KR100350842B1 - Method of stimulating growth in aquatic animals using compositions comprising human growth hormone - Google Patents
Method of stimulating growth in aquatic animals using compositions comprising human growth hormone Download PDFInfo
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- KR100350842B1 KR100350842B1 KR1020010050532A KR20010050532A KR100350842B1 KR 100350842 B1 KR100350842 B1 KR 100350842B1 KR 1020010050532 A KR1020010050532 A KR 1020010050532A KR 20010050532 A KR20010050532 A KR 20010050532A KR 100350842 B1 KR100350842 B1 KR 100350842B1
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- growth hormone
- human growth
- fish
- yeast
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Abstract
본 발명은 인간 성장호르몬을 이용하여 어류의 성장을 촉진시키는 방법에 관한 것이다. 구체적으로, 본 발명은 인간 성장호르몬을 발현시키는 재조합 발현벡터를 만들고, 그 발현벡터를 효모에 도입하며, 상기 발현벡터로 형질전환된 효모로부터 재조합 인간 성장호르몬을 생성하고, 상기 재조합 인간 성장호르몬을 고농도로 함유하고 있는 효모 배양액 또는 효모 배양액과 효모균체 분쇄물을 사료에 혼합하여 어류에게 경구 투여함으로써 어류의 성장을 촉진시키는 방법에 관한 것이다.The present invention relates to a method for promoting the growth of fish using human growth hormone. Specifically, the present invention creates a recombinant expression vector expressing human growth hormone, introduces the expression vector into yeast, generates recombinant human growth hormone from the yeast transformed with the expression vector, the recombinant human growth hormone The present invention relates to a method of promoting growth of fish by orally administering to a fish by mixing yeast culture medium or yeast culture medium containing a high concentration and yeast cell ground powder.
Description
본 발명은 인간 성장호르몬을 이용하여 어류의 성장을 촉진하는 방법에 관한 것이다. 구체적으로, 본 발명은 인간 성장호르몬을 발현시키는 재조합 발현벡터를 만들고, 그 발현벡터를 효모에 도입하며, 상기 발현벡터로 형질전환된 효모로부터 재조합 인간 성장호르몬을 생성하고, 상기 재조합 인간 성장호르몬을 고농도로 함유하고 있는 효모 배양액 또는 효모 배양액과 효모균체 분쇄물을 사료에 혼합하여 어류에게 경구 투여함으로써 어류의 성장을 촉진시키는 방법에 관한 것이다.The present invention relates to a method for promoting the growth of fish using human growth hormone. Specifically, the present invention creates a recombinant expression vector expressing human growth hormone, introduces the expression vector into yeast, generates recombinant human growth hormone from the yeast transformed with the expression vector, the recombinant human growth hormone The present invention relates to a method of promoting growth of fish by orally administering to a fish by mixing yeast culture medium or yeast culture medium containing a high concentration and yeast cell ground powder.
성장호르몬(growth hormone: GH)은 척추동물의 시상하부에서 생성되는 호르몬이다. 191개의 아미노산으로 구성되어 있으며 척추동물의 초기발생과 정상적인 성장에 관여한다 (Palmiter, R. D. et al., Nature, 300, 611-615 (1982)). 성장호르몬에 관한 연구는 1920년대에 포유류의 뇌하수체 호르몬에 관한 연구로부터 시작되었으며, 1966년에는 처음으로 인간 성장호르몬의 아미노산 염기서열이 규명되었고 이후로 조류, 소, 말, 돼지의 아미노산 염기서열이 밝혀졌다.Growth hormone (GH) is a hormone produced in the hypothalamus of vertebrates. Consists of 191 amino acids and is involved in the early development and normal growth of vertebrates (Palmiter, R. D. et al., Nature, 300, 611-615 (1982)). The study of growth hormone began with the study of the pituitary hormones of mammals in the 1920s, and in 1966 the first amino acid sequence of human growth hormone was identified, and since that time, the amino acid sequences of birds, cattle, horses, and pigs were revealed. lost.
포유동물의 성장호르몬을 어류의 성장을 촉진하는데 사용하고자 하는 노력은 1957년 힉스 등에 의해 뇌하수체로부터 직접 분리된 성장호르몬을 가지고 처음 시도되었다 (Higgs, D. et al., Gen. Comp. Endocrinol. 27, 240-257 (1975)). 이후에도 온코린쿠스 케타(Oncorhynchus keta)의 뇌하수체(pituitary gland)로부터 성장호르몬을 추출하여 이를 무지개 송어(rainbow trout)에 주사로 투여한 예 (미국특허 제4,645,755호)를 포함하여, 많은 사람들이 뇌하수체로부터 직접 분리된 포유류 및 어류의 성장호르몬들을 가축과 어류의 성장을 촉진시키는데 이용하고자 노력하였다. 그러나 천연의 원료로부터 성장호르몬을 분리하여 어류의 성장 촉진용으로 사용하는 것은 원료의 공급에 한계가 있고 분리되는 양도 적으며 이를 추출하는데 많은 비용이 들기 때문에 현실적인 제약이 많았다. 이후, 개발된 유전자 재조합 기술은 성장호르몬의 다량 제조를 가능하게 함으로써 성장호르몬을 가축 및 어류의 성장 촉진제로 사용하는데 기여하였다. 그러나, 아직까지도 성장호르몬을 어류의 성장촉진을 위해 사용할 수 있는 경제적이고 실용적인 방법은 개발되지 못하였다고 할 수 있다.Efforts to use mammalian growth hormones to promote fish growth were first attempted in 1957 with growth hormones isolated directly from the pituitary gland (Higgs, D. et al., Gen. Comp. Endocrinol. 27). , 240-257 (1975). Subsequently, many people, including the example of extracting growth hormone from the pituitary gland of Oncorhynchus keta and administering it to the rainbow trout by injection (US Pat. No. 4,645,755), have received many people from the pituitary gland. Efforts have been made to use the growth hormones of mammals and fish isolated directly to promote the growth of livestock and fish. However, the separation of growth hormone from natural raw materials and the use of fish for promoting the growth of fish are limited because the supply of raw materials is limited, the amount of separation is small, and the cost of extraction is high. Later, the developed recombinant technology enabled the production of large amounts of growth hormone, thereby contributing to the use of growth hormone as a growth promoter for livestock and fish. However, economic and practical methods that can use growth hormone to promote fish growth have not yet been developed.
미국의 식품의약품국(FDA)이 성장호르몬을 사용한 소가 인체에 해가 없다고발표한 이후 유전공학적인 방법에 의해 생산된 성장호르몬은 상업적으로 많이 응용되었고 그와 관련 기술도 많이 개발되었다. 맥린 등은 소의 성장호르몬 (somatotropin)이 연어과 어류, 무지개 송어, 틸라피아 및 잉어에서 성장을 촉진하는 효과가 있다고 보고하였으며 (McLean et al., Aquaculture 91, 197-203 (1990)), 미국특허 제5,122,513호는 천연에서 분리되거나 또는 유전자 재조합에 의해 만들어진 소의 태반에서 유래하는 락토젠(bovine placental lactogen)을 급여하여 어류의 성장을 촉진시키는 방법을 기술하였다. 미국특허 제4,894,362호는 장어 성장호르몬을 유전자 재조합 방법으로 생산하여 이를 무지개 송어에 복강내 주사하여 성장 촉진효과를 조사한 것을 보고하였다.After the US Food and Drug Administration (FDA) announced that cows using growth hormone are harmless to humans, growth hormone produced by genetic engineering methods has been widely applied commercially and related technologies have been developed. McLean et al. Reported that bovine growth hormone (somatotropin) has the effect of promoting growth in salmon, fish, rainbow trout, tilapia and carp (McLean et al., Aquaculture 91, 197-203 (1990)), US Patent No. 5,122,513 No. describes a method of promoting the growth of fish by feeding bovine placental lactogen derived from the placenta of cattle isolated from nature or made by genetic recombination. U.S. Patent No. 4,894,362 reported that eel growth hormone was produced by genetic recombination and examined for growth promoting effect by intraperitoneal injection into rainbow trout.
대한민국특허 제1999-38718호는 유전자 재조합 기법으로 우럭(Sebastes schelegeli)의 성장호르몬을 대장균에서 대량 발현시켜 무지개 송어 등에게 급여하는 방법에 관하여 기술하고 있고, 대한민국특허 제10-201353호, 제10-201354호 및 제10-201355호는 분리된 재조합 소 성장호르몬을 투여하여 잉어, 광어 및 장어의 성장으로 촉진시키는 방법에 관하여 기술하고 있다.Korean Patent No. 1999-38718 describes a method of feeding a rainbow trout by expressing a growth hormone of Sebastes schelegeli in Escherichia coli by a gene recombination technique. Korean Patent Nos. 10-201353, 10- 201354 and 10-201355 describe methods of administering isolated recombinant bovine growth hormone to promote the growth of carp, flatfish and eel.
그러나, 상기 기술들은 모두 성장호르몬을 세균(bacteria), 특히 대장균 (E. coli)에서 발현시킨 것으로 이들 성장호르몬을 어류에 주사하거나 먹이기 전에 분리 정제하는 과정을 거쳐야 하는 불편함이 있다. 이에 본 발명자들은 좀 더 안전하고, 경제적이며 편리한 방법으로 어류의 성장을 촉진시키는 방법을 연구하던 중, 성장호르몬을 효모에서 발현시켜 그 배양 상등액 또는 배양 상등액과 효모 균체를 모두 수거하여 어류에게 급여하므로서 어류의 성장을 촉진시킬 수 있다는 것과, 지금껏 한번도 시도되지 아니하였던 것으로서 인간 성장호르몬을 어류에 투여한 결과 어류의 성장이 현저히 촉진된다는 놀라운 사실을 발견하고 본 발명을 완성하게 되었다.However, all of these techniques express growth hormones in bacteria, particularly E. coli, which is inconvenient to undergo separation and purification before injecting or feeding these growth hormones to fish. Therefore, while the present inventors have been studying how to promote the growth of fish in a safer, economical and convenient way, by expressing growth hormone in yeast, the culture supernatant or the culture supernatant and yeast cells are collected and fed to the fish. The present invention has been completed by discovering the remarkable fact that it is possible to promote the growth of fish, and that administration of human growth hormone to fish has not been attempted so far.
본 발명은 인간 성장호르몬을 함유하는 것을 특징으로 하는 어류 성장 촉진용 조성물을 제공한다. 상기 성장 촉진용 조성물은 바람직하게는 인간 성장 호르몬을 함유하는 것을 특징으로 하는 사료 조성물이다.The present invention provides a composition for promoting fish growth, characterized in that it contains human growth hormone. The growth promoting composition is preferably a feed composition comprising a human growth hormone.
본 발명은 나아가 상기 성장 촉진용 조성물이 재조합 벡터로 형질전환된 효모에 의해 생산된 재조합 인간 성장호르몬을 함유하는 것을 특징으로 하는 조성물을 제공한다.The present invention further provides a composition comprising the recombinant human growth hormone produced by the yeast transformed with the recombinant vector.
본 발명은 인간 성장호르몬을 어류에 투여하는 것을 특징으로 하는 어류의 성장을 촉진시키는 방법을 제공한다. 본 발명은 인간 성장호르몬을 사료에 첨가하여 경구 투여함을 특징으로 하는 어류 성장 촉진방법을 제공한다.The present invention provides a method for promoting the growth of fish, comprising administering human growth hormone to the fish. The present invention provides a method for promoting fish growth, characterized in that oral administration of human growth hormone to feed.
본 발명은 상기 인간 성장호르몬이 농축된 효모 배양액 또는 효모 배양액과 효모 균체를 함유하는 것을 특징으로 하는 어류 성장 촉진용 조성물 및 상기 조성물을 사용하여 어류의 성장을 촉진하는 방법을 제공한다.The present invention provides a composition for promoting fish growth, and a method for promoting fish growth using the composition, wherein the human growth hormone contains a yeast culture medium or a yeast culture medium in which the growth hormone is concentrated.
도 1은 인간 성장호르몬과 넙치의 성장호르몬 염기서열의 상동성을 비교하여 나타낸 것이다.Figure 1 shows the homology between the growth hormone nucleotide sequence of human growth hormone and halibut.
도 2는 인간 성장호르몬을 발현시키는 발현벡터 pYINU-hGH의 제작과정을 나타내는 개요도이다.Figure 2 is a schematic diagram showing the manufacturing process of the expression vector pYINU-hGH expressing human growth hormone.
도 3은 인간 성장호르몬의 넙치 성장촉진 효과를 보여주는 사진이다.Figure 3 is a photograph showing the growth effect of the halibut growth hormone of human.
본 발명은 인간 성장호르몬을 함유하는 것을 특징으로 하는 어류 성장 촉진용 조성물 및 상기 조성물을 사용하여 어류의 성장을 촉진시키는 방법을 제공한다. '인간 성장호르몬(Human growth hormone hGH)'은 뇌하수체전엽 호르몬의 하나로 인간의 성장과 발달을 조절한다. 인간 성장호르몬은 분자량이 약 22,000Da이며 장골의 생장촉진(somatogenesis), 젖분비(lactation), 대식세포의 활성화, 단백질 생합성 촉진과 같은 다양한 생물학적 효과를 지니고 있다 (Chawla, Annu. Rev. Med., 34, 519-547 (1983); Edwards et al., Science, 39, 769-771 (1988); Isaksson et al., Annu. Rev. Physiol., 47, 483-499 (1985)). 인간 성장호르몬의 DNA와 아미노산 서열은 공지되어 있으며 유전공학적 방법으로 형질전환된 E. coli, 효모 또는 동물세포에 의해 발현된 바 있다(Chang et al., Gene, 55, 189-196 (1987); Goeddel et al., Nature, 281, 544-548 (1979); Gray et al., Gene, 39, 247-254 (1985); 미국특허공보 제5,496,713호). 인간 성장호르몬은 주로 왜소증의 치료에 사용되어 왔으며 본 발명자들이 알고 있는 한 동물이나 어류의 성장을 촉진하기 위한 용도로 사용된 적은 없다.The present invention provides a composition for promoting fish growth, comprising a human growth hormone, and a method for promoting the growth of fish using the composition. Human growth hormone (hGH) is an anterior pituitary hormone that regulates human growth and development. Human growth hormone has a molecular weight of about 22,000 Da and has various biological effects, such as iliac somatogenesis, lactation, macrophage activation, and protein biosynthesis (Chawla, Annu. Rev. Med., 34, 519-547 (1983); Edwards et al., Science, 39, 769-771 (1988); Isaksson et al., Annu. Rev. Physiol., 47, 483-499 (1985)). The DNA and amino acid sequences of human growth hormone are known and have been expressed by E. coli, yeast or animal cells transformed by genetic engineering methods (Chang et al., Gene, 55, 189-196 (1987); Goeddel et al., Nature, 281, 544-548 (1979); Gray et al., Gene, 39, 247-254 (1985); US Pat. No. 5,496,713). Human growth hormone has been used mainly for the treatment of dwarfism and has not been used to promote the growth of animals or fish as far as the inventors know.
어류 성장호르몬에 관한 연구로는 1985년 세카인 등에 의하여 연어 성장호르몬의 cDNA 서열이 처음 밝혀 졌으며, 1986년 무지개 송어 성장호르몬 및 1988년에 장어(뱀장어) 성장호르몬 cDNA 서열들이 밝혀졌고 (Sekine, S., et at., Proc. Natl. Acad. Sci. Acad. USA., 82, 4306-4310 (1985); Agellon, L. B. and Chen, T. T., DNA, 5, 463-471; Saito, N., et al., Gene, 73, 545-552 (1988)), 장어 성장호르몬 및 연어 성장호르몬은 대장균에서 유전자 재조합 기술에 의하여 대량 발현된 바 있다 (미국특허 제4,894,362호; 미국특허 제4,689,402호).Studies on fish growth hormones first revealed the cDNA sequence of salmon growth hormone by Secain et al. In 1985, the rainbow trout growth hormone in 1986 and the eels (eel) growth hormone cDNA sequence in 1988 (Sekine, S , et at., Proc. Natl. Acad. Sci. Acad. USA., 82, 4306-4310 (1985); Agellon, LB and Chen, TT, DNA, 5, 463-471; Saito, N., et al., Gene, 73, 545-552 (1988)), eel growth hormone and salmon growth hormone have been mass-expressed in E. coli by genetic recombination techniques (US Pat. No. 4,894,362; US Pat. No. 4,689,402).
상기의 예에서 처럼 대장균에서 어류의 성장호르몬을 발현시킨 경우에는 대장균에서 유래하는 엔토톡신의 독성을 피하기 위해 이를 분리 정제하여야 하는 단점이 있다. 또한 대부분의 재조합 성장호르몬들은 불용성 응집체(inclusion body) 형태로 생산되기 때문에 호르몬을 활성화 시키기 위하여 리폴딩(refolding)과정을 거쳐야 하는데 이 방법이 복잡하고 경제적이지 못하다.When the growth hormone of the fish is expressed in E. coli as in the above example, there is a disadvantage in that the purified purified to avoid the toxicity of entotoxin derived from E. coli. In addition, since most recombinant growth hormones are produced in the form of insoluble aggregate (inclusion body), the refolding process is required to activate the hormone, which is not complicated and economical.
어류의 성장호르몬과 인간 성장호르몬은 상당한 서열의 이질성을 보인다. 예를 들어, 진뱅크(GeneBank)에 등록된 넙치와 인간 성장호르몬의 서열을 PUPMED BLAST Search 프로그램을 이용하여 상동성 검색을 한 결과에 의하면 상호간에 약 40%의 서열 유사성만이 존재한다(도 1). 그러나, 이러한 서열의 이질성에도 불구하고 본 발명자들은 놀라웁게도 인간 성장호르몬이 어류의 성장을 촉진하는 효과가 뛰어남을 발견하였다 (도 2, 도 3).Growth hormones and human growth hormones in fish show considerable heterogeneity. For example, the homology search of the flounder and human growth hormones registered in GeneBank using the PUPMED BLAST Search program shows that only about 40% of sequence similarity exists between each other (FIG. 1). ). However, despite the heterogeneity of these sequences, the inventors have surprisingly found that human growth hormone is superior in promoting fish growth (FIGS. 2 and 3).
본 발명의 목적을 달성하기 위하여 사람에게서 기원하는 천연의 성장호르몬 뿐만이 아니라 그의 기능적 동등물 또는 기능적 유도체를 사용할 수 있다. 따라서, 본원 발명의 목적상 본원에서 지칭하는 인간 성장호르몬의 범위에는 그의 기능적 동등물과 기능적 유도체가 포함된다. 인간 성장호르몬의 "기능적 동등물"이란 천연형 인간 성장호르몬과 실질적으로 동질의 생리 활성을 나타내며 천연의 인간 성장호르몬과 적어도 60% 이상의, 바람직하게는 70%, 더욱 바람직하게는 90% 이상의 서열 상동성을 갖는 단백질을 말한다. 상기 기능적 동등물에, 예를 들어, 천연형 단백질 아미노산 중 일부 또는 전부가 치환되거나, 아미노산의 일부가 결실 또는 부가된 아미노산 서열 변형체가 포함된다. 아미노산의 치환은 바람직하게는 보존적 치환이다. 천연에 존재하는 아미노산의 보존적 치환의 예는 다음과 같다; 지방족 아미노산(Gly, Ala, Pro), 소수성 아미노산 (Ile, Leu, Val), 방향족 아미노산(Phe, Tyr, Trp), 산성 아미노산 (Asp, Glu), 염기성 아미노산 (His, Lys, Arg, Gln, Asn) 및 황함유 아미노산(Cys, Met). 아미노산의 결실은 바람직하게는 인간 성장호몬의 생리 활성에 직접 관여하지 않는 부분에 위치한다.In order to achieve the object of the present invention, not only natural growth hormone derived from human but also functional equivalent or functional derivative thereof can be used. Thus, for the purposes of the present invention, the scope of human growth hormone referred to herein includes functional equivalents and functional derivatives thereof. A "functional equivalent" of human growth hormone is substantially homologous to a native human growth hormone and is at least 60% or greater, preferably 70%, more preferably 90% or more in sequence with native human growth hormone. Refers to proteins having the same sex. Such functional equivalents include, for example, amino acid sequence variants in which some or all of the naturally occurring protein amino acids are substituted, or some of the amino acids are deleted or added. Substitutions of amino acids are preferably conservative substitutions. Examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located at a portion that is not directly involved in the physiological activity of human growth hormone.
본 발명의 '기능적 유도체'는 인간 성장호르몬의 물리 화학적 성질을 증가 또는 감소시키기 위한 물리 화학적 변형을 가한 단백질을 말한다. 인간 성장호르몬의 온도 안정성, 저장성, 용해도, pH 안정성 등을 변경시키기 위한 변형 또는 예를 들어 인간 성장호르몬 수용체와의 결합력 정도를 변경시키기 위하여 변형을 가한 유도체가 본 발명의 범위에 포함된다. 상기와 같은 단백질의 기능적 유도체를 만드는 방법은 공지되어 있다.The 'functional derivative' of the present invention refers to a protein to which physicochemical modifications are added to increase or decrease the physicochemical properties of human growth hormone. Modifications to alter the temperature stability, shelf life, solubility, pH stability, etc. of human growth hormone, or derivatives that have been modified to modify the degree of binding to, for example, human growth hormone receptors, are included within the scope of the present invention. Methods of making functional derivatives of such proteins are known.
인간 성장호르몬은 뇌하수체로부터 직접 분리하거나 화학 합성하거나 또는 유전자 재조합 기술에 의하여 발현시킴으로써 제조할 수 있다. 인간 성장호르몬을 뇌하수체에서 분리하는 방법은 미국특허 제4,332,717호 등에 기재된 바와 같이 공지되어 있다. 예를 들어, 중성 또는 약염기성 완충용액에 냉동된 인간 뇌하수체를 첨가하여 혼합하고 균질화 다음 상기 혼합액을 원심분리하여 상등액을 획득한다. 상등액에 중성 또는 약산성 완충용액을 혼합한 다음 겔 필터레이션 또는 이온교환 크로마토그래피와 같은 컬럼 크로마토그래피를 사용하여 정제함으로써 인간 성장호르몬을 제조할 수 있다.Human growth hormone can be prepared by direct isolation from the pituitary gland, by chemical synthesis, or by expression by genetic recombination techniques. Methods for separating human growth hormone from the pituitary gland are known as described in US Pat. No. 4,332,717 or the like. For example, a frozen human pituitary gland is added to a neutral or weakly basic buffer solution, mixed, homogenized and centrifuged to obtain a supernatant. Human growth hormone can be prepared by mixing the supernatant with neutral or weakly acidic buffer and purifying by column chromatography such as gel filtration or ion exchange chromatography.
인간 성장호르몬은 당 분야에 공지된 폴리펩타이드 화학 합성 방법에 의하여 제조할 수 있다. 펩타이드는 통상의 단계적인 액체 또는 고체상 합성, 단편 응축, F-MOC 또는 T-BOC 화학법을 이용하여 제조할 수 있다 (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, (1997); A Practical Approach, Atherton ＆ Sheppard, Eds., IRL Press, Oxford, England, (1989)).Human growth hormone can be prepared by polypeptide chemical synthesis methods known in the art. Peptides can be prepared using conventional stepwise liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC). Press, Boca Raton Florida, (1997); A Practical Approach, Atherton & Sheppard, Eds., IRL Press, Oxford, England, (1989)).
특히, 바람직한 폴리펩타이드의 제조방법은 고체상 합성방법(solid phase synthesis)을 이용하는 것이다. 인간 성장호르몬은 보호된 아미노산간의 응축반응 (condensation reaction)에 의하여 통상의 고체상 방법으로, C-말단으로부터 시작하여 그 서열에 따라 순차적으로 진행하면서 합성할 수 있다. 응축 반응 후 보호기 및 C-말단 아미노산이 연결된 담체를 산분해 (acid decomposition) 또는 아미놀리시스 (aminolysis)와 같은 공지의 방법에 의해 제거할 수 있다. 상기 언급된 펩타이드 합성법은 관련 서적에 상세히 기술되어 있다 (Gross and Meienhofer's, The Peptides, vol 2., Academic Press (1980)).In particular, a preferred method of preparing a polypeptide is to use solid phase synthesis. Human growth hormone can be synthesized by a condensation reaction between protected amino acids in a conventional solid phase method, starting from the C-terminus and proceeding sequentially according to the sequence. After the condensation reaction, the carrier to which the protecting group and the C-terminal amino acid are linked can be removed by a known method such as acid decomposition or aminolysis. The peptide synthesis described above is described in detail in the related book (Gross and Meienhofer's, The Peptides, vol 2., Academic Press (1980)).
본 발명 펩타이드의 합성을 위해 사용할 수 있는 고체상의 담체는 이 분야에서 통상 사용되는 담체로서 예를 들어, 치환된 벤질 형태의 폴리스티렌 레진 (polystyrene resins of substituted benzyl type), 히드록시메틸페닐아세틱 아미드 형태의 폴리스티렌 레진, 치환된 벤즈히드릴폴리스티렌 레진, 펩타이드에 결합할 수 있는 기능기를 가진 폴리아크릴아미드 레진 등을 사용할 수 있다. 아미노산 응축 또한 통상의 방법일 수 있으며, 예를 들어 디시클로헥실카보디이미드(DDC), 산 무수물(acid anhydride) 및 활성화 에스테르 (activated ester) 방법이 사용될 수 있다.Solid phase carriers that can be used for the synthesis of the peptides of the present invention are carriers commonly used in the art, for example, polystyrene resins of substituted benzyl type, hydroxymethylphenylacetic amide, Polystyrene resins, substituted benzhydrylpolystyrene resins, polyacrylamide resins having functional groups capable of binding to peptides, and the like can be used. Amino acid condensation can also be a conventional method, for example dicyclohexylcarbodiimide (DDC), acid anhydride and activated ester methods can be used.
최초 보호 아미노산의 보호기들은 통상의 펩타이드 합성에 사용되는 보호기들로서 산 분해, 환원 또는 아미놀리시스와 같은 통상의 방법에 의해 쉽게 제거되는 것들이다. 아미노 보호기들의 구체예로는 포밀; 트리플루오로아세틸; 벤질옥시카보닐; (오르쏘- 또는 파라-) 클로로벤질옥시카르보닐 및 (오르쏘- 또는 파라-) 브로모벤질옥시카르보닐과 같은 치환된 벤질옥시카르보닐; t-부톡시카보닐 및 t-아밀록시카보닐과 같은 지방족 옥시카보닐 등이 있다. 아미노산의 카복실기들은 에스테르기로 전환시키므로써 보호할 수 있다. 에스테르기로는 벤질 에스테르, 메톡시벤질 에스테르와 같은 치환된 벤질 에스테르; 시클로헥실 에스테르, 시클로헵틸 에스테르 또는 t-부틸 에스테르와 같은 알킬 에스테르 등이 있다. 구아니디노기는 보호기가 필요하지 않지만, 니트로; 또는 토실, 메톡시벤젠슬포닐 또는 메시틸렌술포닐과 같은 아릴술포닐에의해 보호될 수 있다. 이미다졸의 보호기로는 토실, 벤질 및 디니트로페릴 등이 있다. 트립토판의 인돌기는 보호기가 없어도 되며 포밀 등으로 보호기를 붙일수도 있다.The protecting groups of the original protecting amino acids are those protecting groups used in conventional peptide synthesis and those which are easily removed by conventional methods such as acid degradation, reduction or aminolsis. Specific examples of amino protecting groups include formyl; Trifluoroacetyl; Benzyloxycarbonyl; Substituted benzyloxycarbonyl such as (ortho- or para-) chlorobenzyloxycarbonyl and (ortho- or para-) bromobenzyloxycarbonyl; aliphatic oxycarbonyl such as t-butoxycarbonyl and t-amyloxycarbonyl and the like. Carboxyl groups of amino acids can be protected by conversion to ester groups. Ester groups include substituted benzyl esters such as benzyl ester, methoxybenzyl ester; Alkyl esters such as cyclohexyl esters, cycloheptyl esters or t-butyl esters and the like. Guanidino groups do not require protecting groups, but nitro; Or arylsulfonyl such as tosyl, methoxybenzenesulfonyl or mesitylenesulfonyl. Protective groups for imidazoles include tosyl, benzyl and dinitroperyl. Tryptophan indole may not have a protecting group or may be attached with formyl.
탈보호 및 담체로부터 결과 펩타이드를 분리하는 것은 여러 스캐빈저 (scavenger) 하에 무수 하이드로 플루오라이드에 의해 수행될 수 있다. 스캐빈저의 예로는 아니솔, (오르쏘-, 메타-, 파라-) 크레솔, 디메틸술파이드, 씨오크레솔, 에탄엔디올 및 머캅토피리딘 등을 들 수 있는데 이들은 펩타이드 합성에서 통상 사용되는 것들이다.Deprotection and separation of the resulting peptides from the carrier can be performed with anhydrous hydrofluoride under various scavengers. Examples of scavengers include anisole, (ortho-, meta-, para-) cresol, dimethyl sulfide, thiocresol, ethaneenediol and mercaptopyridine, which are commonly used in peptide synthesis. to be.
본 발명의 폴리펩타이드는 예를 들면 추출법, 재결정법, 다양한 크로마토크래피(겔 여과법, 이온 교환, 침전, 흡착, 역전상), 전기영동, 역류 분배법등의 본분야에 공지된 방법으로 분리 및 정제할 수 있으며, 역전상 고성능 액체 크로마토그래피가 가장 효과적이다. 고체상법의 장점은 각 단계에서 생성되는 생성물이 비드에 결합되어 있기 때문에, 중간물질의 정제가 필요하지 않다는 것이다.Polypeptides of the present invention are isolated and purified by methods known in the art, for example, extraction, recrystallization, various chromatography (gel filtration, ion exchange, precipitation, adsorption, reverse phase), electrophoresis, countercurrent distribution, etc. Reversed-phase high performance liquid chromatography is most effective. The advantage of the solid phase method is that purification of the intermediate is not necessary because the product produced in each step is bound to the beads.
본 발명의 인간 성장호르몬은 유전자 재조합 기술에 의하여 제조할 수 있다. 인간 성장호르몬을 코드하는 핵산서열을 포함하는 발현벡터를 제조하여, 그 발현벡터를 적당한 숙주세포에서 발현시키고, 숙주세포로부터 펩타이드를 분리한다. 유전공학적으로 폴리펩타이드를 제조하는 방법은 당업계에 공지되어 있다 (Maniatis et al., Molecula Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982); Sambrook et al., supra; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Methods in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif. (1991); Hitzeman et al., J. Biol. Chem., 255, 12073-12080 (1980)). 구체적으로 인간 성장호르몬을 유전자 재조합 기술에 의하여 제조한 예들이 공지되어 있다 (Chang et al., Gene, 55, 189-196 (1987); Goeddel et al., Nature, 281, 544-548 (1979); Gray et al., Gene, 39, 247-254 (1985); 미국특허공보 제5,496,713). 위 어느 방법에 의하여 제조된 인간 성장호르몬이라도 본 발명의 어류의 성장을 촉진한다는 용도에 사용할 수 있다. 가장 바람직한 인간 성장호르몬은 후술하는 바와 같이 효모, 특히 사카로마이세스 세레비지애에서 발현, 제조되는 것이다. 따라서, 본 발명은 인간 성장호르몬이 재조합 벡터로 형질전환된 효모에 의하여 생산된 재조합 인간 성장호르몬임을 특징으로 하는 어류 성장 촉진용 조성물을 제공한다. 사카로마이세스 세레비지애를 이용하여 인간 성장호르몬을 제조하는 방법을 실시예 1 내지 3에 예시하였다.Human growth hormone of the present invention can be produced by genetic recombination technology. An expression vector comprising a nucleic acid sequence encoding human growth hormone is prepared, the expression vector is expressed in a suitable host cell, and the peptide is separated from the host cell. Methods for preparing polypeptides genetically are known in the art (Maniatis et al., Molecula Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982); Sambrook et al., Supra; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Methods in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif. (1991); Hitzeman et al., J. Biol. Chem., 255, 12073-12080 (1980 )). Specifically, examples of producing human growth hormone by genetic recombination techniques are known (Chang et al., Gene, 55, 189-196 (1987); Goeddel et al., Nature, 281, 544-548 (1979)). Gray et al., Gene, 39, 247-254 (1985); US Pat. No. 5,496,713. Human growth hormone prepared by any of the above methods can be used for the purpose of promoting the growth of the fish of the present invention. Most preferred human growth hormone is expressed and produced in yeast, in particular Saccharomyces cerevisiae, as described below. Accordingly, the present invention provides a composition for promoting fish growth, wherein the human growth hormone is a recombinant human growth hormone produced by a yeast transformed with a recombinant vector. A method for producing human growth hormone using Saccharomyces cerevisiae is illustrated in Examples 1 to 3.
본 발명은 인간 성장호르몬을 함유하는 것을 특징으로 하는 어류 성장 촉진용 '조성물'을 제공한다. 본 발명의 조성물은 바람직하게는 어류 성장 촉진용 사료 조성물이다. 인간 성장호르몬을 통상의 사료와 혼합하여 인간 성장호르몬이 함유된 성장촉진용 어류 사료 조성물을 제조한다. 어류양식에 사용되는 사료는 일반 사료원을 혼합하여 열과 압력을 가하여 펠렛형태로 제작한 배합사료(extruded pellet, EP), 양식어류의 먹이로 사용 가능한 어류를 펠렛형태로 만들어 그대로 사료로 사용하는 생사료 및 생사료와 가루사료(일반 사료원을 단순히 혼합한 사료) 혼합하여 펠렛형태로 만든 습사료(moisture pellet, MP)가 있다. 본 발명 성장촉진용 어류 사료는 인간 성장호르몬 분말을 통상의 어류 사료와 잘 혼합하여 펠렛 등의 형태로 제형화 할 수 있다. 예를 들어, 일반 배합사료 제작시 인간성장호르몬을 보조사료로 첨가하여 제조할 수 있으며, 또는 인간 성장호르몬을 물 또는 어간유에 용해시킨 후(용해가 가능한지는 확인 해야함), 배합사료에 골고루 적시는 방법 또는 스프레이로 사료에 분무하는 방법으로 배합사료에 잘 혼합하여 제조할 수 있다. 생사료와 습사료에는 가루형태인 인간 성장호르몬을 첨가하여 펠렛기안에서 충분히 혼합한 후에 펠렛형태로 제조할 수 있다.The present invention provides a composition for promoting fish growth, characterized by containing human growth hormone. The composition of the present invention is preferably a feed composition for promoting fish growth. Human growth hormone is mixed with a conventional feed to prepare a growth promoting fish feed composition containing human growth hormone. The feed used for fish farming is made of extruded pellets (EP) made of pellets by applying heat and pressure by mixing general feed sources, and raw feeds made of pellets made of fish that can be used for feeding fish. And wet feed (moisture pellet, MP) made of pellets by mixing raw feed and powdered feed (simply mixed with a general feed source). The growth-promoting fish feed of the present invention can be formulated in the form of pellets by mixing human growth hormone powders well with conventional fish feed. For example, it may be prepared by adding human growth hormone as a supplementary feed when preparing a general formula feed, or after dissolving human growth hormone in water or fish liver oil (make sure that it is soluble), soak the mixture evenly. It can be prepared by mixing well with the compound feed by spraying the feed method or spray method. Raw feed and wet feed can be prepared in pellet form after sufficient mixing in pellets by adding human growth hormone in powder form.
본 발명은 인간 성장 호르몬을 이용하여 어류의 성장을 촉진시키는 방법을제공한다. 본 발명자들은 인간 성장호르몬의 서열이 어류의 성장 호르몬과 많이 다름에도 불구하고 어류의 성장을 촉진하는 놀라운 효과가 있음을 발견하였다. 본 발명의 실시예에서 재조합 인간 성장호르몬을 각각 10, 20, 40 mg/ Kg 어체중/주 (Yeast-FGH10,Yeast-FGH20,Yeast-FGH40) 가 되도록 함유한 실험사료를 제조하여 이를 4주 동안 급여하였다. 그 결과 대조구에 비해 인간 성장호르몬을 첨가한 모든 실험구의 증체율이 유의적으로 높게 나타났으며 (P<0.05), Yeast-FGH20를 급여한 경우가 가장 높은 증체율을 보였다. 사료효율도 대조구에 비해 인간 성장호르몬을 첨가한 모든 실험구가 유의적으로 높은 결과를 나타냈으며(P<0.05), Yeast-FGH20을 급여한 경우 가장 높은 결과를 나타냈다. 생존율은 모든 실험구간에 유의적인 차이가 없었으며 한 마리의 폐사도 일어나지 않았다(표 2, 도 3). 즉, 대조구에 비해 본원 발명의 인간 성장호르몬을 함유한 사료를 4주간 급여한 경우 인간 성장호르몬을 함유하지 않은 기본사료를 급여한 경우에 비하여 어류의 성장률이 최대 22%, 사료효율이 20% 증진되어 성장 촉진효과가 관찰되었고, 6주간 실험에서는 37%의 성장 촉진효과를 나타내었다. 어류의 성장을 촉진시키기 위하여 투여하여야 할 인간 성장호르몬의 투여량 및 투여방법은 어종, 어류의 발생 정도, 투여 방법 등에 따라 조절되어야 하며, 그러한 조건은 당업자가 위 실시예의 결과를 기준으로 예측하거나 단순 실험의 반복으로 확인할 수 있다.The present invention provides a method of promoting the growth of fish using human growth hormone. The inventors have found that there is a surprising effect of promoting the growth of fish despite the fact that the sequence of human growth hormone differs greatly from the growth hormone of fish. In the embodiment of the present invention prepared experimental feed containing the recombinant human growth hormone to 10, 20, 40 mg / Kg body weight / week (Yeast-FGH 10, Yeast-FGH 20, Yeast-FGH 40 ), respectively Salary for 4 weeks. As a result, all of the experimental groups containing human growth hormone were significantly higher than the control group (P <0.05), and Yeast-FGH 20 was the highest. Feed efficiency was significantly higher in all experimental groups containing human growth hormone than the control group (P <0.05), and the highest results were obtained when Yeast-FGH 20 was fed. Survival was not significantly different in all experiments and no mortality occurred (Table 2, Figure 3). In other words, compared to the control group, the feed containing human growth hormone of the present invention was fed for 4 weeks, the growth rate of fish was increased up to 22% and the feed efficiency was increased by 20% compared with the feeding of basic feed containing no human growth hormone. The growth promoting effect was observed, and the 6 week experiment showed 37% growth promoting effect. The dose and method of administration of human growth hormone to be administered to promote the growth of fish should be adjusted according to the species of fish, the incidence of fish, the method of administration, etc. This can be confirmed by repeating the experiment.
본 발명의 인간 성장호르몬을 어류에 적용하는 가장 바람직한 방법은 성장호르몬을 어류의 사료에 첨가하여 경구 투여하는 것이다. 성장호르몬을 어류에 투여하는 방법에는 크게 주사법(injection), 입욕법(immersion) 및 경구투여법(oral administration)이 있다. 주사법은 너무 번거로우며 주사시 많은 스트레스를 받아 성장저하와 같은 부작용이 있을 수 있다. 입욕법의 경우 그 효과가 육성어의 경우 확실치 않을 뿐만이 아니라 이에 드는 비용도 적지 않다. 본 발명의 목적을 달성할 수 있는 한 본 발명의 인간 성장호르몬을 함유하는 조성물은 상기 어떠한 방법으로 어류에 투여되어도 무관하나 가장 바람직한 방법은 이를 사료에 첨가하여 어류에 경구 투여하는 것이다.The most preferred method of applying the human growth hormone of the present invention to fish is by orally administering the growth hormone to the feed of the fish. There are largely methods of administering growth hormone to fish, such as injection, immersion, and oral administration. Injections are too cumbersome and have a lot of stress during injections, which can have side effects such as slowing growth. In the case of the bathing method, the effect is not sure in the case of rearing fish, and the cost is not small. As long as the object of the present invention can be achieved, the composition containing the human growth hormone of the present invention may be administered to fish by any of the above methods, but the most preferred method is to add it to a feed and orally administer the fish.
어류의 경우에는 포유동물과 달리 단백질이나 펩타이드의 경구투여가 가능하다는 것을 보여주는 많은 실험 결과들이 있으며(Pickford, G. E and Atz, J W., The physiology of the pituitary gland of fishers, New York : New York Zoological Society(1957): Asy, R., Protein digestion and absorption, In.'nutrition and feeding in fish (Cowey, C. B, Macki, A. M., Bull, J.G., eds), 69-93, London, Academic Press (1985); Mclean, E., and Donaldson, E. M., Absorptiion of bioactive proteins by the gastrointestinal tract of fish: a review. J. Aquatic Animal Health 2, 1-11(1990)) 이에 따라 경구투여에 의한 성장 호르몬의 전달 가능성과 어류에 대한 성장효과가 실험적으로 입증되었다 (Donaldson, E. M., Fagerulund, U. H. M., Higgs D., and McBride, J. R., In: Enhancement of growth in fish(Hoar, W. S., Randall, D. J., and Brett, J. J., eds.), fish physiology, Vol VIII: 456-497, New York: Academic Press(1979);McLean at al., Fish Physiology and Biochemistry, 11, 363-369 (1990); Kawauchi, H. et al., Oceanis, 18(1), 109-120 (1992)).In the case of fish, there are many experimental results showing that oral administration of proteins or peptides is possible unlike mammals (Pickford, G. E and Atz, J W., The physiology of the pituitary gland of fishers, New York: New York) York Zoological Society (1957): Asy, R., Protein digestion and absorption, In.'nutrition and feeding in fish (Cowey, C. B, Macki, AM, Bull, JG, eds), 69-93, London, Academic Press (1985); Mclean, E., and Donaldson, EM, Absorptiion of bioactive proteins by the gastrointestinal tract of fish: a review.J. Aquatic Animal Health 2, 1-11 (1990)) Hormonal delivery potential and growth effects on fish have been experimentally demonstrated (Donaldson, EM, Fagerulund, UHM, Higgs D., and McBride, JR, In: Enhancement of growth in fish (Hoar, WS, Randall, DJ, and Brett, JJ, eds.), Fish physiology, Vol VIII: 456-497, New York: Academic Press (1979); McLean at al., Fish Physiology and Biochemistry, 11, 363-369 (1990); Kawauchi, H. et al., Oceanis, 18 (1), 109-120 (1992)).
인간 성장호르몬의 투여량은 어종이나 어류의 발생과정 또는 무게 등에 따라 달라질 수 있으며, 당업자는 통상의 기술을 이용하여 적정량을 산정 할 수 있다. 본 발명에서 넙치를 상대로 한 실험에서는 20mg/Kg 어체중/주 의 량이 어류의 체중을 증가시키는데 가장 효과적인 것으로 나타났다. 본 발명의 인간 성장호르몬은 2 내지 40, 바람직하게는 15 내지 30, 더욱 바람직하게는 15 내지 25 mg/Kg 어체중 / 주 의 량으로 투여한다. 기존에 성장 호르몬은 어류에 투여하여 어류의 성장을 촉진시키는 경우에는 통상 1주 또는 2주 단위로 한번씩 처리하는 것을 특징으로 하였으나 본 발명의 경우에는 이를 어류의 사료에 혼합하여 매일 일정량을 공급하는 것이 특히 바람직하다.The dosage of human growth hormone may vary depending on the species or the development or weight of the fish, and those skilled in the art can calculate an appropriate amount using conventional techniques. In the experiments with the flounder in the present invention, the 20mg / Kg body weight / week was found to be the most effective to increase the weight of the fish. Human growth hormone of the present invention is administered in 2 to 40, preferably 15 to 30, more preferably 15 to 25 mg / Kg body weight / week dose. Conventionally, when growth hormone is administered to fish to promote the growth of fish, it is characterized in that it is usually treated once every 1 or 2 weeks, but in the case of the present invention, it is mixed with the feed of fish to supply a certain amount every day. Particularly preferred.
본 발명은 인간 성장호르몬이 농축된 효모 배양액 또는 효모 배양액과 효모 균체를 포함하는 것을 특징으로 하는 성장 촉진용 조성물 및 상기 성장 촉진용 조성물을 어류에 경구 투여함을 특징으로 하는 어류 성장 촉진 방법을 제공한다. 상기 성장 촉진용 조성물은 바람직하게는 인간 성장호르몬이 농축된 효모 배양액 또는 효모 배양액과 효모 균체를 포함하는 것을 특징으로 하는 성장 촉진용 사료 조성물이다.The present invention provides a growth promoting composition comprising a yeast culture solution or a yeast culture medium in which human growth hormone is concentrated, and a yeast cell, and a method for promoting fish growth oral administration to the fish. do. The growth promoting composition is preferably a growth promoting feed composition comprising a yeast culture solution or yeast culture medium and human yeast growth medium is concentrated.
사카로마이세스 세레비지애는 현재 호스트-벡터(host-vector)계가 가장 잘 확립되어 있는 효모이며 인체에 무해하고 엔도톡신(endotoxin)을 생산하지 않는GRAS(generally recognized as safe) 미생물이다. 재조합 인간 성장호르몬이 대장균 숙주에서 생산된 경우가 있다 (미국특허공보 US 5,496,713호). 그러나, 대장균을 숙주로 사용하는 경우에는 재조합 인간 성장호르몬이 대부분 불용성 응집체(inclusion body)로 대장균 내에 축적되기 때문에 이를 활성화시키기 위하여 리폴딩(refolding)과정을 거쳐야하고 대장균으로부터 만들어진 엔도톡신(endotoxi n) 때문에 성장호르몬을 대장균 배양액으로부터 분리하여야한다.Saccharomyces cerevisiae is a yeast whose host-vector system is currently well established and is harmless to the human body and does not produce endotoxin. It is a generally recognized as safe (GRAS) microorganism. Recombinant human growth hormone is sometimes produced in E. coli hosts (US Pat. No. 5,496,713). However, when E. coli is used as a host, recombinant human growth hormone is mostly accumulated in E. coli as an insoluble inclusion body, and thus requires refolding to activate it, and due to endotoxi n produced from E. coli. Growth hormone should be separated from E. coli culture.
본 발명자들은 재조합 인간 성장호르몬을 효모, 더욱 바람직하게는 사카로마이세스 세레비지애에서 발현시킨 후 그 발효배양액 전체를 어류의 성장촉진용 조성물로 사용하는 방법을 개발하였다. 이때 인간 성장호르몬은 바람직하게는 분비 단백질 형태로 발현되는 것이며, 상기 인간 성장호르몬이 분비된 효모 배양액 또는 효모 배양액과 효모균체 분쇄물을 함께 어류의 성장촉진용 조성물로 사용할 수 있다. 본 발명의 성장촉진용 조성물은 바람직하게는 양어 사료에 첨가하여 어류에 경구투여한다. 따라서, 본 발명은 상기 인간 성장호르몬이 농축된 효모 배양액 또는 효모 배양액과 효모 균체, 더욱 바람직하게는 인간 성장호르몬이 농축된 효모 배양액 또는 효모 배양액과 효모균체 파쇄물을 사료에 첨가하여 급여함을 특징으로 하는 어류 성장 촉진방법을 제공한다.The present inventors have developed a method of expressing recombinant human growth hormone in yeast, more preferably Saccharomyces cerevisiae, and then using the entire fermentation broth as a composition for promoting growth of fish. In this case, the human growth hormone is preferably expressed in the form of a secreted protein, and the yeast culture medium or yeast culture medium in which the human growth hormone is secreted may be used as a composition for promoting growth of fish together. The growth promoting composition of the present invention is preferably orally administered to fish in addition to the fish feed. Therefore, the present invention is characterized in that the yeast culture solution or yeast culture medium and yeast culture medium enriched with human growth hormone, and more preferably the yeast culture medium or yeast culture medium and yeast cell lysate enriched in human growth hormone is added to feed It provides a method of promoting fish growth.
본 발명의 실시예에서는 인간 성장호르몬을 제조하기 위하연 효모에서 분비 능력이 뛰어난 것으로 알려진 이누리나제 시그날 서열을 이용하였다 (대한민국특허 제145452호; 미국특허공보 제5,712,113호). 그러나, 효모에서 인간 성장호르몬을 발현할 수 있는 어떠한 방법이라도 본 발명의 목적을 위하여 적합하게 사용할 수있다. 시그날 서열이 부착되거나 또는 부착되지 않은 성장호르몬 유전자를 발현하는 벡터를 제조하여 이를 효모에 형질전환 시킨 후 그 효모를 배양하여 재조합 인간성장호르몬을 제조한다. 인간 성장호르몬의 발현을 최대화하기 위하여 발현 인듀서를 사용할 수 있다.In an embodiment of the present invention, an inulinase signal sequence known to have excellent secretion ability in yeast yeast to produce human growth hormone was used (Korean Patent No. 145452; US Patent Publication No. 5,712,113). However, any method capable of expressing human growth hormone in yeast may be suitably used for the purposes of the present invention. A recombinant human growth hormone is prepared by preparing a vector expressing a growth hormone gene with or without a signal sequence attached thereto, transforming it into yeast, and culturing the yeast. Expression inducers can be used to maximize the expression of human growth hormone.
구체적으로, 본 발명 실시예의 이누리나제 시그날 서열의 유전자는 pYGILp10을 주형으로하여 PCR로 증폭하여 제작하였다. 인간 성장호르몬 유전자는 인간 유래의 성장호르몬 유전자를 가진 pT2GH (Shin et al., J. Biotechnol., 62, 143-151 (1998))를 주형으로 PCR로 증폭한 후 플라스미드 pBluescript KS+ (Novagen, 독일)를 제한효소 EcoRⅠ과 SalⅠ로 처리하여 아가로스 젤에서 분리한 다음, 위에서 얻어진 이누리나제 단편과 인간 성장호르몬 단편을 연결하여 pBINU-hGH를 제작하였다.Specifically, the gene of the inulinase signal sequence of the embodiment of the present invention was prepared by amplifying by PCR using pYGILp10 as a template. The human growth hormone gene was amplified by PCR with pT2GH (Shin et al., J. Biotechnol., 62, 143-151 (1998)) having a human growth hormone gene as a template, followed by plasmid pBluescript KS + (Novagen, Germany). Was isolated from agarose gel by treatment with restriction enzymes EcoR I and Sal I, and pBINU-hGH was prepared by linking the inulinase fragment obtained above with the human growth hormone fragment.
플라스미드 YEGa-HIR525(KCTC 8518P)를 EcoRⅠ과 SalⅠ으로 절단하여 약 5,500bp를 갖는 단편을 분리하고, 그 분리한 단편과 pBINU-hGH를 EcoRⅠ과 SalⅠ으로 절단하여 형성된 시그날 서열을 포함하는 hGH 단편을 리가제(T4 DNA ligase, 베링거 만하임사, 독일)로 연결하여 플라스미드 pYINU-hGH를 제작하였다. 플라스미드 pYINU-hGH는 GAL10 프로모터::이누리나제 시그날 서열::hGH유전자::GAL7 터미네이터를 순차적으로 포함하고 있다. 플라스미드 pYINU-hGH의 제작과정을 도 2에 나타내었다.The plasmid YEGa-HIR525 (KCTC 8518P) was digested with EcoR I and Sal I to isolate a fragment having about 5,500 bp, and the hGH fragment containing the isolated fragment and the signal sequence formed by cleaving pBINU-hGH with Eco R I and Sal I were ligated. The plasmid pYINU-hGH was prepared by linking with T4 DNA ligase (Beringer Mannheim, Germany). Plasmid pYINU-hGH sequentially contains the GAL10 promoter :: inurinase signal sequence :: hGH gene :: GAL7 terminator. The construction of the plasmid pYINU-hGH is shown in FIG. 2.
상기 인간 성장호르몬을 발현하는 벡터를 사카로마이세스 세레비지애 2805 (MATαpep4::HIS3 prb1 can1 his3-200.ura3-52)에 형질전환시켰다 (Sambrook, J.,Fritsch, E. E., Maniatis, T., Molecular Cloning: A Laboratory Manual., 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring, New York (1980)). pYINU-hGH 벡터를 열충격법으로 효모에 도입시켜 사카로마이세스 세레비지애 2805 균주 2805/pYINU-hGH5/pYINU-hGH를 얻었다.The vector expressing human growth hormone was transformed into Saccharomyces cerevisiae 2805 ( MATαpep4 :: HIS3 prb1 can1 his3-200.ura3-52 ) (Sambrook, J., Frisch , EE, Maniatis, T. , Molecular Cloning: A Laboratory Manual., 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring, New York (1980)). The pYINU-hGH vector was introduced into the yeast by thermal shock to obtain Saccharomyces cerevisiae 2805 strain 2805 / pYINU-hGH5 / pYINU-hGH.
형질전환된 사카로마이세스 세레비지애 2805/pYINU-hGH를 배양하여 인간 성장호르몬을 제조하였다. 재조합 인간 성장호르몬의 고발현을 위한 인듀서 (inducer)로는 갈락토오스를 사용하였다. 형질전환된 효모로부터 인간 성장호르몬이 발현되었음은 웨스턴블롯으로 이를 확인하였고, 발현된 성장호르몬의 양은 덴시토미터를 이용하여 정량하였다. 이렇게 생산된 효모 배양액을 원심분리하여 인간 성장호르몬이 농축되어 있는 배양 상등액과 효모균체로 분리하였다. 상등액에 존재하는 재조합 인간 성장호르몬을 농축하고, 분리된 효모균체는 세포벽을 분쇄하여 농축한 재조합 인간 성장호르몬과 분쇄한 효모균체를 함께 동결 건조하여 재조합 인간 성장호르몬 분말(Yeast-FGH)을 제조하였다. 재조합 인간 성장호르몬 분말은 위에서 설명한 양어사료를 만드는데 사용하였다.Human growth hormone was prepared by culturing the transformed Saccharomyces cerevisiae 2805 / pYINU-hGH. Galactose was used as an inducer for the high expression of recombinant human growth hormone. It was confirmed by Western blot that the human growth hormone was expressed from the transformed yeast, and the amount of the growth hormone expressed was quantified using a densitometer. The yeast culture solution thus produced was centrifuged and separated into culture supernatant and yeast cells in which human growth hormone was concentrated. The recombinant human growth hormone present in the supernatant was concentrated, and the isolated yeast cells were lyophilized together with the concentrated human growth hormone and the crushed yeast cells by crushing the cell wall to prepare a recombinant human growth hormone powder (Yeast-FGH). . Recombinant human growth hormone powder was used to make the fish farm described above.
이하, 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 단, 하기 실시예들은 본 발명을 예시하는 것으로 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.
<실시예 1><Example 1>
인간 성장호르몬 발현벡터 pYINU-hGH의 제작Construction of Human Growth Hormone Expression Vector pYINU-hGH
인간 성장호르몬 발현벡터 pYINU-hGH를 이누리나제 시그날 서열을 이용하여 제작하였다. 이누리나제 시그날 서열 유전자의 제작을 위하여 이누리나제 유전자를 가진 pYGILp10을 주형으로 하여 PCR로 증폭하여 제작하였다. 이때 사용한 프라이머는 다음과 같다.Human growth hormone expression vector pYINU-hGH was constructed using the inulinase signal sequence. For the production of the inulinase signal sequence gene, pYGILp10 having the inulinase gene was used as a template and amplified by PCR. The primer used at this time is as follows.
프라이머 1(센스, 서열번호 1)Primer 1 (sense, SEQ ID NO: 1)
5'-GAA TTC ATG AAG TTA GCA TAC TTC CTC TTG-3'5'-GAA TTC ATG AAG TTA GCA TAC TTC CTC TTG-3 '
프라이머 2(안티센스, 서열번호 2)Primer 2 (antisense, SEQ ID NO: 2)
5'- TCT CTT GTA ATT AAT AAC TGA AGC ACT GAC-3'5'- TCT CTT GTA ATT AAT AAC TGA AGC ACT GAC-3 '
상기 프라이머의 N-말단에는 발현벡터에 삽입될 수 있도록 EcoRI 제한효소 절단부위를 도입하였고 인간 성장호르몬과 연결될 C-말단에는 제한효소 AsnI 절단부위를 도입하였다. PCR 반응은 94℃, 30초간 열변성한 후 55℃, 30초간 프라이머 어닐링(annealing)하고 72℃, 30초, 30사이클로 DNA 합성신장을 수행하였다. PCR로 얻어진 단편(75bp)을 제한효소 EcoRI 과 AsnI으로 절단하여 얻어진 이누리라제 단편을 이누아가로스 젤(agarose gel)에서 분리하였다.An EcoRI restriction enzyme cleavage site was introduced at the N-terminus of the primer so as to be inserted into an expression vector, and a restriction enzyme AsnI cleavage site was introduced at the C-terminus to be linked to human growth hormone. The PCR reaction was thermally denatured at 94 ° C. for 30 seconds, followed by primer annealing at 55 ° C. for 30 seconds, and DNA synthesis extension at 72 ° C., 30 seconds, and 30 cycles. A fragment obtained by PCR (75bp) was digested with restriction enzymes EcoRI and AsnI, and the inuriase fragment obtained was isolated from an agarose gel.
인간 성장호르몬 유전자는 인간 유래의 성장호르몬 유전자를 가진 pT2GH (Shin et al., J. Biotechnol., 62, 143-151 (1998))를 주형으로 위와 동일한 조건에서 PCR를 실시하여 제작하였다. PCR에 사용된 프라이머는 다음과 같다.Human growth hormone gene was prepared by PCR using pT2GH (Shin et al., J. Biotechnol., 62, 143-151 (1998)) having a growth hormone gene of human origin under the same conditions as above. Primers used for PCR are as follows.
프라이머 3(센스, 서열번호 3)Primer 3 (sense, SEQ ID NO: 3)
5'-TCA GTT ATT AAT TAC AAG AGA TTC CCA GCC ATG TCC TTG TCC-3'5'-TCA GTT ATT AAT TAC AAG AGA TTC CCA GCC ATG TCC TTG TCC-3 '
프라이머 4(안티센스, 서열번호 4)Primer 4 (antisense, SEQ ID NO: 4)
5'-GTC GAC CTA GAA GCC ACA GCT GCC CTC CAC AGA-3'5'-GTC GAC CTA GAA GCC ACA GCT GCC CTC CAC AGA-3 '
프라이머 3에는 이누리나제 시그날 서열에 연결될 수 있도록 AsnⅠ제한효소 절단부위를 도입하였고 프라이머 4에는 제한효소 SalⅠ 절단부위를 도입하였다. PCR로 획득된 단편(585bp)을 제한효소 ASnI 과 SalI 으로 절단하였다. 그 결과 얻어진 인간 성장호르몬 단편을 아가로스 젤로 분리하였다.In primer 3, an AsnI restriction enzyme cleavage site was introduced so as to be linked to the inulinase sequence, and primer 4 was introduced with a restriction enzyme SalI cleavage site. The fragment (585bp) obtained by PCR was digested with restriction enzymes ASnI and SalI. The resulting human growth hormone fragment was isolated by agarose gel.
플라스미드 pBluescript KS+ (Novagen, 독일)를 제한효소 EcoRⅠ과 SalⅠ로 처리하여 아가로스 젤에서 분리한 다음, 위에서 얻어진 이누리나제 단편과 인간 성장호르몬 단편을 연결하여 pBINU-hGH를 제작하였다. 상기 pBINU-hGH는 합성한 인간 성장호르몬의 유전자 염기서열 확인에 사용하였다.The plasmid pBluescript KS + (Novagen, Germany) was treated with the restriction enzymes EcoR I and Sal I, isolated from the agarose gel, and pBINU-hGH was prepared by linking the inulinase fragment obtained above with the human growth hormone fragment. The pBINU-hGH was used to confirm the gene sequence of the synthetic human growth hormone.
플라스미드 YEGa-HIR525(KCTC 8518P)를 EcoRⅠ과 SalⅠ으로 절단하여 약 5,500bp를 갖는 단편을 분리하였다. 그 분리한 단편과 pBINU-hGH를 EcoRⅠ과 SalⅠ으로 절단하여 형성된 hGH 단편을 리가제(T4 DNA ligase, 베링거 만하임사, 독일)로 연결하여 플라스미드 pYINU-hGH를 제작하였다. 플라스미드 pYINU-hGH는 GAL10 프로모터::이누리나제 시그날 서열::hGH유전자::GAL7 터미네이터를 순차적으로 포함하고 있다. 플라스미드 pYINU-hGH의 제작과정을 도 2에 나타내었다.Plasmid YEGa-HIR525 (KCTC 8518P) was digested with EcoR I and Sal I to isolate fragments with about 5,500 bp. The isolated fragment and the hGH fragment formed by cleaving pBINU-hGH with EcoR I and Sal I were linked with ligase (T4 DNA ligase, Boehringer Mannheim, Germany) to prepare plasmid pYINU-hGH. Plasmid pYINU-hGH sequentially contains the GAL10 promoter :: inurinase signal sequence :: hGH gene :: GAL7 terminator. The construction of the plasmid pYINU-hGH is shown in FIG. 2.
<실시예 2><Example 2>
사카로마이세스 세레비지애 2805의 형질전환Transformation of Saccharomyces cerevisiae 2805
Ito 등의 방법(Ito et al., J. Bacteriol. 153, 163-168 (1983))에 따라 실시예 1에서 제작된 플라스미드 pYINU-hGH를 가지고 사카로마이세스 세레비지애 (Saccharomyces serevisiae) 2805 (MATαpep4::HIS3 prb1 can1 his3-200.ura3-52)를 형질전환시켰다 (Sambrook, J., Fritsch, E. E., Maniatis, T., Molecular Cloning: A Laboratory Manual., 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring, New York (1980)).Saccharomyces serevisiae 2805 (with plasmid pYINU-hGH prepared in Example 1 according to Ito et al. (Ito et al., J. Bacteriol. 153, 163-168 (1983)) MATαpep4 :: HIS3 prb1 can1 his3-200.ura3-52 ) (Sambrook, J., Fritsch, EE, Maniatis, T., Molecular Cloning: A Laboratory Manual., 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring, New York (1980)).
YPD 배지 (효모 추출액 1%, 박토 펩톤 2%, 포도당 2%) 10 ml에서 30℃로 하룻 밤동안 진탕 배양하여 얻은 사카로마이세스 세레비지애 2805 배양액 300 ㎕를 다시 YPD 배지 10 ml에 접종하여 흡광도가 1.0 (OD600=1.0)이 될 때까지 30℃에서 진탕 배양하였다. 배양액 1 ml을 5000 rpm에서 5분간 원심분리하여 배지를 제거한 후 Tris-EDTA 완충액 (10 mM Tri-HCl, 1 mM EDTA, pH 8.0) 1 ml로 균체를 세척하였다. 500 ㎕의 완충액과 0.2 M 염화리튬(LiCl2) 500 ㎕를 넣어 잘 현탁한 후 30℃에서 1시간 진탕 배양하고, 진탕액 100 ㎕에 실시예 1에서 제작한 플라스미드 pYINU-hGH 3 ㎕, 70% 폴리에틸렌글리콜 4000 용액 200 ㎕를 각각 첨가하여 30℃에서 30분 동안 진탕 배양한 후 45 ℃에서 15분간 열충격을 가하였다. 15,000 rpm에서 1분간 원심분리하여 상층액을 버리고, 0.8% 식염수로 침전물을 세척하였다. 100 ㎕의 식염수에 현탁시킨 현탁액을 최소선택배지 (minimal selection media; 아미노산 부재의 효모질소 기질 0.67%, 포도당 2%, 카사미노애시드(casamino acid) 0.5%, 아가로스 2%)에 도말한 후 30℃에서 3일간 배양하여 형질전환된 사카로마이세스 세레비지애 2805 균주 2805/pYINU-hGH를 얻었다.300 μl of Saccharomyces cerevisiae 2805 culture obtained by shaking culture overnight at 10 ° C. in 10 ml of YPD medium (1% yeast extract, 2% bacterium peptone, 2% glucose) was inoculated again in 10 ml of YPD medium. Shaking was incubated at 30 ° C. until the absorbance became 1.0 (OD 600 = 1.0). 1 ml of the culture was centrifuged at 5000 rpm for 5 minutes to remove the medium, and the cells were washed with 1 ml of Tris-EDTA buffer (10 mM Tri-HCl, 1 mM EDTA, pH 8.0). 500 μl of buffer and 500 μl of 0.2 M lithium chloride (LiCl 2 ) were added and suspended well, followed by shaking culture for 1 hour at 30 ° C., 3 μl of the plasmid pYINU-hGH prepared in Example 1 to 100 μl of shake solution. 200 μl of polyethylene glycol 4000 solution was added thereto, followed by shaking culture at 30 ° C. for 30 minutes, followed by thermal shock at 45 ° C. for 15 minutes. The supernatant was discarded by centrifugation at 15,000 rpm for 1 minute and the precipitate was washed with 0.8% saline. The suspension suspended in 100 μl saline was plated in minimal selection media (0.67% yeast nitrogen substrate without amino acid, 2% glucose, 0.5% casamino acid, 2% agarose) Incubation at 3 ° C. for 3 days yielded transformed Saccharomyces cerevisiae 2805 strain 2805 / pYINU-hGH.
<실시예 3><Example 3>
사카로마이세스 세레비지애 2805/pYINU-hGH 를 이용한 재조합 인간 성장호르몬의 제조Preparation of Recombinant Human Growth Hormone Using Saccharomyces cerevisiae 2805 / pYINU-hGH
형질전환된 사카로마이세스 세레비지애 2805/pYINU-hGH 콜로니 하나를 선택하여 10ml의 YNBCAD(0.67% yeast nitrogen base without amino acids, 2%포도당, 05% casamino acid)에 접종하고 30℃에서 16시간 배양하여 1차 접종원을 제조하였다. 상기 1차 접종원을 새로운 YNBCAD배지에 5-10%의 농도로 접종하고 동일한 방법으로 배양하여 2차 접종원을 제조하였다. 고농도의 재조합 인간 성장호르몬을 생산하기 위해 세포 성장에 맞추어 영양원을 공급해주는 유가식 배양을 실시하였다. 유가식 배양시 초기배지 및 공급배지의 조성은 다음과 같다. 초기배지로는 포도당(glucose) 15g/L, 효모추출물(yeast extract) 3g/L, 카사미노 애시드 (casamino acid) 3g/L, 황산암모늄(ammonium sulfate) 3g/L, 이인산 칼륨 (potassium diphosphate)1.5g/L, 황산마그네슘(magnesium sulfate) 0.5g/L, 염화칼슘(calcium chloride) 0.2g/L를 사용하였다. 공급배지로는 포도당 400g/L, 갈락토오스(glactose) 200g/L, 효모추출물 30g/L, 카사미노 애시드 25g/L, 황산 암모늄 25g/L, 이인산 칼륨 15g/L, 황산 마그네슘 5.7g/L, 염화칼슘 1.8g/L, 기타 미량원소를 함유하고 있는 트레이서(tracer; (NH4)2MoO4·2H2O 3μM, H3BO3400μM, CuSO4·5H2O 10μM, MnCl3·4H2O 80μM, ZnSO4·7H2O 10μM) 10m/L를 사용하였다.One transformed Saccharomyces cerevisiae 2805 / pYINU-hGH colony was selected and inoculated in 10 ml of YNBCAD (0.67% yeast nitrogen base without amino acids, 2% glucose, 05% casamino acid) and 16 hours at 30 ° C. The primary inoculum was prepared by culturing. The primary inoculum was inoculated in fresh YNBCAD medium at a concentration of 5-10% and cultured in the same manner to prepare a secondary inoculum. In order to produce high levels of recombinant human growth hormone, a fed-batch culture was performed to supply nutrients according to cell growth. The composition of the initial medium and feed medium in the fed-batch culture is as follows. Initial medium was glucose 15g / L, yeast extract 3g / L, casamino acid 3g / L, ammonium sulfate 3g / L, potassium diphosphate 1.5 g / L, magnesium sulfate 0.5 g / L, and calcium chloride 0.2 g / L were used. Feed medium includes glucose 400g / L, galactose 200g / L, yeast extract 30g / L, casamino acid 25g / L, ammonium sulfate 25g / L, potassium diphosphate 15g / L, magnesium sulfate 5.7g / L, Tracer containing 1.8 g / L calcium chloride and other trace elements; (NH 4 ) 2 MoO 4 · 2H 2 O 3μM, H 3 BO 3 400μM, CuSO 4 · 5H 2 O 10μM, MnCl 3 · 4H 2 O 80 μM, ZnSO 4 7H 2 O 10 μM) 10 m / L was used.
상기 초기배지 2L를 5L 발효조에 조제하여 멸균한 다음 사카로마이세스 세레비지애 2805/pYINU-hGH의 2차 접종원 100ml를 접종한 후 회전속도 300rpm, 통기량 1vvm, 배양온도 30℃, pH 5.5에서 회분식 배양하였다. 초기배지의 탄소원이 고갈되는 시점에서 공급배지를 사카로마이세스 세레비지애의 성장속도에 맞추어 기하급수적으로 공급(exponential feeding)하면서 유가식 배양하였다. 배양결과, 총 3L의 효모배양액을 획득하였다. 형질전환된 효모로부터 인간 성장호르몬이 발현되었음은 웨스턴블롯으로 이를 확인하였고, 발현된 성장호르몬의 양은 웨스턴블롯에서 확인된 밴드의 두께 및 크기를 덴시토미터(densitometer)로 측정하여 정량하였다.2L of the initial medium was prepared in a 5L fermenter, sterilized, and then inoculated with 100 ml of a second inoculum of Saccharomyces cerevisiae 2805 / pYINU-hGH, followed by a rotational speed of 300 rpm, aeration rate of 1vvm, incubation temperature of 30 ° C, and pH 5.5. Batch culture was performed. When the carbon source of the initial medium was depleted, the feeding medium was fed by feeding on the growth rate of Saccharomyces cerevisiae with exponential feeding. As a result of the culture, a total of 3L yeast culture solution was obtained. The expression of human growth hormone from the transformed yeast was confirmed by Western blot, and the amount of growth hormone expressed was quantified by measuring the thickness and size of the band identified in the western blot using a densitometer.
그 결과, 생성된 성장호르몬의 총량은 2.5g이였다As a result, the total amount of growth hormone produced was 2.5 g.
<실시예 4><Example 4>
재조합 인간 성장호르몬을 함유한 동결건조 효모 분말의 제조Preparation of Lyophilized Yeast Powder Containing Recombinant Human Growth Hormone
실시예 3에서 획득한 효모 배양액 3L를 7000rpm, 15℃, 15분간 원심분리하여 인간 성장호르몬이 농축되어 있는 배양 상등액과 효모균체로 분리하였다. 상등액에 존재하는 재조합 인간 성장호르몬을 10K 한외여과막 (Satorious)을 이용하여 농축하였다. 분리된 효모균체는 세포파쇄기 다이노밀(dyno-mill, 신양인터내셔널)를 이용하여 효모의 세포벽을 분쇄하였다. 농축한 재조합 인간 성장호르몬 2.5g과 분쇄한 효모 120g를 함께 동결 건조하여 재조합 인간 성장호르몬 분말(Yeast-FGH)을 제조하였다.3 L of the yeast culture solution obtained in Example 3 was centrifuged at 7000 rpm, 15 ° C. for 15 minutes to separate the culture supernatant and yeast cells in which human growth hormone was concentrated. Recombinant human growth hormone present in the supernatant was concentrated using 10K ultrafiltration membrane (Satorious). The isolated yeast cells were crushed cell walls of yeast using a cell breaker dyno-mill (Shinyang International). 2.5 g of concentrated human growth hormone and 120 g of crushed yeast were lyophilized to prepare a recombinant human growth hormone powder (Yeast-FGH).
<실시예 5>Example 5
재조합 인간 성장호르몬을 함유한 동결건조 효모분말의 넙치 성장 촉진 효과Growth Promotion Effects of Lyophilized Yeast Powder Containing Recombinant Human Growth Hormone
실시예 3의 재조합 인간 성장호르몬을 함유한 동결건조 분말의 급여에 따른 넙치 성장 촉진효과를 조사하였다. 실험어로는 넙치 치어를 사용하였다. 넙치 치어는 예비사육 후 60L의 사각 수조에 25마리씩(평균무게 6.0±0.1g) 각 실험군 당 3반복으로 무작위 배치하였다. 사육수는 해산어류를 양식하는 일반적인 방법인 바다에서 물을 끌어올려 어류가 있는 탱크로 들어간 다음 다시 바다로 흘러 나가게 유수식으로 공급하였으며 유수량은 2-4L/min으로 조절하였다. 각 수조에 충분한산소공급을 위해 에어스톤(airstone)을 설치하였으며 수온은 전 실험기간동안 자연 수온에 의존하였다. 일일 사료공급량은 어체중의 3%(건물량 기준)로 1일 2회(10:00, 16:00h) 공급하였다.The effect of promoting the flounder growth according to the feeding of the lyophilized powder containing the recombinant human growth hormone of Example 3 was investigated. The experimental fish was used for flounder fry. After the preliminary breeding, the flounder chicks were randomly placed in three replicates for each experimental group, 25 rats (average weight 6.0 ± 0.1 g) in a 60 L square tank. Breeding water was fed from the sea, which is a common method of farming marine fish, into a tank containing fish, and then flowed back into the sea, and the flow rate was adjusted to 2-4 L / min. Airstones were installed to provide sufficient oxygen to each tank, and the water temperature depended on the natural water temperature throughout the experiment. The daily feed was 3% of the body weight (based on building volume) twice a day (10:00, 16: 00h).
기본 실험사료는 단백질원으로 북양어분(white fish meal, Han Chang Fishmeal Co., Pusan, Korea), 젤라틴 및 카제인(Gelatin and Casein, United States Biochemical, Cleveland, Ohio 44122)을 사용하였고, 지질원으로는 오징어 간유(squid liver oil), 이피에이(EPA), 디에이치에이(DHA) 농축유(E-Wha oil Co., Ltd., Pusan, Korea), 옥수수기름(corn oil)을 사용하였으며, 탄수화물원으로는 밀가루(wheat meal, Young Nam Flour Mills Co., Pusan, Korea)를 사용하였다. 그밖에 비타민 혼합물(vitamin premix, vitA 3000IU, vitD32400IU, vitE 120IU, menadione sodium bisulfate 6mg/kg, vitB-HCl 15mg/kg, vitB230mg/kg, vitB6-HCl 15mg/kg, vitB120.06mg/kg, vitC 300mg/kg, calcium pantothenate 150mg/kg, nicotin amide 150mg/kg, inositol 150mg/kg, d-biotin 1.5mg/kg, choline chloride 3000mg/kg, pancreatin 12.5mg/kg)과 미네랄 혼합물(mineral premix, MnSO4320mg/kg, ZnSO4270mg/kg, FeSO4750mg/kg, CuSO460mg/kg, CoSO47mg/kg, MgSO417.25mg/kg, K2SO4212.24mg/kg, NaCl 51.88mg/kg, K2HPO4136.09mg/kg, NaSeO30.013mg/kg, KI 0.15mg.kg)을 사용하였다. 또한, 부형제 및 점착제로서 셀룰로스를 사용하였다. 기본 실험사료의 조성을 표 1에 나타내었다.Basic feed was used as protein source, white fish meal (Han Chang Fishmeal Co., Pusan, Korea), gelatin and casein (Gelatin and Casein, United States Biochemical, Cleveland, Ohio 44122). Squid liver oil, EPA, DHA concentrated oil (E-Wha oil Co., Ltd., Pusan, Korea) and corn oil were used. Wheat flour (wheat meal, Young Nam Flour Mills Co., Pusan, Korea) was used. Other vitamin mixtures (vitamin premix, vitA 3000IU, vitD 3 2400IU, vitE 120IU, menadione sodium bisulfate 6mg / kg, vitB-HCl 15mg / kg, vitB 2 30mg / kg, vitB 6 -HCl 15mg / kg, vitB 12 0.06mg / kg, vitC 300mg / kg, calcium pantothenate 150mg / kg, nicotin amide 150mg / kg, inositol 150mg / kg, d-biotin 1.5mg / kg, choline chloride 3000mg / kg, pancreatin 12.5mg / kg) and mineral mixture (mineral premix , MnSO 4 320mg / kg, ZnSO 4 270mg / kg, FeSO 4 750mg / kg, CuSO 4 60mg / kg, CoSO 4 7mg / kg, MgSO 4 17.25mg / kg, K 2 SO 4 212.24mg / kg, NaCl 51.88mg / kg, K 2 HPO 4 136.09mg / kg, NaSeO 3 0.013mg / kg, KI 0.15mg.kg). In addition, cellulose was used as an excipient and an adhesive. The composition of the basic experimental feed is shown in Table 1.
인간 성장호르몬을 함유한 사료는 위에서 만든 기본 실험사료에 실시예 4의 재조합 인간 성장호르몬 분말을 각각 재조합 인간 성장호르몬이 10(Yeast-FGH10), 20(Yeast FGH20) 40(Yeast-FGH40) mg/kg 어체중(Body weight)/주(week)되도록 첨가하여 제조하였다. 기본 실험사료와 인간 성장호르몬 분말을 잘 혼합한 다음 펠렛 제조기(보경상회, 부산)로 압출·성형하여 밀봉상태로 -20℃에서 냉동 보관하여 사용하였다.For the feed containing human growth hormone, the recombinant human growth hormone powder of Example 4 was added to the recombinant human growth hormone powder 10 (Yeast-FGH 10 ) and 20 (Yeast FGH 20 ) 40 (Yeast-FGH 40). ) mg / kg body weight / week. After mixing the basic experimental feed and human growth hormone powder well, it was extruded and molded into a pellet maker (Bo Gyeongsang, Busan) and frozen and stored at -20 ° C in a sealed state.
사료에 재조합 인간 성장호르몬 분말을 첨가할수록 단백질 및 에너지 함량이 높아지기 때문에 알라닌, 옥수수기름 및 셀룰로스를 사용하여 각 사료의 조단백과 가용에너지가 동일한 양이 되도록 조절하였다. 모든 실험사료의 조단백 함량은 50%, 가용에너지는 17.6 KJ/g(단백질, 탄수화물, 지질 : 16.7, 16.7, 37.7J/g)이었다. 실험사료의 단백질, 수분 및 회분은 AOAC(Association of Official Analytical Chemists, 1995) 방법에 따라 분석하였고, 조지방은 소스텍 시스템(Soxtec system) 1046 (Tecator AT, Sweden)으로 분석하였다.Since the amount of protein and energy increased as the recombinant human growth hormone powder was added to the feed, alanine, corn oil and cellulose were used to adjust the crude protein and available energy of each feed to the same amount. Crude protein content of all experimental feeds was 50% and available energy was 17.6 KJ / g (protein, carbohydrate, lipid: 16.7, 16.7, 37.7J / g). Proteins, moisture, and ash of the experimental diet were analyzed according to the Association of Official Analytical Chemists (AOAC) method, and crude fat was analyzed by Soctec system 1046 (Tecator AT, Sweden).
어체의 무게 측정은 4주째와 6주째 각 수조별로 전체무게를 측정하였다. 모든 통계 자료는 컴퓨터에 프로그램된 스태티스틱스(Statistix) 3.1(Analytical Software, St. Paul, Mn. USA)로 분산분석(ANOVA)을 실시하여 최소유의차검정(LSD: Least Significant Difference)으로 평균간의 유의성(P=0.05)을 검정하였다. 실험어의 증체율(Weight Gain: WG), 사료효율(Feed Efficiency: FE) 및 생존율 (Sruvival Rate: SR)은 다음과 같이 계산하였다.The weight of the fish was measured at each tank at 4th and 6th weeks. All statistical data were analyzed by ANOVA with Statisix 3.1 (Analytical Software, St. Paul, Mn. USA) programmed on a computer, and then analyzed by means of Least Significant Difference (LSD). Significance (P = 0.05) was tested. The weight gain (WG), feed efficiency (FE) and survival rate (SR) of the experimental fish were calculated as follows.
증체율(Weight gain, %)=(최종 체중-초기 체중) × 100 / 초기 체중Weight gain (%) = (final weight-initial weight) × 100 / initial weight
사료효율(Feed Efficiency, %)= 증체량(g) × 100/ 건물 사료 섭취량(g)Feed Efficiency (%) = weight gain (g) × 100 / dry feed intake (g)
생존률(Survival rate, %)=최종 실험어수 × 100/초기 실험어수Survival rate (%) = final experimental number × 100 / initial experimental number
실험 결과, 재조합 인간 성장호르몬을 함유한 실험사료를 4주 동안 급여한 경우 대조구에 비해 모든 실험군의 증체율이 유의적으로 높게 나타났다 (P<0.05). Yeast-FGH20를 급여한 경우가 가장 높은 증체율을 보였다. 사료효율도 대조구에 비해 재조합 인간 성장호르몬을 첨가한 모든 실험구가 유의적으로 높은 결과를 나타냈으며(P<0.05), Yeast-FGH20을 급여한 경우 가장 높은 결과를 나타냈다. 생존율은 대조구 및 모든 실험구간에 유의적인 차이가 없었으며 한 마리의 폐사도 일어나지 않았다(표 2). 즉, 대조구에 비해 본원 발명의 인간 성장호르몬을 첨가한 사료를 급여한 경우 인간 성장호르몬을 첨가하지 않은 기본사료를 급여한 경우에 비하여 어류의 성장률이 최대 22%, 사료효율이 20% 증진되어 성장촉진효과가 우수함을 알 수 있었다 (도 3).As a result, when the experimental feed containing recombinant human growth hormone was fed for 4 weeks, the increase rate of all experimental groups was significantly higher than that of the control group (P <0.05). Yeast-FGH 20 was the highest gain rate. Feed efficiency was significantly higher in all experimental groups containing recombinant human growth hormone than the control group (P <0.05), and the highest results were obtained when Yeast-FGH 20 was fed. Survival was not significantly different between the control and all experiments, and no mortality occurred (Table 2). In other words, compared to the control group, when the feed containing the human growth hormone of the present invention is fed, the growth rate of fish is increased by up to 22% and the feed efficiency is increased by 20% compared to the case of feeding the basic feed without adding the human growth hormone. It can be seen that the promoting effect is excellent (Fig. 3).
(실험값들은 3개의 어류군으로부터 얻어진 값들의 평균이며, 각 컬럼의 평균값들은 유의적을 다른 값들이다 (P<0.05))(Experimental values are the average of values from three fish groups, and mean values for each column are significantly different values (P <0.05))
본 발명에서는 효모에서 인간 성장호르몬을 재조하여 인간 성장호르몬 및 효모균체를 함유하는 분말을 만들고 이를 사료에 첨가하여 어류에게 경구 투여할 수 있는 사료 조성물을 제조하였다. 본 발명의 사료 조성물은 어류의 성장을 촉진하는데 유용하게 사용될 수 있다.In the present invention, by preparing a human growth hormone in yeast, a powder containing human growth hormone and yeast cells was prepared and added to the feed to prepare a feed composition that can be administered orally to fish. The feed composition of the present invention can be usefully used to promote the growth of fish.
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