KR20010081456A - The production method of transgenic porcine producing human erythropoietin and the transgenic porcine - Google Patents
The production method of transgenic porcine producing human erythropoietin and the transgenic porcine Download PDFInfo
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- KR20010081456A KR20010081456A KR1020000006888A KR20000006888A KR20010081456A KR 20010081456 A KR20010081456 A KR 20010081456A KR 1020000006888 A KR1020000006888 A KR 1020000006888A KR 20000006888 A KR20000006888 A KR 20000006888A KR 20010081456 A KR20010081456 A KR 20010081456A
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Abstract
Description
본 발명은 유전공학적인 방법 즉, 어떤 유전자(DNA;deoxyribonucleic acid)의 일부를 꺼내어 그것을 다른 DNA 분자에 이어 붙여서 유전자를 만들어 내는 조작인 유전자 재조합(gene recombination) 기술을 이용하여 돼지의 유선에서 인간의 조혈촉진제(EPO::erythropoietin)를 대량으로 생산할 수 있는 형질전환 돼지(웅성)를 생산하는 것에 관한 것으로서, 더 상세하게는 인간의 신장에 존재하는 조혈촉진제(EPO) 유전자를 돼지의 수정란에 주입, 형질전환시키므로서 인간의 조혈촉진제 (EPO)유전자를 가지는 돼지(일명 "새롬이"라 한다)를 생산하고, 그 돼지의 젖을 통하여 고가의 의약품인 인간의 조혈촉진제(EPO)를 대량으로 분비할 수 있도록 하므로서 인류건강의 진일보에 기여함은 물론 고가의 의약품 수출을 통하여 국가경제 및 국가경쟁력 기반구축에 크게 기여할 수 있는 발명이다.The present invention is a genetic engineering method, that is, a genetic recombination technique that takes a part of a gene (DNA; deoxyribonucleic acid) and attaches it to another DNA molecule to make a gene. The present invention relates to the production of transgenic pigs (males) capable of producing a large amount of hematopoietic promoters (EPO :: erythropoietin), and more specifically, the injection of hematopoietic promoter (EPO) genes present in human kidneys into pig embryos, By transforming, pigs with human hematopoietic promoters (EPO) genes (also called "Sarom") are produced, and the pigs' milk can be used to secrete large amounts of expensive human hematopoietic promoters (EPO). By doing so, it can contribute to the progress of human health and can contribute greatly to the establishment of the national economy and national competitiveness through the export of expensive medicines. Invention.
일반적으로 인간 적혈구의 수명은 약 120일로 전체 적혈구중 약 120분지 1일이 매일 세망내피 조직계에서 파괴되며 동시에 상대적으로 일정한 수의 적혈구가 매일 생성되어 적혈구의 농도가 일정하게 유지된다 (Guyton, Textbook of Medical Physiology, pp56∼60, W.B.Saunders Co., Philadelphia(1976)).In general, the lifespan of human erythrocytes is about 120 days, and about 120 minutes of the total erythrocytes are destroyed in the reticuloendothelial system every day, while a relatively constant number of erythrocytes are generated daily to maintain a constant concentration of erythrocytes (Guyton, Textbook of Medical Physiology, pp 56-60, WBSaunders Co., Philadelphia (1976).
적혈구는 골수에서 적아구의 성숙 및 분화에 의하여 생성되며 조혈촉진제 (EPO)는 덜 분화된 세포에 작용하여 적혈구로의 분화를 유도하는 인자이다. (Guyton, 상기와 동일).Erythrocytes are produced by the maturation and differentiation of erythrocytes in the bone marrow and hematopoietic promoters (EPO) act on less differentiated cells to induce differentiation into erythrocytes. (Guyton, same as above).
조혈촉진제(EPO)는 1950년대에 빈혈 동물의 혈장을 정상 동물에 주입할 때 새로 형성되는 적혈구에59Fe가 많이 삽입되는 것이 관찰됨으로서 발견되었는데 (Borsook, et, al., Blood, 9, 734(1954)), 대기중 산소가 부족하거나 출혈 또는 빈혈상태의 세포수가 증가될 경우 성인의 신장에서 주로 생성되는 당단백질로 적혈구 생성(erythropoiesis)을 조절하고 적혈구 수를 유지하는 데에도 중요한 역할을 한다.(Carnot, et al., Comot. Rend. 143, 384(1906); Kranz. S.B., Blood 77, 419(1991); Goldwasser, E. et al., in Peptide Growth Factors and their Receptors I, Sporn, M.B. and A.B. Roberts, eds., Springer-Verlag, Berlin, p. 747(1990)).Stem promoters (EPO) is to be 59 Fe is inserted a lot of red cells that are newly formed when injecting the plasma of anemic animals to normal animals in the 1950s, it was found by being observed (Borsook, et, al., Blood, 9, 734 ( 1954), glycoproteins produced mainly in the kidneys of adults in the absence of oxygen in the atmosphere or increased cell numbers in bleeding or anemia play an important role in regulating erythropoiesis and maintaining erythrocyte counts. (Carnot, et al., Comot.Rend. 143, 384 (1906); Kranz. SB, Blood 77, 419 (1991); Goldwasser, E. et al., In Peptide Growth Factors and their Receptors I, Sporn, MB and AB Roberts, eds., Springer-Verlag, Berlin, p. 747 (1990)).
당업자에게 잘 알려진 바와 같이, 적혈구 생성을 조절하는 호르몬인 천연형의 조혈촉진제(EPO)는 태아의 간에서 분비되기 시작하고 임신 120-140일 사이에 신장으로 이동하기 시작하여 분만후 40일에는 완전히 이동이 완료되며 성인의 경우에는 신장에서 주로 생성되나 간에서도 전체량의 약 10% 정도가 생성되는 것으로 알려져 있으며 이외에 적은 양은 골수(bone marrow)의 거식세포(macrophage)에서도 생성되는 것으로 알려져 있다.As is well known to those skilled in the art, natural hematopoietic promoters (EPO), hormones that regulate red blood cell production, begin to be secreted from the liver of the fetus and begin to migrate to the kidney between 120 and 140 days of gestation and are completely at 40 days postpartum. The migration is completed and in adults it is mainly produced in the kidneys, but about 10% of the total amount is known to be produced in the liver, in addition to the small amount is known to be produced in the macrophage of the bone marrow (bone marrow).
조혈촉진제(EPO)는 정상인의 혈액 1㎖당 약 15 내지 30 mU 혹은 0.01 mM으로 유지되며(Garcia, J. F., Lab. Clin. Med. 99, 624-635(1982)), 재생불량성 빈혈 등의 질환이 있는 환자에서는 정상인보다 높은 농도의 조혈촉진제(EPO)가 생성되어 이들의 혈액이나 뇨가 조혈촉진제(EPO)의 생산에 이용되기도 한다.(White, et al., Rec. Prog. Horm. Res. 16, 219(1960)); Espada, et al., Biochem. Med. 3, 475(1970); Fisher, Pharmacol. Rev. 24, 459(1972)).Hematopoietic promoters (EPO) are maintained at about 15 to 30 mU or 0.01 mM per 1 ml of normal human blood (Garcia, JF, Lab. Clin. Med. 99, 624-635 (1982)) and diseases such as aplastic anemia In patients with this condition, hematopoietic promoters (EPO) are produced at higher concentrations than normal people, and their blood or urine may be used for the production of hematopoietic promoters (EPO) (White, et al., Rec. Prog. Horm. Res. 16, 219 (1960); Espada, et al., Biochem. Med. 3, 475 (1970); Fisher, Pharmacol. Rev. 24, 459 (1972).
조혈촉진제(EPO)는 당단백질 호르몬의 일종으로 분자량은 약 30Kd이고, 24, 38 및 83번 아미노산에 N-Linked 당쇄첨가부위를 가지고 있고, 또한 126번 아미노산에 하나의 O-Linked 당쇄첨가 결합부위를 가지고 있으며(P.S.E.B.M. 216, 358-369(1997)), 종래에는 동물의 세포에서 생산한 재조합 조혈촉진제(EPO)를 사용하고 있으나 분비량이 너무 적고 또한 생리활성도 천연형과는 동일하지 않고 낮은 문제점을 가지고 있다.Hematopoietic promoter (EPO) is a glycoprotein hormone that has a molecular weight of about 30 Kd, has an N-linked sugar chain at amino acids 24, 38 and 83, and an O-linked sugar chain at the amino acid 126. (PSEBM 216, 358-369 (1997)), but conventionally used recombinant hematopoietic promoters (EPO) produced in animal cells, but the secretion amount is too small and the physiological activity is not the same as the natural type, and low problems Have.
조혈촉진제(EPO)는 빈혈, 특히 신장성 빈혈의 임상적 치료에 유망한 치료제이나 항원성의 문제로 인해 가능한 인간 유래의 원료로부터 제조하는 것이 바람직하며, 따라서 상기에 설명한 것과 같이 대량의 조혈촉진제(EPO)를 분비하는 재생불량성 빈혈 등의 질병을 앓고 있는 환자의 혈액이나 뇨를 이용하여 얻을 수 있다. 그러나 그 양은 전체적으로 극히 제한돼 있는 실정이다.Hematopoietic promoters (EPO) are preferably prepared from human-derived raw materials due to promising therapeutics or antigenic problems for the clinical treatment of anemia, especially renal anemia, and therefore a large amount of hematopoietic promoters (EPO) as described above. It can be obtained by using blood or urine of a patient suffering from diseases such as aplastic anemia that secrete thyroid. However, the amount is extremely limited overall.
또한 조혈촉진제(EPO)는 동물인 양의 혈장으로부터도 회수될 수 있으나 양의 혈장으로부터 분리된 조혈촉진제(EPO)는 어느정도 만족스러운 역가 및 안정한 수용성 제재를 제공하나 역시 인체의 항원으로 작용할 수 있는 문제를 내포한다.Hematopoietic promoters (EPO) can also be recovered from animal plasma, but hematopoietic promoters (EPO) isolated from sheep plasma provide somewhat satisfactory titers and stable water-soluble agents, but can also act as antigens for the human body. Implies
국외에서는 쿠바의 바이오테크놀로지사가 인간 조혈촉진제(hEPO) cDNA를 이용 토끼의 유선조직에서 조혈촉진제(hEPO)를 발현하는 형질전환 동물을 생산한 연구실적이 있으며, 핀랜드의 쿠오피에크르(Kuopioeogkr)사는 쥐의 유선에서 조혈촉진제(hEPO) cDNA 형질전환 동물을 생산한 연구실적이 있으나 돼지에서의 형질전환 동물 생산은 아직까지 보고된 바 없으며, 국내에서는 대한민국 특허공고 제93-5917호와 같이 사람의 조혈촉진제(EPO) 유전자를 클로닝(복제)하여 동물세포 또는 곤충세포에서 발현시키는 방법이 개발되기도 하였으나 이 방법에서는 조혈촉진제 (EPO)의 발현량이 미약하거나 글리코실화가 정확히 일어나지 않아 생체내에서 빨리 분해하여 버리는 단점이 있으며, 대한민국 특허출원 제94-12082호는 변형된 재조합인간적혈구조혈인자(rhEPO) 유전자를 포함하는 발현벡터를 개발하였으며 이 발현벡터로 형질전환시킨 동물 세포주 COS-7(ATCC CRL 1651, African Green Monkey Kidney Cell)에서는 재조합인간적혈구조혈인자(rhEPO)를 대량으로 생산할 수 있었으나 이 방법에서는 계속적으로 형질전환을 시켜야 한다는 번거로움으로 인하여 대량생산에는 부적합한 문제가 있었다.Overseas, Cuban biotechnology company used human hematopoietic promoter (hEPO) cDNA to produce transgenic animals expressing hematopoietic promoter (hEPO) in rabbit's mammary gland tissue, and Kuopioeogkr of Finland Although there have been studies on the production of hemopoietic promoter (hEPO) cDNA transgenic animals in the mammary gland, the production of transgenic animals in pigs has not been reported so far. In Korea, as in Korean Patent Publication No. 93-5917, human hematopoiesis has been reported. Cloning (cloning) of the promoter (EPO) gene has been developed to express it in animal cells or insect cells.However, in this method, the hematopoietic promoter (EPO) expression level is weak or glycosylation does not occur precisely and is rapidly degraded in vivo. Korean Patent Application No. 94-12082 discloses a modified recombinant human hematopoietic factor (rhEPO) gene. The expression vector was developed. The animal cell line COS-7 (ATCC CRL 1651, African Green Monkey Kidney Cell) transformed with the expression vector was able to produce a large amount of recombinant human hematopoietic factor (rhEPO). Due to the hassle of transforming, there was an unsuitable problem in mass production.
또한 재조합인간적혈구조혈인자(rhEPO)를 대량 발현벡터에 의하여 형질전환된 영구 세포주는 재조합인간적혈구조혈인자(rhEPO)를 동물세포 내에서 효과적이고 안정적으로 대량 생산할 수 있는 대한민국 특허등록번호 제0184778호의 재조합인간적혈구조혈인자(rhEPO)의 제조방법에 대한 것이 있으나 돼지의 유즙속으로 인간 적혈구조혈인자(rhEPO)를 생산하는 본 발명과는 상이한 방법이다.In addition, a permanent cell line transformed with a recombinant human erythropoietic factor (rhEPO) by a mass expression vector can be recombined with Korean Patent Registration No. 0184778, which can efficiently and stably produce recombinant human erythropoietic factor (rhEPO) in animal cells. There is a method for producing human erythropoietic factor (rhEPO), but is different from the present invention for producing human erythropoietin (rhEPO) in pig milk.
본 발명은 상기한 바와 같은 종래의 문제점을 해결하기 위하여 창안된 것으로서, 본 발명의 주 목적은 인간의 조혈인자인 조혈촉진제(EPO)를 유용하게 생산하는 형질전환 동물을 생산하는 데 있다.The present invention was devised to solve the conventional problems as described above, and a main object of the present invention is to produce a transgenic animal usefully producing a hematopoietic promoter (EPO), which is a human hematopoietic factor.
본 발명의 또 다른 목적은 인간의 조혈인자인 조혈촉진제(EPO)를 형질전환 동물인 돼지의 젖을 통해 대량으로 생산하고자 하며, 그를 통해 인간의 건강복지를 증진시키고, 축산을 고부가가치 산업으로 전환시키고자 하는 데 목적이 있다.Another object of the present invention is to produce a large amount of hematopoietic promoter (EPO), a human hematopoietic factor through the milk of a transgenic animal, thereby promoting human health welfare, converting livestock into a high value-added industry The purpose is to sleep.
본 발명의 또 다른 목적은 고가의 의약품인 조혈촉진제(EPO)를 대량으로 생산함으로서 세계시장으로의 수출을 통해 경제성을 증대시키고자 하는 데 있다.Another object of the present invention is to increase the economics through export to the world market by producing a large amount of expensive hematopoietic accelerator (EPO).
도 1 은 형질전환 돼지 생산과정을 나타내는 계통도,1 is a schematic diagram showing a process of producing a transgenic pig,
도 2 는 PCR(polymerare chain reaction) 사진,Figure 2 is a polymerase chain reaction (PCR) photograph,
도 3 은 형질전환된 돼지의 cDNA 염기서열을 보여주는 도이다.Figure 3 is a diagram showing the cDNA sequence of the transformed pig.
상기한 목적을 달성하기 위하여, 본 발명에서는 인간의 신장에 존재하는 조혈촉진제(EPO) 유전자를 돼지의 수정란에 주입, 형질전환시키므로서 인간의 조혈촉진제(EPO)유전자를 가지는 형질전환 돼지(일명 : 새롬이)를 생산하고, 그 돼지의 젖을 통하여 고가의 의약품인 인간의 조혈촉진제(EPO)를 대량으로 분비할 수 있도록 하는 것을 특징으로 하는 방법을 제공한다.In order to achieve the above object, in the present invention, a transgenic pig having a human hematopoietic promoter (EPO) gene by injecting and transforming a hematopoietic promoter (EPO) gene present in human kidney into a fertilized egg of pigs (aka: Salom) produces and provides a method for producing a large amount of expensive hematopoietic promoters (EPO) through the pig's milk.
이하 도면을 참고하여 본 발명의 형질전환 돼지의 생산에 대하여 좀 더 상세하게 설명하고자 하며, 하기에서 본 발명을 설명함에 있어, 관련된 공지 기능 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명은 생략할 것이다. 그리고 후술되는 용어들은 본 발명에서의 기능을 고려하여 정의된 용어들로서 이는 사용자, 운용자의 의도 또는 관례 등에 따라 달라질 수 있다. 그러므로 그 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다.Hereinafter, the production of the transgenic pigs of the present invention will be described in more detail with reference to the drawings, and in the following description of the present invention, a detailed description of related known functions or configurations will unnecessarily obscure the gist of the present invention. If it can be determined that the detailed description will be omitted. Terms to be described later are terms defined in consideration of functions in the present invention, and may be changed according to intentions or customs of users or operators. Therefore, the definition should be made based on the contents throughout the specification.
도 1은 본 발명이 성립되기까지의 과정을 간략히 도식화 한 것으로서, 이에 따라 설명하면, 본 발명의 인간의 조혈촉진제(EPO) 생산용 형질전환 돼지를 생산하기 위하여 먼저 본 발명의 유전자 재조합체인 조혈촉진제(EPO)를 제조하고자 인간의 조혈촉진제(EPO) 게놈유전자(genome DNA)는 대한민국 국립경상대학교 축산과학부 김진회 교수로부터 제공받았으며, 이를 생쥐의 유선으로부터 2.6kb의 WAP 촉진자 (promoter)를 매우 적은양의 유전자를 많이 복사하여 증폭시키는 중합효소연쇄반응 (PCR;polymerare chain reaction)을 이용하여 크로닝(cloning, 유전적복제)후, 인간적혈구조혈인자(hEPO)와 SV40 poly A 유전자를 준비하여 플라스미드 (plasmid) 조혈촉진제(EPO) 형질전환 발현 벡터를 구축하였으며 그 결과, 다음 <표 1>과 같이 되었다.Figure 1 is a schematic diagram of the process until the present invention was established, according to the description, in order to produce a transgenic pig for the production of human hematopoietic promoter (EPO) of the present invention, the hematopoietic promoter which is a genetic recombinant of the present invention first To prepare (EPO), a human hematopoietic promoter (EPO) genome DNA was provided by Professor Kim Jin Hoe of the Faculty of Animal Science, Gyeongsang National University, Korea, which produced a very small amount of 2.6kb of WAP promoter from mouse mammary gland. After cloning using a polymerare chain reaction (PCR) that amplifies and copies a large number of genes, human erythropoietic factor (hEPO) and SV40 poly A genes are prepared and plasmid A hematopoietic promoter (EPO) transformed expression vector was constructed. As a result, it was as shown in <Table 1>.
돼지의 수정란은 과배란 유도제 호르몬인 임마혈청성 성선자극 호르몬(eCG) 및 태반윰모성 성선자극 호르몬(hCG)을 근육내 주사하여 과배란을 유기하여 발정 확인 자연종부를 시킨 후, 1 세포기 수정란을 회수하여 상기에서 나타난 유전자 (DNA)를 매니퓰래이터(manipulator)를 이용하여 웅성 전핵에 주입한 후 즉시 대리모돈에 이식시키고 상기 대리모돈을 통하여 분만된 돼지중 1마리의 꼬리, 혈액, 정액(정자)에서 인간의 조혈촉진제 유전자(EPO DNA)를 코드하는 유전자(DNA)가 전이된 것을 도 2의 중합효소연쇄반응(PCR;polymerare chain reaction) 사진 결과를 통하여 확인하였다.Porcine fertilized eggs were intramuscularly injected with ovum serum gonadotropin (eCG) and placental maternal gonadotropin (hCG), which are hyperovulation inducer hormones, to induce overovulation and to establish natural estrus after confirmation of estrus. The above-described gene (DNA) was injected into the male pronucleus using a manipulator, and then immediately implanted into surrogate sows, and the tail, blood, and semen (sperm) of one of the pigs delivered through the surrogate sows. It was confirmed through the polymerare chain reaction (PCR) photographic result of FIG. 2 that the gene (DNA) encoding human hematopoietic promoter gene (EPO DNA) was transferred from.
다음의 <표 2>는 상기의 중합효소연쇄반응(PCR;polymerare chain reaction) 에 따른 프라이머(primer) 시퀀스(sequence)를 보여주며,Table 2 shows primer sequences according to the polymerare chain reaction (PCR).
<표 3>은 조혈촉진제(EPO) 형질전환 돼지에서 채취한 혈액을 분석한 결과를 나타낸다.Table 3 shows the results of analysis of blood collected from hematopoietic promoters (EPO) transgenic pigs.
상기 대리모돈을 통하여 분만된 돼지에서 인간의 조혈촉진제 유전자(EPO DNA)를 코드하는 DNA가 전이된 것을 도 2의 중합효소연쇄반응(PCR;polymerare chain reaction) 사진 결과를 통하여 확인하였는 바 그 cDNA의 염기서열은 다음과 같다.The transfer of DNA encoding human hematopoietic promoter gene (EPO DNA) in pigs delivered through surrogate sows was confirmed through the polymerare chain reaction (PCR) photograph of FIG. 2. The nucleotide sequence is as follows.
(서열목록)(Sequence list)
서열번호 : 1SEQ ID NO: 1
서열의 길이 : 582Sequence length: 582
서열의 타입 : 핵산Type of sequence: nucleic acid
쇄의 수 : 2 본쇄Number of chains: 2 chains
토폴로지 : 선형Topology: linear
분자의 타입 : cDNAType of molecule: cDNA
기원origin
생물명 : 인간의 간 유전자(DNA)로부터 분리된 EPO cDNABiological name: EPO cDNA isolated from human liver gene (DNA)
서열의 특징Characteristic of the sequence
특징을 나타내는 기호 : sig peptideCharacteristic symbols: sig peptide
존재위치 : 1-81Location: 1-81
특징을 나타내는 기호 : mat peptideCharacteristic symbols: mat peptide
존재위치 : 82-579Location: 82-579
특징을 나타내는 기호 : terminatorCharacteristic symbols: terminator
존재위치 : 580-582Location: 580-582
(서열 1)(SEQ ID NO: 1)
ATG GGG GTG CAC GAA TGT CCT GCC TGG CTG TGG CTT CTC CTG TCC 45ATG GGG GTG CAC GAA TGT CCT GCC TGG CTG TGG CTT CTC CTG TCC 45
Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu SerMet Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser
-27 -20-27 -20
CTG CTG TCG CTC CCT CTG GGC CTC CCA GTC CTG GGC GCC CCA CCA 90CTG CTG TCG CTC CCT CTG GGC CTC CCA GTC CTG GGC GCC CCA CCA 90
Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Prp ProLeu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Prp Pro
-10 +1-10 +1
CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG GAG 135CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG GAG 135
Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu GluArg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu
1010
GCC AAG GAG GCC GAG AAT ATC ACG ACG GGC TGT GCT GAA CAC TGC 180GCC AAG GAG GCC GAG AAT ATC ACG ACG GGC TGT GCT GAA CAC TGC 180
Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His CysAla Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys
20 3020 30
AGC TTG AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT AAT TTC 225AGC TTG AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT AAT TTC 225
Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn PheSer Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe
4040
TAT GCC TGG AAG AGG ATG GAG GTC GGG CAG CAG GCC GTA GAA GTC 270TAT GCC TGG AAG AGG ATG GAG GTC GGG CAG CAG GCC GTA GAA GTC 270
Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu ValTyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val
50 6050 60
TGG CAG GGC CTG GCC CTG CTG TCG GAA GCT GTC CTG CGG GGC CAG 315TGG CAG GGC CTG GCC CTG CTG TCG GAA GCT GTC CTG CGG GGC CAG 315
Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly GlnTrp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln
7070
GCC CTG TTG GTC AAC TCT TCC CAG CCG TGG GAG CCC CTG CAG CTG 360GCC CTG TTG GTC AAC TCT TCC CAG CCG TGG GAG CCC CTG CAG CTG 360
Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln LeuAla Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu
80 9080 90
CAT GTG GAT AAA GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG 405CAT GTG GAT AAA GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG 405
His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr LeuHis Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu
100100
CTT CGG GCT CTG GGA GCC CAG AAG GAA GCC ATC TCC CCT CCA GAT 450CTT CGG GCT CTG GGA GCC CAG AAG GAA GCC ATC TCC CCT CCA GAT 450
Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro AspLeu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp
110 120110 120
GCG GCC TCA GCT GCT CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC 495GCG GCC TCA GCT GCT CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC 495
Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr PheAla Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe
130130
CGC AAA CTC TTC CGA GTC TAC TCC AAT TTC CTC CGG GGA AAG CTG 540CGC AAA CTC TTC CGA GTC TAC TCC AAT TTC CTC CGG GGA AAG CTG 540
Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys LeuArg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu
140 150140 150
AAG CTG TAC ACA GGG GAG GCC TGC AGG ACA GGG GAC AGA TGA 582AAG CTG TAC ACA GGG GAG GCC TGC AGG ACA GGG GAC AGA TGA 582
Lys Leu Tyr Thr Gly Gly Ala Cys Arg Thr Gly Asp ArgLys Leu Tyr Thr Gly Gly Ala Cys Arg Thr Gly Asp Arg
160160
본 발명은 다양하게 변형될 수 있고 여러 가지 형태를 취할 수 있으며 상기 발명의 상세한 설명에서는 그에 따른 특별한 실시예에 대해서만 기술하였다. 하지만 본 발명은 상세한 설명에서 언급되는 특별한 형태로 한정되는 것이 아닌 것으로 이해되어야 하며, 오히려 첨부된 청구범위에 의해 정의되는 본 발명의 정신과 범위 내에 있는 모든 변형물과 균등물 및 대체물을 포함하는 것으로 이해되어야 한다.The present invention can be variously modified and can take various forms and only the specific embodiments thereof are described in the detailed description of the invention. It is to be understood, however, that the present invention is not limited to the specific forms referred to in the description, but rather includes all modifications, equivalents, and substitutions within the spirit and scope of the invention as defined by the appended claims. Should be.
이와 같이 본 발명은, 인간의 조혈인자인 조혈촉진제(EPO)를 유용하게 생산하는 형질전환 동물을 생산할 수 있을뿐만 아니라, 상기 형질전환된 돼지의 젖을 통해 인간의 조혈인자인 조혈촉진제(EPO)를 대량으로 생산할 수 있으며, 고가의 의약품인 조혈촉진제(EPO)를 대량으로 생산함으로서 수출 등을 통해 경제성을 증대시킬 수 있는 매우 유익한 발명임이 명백하다.As such, the present invention can not only produce a transgenic animal usefully producing hematopoietic promoter (EPO), which is a human hematopoietic factor, but also a hematopoietic promoter (EPO) which is a human hematopoietic factor through the milk of the transformed pig. It is obvious that it is a very advantageous invention that can be produced in large quantities and can increase economics through export by producing hematopoietic promoters (EPO), which are expensive drugs, in large quantities.
본 발명의 또 다른 효과는 조혈촉진제의 대량 생산을 통하여 인간의 건강복지를 증진시키고, 축산업을 고부가가치 산업으로 전환시킬 수 있다.Another effect of the present invention is to promote human health and welfare through the mass production of hematopoietic accelerators, and to turn the livestock industry into a high value-added industry.
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GB0220971A GB2376024B (en) | 2000-02-14 | 2000-06-28 | The production method of transgenic porcine producing human eryhropoietin and the transgenic porcine |
PCT/KR2000/000675 WO2001059074A1 (en) | 2000-02-14 | 2000-06-28 | The production method of transgenic porcine producing human erythropoietin and the transgenic porcine |
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KR100453571B1 (en) * | 2001-07-24 | 2004-10-20 | 대한민국 | Transgenic porcine capable of producing human erythropoietin and preparation method thereof |
KR100769291B1 (en) * | 2006-02-13 | 2007-10-24 | 조아제약주식회사 | Mammary gland-specific human erythropoietin expression vector transgenic animal and method for producing human erythropoietin using same |
US8420388B2 (en) | 2008-06-30 | 2013-04-16 | Cho-A Pharm. Co., Ltd. | Gene of porcine beta casein, a promoter of the same and the use thereof |
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CN1283785C (en) * | 2002-05-20 | 2006-11-08 | 上海杰隆生物工程股份有限公司 | Method of producing human forcing erythrogenin using transgene animal mammary gland |
GB0322196D0 (en) * | 2003-09-23 | 2003-10-22 | Cxr Biosciences Ltd | Excretable reporter systems |
CA2556266A1 (en) | 2004-02-13 | 2005-08-25 | Stem Cell Therapeutics Corp. | Use of luteinizing hormone (lh), and chorionic gonadotropin (hcg) for proliferation of neural stem cells and neurogenesis |
WO2006079155A1 (en) * | 2005-01-25 | 2006-08-03 | Apollo Life Sciences Limited | Molecules and chimeric molecules thereof |
WO2007106986A1 (en) * | 2006-03-17 | 2007-09-27 | Stem Cell Therapeutics Corp. | Dosing regimes for lh or hcg and epo for treatment of neurological disorders |
KR100935335B1 (en) * | 2007-09-12 | 2010-01-07 | 차의과학대학교 산학협력단 | Process for preparing hematopoietic cells in vivo using immunosuppressed erythropoietin-produceable transgenic porcines and animal bioreactor for the expansion of hematopoietic cells |
JP5507555B2 (en) | 2008-06-30 | 2014-05-28 | チョ−エー・ファーム・カンパニー・リミテッド | Porcine αS1 casein gene, promoter thereof, and use thereof |
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KR850004274A (en) * | 1983-12-13 | 1985-07-11 | 원본미기재 | Method for preparing erythropoietin |
US5831141A (en) * | 1991-01-11 | 1998-11-03 | United States Of America As Represented By The Department Of Health And Human Services | Expression of a heterologous polypeptide in mammary tissue of transgenic non-human mammals using a long whey acidic protein promoter |
US5959171A (en) * | 1994-08-17 | 1999-09-28 | Pharming B.V. | Method for the production of biologically active polypeptides in a mammal's |
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KR100453571B1 (en) * | 2001-07-24 | 2004-10-20 | 대한민국 | Transgenic porcine capable of producing human erythropoietin and preparation method thereof |
KR100769291B1 (en) * | 2006-02-13 | 2007-10-24 | 조아제약주식회사 | Mammary gland-specific human erythropoietin expression vector transgenic animal and method for producing human erythropoietin using same |
US8420388B2 (en) | 2008-06-30 | 2013-04-16 | Cho-A Pharm. Co., Ltd. | Gene of porcine beta casein, a promoter of the same and the use thereof |
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