KR100375672B1 - Single chain bovine Follicle-stimulating hormone and producing method thereof - Google Patents
Single chain bovine Follicle-stimulating hormone and producing method thereof Download PDFInfo
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- 108010079345 Follicle Stimulating Hormone Proteins 0.000 title claims abstract description 27
- 229940028334 follicle stimulating hormone Drugs 0.000 title claims abstract description 27
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- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
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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
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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Abstract
본 발명은 유전자재조합에 의하여 단일체인으로 형성된 소의 난포자극호르몬, 상기 호르몬을 코딩하는 핵산분자 및 상기 호르몬을 제조하는 방법에 관한 것으로, α단체와 β단체의 2부분으로 구성된 난포자극호르몬을 단일체인으로 형성함으로써 대량의 호르몬을 용이하게 제조할 수 있으며, 본 발명의 단일체인 난포자극호르몬은 천연형의 난포자극호르몬보다 활성이 강하여 동물약으로서 유용하게 이용될 수 있다.The present invention relates to a follicle stimulating hormone of a cow formed in a single chain by genetic recombination, a nucleic acid molecule encoding the hormone and a method for producing the hormone, a single follicle stimulating hormone composed of two parts of α group and β group By forming a large amount of hormones can be easily produced, the follicle stimulating hormone of the present invention is stronger than the natural type follicle stimulating hormone can be usefully used as an animal medicine.
Description
본 발명은 난포자극호르몬 및 그 제조방법에 관한 것으로, 보다 상세하게는 유전자재조합에 의하여 단일체인으로 형성된 난포자극호르몬 및 그러한 단일체인 호르몬을 제조하는 방법에 관한 것이다.The present invention relates to a follicle stimulating hormone and a method for producing the same, and more particularly to a method for producing a follicle stimulating hormone formed in a single chain by genetic recombination and a hormone that is a single body.
천연형의 난포자극호르몬(Follicle-stimulating hormone, 이하 "FSH"라 함)은 뇌하수체에서 분비되는 성선자극호르몬으로서 당단백질 호르몬계에 속한다.Natural follicle-stimulating hormone (hereinafter referred to as "FSH") is a gonadotropin secreted by the pituitary gland and belongs to the glycoprotein hormone system.
이 호르몬은 뇌하수체 유래의 항체형성호르몬(LH), 융모성성선자극호르몬(hCG), 임마혈청성선자극호르몬(eCG)과 마찬가지로 α단체와 β단체가 비공유결합으로 결합하여 구성된다.This hormone, like the antibody-forming hormone (LH) derived from the pituitary gland, chorionic gonadotropin (hCG), and γ serum gonadotropin (eCG), is composed of noncovalent bonds of α and β groups.
소의 난포자극호르몬(bovine Follicle-stimulating hormone, 이하 "bFSH"라함)은 소의 뇌하수체로부터 분비되어 소의 발정유기에 관여하는 성선자극호르몬으로서 α단체와 β단체로 구성된다.Bovine Follicle-stimulating hormone (hereinafter referred to as "bFSH") is a gonadotropin secreted from the pituitary gland of the cow and is involved in the estrus of the cow.
bFSH의 α단체는 24개의 아미노산으로 구성된 시그널 펩티드(signal peptide)와 96개의 아미노산으로 구성된 펩티드로 구성되며, 56 및 82번에 N말단결합 당쇄첨가결합부위를 가지고 있다[Biochemistry.22, 4856(1983) 참조]. bFSH의 α단체를 코딩하는 cDNA의 핵산염기서열은 서열목록 1에 기재한 바와 같으며, bFSH의 α단체의 아미노산서열은 서열목록 2에 기재한 바와 같다.The α group of bFSH consists of a signal peptide consisting of 24 amino acids and a peptide consisting of 96 amino acids, and has N-terminal sugar chain binding sites at 56 and 82 [Biochemistry. 22, 4856 (1983). ) Reference]. The nucleic acid base sequence of the cDNA encoding the α group of bFSH is as described in SEQ ID NO: 1, and the amino acid sequence of the α group of bFSH is as described in SEQ ID NO: 2.
bFSH의 β단체는 20개의 아미노산으로 구성된 시그널 펩티드와 109개의 아미노산으로 구성된 펩티드로 구성되어 있다[PNAS. USA. 83, 6618(1986) 참조]. bFSH의 β단체를 코딩하는 cDNA의 핵산염기서열은 서열목록 3에 기재된 바와 같으며, bFSH의 β단체의 아미노산서열은 서열목록 4에 기재한 바와 같다.The β group of bFSH consists of a signal peptide consisting of 20 amino acids and a peptide consisting of 109 amino acids [PNAS. USA. 83, 6618 (1986). The nucleic acid base sequence of the cDNA encoding the β group of bFSH is as described in SEQ ID NO: 3, and the amino acid sequence of the β group of bFSH is as described in SEQ ID NO: 4.
상기와 같은 소의 bFSH는 소의 과배란 및 발정유지를 위하여 필요하지만, 종래에는 bFSH를 국내에서 생산하지 못하였으므로 수입해서 사용하여야만 한다는 문제점이 있다.The bFSH of the cow as described above is necessary for the over-ovulation and estrous maintenance of the cow, but there is a problem that must be imported and used because the conventional bFSH was not produced in Korea.
비용절감을 위하여 bFSH 대신에 도축장에서 도살된 돼지의 뇌하수체로부터 FSH를 정제하여 사용하기도 하지만, FSH를 분리 및 정제하는 공정이 복잡하고 수율이 낮아 대량생산이 어려운 문제점이 있다.In order to reduce costs, instead of bFSH, FSH may be purified from the pituitary gland of a pig slaughtered in a slaughterhouse, but the process of separating and purifying FSH is difficult and the yield is low.
상기의 문제점을 해결하기 위한 본 발명은 유전자재조합에 의하여 단일체인으로 형성되어 발현세포계에서 용이하게 발현되므로 대량생산이 용이하며, 호르몬의 생물활성이 천연형 호르몬에 비하여 우수한 단일체인 소의 난포자극호르몬을 제공하는 것을 목적으로 한다.The present invention for solving the above problems is formed in a single chain by genetic recombination is easily expressed in the expression cell system is easy to mass production, the biological activity of the hormone is a monolithic cow follicle stimulating hormone superior to the natural hormone It aims to provide.
또한 본 발명은 상기 단일체인 소의 난포자극호르몬을 제조하는 방법을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a method for producing a follicle stimulating hormone of bovine cattle.
도 1은 α단체의 cDNA와 β단체의 cDNA를 결합시키기 위한 과정을 도시한 것,1 shows a process for combining cDNA of α group and cDNA of β group,
도 2는 pcDNA3-bFSH의 플라스미드지도.2 is a plasmid map of pcDNA3-bFSH.
상기의 목적을 달성하기 위한 본 발명은 유전자재조합에 의하여 소의 난포자극호르몬의 α단체와 β단체를 단일체인으로 만든, 아미노산서열이 서열목록 6에서의 위치 1-225 또는 21-225와 동일하거나 실질적으로 유사한 단일체인 소의 난포자극호르몬에 그 특징이 있다.In order to achieve the above object, the present invention provides a single chain of α group and β group of bovine follicle stimulating hormone by genetic recombination, wherein the amino acid sequence is identical to or substantially identical to positions 1-225 or 21-225 in SEQ ID NO: 6. Similar characteristics are found in bovine follicle stimulating hormones.
본 발명의 재조합 단일체인 bFSH의 대표적인 아미노산서열을 서열목록 6에 기재하고 있다.A representative amino acid sequence of bFSH, the recombinant monolith of the present invention, is shown in SEQ ID NO: 6.
서열목록 6에 기재한 바와 같이 bFSH는 위치 1-20의 시그널 펩티드와 위치 21-225의 205개의 아미노산으로 구성된 펩티드로 구성되며, 21번부터 129번까지의 아미노산은 β단체에 해당하고 130번부터 225번까지의 아미노산서열은 α단체에 해당한다. 또한 25번과 42번의 아스파라긴(Asn)에는 N-말단결합 당쇄가 결합되고, 185번과 211번의 Asn에는 각각 하나의 N-말단결합 당쇄가 결합되어 있다.As shown in SEQ ID NO: 6, bFSH consists of a peptide consisting of a signal peptide at positions 1-20 and a peptide consisting of 205 amino acids at positions 21-225, and amino acids 21-129 correspond to β groups and from 130 The amino acid sequence up to 225 corresponds to the α group. In addition, N-terminal sugar chains are bonded to Asparagine (Asn) at Nos. 25 and 42, and one N-terminal sugar chain is bonded to Asn at Nos. 185 and 211, respectively.
그러나 상기 서열목록 6에서 위치 1-225 또는 21-225와 동일한 아미노산서열만이 아니라 실질적으로 유사한 아미노산서열도 본 발명의 범위에 포함된다.However, not only the same amino acid sequence as positions 1-225 or 21-225 in SEQ ID NO: 6 but also substantially similar amino acid sequences are included in the scope of the present invention.
또한 본 발명은 상기 단일체인 소의 난포자극호르몬을 코딩하는 핵산염기서열을 가지는 단리된 핵산분자에 그 특징이 있다.In addition, the present invention is characterized by an isolated nucleic acid molecule having a nucleic acid base sequence encoding the follicle stimulating hormone of the bovine.
대표적인 핵산분자의 서열을 서열목록 5에 기재하고 있다. 서열목록 5에 기재한 DNA는 678개의 핵산염기로 구성되며, 위치 1-60은 시그널 펩티드, 위치 61-675는 펩티드를 코딩하며, 위치 676-687은 터미네이터를 나타낸다. 또한 위치 61-675중 61-397은 β단체에 대한 아미노산서열을 나타내고, 398-675가 α단체에 대한 아미노산서열을 나타낸다.Representative nucleic acid sequences are shown in SEQ ID NO: 5. The DNA set forth in SEQ ID NO: 5 consists of 678 nucleic acid bases, positions 1-60 encode signal peptides, positions 61-675 encode peptides, and positions 676-687 represent terminators. In addition, 61-397 in positions 61-675 represent the amino acid sequence for β group, and 398-675 represents the amino acid sequence for α group.
그러나 상기 서열목록 5에 기재된 핵산염기서열에서 위치 1-675 또는 61-675와 동일한 것뿐 아니라 실질적으로 유사한 핵산염기서열도 본 발명의 범위에 포함된다.However, not only the same nucleic acid sequences as those of positions 1-675 or 61-675 in the nucleic acid sequences set forth in SEQ ID NO: 5 are included in the scope of the present invention.
또한 본 발명은 소의 난포자극호르몬의 α단체 및 β단체를 코딩하는 핵산분자를 중합연쇄반응(PCR)에 의하여 증폭시키는 단계와; 상기 증폭된 α단체를 코딩하는 핵산분자와 β단체를 코딩하는 핵산분자를 클로닝시키는 단계와; 클로닝된 α단체를 코딩하는 핵산분자와 β단체를 코딩하는 핵산분자를 중합연쇄반응에 의하여 결합시키는 단계와; 상기 결합된 핵산분자를 포함하는 발현벡터를 형성하는 단계와; 상기 발현벡터를 도입하여 진핵세포를 형질전환하는 단계와; 상기 형질전환된 진핵세포에서 소의 난포자극호르몬을 발현시키는 단계와; 발현된 소의 난포자극호르몬을 분리하는 단계를 포함하는 단일체인 소의 난포자극호르몬의 제조방법에 그 특징이 있다.The present invention also comprises amplifying a nucleic acid molecule encoding α group and β group of bovine follicle stimulating hormone by polymerase chain reaction (PCR); Cloning the nucleic acid molecule encoding the amplified α-group and the nucleic acid molecule encoding the β-group; Binding a nucleic acid molecule encoding a cloned α group and a nucleic acid molecule encoding a β group by a polymerization chain reaction; Forming an expression vector comprising the bound nucleic acid molecule; Transforming eukaryotic cells by introducing the expression vector; Expressing bovine follicle stimulating hormone in said transformed eukaryotic cell; There is a feature of the method for producing a follicle stimulating hormone of bovine monolith comprising the step of separating the expressed bovine follicle stimulating hormone.
상기 방법에서 발현벡터로는 플라스미드를 사용하는 것이 바람직하다.In the above method, it is preferable to use a plasmid as the expression vector.
또한 발현벡터를 도입하여 호르몬을 발현시키기 위한 숙주세포인 진핵세포는 포유류 동물세포유래의 배양가능한 세포인 것이 바람직하며, CHO-K1세포를 사용하는 것이 특히 바람직하다.In addition, eukaryotic cells, which are host cells for introducing hormones by introducing expression vectors, are preferably cultureable cells derived from mammalian animal cells, and particularly preferably CHO-K1 cells.
이하 본 발명을 실시예에 의하여 보다 상세하게 설명한다. 그러나 다음의 실시예는 본 발명을 예시하는 것으로 본 발명의 범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples, however, illustrate the invention and do not limit the scope of the invention.
<실시예 1><Example 1>
단일체인 bFSH의 제조Preparation of monochain bFSH
(1)단일체인 bFSH의 전이벡터(transfer vector)의 형성(1) Formation of Transfer Vector of Monolithic bFSH
먼저 bFSH의 α단체를 코딩하는 DNA와 β단체를 코딩하는 DNA를 연결하기 위하여 도 1에 도시한 바와 같은 중합연쇄반응(polymerase chain reaction, 이하 "PCR"이라 함)을 실시하였다.First, a polymerase chain reaction (hereinafter, referred to as "PCR") as shown in FIG. 1 was performed to connect DNA encoding α group of bFSH and DNA encoding β group.
이때 Primer로는 하기와 같은 염기서열을 갖는 4종의 올리고핵산염기를 사용하였다.In this case, four oligonucleotide groups having the following nucleotide sequences were used as the primer.
Primer-1, M13 RV, 5'-CAGGAAACAGCTATGAC-3'Primer-1, M13 RV, 5'-CAGGAAACAGCTATGAC-3 '
Primer-2, 5'-TCCATCAGGAAATTCTTTGATTTCCCT-3'Primer-2, 5'-TCCATCAGGAAATTCTTTGATTTCCCT-3 '
Primer-3, 5'-GAAATCAAAGAATTTCCTGATGGAGAG-3'Primer-3, 5'-GAAATCAAAGAATTTCCTGATGGAGAG-3 '
Primer-4, M13 M4, 5'-GTTTTCCCAGTCACGAC-3'Primer-4, M13 M4, 5'-GTTTTCCCAGTCACGAC-3 '
bFSH의 α단체의 cDNA 및 β단체의 cDNA{Biochemistry. 22, 4856(1983) 및 PNAS. USA. 83, 6618(1986) 참조}를 PCR로 증폭하였다. β단체의 cDNA에 대한 PCR은 Primer 1과 Primer 2를 사용하여 실행하였으며, α단체의 cDNA에 대한 PCR은 Primer 3와 Primer 4를 사용하여 실행하였다.cDNA of α group of bFSH and cDNA of β group {Biochemistry. 22, 4856 (1983) and PNAS. USA. 83, 6618 (1986)} was amplified by PCR. PCR for β-group cDNA was performed using Primer 1 and Primer 2, and PCR for α-group cDNA was performed using Primer 3 and Primer 4.
PCR에 의하여 증폭된 α단체의 cDNA와 β단체의 cDNA를 pUC119(Takara,Japan)에 클로닝하였다. 클로닝된 DNA를 Primer 1과 Primer 4를 사용하여 PCR을 실행하여 α단체의 cDNA와 β단체의 cDNA가 결합된 단일체인 bFSH를 형성하였다.The cDNA of α group and the cDNA of β group amplified by PCR were cloned into pUC119 (Takara, Japan). The cloned DNA was subjected to PCR using Primer 1 and Primer 4 to form bFSH, a single entity in which cDNA of α group and cDNA of β group were combined.
형성된 단일체인 bFSH를 Kpnl/Xbal으로 절단하여, pUC119 벡터의 같은 부위에 연결하였다. 연결 후 잘못된 염기서열을 확인하기 위하여 전체염기서열을 확인하였다.The monolith formed, bFSH, was cleaved with Kpnl / Xbal and linked to the same site of the pUC119 vector. After linking, the entire base sequence was checked to identify the incorrect sequence.
염기서열을 확인한 후 Kpnl/Xbal로 절단하고 발현벡터 pcDNA3(Invitro사)의 같은 부위에 연결하여 도 2에 도시한 바와 같은 발현벡터 pcDNA3-bFSH를 형성하였다. 발현벡터의 방향을 제한효소로 확인하였다.After confirming the nucleotide sequence, Kpnl / Xbal was cut and linked to the same site of the expression vector pcDNA3 (Invitro) to form the expression vector pcDNA3-bFSH as shown in FIG. 2. The direction of the expression vector was confirmed by restriction enzymes.
(2)세포배양(2) Cell culture
상기 발현벡터 pcDNA3-bFSH를 리포조움제제인 리포펙트아민을 사용하여CHO-K1세포에 도입하였다.The expression vector pcDNA3-bFSH was introduced into CHO-K1 cells using lipofectamine, which is a liposome preparation.
50㎍/㎖의 페니실린, 50㎍/㎖의 스트렙토마이신, 2mM의 글루타민(2mM) 및 10% FCS를 포함하는 안정한 클론 증식배지인 Ham F-12배지에 800㎍/㎖의 G418을 첨가한 배지에 발현벡터 pcDNA3-bFSH가 도입된 CHO-K1세포를 넣고 5% CO2와 95% 공기가 혼합된 분위기 하의 37℃에서 2주간 배양하였다. 배양 후 안정한 세포만을 선별하였다.To a medium in which 800 μg / ml G418 was added to Ham F-12 medium, a stable clone growth medium containing 50 μg / ml penicillin, 50 μg / ml streptomycin, 2 mM glutamine (2 mM) and 10% FCS. CHO-K1 cells into which the expression vector pcDNA3-bFSH was introduced were added and incubated for 2 weeks at 37 ° C. under a mixed atmosphere of 5% CO 2 and 95% air. After incubation, only stable cells were selected.
(3)재조합 단일체인 bFSH의 발현(3) Expression of bFSH, a Recombinant Monomer
선별된 안정세포(1×106)를 50units/ml의 페니실린과 50㎍/㎖의 스트렙토마이신을 포함한 20ml의 CHO-S-SFM-11 배지에 넣고 37℃에서 48시간 배양하였다.Selected stable cells (1 × 10 6 ) were placed in 20 ml of CHO-S-SFM-11 medium containing 50 units / ml penicillin and 50 µg / ml streptomycin and incubated at 37 ° C. for 48 hours.
배양후 배양상층을 모아서 100,000xg에서 60분간 원심분리하여 세포찌꺼기등을 제거하였다. 원심분리한 분획중 상층분획에 단일체인 bFSH가 발현된다.After incubation, the culture supernatant was collected and centrifuged at 100,000xg for 60 minutes to remove cell debris. The monolayer bFSH is expressed in the upper fraction of the centrifuged fraction.
<실시예 2><Example 2>
단일체인 bFSH의 활성측정Activity measurement of single chain bFSH
(1)RIA를 이용한 단일체인 bFSH의 정량(1) Quantification of bFSH, a homologue using RIA
천연형 bFSH와 본 발명의 단일체인 bFSH를 폴리클로날항체(A558/P1H)를 사용하여 RIA(Radio Immuno Assay)에 의하여 정량하였다.Native bFSH and the monolithic bFSH of the present invention were quantified by RIA (Radio Immuno Assay) using polyclonal antibody (A558 / P1H).
200㎕의 0.02M 인산버퍼[0.017M Na2HPO4·12H2O, 0.003M KH2PO4, 0.9%(w/v) NaCl, 0.5%(w/v)BSA, 0.02%(w/v) NaN3], 100㎕의 천연형 bFSH 표준품(0.09, 0.19, 0.390, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50ng), 100㎕의 토끼의 항bFSH 폴리클로날항체(7㎍/ml) 및 100㎕의 클로라민-T(Chloramine-T)방법에 따라 표식한125I bFSH 탐식자(20,000cpm/100㎕)를 각 튜브에 첨가하여 4℃에서 하룻밤 배양하였다.200 μl of 0.02 M phosphate buffer [0.017 M Na 2 HPO 4 .12H 2 O, 0.003 M KH 2 PO 4 , 0.9% (w / v) NaCl, 0.5% (w / v) BSA, 0.02% (w / v ) NaN 3 ], 100 μl native bFSH standard (0.09, 0.19, 0.390, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50 ng), 100 μl rabbit anti-bFSH polyclonal antibody (7 μg / ml ) And 125 l bFSH probe (20,000 cpm / 100 μl) labeled according to 100 μl Chloramine-T method were added to each tube and incubated overnight at 4 ° C.
배양한 후 500㎕의 항토끼침강항체를 각 튜브에 넣고 다시 30분간 배양하였다. 30분간 배양한 후 2ml의 인산버퍼를 첨가하고 3,000rpm에서 15분간 원심분리하여 상층을 제거한 후 카운터로 측정하여 정량하였다.After incubation, 500 μl of anti-rabbit precipitated antibody was placed in each tube and incubated for another 30 minutes. After culturing for 30 minutes, 2 ml of phosphate buffer was added, and the upper layer was removed by centrifugation at 3,000 rpm for 15 minutes, and then quantified by measuring with a counter.
(2)세포내 cAMP 정량에 의한 단일체인 bFSH의 활성측정(2) Determination of activity of bFSH, a monolith by intracellular cAMP quantification
난포자극호르몬의 수용체를 발현하는 세포(HEK293-FSHR:HEK293-FSHR)를 이용하여 재조합호르몬의 생리활성을 확인하였다.The physiological activity of the recombinant hormone was confirmed using cells expressing the receptor of follicle stimulating hormone (HEK293-FSHR: HEK293-FSHR).
6개 배양조(well)에 3×105세포를 넣고 인큐베이터에서 약 72시간동안 배양한 후 WA/BSA배양액으로 2회 세척하였다. WA/BSA 배양액으로 세척한 후 0.5mM MIX 1ml를 첨가하고 천연형 및 재조합 bFSH 0.75-250ng을 첨가하여 30분간 배양하였다.3 × 10 5 cells were put in six wells, incubated in an incubator for about 72 hours, and washed twice with WA / BSA culture. After washing with WA / BSA culture, 1 ml of 0.5 mM MIX was added and incubated for 30 minutes by adding 0.75-250 ng of native and recombinant bFSH.
배양이 완료된 후 얼음 위에 두고 최종농도 5%가 되도록 트리클로로아세트산(Trichloroacetic acid)을 첨가하고 분석시까지 -20℃에 보관하였다.After the incubation was completed, put on ice and trichloroacetic acid (Trichloroacetic acid) was added to the final concentration of 5% and stored at -20 ℃ until analysis.
cAMP는 cAMP 효소 면역분석 키트(Cyclic AMP Enzyme Immunoassay Kit)(Cayman Chemical, MI, USA)를 사용하여 정량하였다.cAMP was quantified using the Cyclic AMP Enzyme Immunoassay Kit (Cayman Chemical, MI, USA).
즉, 먼저 각 배양조에 버퍼 50㎕, 시료 50㎕, cAMP 아세틸콜린에스테라제 탐식자(Cyclic AMP Acetylcholinesterase Tracer) 50㎕, cAMP 항혈청(Cyclic AMP Antiserum) 50㎕를 첨가하여 18시간 배양한 후 배양조를 세척용 버퍼로 5회 세척하였다. 세척 후 200㎕의 엘만시약(Ellman's Reagent)을 첨가하여 60분간 방치한 후 405nm에서 플레이트를 읽어 cAMP의 활성을 측정하였다.In other words, 50 μl of buffer, 50 μl of sample, 50 μl of Cyclic AMP Acetylcholinesterase Tracer and 50 μl of Cyclic AMP Antiserum were added to each culture tank, followed by incubation for 18 hours. Was washed 5 times with washing buffer. After washing, 200 μl of Elman reagent (Ellman's Reagent) was added thereto, and left for 60 minutes. Then, the plate was read at 405 nm to measure the activity of cAMP.
활성을 측정한 결과를 하기 표 1에 나타내었다.The results of measuring the activity are shown in Table 1 below.
상기와 같은 본 발명은 α단체와 β단체의 2부분으로 구성된 난포자극호르몬을 단일체인으로 형성함으로써 대량의 호르몬을 용이하게 제조할 수 있는 효과가있다.The present invention as described above has the effect of easily producing a large amount of hormones by forming a follicle stimulating hormone consisting of two parts of α group and β group in a single chain.
또한 본 발명의 단일체인 난포자극호르몬은 천연형의 난포자극호르몬보다 활성이 강하여 동물약으로서 유용하게 이용될 수 있다.In addition, the follicle stimulating hormone of the present invention is stronger than the natural type follicle stimulating hormone can be usefully used as an animal medicine.
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