression ability in an experimental animal should be preceded. Mammary gland specific expression vectors consist of promoters of useful protein genes and genes of useful proteins to be produced. Generally, the genes of useful proteins are known to be much more efficient than cDNA genes. Is much more difficult to handle than cDNA genes, and it is difficult to use, such as when it is not cloned [L.-M. Houdebine, J Biotech., 34, 269 (1994); NS Rudolph, TIBTECH., 17, 367 (1999).

OPG의 게놈 유전자 역시 26 kb로서 매우 길기 때문에 유전자의 확보와 활용이 어려운 실정이다. 특히, OPG의 cDNA 유전자를 이용한 발현 연구에서는 발현양이 매우 낮은 수준을 보이고 있음이 일반적인데, 본 발명은 hGH 유전자의 일부를 함께 사용함으로써 이런 단점을 극복할 수 있었다는 것이다.The genomic gene of OPG is also very long as 26 kb, so it is difficult to secure and utilize the gene. In particular, the expression of the OPG cDNA gene is generally shown to show a very low level of expression, the present invention was able to overcome this disadvantage by using a portion of the hGH gene.

이에, 본 발명자들은 1.2 kb에 불과한 cDNA 유전자를 활용한 발현 벡타를 제작하고, 형질 전환 생쥐에서 유선 특이적으로 생물학적 활성을 가진 hOPG를 대량 발현시킴으로써 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by preparing an expression vector using a cDNA gene of only 1.2 kb and expressing large amounts of hOPGs with mammary gland specific biological activity in transgenic mice.

따라서, 본 발명은 포유동물의 유선에서 조직 특이적으로 고농도의 hOPG를 함유한 유즙을 안정적으로 생산하기 위한 신규 플라스미드 pGHOPG(KCTC 1019BP)와 이를 사용하여 생물학적 활성을 가진 hOPG를 생산하는 방법을 제공하는데 그 목적이 있다.Accordingly, the present invention provides a novel plasmid pGHOPG (KCTC 1019BP) for stably producing milk containing tissue-specific high concentrations of hOPG in mammalian mammary gland and a method for producing biologically active hOPG using the same. The purpose is.

도 1은 본 발명에 따른 플라스미드 pGHOPG(KCTC 1019BP)의 제작과정을 나타낸 것이다.Figure 1 shows the manufacturing process of the plasmid pGHOPG (KCTC 1019BP) according to the present invention.

도 2는 본 발명에 따른 형질 전환된 생쥐의 유선에서 hOPG mRNA를 노던 블럿(northern blot)으로 확인한 결과를 나타낸 것이다.Figure 2 shows the results confirmed by the Northern blot (northern blot) hOPG mRNA in the mammary gland of the transformed mouse according to the present invention.

도 3은 형질 전환된 생쥐의 여러 조직들을 대상으로 hOPG mRNA 발현이 조직 특이적으로 발현되는지를 알아보기 위해 노던 블럿(northern blot)으로 확인한 결과를 나타낸 것이다.Figure 3 shows the results confirmed by the Northern blot (northern blot) to determine whether the hOPG mRNA expression is tissue-specific expression in various tissues of the transformed mouse.

도 4는 형질 전환된 생쥐의 유즙에서 생산된 hOPG의 분비형태를, hOPG 특이적 항체를 이용한 웨스턴 블럿(western blot)으로 확인한 결과이다.4 is a result of confirming the secreted form of hOPG produced in the milk of the transformed mice by Western blot using hOPG-specific antibodies.

도 5는 유즙에 함유된 hOPG의 생물학적 활성을 확인한 TRAP 활성도 분석 결과이다.5 is a result of TRAP activity analysis confirming the biological activity of hOPG contained in milk.

본 발명은 5.5 kb 크기의 흑염소 유래의 베타-카제인 유전자 및 인체 성장 호르몬 유전자의 엑손 1, 2, 인트론 1과 전사종결요소, 그리고 인체 OPG cDNA를 포함하는 hOPG를 생산하는 재조합 플라스미드 pGHOPG(KCTC 1019BP)를 그 특징으로 한다.The present invention provides a recombinant plasmid pGHOPG (KCTC 1019BP) which produces hOPG comprising a beta-casein gene derived from a goat goat of 5.5 kb and exons 1, 2, intron 1 and transcription terminator of human growth hormone gene, and human OPG cDNA. It is characterized by.

또한, 본 발명은 상기의 신규 플라스미드 pGHOPG(KCTC 1019BP)를 이용하여 포유동물(인간 제외)에 형질 전환시키고, 형질전환된 동물의 유선에서 조직 특이적으로 생물학적 활성을 가진 hOPG를 생산하는 방법을 또 다른 특징으로 한다.The present invention also provides a method for transforming mammals (except humans) using the novel plasmid pGHOPG (KCTC 1019BP), and producing hOPG having tissue-specific biological activity in the mammary gland of the transformed animal. It is another feature.

이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다.Referring to the present invention in more detail as follows.

본 발명은 선출원한 인체 성장 호르몬 생산을 위한 형질 전환용 플라스미드 pGbc5.5hGH[대한민국 출원번호 제 1999-34972호]를 변형하여 hOPG를 포유동물(인간 제외)의 유선에서만 조직 특이적으로 발현시키기 위한 신규 플라스미드 pGHOPG(KCTC 1019BP)를 제조한 바, 상기의 플라스미드를 제조하는 과정을 구체적으로 설명하면 다음과 같다.The present invention is a novel transformation for transforming the plasmid pGbc5.5hGH [Korean Patent Application No. 1999-34972] for the production of human growth hormone, to specifically express tissue-specific expression of hOPG only in the mammary gland of mammals (except humans). When the plasmid pGHOPG (KCTC 1019BP) was prepared, a process of preparing the plasmid described above will be described in detail.

선출원한 pGbc5.5hGH 플라스미드의 인체 성장 호르몬 유전자 부위의 엑손 1과 엑손 2에 존재하는 단백질 합성 개시신호인 ATG 서열을 포함하는 부분을 각각SalI과HpaI 인식서열로 대체하여 인체 성장 호르몬의 합성을 불가능하게 한 후HpaI 위치에 hOPG cDNA를 삽입하였고, 그 이하 인체 성장 호르몬의 엑손 2부터 엑손 5에 존재하는PvuII 유전자 부위를 제거하여 신규 플라스미드 pGHOPG(KCTC 1019BP)를 제조하였으며, 이를 한국생명공학연구원 유전자은행에 2001년 5월 25일자로 수탁번호 KCTC 1019BP로 기탁하였다.Human growth hormone was synthesized by substituting Sal I and Hpa I recognition sequences, respectively, of the pGbc5.5hGH plasmid containing the ATG sequence, a protein synthesis initiation signal in exon 1 and exon 2 of the human growth hormone gene region. After disabling , hOPG cDNA was inserted at the Hpa I position, and a new plasmid pGHOPG (KCTC 1019BP) was prepared by removing the Pvu II gene region present in exon 2 to exon 5 of human growth hormone thereafter. It was deposited with Researcher Gene Bank under accession number KCTC 1019BP dated May 25, 2001.

한편, 본 발명은 상기 신규 플라스미드 pGHOPG(KCTC 1019BP)를 사용하여 포유동물(인간 제외)을 형질 전환하는 방법에 또 다른 특징이 있는 바, 이를 상세히 설명하면 다음과 같다.On the other hand, the present invention has another feature in the method of transforming mammals (except humans) using the novel plasmid pGHOPG (KCTC 1019BP), described in detail as follows.

본 발명은 상기 신규 플라스미드 pGHOPG(KCTC 1019BP)를 생쥐 수정란에 미세 주입하고, 미세 주입한 수정란을 대리모에 이식함으로써 형질 전환된 생쥐를 생산한다. 그런 다음, 형질 전환된 생쥐를 번식시켜 얻은 형질 전환 생쥐 암컷을대상으로 비유기의 유즙을 채취하여 hOPG의 발현 능력 및 함량, 생물학적 활성 여부를 알아본다.The present invention produces the transformed mice by micro-injection of the novel plasmid pGHOPG (KCTC 1019BP) into mouse fertilized eggs and transplanting the micro-implanted fertilized eggs into surrogate mothers. Then, the organic milk is collected from the transgenic mouse females obtained by breeding the transgenic mice to examine the expression ability, content, and biological activity of hOPG.

결과적으로, 본 발명의 형질 전환된 생쥐 4계통의 유즙에서 hOPG를 생산하고 있었으며, 그 중 두 계통에서는 hOPG를 각각 1.6, 2.0 ㎎/㎖의 높은 발현 수준을 나타낸다. 또한, 본 발명에서는 다른 조직에서는 발현되지 않는 매우 엄격한 유선 조직 특이성을 유지하고 있으며, 유즙에서의 단백질 크기도 인체내에서 발견되는 hOPG와 동일한 크기인 55 kDa의 정상적인 크기를 보였다. 뿐만 아니라, 유즙에 발현된 hOPG는 파골세포의 분화를 억제함으로써, 생물학적 활성을 가지고 있음이 확인되었다.As a result, the milk of four strains of the transformed mouse of the present invention was producing hOPG, two of which show a high expression level of 1.6, 2.0 mg / ㎖ hOPG, respectively. In addition, the present invention maintains a very strict mammary tissue specificity that is not expressed in other tissues, the protein size in milk also showed a normal size of 55 kDa, the same size as hOPG found in the human body. In addition, it was confirmed that hOPG expressed in milk has biological activity by inhibiting the differentiation of osteoclasts.

이하 본 발명을 실시예에 의거하여 상세히 설명하겠는 바, 본 발명이 다음 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples.

실시예 1 : OPG cDNA 발현 벡타(pGHOPG)의 제작Example 1 Preparation of OPG cDNA Expression Vector (pGHOPG)

① OPG cDNA의 PCR 합성과 합성한 cDNA 염기서열① PCR synthesis of OPG cDNA and cDNA base sequence synthesized

hOPG cDNA를 플라스미드에 도입하기 위하여,SmaI과SalI 제한효소 자리를 각각 포함하는 프라이머 1(서열번호 1: 5'-TCCCGGGGACCACAATGAAC-3')과 프라이머 2(서열번호 2: 5'-GGTCGACTTATAAGCAGCTTATTT-3')를 이용한 PCR(polymerase chain reaction)로 전체 길이 1.2 kb의 hOPG cDNA를 합성하였다. 합성한 cDNA의 염기서열은 서열번호 3과 같다. To introduce hOPG cDNA into the plasmid,SmaI andSalPolymerase chain reaction (PCR) using primer 1 (SEQ ID NO: 5'-TCCCGGGGACCACAATGAAC-3 ') and primer 2 (SEQ ID NO: 2: 5'-GGTCGACTTATAAGCAGCTTATTT-3'), each containing a restriction site 1.2 kb of hOPG cDNA was synthesized. The base sequence of the synthesized cDNA is shown in SEQ ID NO: 3.

② 선출원한 pGbc5.5hGH를 변형한 pGH의 제작② Preparation of modified pGbc5.5hGH pGH

도 1에서 나타난 바와 같이 인체 성장 호르몬 유전자 부위의 엑손 1과 엑손 2에 존재하는 단백질 합성 개시신호인 ATG 서열을 포함하는 부분을 각각SalI과HpaI 인식서열을 포함하는 프라이머 3(서열번호 4: 5'-AGCTGTCGACGCTACAGGTAAG-3'), 프라이머 4(서열번호 5:5'-GGCCAGCTGGTGTTAACGATGGGCGCGGAGGATAGCG-3')로 PCR을 수행하여, ATG 서열을 각각SalI과HpaI 인식서열로 대체하여 인체 성장 호르몬 유전자 부위로부터 단백질합성이 유도되는 것을 방지하였다. 동시에 pGbc5.5hGH를 ClaI, PvuII, XhoI으로 절단하여 엑손 2 이하부터 엑손 5에 위치하고 있는 1.1 kb의PvuII 유전자 부위를 제외한 0.4 kb 의 인체 성장 호르몬 유전자 엑손 1, 2 부위와 0.6 kb의 인체 성장 호르몬 유전자의 전사종결요소를 1.5% 아가로오스 겔에 전기영동 후 NA45 DEAE membrane[Schleicher Schuell]을 이용하여 분리, 정제하였다. 또한, pGbc5.5hGH를ClaI,XhoI으로 잘라 0.8% 아가로오스 겔에 전기영동하여 인체 성장 호르몬 유전자를 제거하고, 흑염소 베타-카제인 유전자의 프로모터 5.5 kb 만을 포함하는 pBluescript II KS[pKS II, Stratagene] 벡터를 분리, 정제하였다. 흑염소 베타-카제인 유전자의 프로모터 5.5 kb 만을 포함하는 pBluescript II KS 벡터에 상기 0.4 kb 의 인체 성장 호르몬 유전자 엑손 1, 2 부위와 0.6 kb의 인체 성장 호르몬 유전자의 전사종결요소를 연결하여 pGH 플라스미드를 제작하였다[도 1].As shown in FIG. 1, primer 3 including Sal I and Hpa I recognition sequences was respectively included in a portion including an ATG sequence which is a protein synthesis initiation signal present in exon 1 and exon 2 of the human growth hormone gene region (SEQ ID NO 4 :). PCR was performed with 5'-AGCTGTCGACGCTACAGGTAAG-3 ') and Primer 4 (SEQ ID NO 5: 5'-GGCCAGCTGGTGTTAACGATGGGCGCGGAGGATAGCG-3'), replacing the ATG sequence with Sal I and Hpa I recognition sequences, respectively, from the human growth hormone gene site. Protein synthesis was prevented from being induced. At the same time, pGbc5.5hGH was cleaved with ClaI, PvuII, and XhoI to remove 0.4 kb of human growth hormone gene exons 1 and 2 and 0.6 kb of human growth hormone, except for 1.1 kb of Pvu II gene located from exon 2 to exon 5. The transcription terminator of the gene was electrophoresed on a 1.5% agarose gel and then isolated and purified using a NA45 DEAE membrane [Schleicher Schuell]. In addition, pGbc5.5hGH was cut into Cla I and Xho I and electrophoresed on a 0.8% agarose gel to remove human growth hormone genes, and pBluescript II KS [pKS II, containing only 5.5 kb of the promoter of the black goat beta-casein gene. Stratagene] vector was isolated and purified. PGH plasmid was prepared by linking the transcription terminators of the 0.4 kb human growth hormone gene exons 1 and 2 and the 0.6 kb human growth hormone gene to the pBluescript II KS vector containing only 5.5 kb of the promoter of the black goat beta-casein gene. 1.

③ 플라스미드 pGHOPG의 제조③ Preparation of plasmid pGHOPG

상기 실시예 1에서 합성한 1.2 kb의 hOPG cDNA를SmaI과HindII로 절단하여 1.2% 아가로오스 겔에 전기영동 후 NA45 DEAE membrane을 이용하여 정제하였다. 정제한 cDNA를 실시예 2에서 제작한 플라스미드 pGH의HpaI 자리에 전사 방향에 맞추어 삽입함으로써, 최종적인 신규 플라스미드 pGHOPG를 제조하여 한국생명공학연구원 유전자은행에 2001년 5월 25일자로 수탁번호 KCTC 1019BP로 기탁하였다.The 1.2 kb hOPG cDNA synthesized in Example 1 was digested with Sma I and Hind II and subjected to electrophoresis on 1.2% agarose gel, followed by purification using NA45 DEAE membrane. By inserting the purified cDNA into the Hpa I site of the plasmid pGH prepared in Example 2 in accordance with the transcription direction, the final new plasmid pGHOPG was prepared and deposited in the Korea Biotechnology Research Institute Gene Bank on May 25, 2001 Accession No. KCTC 1019BP Was deposited.

실시예 2 : 쥐의 형질전환Example 2 Transformation of Mice

플라스미드 pGHOPG를 제한효소NotI과XhoI으로 절단하고, 0.7% 아가로스겔로 전기영동한 후, 일루팁 컬럼(Elutip-D column)[Schleicher Schuell, Germany]을 통과시켜 정제하였다. 정제하여 얻은 hOPG 유전자를 0.1 mM EDTA를 첨가한 10 mM 트리스 용액으로 농도가 4 ㎍/㎖이 되도록 조절하였다. 정제한 4 ㎍/㎖ 농도의 hOPG 유전자를 C57BL/6 ×CBA계 교잡종 생쥐로부터 얻은 1 세포기의 수정란에 미세 주입하고, 미세 주입한 수정란을 가임(pseudo-pregnant) ICR종 대리모에 이식하여 새끼 쥐를 생산하였다. 생산된 새끼 쥐의 귀조직 일부를 절취 및 분해하고, 분해된 조직액은 hOPG cDNA의 합성에 이용한 프라이머를 사용하여 PCR 분석을 실시하였다. 선택된 형질 전환된 생쥐를 #3, #5, #6, #7로 명명하였다.Plasmid pGHOPG was digested with restriction enzymes Not I and Xho I, electrophoresed with 0.7% agarose gel, and purified by passing through an Ellutip-D column [Schleicher Schuell, Germany]. The purified hOPG gene was adjusted to a concentration of 4 μg / ml with 10 mM Tris solution to which 0.1 mM EDTA was added. The purified 4 g / ml concentration of hOPG gene was microinjected into 1-cell fertilized eggs obtained from C57BL / 6 x CBA hybrid hybrid mice, and the microinjected fertilized eggs were implanted into pseudo-pregnant ICR surrogate mothers to give birth to pups. Produced. A part of the ear tissue of the produced mouse was cut and digested, and the digested tissue solution was subjected to PCR analysis using primers used for the synthesis of hOPG cDNA. Selected transformed mice were named # 3, # 5, # 6, # 7.

실시예 3 : 형질 전환된 쥐의 유즙에서 hOPG의 함량 확인Example 3 Confirmation of hOPG Content in Milk of Transgenic Mice

유즙내 hOPG의 함량을 정량분석하기 위하여, 상기 실시예 2에서 얻은 #3,#5, #6, #7의 형질 전환된 생쥐계통의 암컷 쥐들을 번식시켜 수유 10일째 되었을 때, 유즙을 채취하여 유즙내 hOPG의 함량를 다음과 같은 방법으로 확인하였다.In order to quantitatively analyze the content of hOPG in milk, female rats of the transformed mouse system of # 3, # 5, # 6, and # 7 obtained in Example 2 were bred and milked at 10 days of lactation. The content of hOPG in milk was confirmed by the following method.

먼저, 유즙을 채취하기 위해 새끼 쥐들과 격리시킨 3시간 후, 옥시토신 10 IU를 복강주사하고, 동시에 유선을 마사지하면서 유즙을 채취하였다. 채취한 유즙을 14,000 g에서 30분간 원심분리하여 유청을 분리하였다. 분리한 유청을 이용하여 hOPG의 함량을 두 종류의 OPG 특이 항체[RD 사]를 이용한 샌드위치 ELISA(Enzyme linked immunosorbent assay)법으로 정량하였다. #3, #7의 형질 전환된 생쥐계통의 암컷 쥐들은 hOPG를 각각 0.15 ㎍/㎖과 0.06 ㎍/㎖의 낮은 농도로 OPG를 생산하고 있음에 반해, 두 계통 #5 및 #6은 각각 1.6 ㎎/㎖ 및 2.0 ㎎/㎖ 의 고농도로 OPG를 유즙으로 생산하고 있었다. 이상의 분석결과, 모든 계통에서 hOPG가 발현되었으며, 4마리 중 2마리는 hOPG를 높은 수준으로 발현하였다.First, after 3 hours of isolation from the rats to collect milk, oxytocin 10 IU was intraperitoneally injected, and milk was collected while massaging the mammary gland. The whey was separated by centrifugation at 14,000 g for 30 minutes. Using the separated whey, the content of hOPG was quantified by sandwich ELISA (Enzyme linked immunosorbent assay) using two types of OPG-specific antibodies [RD Corporation]. Female mice in the transgenic mouse strains of # 3 and # 7 produced hOPG at low concentrations of 0.15 μg / ml and 0.06 μg / ml, respectively, whereas both strains # 5 and # 6 were 1.6 mg, respectively. OPG was produced in milk at high concentrations of / ml and 2.0 mg / ml. As a result of the above analysis, hOPG was expressed in all lines, and 2 out of 4 mice expressed high levels of hOPG.

실시예 4 : hOPG mRNA의 유선 및 기타 조직에서의 발현성 검증Example 4 Verification of Expression of hOPG mRNA in Mammary Gland and Other Tissues

hOPG mRNA가 유선에서 발현되는지를 확인하기 위하여, #3, #5, #6, #7의 형질 전환된 생쥐계통의 암컷 쥐들을 대상으로 비유기 10일째에 유선을 적출하였고, 조직 특이적으로 발현되는지를 확인하기 위하여 #5, #6의 계통은 유선 외에도 간, 비장, 췌장, 신장, 폐, 심장, 흉선, 침샘 및 뇌 조직을 적출하였다. 샘부룩의 방법에 따라 상기 조직의 전체 RNA를 추출하고, 각각의 추출한 20 ㎍ 의 전체 RNA를 1 % 포름알데히드-아가로스 겔상에서 전기영동한 후, 나일론막으로 옮겼다[Sambrook et al.,Mol. cloning: a laboratory manual, pp. 7.3 - 7.23,CSH Press (1989)]. hOPG cDNA를 [α-32P]dCTP[아머샴 사]로 라벨하고, 이것을 탐침자로 하여 노던 블럿(northern blot)을 하였다.In order to confirm whether hOPG mRNA is expressed in the mammary gland, mammary glands were extracted on the 10th day of fertilization of female mice of the transgenic mouse line of # 3, # 5, # 6, and # 7, and tissue-specific expression was performed. In order to check whether the strains of # 5 and # 6, the liver, spleen, pancreas, kidney, lung, heart, thymus, salivary glands and brain tissues were extracted in addition to the mammary gland. Total RNA of the tissue was extracted according to Samburk's method, and each extracted 20 μg of total RNA was electrophoresed on a 1% formaldehyde-agarose gel and then transferred to a nylon membrane [Sambrook et al., Mol. cloning: a laboratory manual , pp. 7.3-7.23, CSS Press (1989)]. The hOPG cDNA was labeled with [α- 32 P] dCTP [Amersham Co., Ltd.] and Northern blot was used as a probe.

도 2는 상기 노던 블럿의 결과를 나타낸 것으로, 모든 계통의 유선에서 hOPG mRNA가 발현되었다.Figure 2 shows the results of the northern blot, hOPG mRNA was expressed in the mammary gland of all lines.

도 3은 각 조직에서의 노던 블럿의 결과를 나타낸 것으로, hOPG mRNA가 유선에서만 과량 발현되는 매우 엄격한 조직 특이성을 보였다.Figure 3 shows the results of the Northern blot in each tissue, showing very stringent tissue specificity where the hOPG mRNA is overexpressed only in the mammary gland.

실시예 5 : 형질 전환된 쥐의 유즙에서 hOPG의 분비 형태 확인Example 5 Confirmation of Secretory Form of hOPG in Milk of Transgenic Mice

#3, #5, #6, #7 형질전환 생쥐의 유즙내에 함유된 hOPG의 분비형태를 확인하기 위하여, 원심분리하여 얻은 유청 0.5 ㎕를 10 % denaturing-PAGE에서 전기영동하고 니트로셀룰로오즈막으로 이동시킨 후 hOPG 특이적 항체[R D systems 사]를 이용하여 웨스턴 블럿(western blot)을 수행한 결과, 인체 혈청에서 확인된 hOPG 단백질[Simonet et al., Cell., 89, 309 (1997); Tsuda et al., Biochem. Biophys. Res. Commun., 139, 1329 (1997)]과 동일한 55 kDa의 크기로 생산되고 있음을 확인하였다[도 4].To confirm the secretion form of hOPG contained in milk of # 3, # 5, # 6 and # 7 transgenic mice, 0.5 μl of whey obtained by centrifugation was electrophoresed on 10% denaturing-PAGE and transferred to nitrocellulose membrane. Western blot using a hOPG specific antibody [RD systems, Inc.], and confirmed the hOPG protein in human serum [Simonet et al., Cell., 89, 309 (1997); Tsuda et al., Biochem. Biophys. Res. Commun., 139, 1329 (1997)] was produced in the same size 55 kDa [Fig. 4].

실시예 6 : 형질 전환된 쥐의 유즙 내에 함유된 hOPG의 생물학적 활성 조사Example 6 Investigation of Biological Activity of hOPG in Milk of Transgenic Mice

형질전환 생쥐의 유즙내에 함유된 hOPG가 파골세포(osteoclast)의 분화 억제 능력을 가지고 있는가를 알아보기 위하여, 생쥐의 골수세포에 형질전환 생쥐의 유즙을 첨가하여 배양하고, 파골세포 분화시 특이적으로 발현되는 단백질인 TRAP(tartrate-resistant acid phosphatase)의 활성을 측정하여 hOPG의 생물학적 활성을 조사하였다.To determine whether hOPG contained in milk of transgenic mice has the ability to inhibit the differentiation of osteoclasts, cultured by adding milk of transgenic mice to bone marrow cells of mice and expressing specifically when osteoclast differentiation. The biological activity of hOPG was examined by measuring the activity of the protein, TRAP (tartrate-resistant acid phosphatase).

우선, 생쥐의 경골에서 골수세포를 회수하여 파골세포의 분화에 필수적인 M-CSF(macrophage-stimulating factor)와 OPG-L(osteoprotegerin-ligand)을 50 ng/㎖의 농도로 처리하고, 동시에 hOPG를 고농도로 발현하는 형질전환 생쥐 #5와 #6의 유즙을 원심분리하여 얻은 유청을 다양한 비율로 희석하여 첨가하였다. 희석할 유즙의 농도는 ELISA 결과를 바탕으로 하였다. 이를 6일동안 배양한 후 에시드 포스파타제 킷(acid phosphatase kit)[Sigma 사]를 이용하여 TRAP-solution assay를 수행하였고, TRAP의 활성 정도를 ELISA 리더(reader)를 이용하여 405 nm에서 측정하였다. 그 결과, 유즙의 농도 약 0.2 ng/㎖에서 유즙을 처리하기 전에 비해 TRAP의 활성 정도가 반으로 감소하였고, 약 20 ng/㎖ 수준에서 파골세포의 형성을 완전히 억제하는 것으로 보아 형질전환 생쥐의 유즙에 발현된 hOPG가 생물학적 활성을 가지고 있음을 확인하였다[도 5].First, bone marrow cells were recovered from the tibia of the mouse and treated with macrophage-stimulating factor (M-CSF) and osteoprotegerin-ligand (OPG-L) at a concentration of 50 ng / ml, which was essential for the differentiation of osteoclasts, and at the same time high concentration of hOPG Whey obtained by centrifugation of the milk of transgenic mice # 5 and # 6 expressed by the dilution was added in various ratios. The concentration of milk to be diluted was based on ELISA results. After culturing for 6 days, TRAP-solution assay was performed using an acid phosphatase kit (Sigma), and the activity of TRAP was measured at 405 nm using an ELISA reader. As a result, the level of the activity of TRAP was reduced by half at the concentration of milk at about 0.2 ng / ml, and the milk of transgenic mice was found to completely inhibit the formation of osteoclasts at the level of about 20 ng / ml. It was confirmed that hOPG expressed in the biological activity [Fig. 5].

이상에서 상세히 설명하였듯이, 본 발명은 유용한 의약품으로 쓰이는 hOPG를 안정적으로 대량 생산할 수 있고, 엄격한 조직 특이성도 유지되는 형질 전환용 신규 플라스미드 pGHOPG(KCTC 1019BP)와 이를 사용하여 포유동물(인간 제외)을 형질 전환시킴으로써 비유기 때에 유선에서만 지속적으로 hOPG를 생산하는 방법에 관한것으로서, 약 5.5 kb 크기의 흑염소 유래의 베타-카세인 프로모터에 인체 성장 호르몬 유전자의 엑손 1과 2, 인트론 1 및 전사 종결요소, 그리고 hOPG cDNA를 융합하여 신규 플라스미드 pGHOPG(KCTC 1019BP)를 제조하고, 상기의 제조한 플라스미드로 형질 전환된 포유동물(인간 제외)의 유선에서만 조직 특이적으로 hOPG를 다량 분비하며, 특히 상기 형질 전환된 생쥐의 고농도의 hOPG를 생물학적 활성을 가진 형태로 유선에서만 특이적으로 대량 생산할 수 있다.As described in detail above, the present invention is capable of stably mass-producing hOPG, which is used as a useful drug, and transforms mammals (except humans) using the novel plasmid pGHOPG (KCTC 1019BP) for transformation, which maintains strict tissue specificity. A method of continuously producing hOPG only in the mammary gland during the inorganic phase by converting it into a beta-casein promoter derived from a black goat of about 5.5 kb in size with exons 1 and 2 of the human growth hormone gene, intron 1 and transcription terminator, and hOPG. cDNA was fused to prepare a new plasmid pGHOPG (KCTC 1019BP), and secreted large amounts of hOPG specifically in the mammary gland of mammals (except humans) transformed with the plasmid prepared above, particularly of the transgenic mice. High concentrations of hOPG can be produced specifically in the mammary gland in a biologically active form have.

Claims (3)

인체 골 재흡수 억제인자(human osteoprotegerin, hOPG)를 생산하는 재조합 플라스미드 pGHOPG(KCTC 1019BP).Recombinant plasmid pGHOPG (KCTC 1019BP), which produces human osteoprotegerin (hOPG). 플라스미드 pGHOPG(KCTC 1019BP)를 포유동물(인간 제외)의 수정란에 미세주입하여 형질 전환시킨 후, 그 동물의 유즙으로부터 인체 골 재흡수 억제인자(hOPG)를 분리하는 것을 특징으로 하는 인체 골 재흡수 억제인자(hOPG)의 생산방법.Plasmid pGHOPG (KCTC 1019BP) was microinjected into a fertilized egg of a mammal (except human) and transformed, and then human bone resorption inhibitory (hOPG) is isolated from the milk of the animal. Method of producing factor (hOPG). 제 2 항에 있어서, 상기 형질전환 동물의 유즙으로부터 생물학적 활성을 가진 인체 골 재흡수 억제인자(hOPG)를 1 ∼ 4 ㎎/㎖의 고농도로 생산하는 것을 특징으로 하는 인체 골 재흡수 억제인자(hOPG)의 생산방법.3. The human bone resorption inhibitor (hOPG) according to claim 2, wherein the human bone resorption inhibitor (hOPG) having biological activity is produced from the milk of the transgenic animal at a high concentration of 1 to 4 mg / ml. ) Production method.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
KR19980703599A (en) * 1995-12-22 1998-12-05 스티븐 엠. 오드르 Osteoprotegerin
WO2000009714A1 (en) * 1998-08-11 2000-02-24 Amgen Inc. Overexpression of desired proteins in eukaryotic cells mediated by cyclin d1 overexpression
WO2001016299A1 (en) * 1999-08-30 2001-03-08 Mayo Foundation For Medical Education And Research Use of dna encoding osteoprotegerin to prevent or inhibit metabolic bone disorders

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR19980703599A (en) * 1995-12-22 1998-12-05 스티븐 엠. 오드르 Osteoprotegerin
WO2000009714A1 (en) * 1998-08-11 2000-02-24 Amgen Inc. Overexpression of desired proteins in eukaryotic cells mediated by cyclin d1 overexpression
WO2001016299A1 (en) * 1999-08-30 2001-03-08 Mayo Foundation For Medical Education And Research Use of dna encoding osteoprotegerin to prevent or inhibit metabolic bone disorders

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한국수정난이식학회지 2000년도 추계학술대회 및 정기총회 pp.23-26, 2000, 김진회 *

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