JPH0568572A - Blue-green alga synechococcus capable of producing salmon growth hormone - Google Patents
Blue-green alga synechococcus capable of producing salmon growth hormoneInfo
- Publication number
- JPH0568572A JPH0568572A JP3232069A JP23206991A JPH0568572A JP H0568572 A JPH0568572 A JP H0568572A JP 3232069 A JP3232069 A JP 3232069A JP 23206991 A JP23206991 A JP 23206991A JP H0568572 A JPH0568572 A JP H0568572A
- Authority
- JP
- Japan
- Prior art keywords
- synechococcus
- growth hormone
- salmon growth
- blue
- coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Farming Of Fish And Shellfish (AREA)
- Fodder In General (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Feed For Specific Animals (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、遺伝子組換えによって
サケ成長ホルモン(以下、ｓＧＨと言う場合もある）を
ラン藻シネココッカス菌体中に生産させる方法及びそれ
を魚類に投与する方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing salmon growth hormone (hereinafter also referred to as sGH) in a cyanobacterium, Synechococcus by genetic recombination, and a method for administering it to fish.
【０００２】[0002]
【従来の技術】成長ホルモンは遺伝子工学の進歩により
微生物内で大量に生産することが可能になり、ウシ、ブ
タ、ヒツジ、ヤギ、ニワトリなど多数の家畜動物におい
て実用化が進められている。養殖漁業は成長ホルモンの
もうひとつの大きな応用分野であるが、家畜動物に比べ
実用化が遅れている。しかし最近、シロサケに続き、ニ
ジマス、ウナギ、ブリ、マグロ、マダイ、ヒラメの成長
ホルモン遺伝子がクローニングされ、ｓＧＨではその粗
精製物が実際にニジマスに投与されて成長促進効果が確
かめられた。そこで実用上大きな問題になるのは成長ホ
ルモンの投与法である。従来のテストでは腹腔への注射
と成長ホルモン溶液への浸漬が試みられた。しかしこれ
らの方法は大量に養殖されている魚に対してはとても実
用的とは言えず、最も望ましい経口投与の方法が求めら
れていた。2. Description of the Related Art Growth hormone has become capable of being produced in large quantities in microorganisms due to advances in genetic engineering, and is being put to practical use in many domestic animals such as cattle, pigs, sheep, goats, and chickens. Aquaculture is another major application area for growth hormone, but its commercialization is delayed compared to livestock animals. However, recently, following the salmon, the growth hormone genes of rainbow trout, eel, yellowtail, tuna, red sea bream, and Japanese flounder were cloned, and the crude product of sGH was actually administered to rainbow trout to confirm the growth promoting effect. Therefore, the growth hormone administration method becomes a big problem in practical use. Traditional tests have tried injection into the abdominal cavity and immersion in growth hormone solution. However, these methods are not very practical for fish cultivated in large quantities, and the most desirable oral administration method has been required.
【０００３】しかし、成長ホルモンはタンパク質なので
容易に失活したり、微生物によって分解する。また摂食
後においても魚の消化器官で消化されてしまう。したが
って魚にどのように摂食させてどのように血液循環系に
吸収させ、ホルモンの効果を奏せさるかが大きな問題と
なる。ところで、最近 Moriyama ら (J. Comp. Physio
l. B (1990) 160：251-257)によってｓＧＨがニジマス
において腸管の後部で吸収され、循環系に入ることが明
らかにされた。この事実からすれば、成長ホルモンを食
後胃における消化を避けて腸管後部にまで送れればよい
こととなる。そこでこの解決手段としては二つの方法が
考えられる。ひとつは成長ホルモンそのものを適当な材
質でマイクロカプセル化する方法、いまひとつは分解さ
れにくい宿主細胞で成長ホルモンを生産し、得られる菌
体をそのまま魚類に摂取させる方法である。そして、後
者は前者に比べ、分離やマイクロカプセル化の工程を含
まないので経済的に有利である。However, since growth hormone is a protein, it is easily inactivated or decomposed by microorganisms. Even after eating, it is digested by the digestive organs of fish. Therefore, how to feed the fish and how to absorb it into the blood circulation system to exert the effect of hormones becomes a big problem. By the way, recently Moriyama et al. (J. Comp. Physio
l. B (1990) 160 : 251-257) revealed that sGH was absorbed in the posterior part of the intestinal tract in rainbow trout and entered the circulatory system. From this fact, it is sufficient that the growth hormone can be delivered to the posterior intestinal tract while avoiding digestion in the postprandial stomach. Therefore, there are two possible ways to solve this problem. One is a method in which the growth hormone itself is microencapsulated with an appropriate material, and the other is a method in which the growth hormone is produced in a host cell that is not easily decomposed, and the obtained bacterial cells are directly taken up by fish. The latter is economically advantageous as compared with the former because it does not include the steps of separation and microencapsulation.
【０００４】[0004]
【発明が解決しようとする課題】そこで、我々はラン藻
のシネココッカスが、従来宿主として使われている大腸
菌や酵母などと比べて、キチン質に富んだ厚い細胞壁を
持ち、且つ植物プランクトンの一種なので養殖池に生存
することができることに着目し、ラン藻シネココッカス
菌体中で遺伝子組換え技術によりサケ成長ホルモンを生
産せしめ、これを菌体のまま魚類に投与することを目的
として研究開発を行った。そして、鋭意研究の結果シネ
ココッカスに内在するプラスミドのひとつに大腸菌のト
リプトファンプロモーター (Ptrp) をプロモーターとし
てｓＧＨ遺伝子を導入し、シネココッカスを形質転換す
ると細胞中にｓＧＨが生産される事を見出し、本発明を
完成するに至った。Therefore, we have found that the cyanobacteria Synechococcus has a thick cell wall rich in chitin and is a kind of phytoplankton as compared with E. coli and yeast which are conventionally used as hosts. Focusing on the ability to survive in aquaculture ponds, research and development was carried out with the aim of producing salmon growth hormone by genetic recombination technology in the alga Acinetococcus cells and administering it to fish as is. .. As a result of diligent research, it was found that sGH gene was produced in the cells by introducing sGH gene into one of the plasmids inherent in Synechococcus using the tryptophan promoter (Ptrp) of E. coli as a promoter and transforming Synecococcus. It came to completion.
【０００５】[0005]
【課題を解決するための手段】本発明は、ラン藻シネコ
コッカス由来のプラスミドにE.coliのトリプトファンプ
ロモーターとサケ成長ホルモン遺伝子を導入した組み換
えプラスミドにある。さらに、本発明は、組み換えプラ
スミドで形質転換されたラン藻シネココッカスの形質転
換体にある。The present invention resides in a recombinant plasmid obtained by introducing the E. coli tryptophan promoter and the salmon growth hormone gene into a plasmid derived from the cyanobacteria Synechococcus. Furthermore, the present invention is a transformant of the cyanobacteria Synechococcus transformed with the recombinant plasmid.
【０００６】さらに、本発明は、前記組み換えプラスミ
ドで形質転換されたラン藻シネココッカスを培地に生育
せしめて該シネココッカスの菌体中においてサケ成長ホ
ルモンを蓄積せしめることを特徴とするサケ成長ホルモ
ンの製造方法にある。さらに、本発明は前記方法で得ら
れたサケ成長ホルモンを含むラン藻シネココッカスの菌
体を魚類に投与することを特徴とするサケ成長ホルモン
の投与方法にある。[0006] Furthermore, the present invention comprises a method for producing salmon growth hormone, characterized in that the cyanobacteria Synechococcus transformed with the above recombinant plasmid is grown in a medium to accumulate salmon growth hormone in the cells of said Synechococcus. It is in. Furthermore, the present invention provides a method for administering salmon growth hormone, which comprises administering to the fish the bacterium of the cyanobacterium Synechococcus containing the salmon growth hormone obtained by the above method.
【０００７】以下、本発明を詳細に説明する。先ず、遺
伝子工学的手法によりラン藻シネココッカスにサケ成長
ホルモンを産生せしめる方法について説明する。 １) ラン藻シネココッカスを培地中で培養し、内在する
プラスミドのうちの最小サイズのプラスミドを分離精製
する。このプラスミドと大腸菌プラスミドとを連結して
シネココッカス、大腸菌双方で増殖するシャトルベクタ
ーをつくる。このベクターにはマーカーとなる抗生物質
耐性遺伝子（アンピシリン遺伝子）も導入しておく。 ２) このシャトルベクターの、上記の増殖機能が損なわ
れないようなサイトに大腸菌、Ptrpそしてその転写開始
コドンの下流にｓＧＨの構造遺伝子、更にその下流に転
写のターミネーターを導入して組み換えプラスミドを得
る。 ３) この組み換えプラスミドの数μg を先のラン藻シネ
ココッカスの対数増殖期の培養液に加え、１％CO2 含有
空気の雰囲気と照光の条件下で培養する。その結果、組
え換えプラスミドがラン藻シネココッカスの細胞内に取
込まれた形質転換株が得られる。この形質転換株は抗生
物質含有の寒天プレートに培養液を塗布して選抜する。The present invention will be described in detail below. First, a method for producing salmon growth hormone in cyanobacteria Synechococcus by a genetic engineering method will be described. 1) Cyanobacteria Cincococcus is cultivated in a medium, and the minimum size plasmid among the internal plasmids is separated and purified. This plasmid is ligated with an E. coli plasmid to form a shuttle vector that grows in both Synechococcus and E. coli. An antibiotic resistance gene (ampicillin gene) serving as a marker is also introduced into this vector. 2) A recombinant plasmid is obtained by introducing Escherichia coli, Ptrp, and the sGH structural gene downstream of the transcription initiation codon into the site of the shuttle vector where the growth function is not impaired, and further introducing the transcription terminator downstream thereof. .. 3) A few μg of this recombinant plasmid is added to the culture solution of the cyanobacterium Cinecococcus in the logarithmic growth phase, and the mixture is cultured under an atmosphere of 1% CO 2 -containing air and illumination. As a result, a transformant is obtained in which the recombinant plasmid is incorporated into the cells of the cyanobacteria Synechococcus. This transformant is selected by applying a culture solution on an agar plate containing antibiotics.
【０００８】上記ラン藻シネココッカスは多くの株が内
外のカルチャーコレクションに登録されており利用可能
である。海洋性、淡水性、好熱性など、数多くの種があ
り、多くは数種類のプラスミドを保持している。組換え
るプラスミドはそのどれを用いて行ってもよいが、サイ
ズの小さい方が便利である。遺伝子工学の多くの操作は
E.coli系に準拠して行うことが出来る。Many strains of the above-mentioned cyanobacteria Synechococcus are available because they are registered in culture collections inside and outside Japan. There are many species, such as marine, freshwater, and thermophilic, and many carry several types of plasmids. Any of the plasmids used for recombination may be used, but the smaller size is convenient. Many operations in genetic engineering
It can be performed based on the E.coli system.
【０００９】上記Ptrp, ｓＧＨ遺伝子、ターミネーター
の位置関係や配列は、E.coli系の発現と同じように考え
て工夫できる。しかしそれで高発現が保証されるわけで
はなく、最終的には試行錯誤に依るしかない。例えば、
Ptrpはタンデムにしたり、lacプロモーターと並べると
発現レベルが高まる。組え換えプラスミドによるラン藻
シネココッカスの形質転換は通常上記のような自然取込
み法で行うことができる。しかし効率が良くない種では
他の方法、例えば電気パルス法を使ってもよい。その場
合E.coli系に比べてやや高いパルス電場 (〜７KV/cm)を
必要とする。The positional relationship and sequences of the above Ptrp, sGH gene and terminator can be devised in the same manner as the expression in E. coli system. However, this does not guarantee high expression, and ultimately depends on trial and error. For example,
The expression level of Ptrp increases when it is tandem or aligned with the lac promoter. Transformation of cyanobacteria Synechococcus with the recombinant plasmid can be usually performed by the above-mentioned natural uptake method. However, for less efficient species, other methods may be used, such as the electric pulse method. In that case, a slightly higher pulsed electric field (~ 7KV / cm) is required compared to the E. coli system.
【００１０】本発明は大腸菌のトリプトファンプロモー
ターがラン藻シネココッカス中でも機能するという我々
の新しい知見に基づくものであるが、本来シネココッカ
ス遺伝子が持っているプロモーターを使ってｓＧＨ遺伝
子を発現してもよい。シネココッカスはラン藻の中で最
も原始的な単細胞生物のひとつであり、生活環境に対し
て非常に優れた耐久能力を持っている。細胞壁はキチン
質に富み、非常に厚い。また、一様に小孔 (〜70nm) が
分布している。細胞壁を構成するペプチドグリカン層
(１-10nm)は１00℃の４％ＳＤＳでも溶解しない。した
がって、細胞壁はｓＧＨにとってマイクロカプセルのよ
うなもので、これによって保護されると同時に、外にし
み出ることも可能になる。ここではサケ成長ホルモンに
注目して、経口から腸への運搬体としたが、腸管で吸収
するのは魚だけでなく、哺乳動物においても同じである
ので、本発明は成長ホルモンの経口投与法としてひろく
家畜動物一般に適用できる可能性がある。また腸管から
吸収されるのは成長ホルモンに限らずタンパク質一般に
及ぶと考えられる。従って他の生理活性タンパク質の経
口投与法ともなる可能性がある。The present invention is based on our new finding that the E. coli tryptophan promoter also functions in the cyanobacteria Synechococcus. However, the sGH gene may be expressed using the promoter originally possessed by the Synecococcus gene. Synechococcus is one of the most primitive unicellular organisms in cyanobacteria, and has extremely excellent endurance to living environment. The cell wall is rich in chitin and very thick. In addition, small holes (~ 70 nm) are distributed uniformly. Peptidoglycan layer that constitutes the cell wall
(1-10 nm) does not dissolve even in 4% SDS at 100 ° C. Thus, the cell wall is like a microcapsule for sGH, which protects it while allowing it to exude. Here, we focused on salmon growth hormone and used it as a carrier from the oral to the intestine, but since it is the same not only in fish but also in mammals that is absorbed in the intestinal tract, the present invention provides a method for oral administration of growth hormone. As a whole, it may be applicable to domestic animals in general. In addition, it is considered that proteins that are absorbed from the intestinal tract are not limited to growth hormones, but generally proteins. Therefore, it may be an oral administration method for other physiologically active proteins.
【００１１】本発明で得られるサケ成長ホルモンを含む
ラン藻シネココッカス菌体を投与する魚類としては特に
限定はされないが、例えばニジマス、サケ等があげられ
る。また、このサケ成長ホルモンを含むラン藻シネココ
ッカス菌体の魚類への投与は、菌体をそのままでもよ
く，あるいは魚の餌に混合して投与してもよい。そし
て、その投与量は凡そ10-6／ｇ週である。The fish to which the cyanobacterial Cinecoccus fungus body containing the salmon growth hormone obtained in the present invention is administered is not particularly limited, and examples thereof include rainbow trout and salmon. In addition, the cyanobacterial Cinecoccus fungus body containing the salmon growth hormone may be administered to the fish as it is, or may be mixed with fish feed and administered. The dose is about 10 −6 / g week.
【００１２】[0012]
【発明の効果】本発明によりラン藻シネココッカス菌体
中にサケ成長ホルモンを効率的に製造することができ
た。そして、このサケ成長ホルモンを含むラン藻シネコ
コッカス菌体を魚類に投与することにより魚類の成育を
顕著に促進することができた。以下、本発明を実施例に
より具体的に説明する。ただし、本発明はこれら実施例
によりその技術的範囲が限定されるものではない。INDUSTRIAL APPLICABILITY According to the present invention, salmon growth hormone could be efficiently produced in Cyanococcus cyanobacteria. Then, the growth of the fish could be remarkably promoted by administering the cyanobacterial Synechococcus cells containing the salmon growth hormone to the fish. Hereinafter, the present invention will be specifically described with reference to examples. However, the technical scope of the present invention is not limited by these examples.
【００１３】[0013]
【実施例】図１に従ってSynecococcus sp. PCC7002の内
在プラスミドｐＡＱ１を用いるｓＧＨ発現のプラスミド
ベクターの構築を示す。この株はパスツール研究所カル
チャーコレクションに PCC7002として保存されている。 （１) Synecococcus sp. PCC7002の菌株を培地A (J. Ph
ysiol., 9, 427 (1973）) ３00mlに加え、500ml のエー
レンマイヤーフラスコ中で蛍光燈照射下、１％CO 2 の空
気を通じながら、ＯＤ550 2.0に至るまで培養した。 （２) 得られる培養液を遠心分離にかけ集菌し、菌体0.
2g を得た。この菌体0.2g からアルカリ法 (村松正實
編：ラボマニュアル遺伝子工学、丸善 (1988))によりプ
ラスミド30μｇを分離した。このプラスミド溶液をアガ
ロース電気泳動にかけて、最小サイズのバンドを切り取
り、ジーンクリーン (バイオラッド) で精製してｐＡＱ
１ ３μｇを得た。 （３) E.coliプラスミドｐＢＲ３２２ (ファルマシア社
製) ５μｇに制限酵素 HindIII (宝酒造社製) 10ユニッ
トを加え、処理して該プラスミドを切断し、アルカリフ
ォスファターゼ(Calf in stestine タカラ) で脱リン酸
化処理をし、次いでジーンクリーンで精製した。またｐ
ＡＱ１ 3μｇをHindIII 5 ユニットで切断し、ジーンク
リーンで精製した。この両者にＴ４ＤＮＡリガーゼ (宝
酒造社製)10ユニットを加え、処理して両者を接続し
た。このライゲーション混合液を用いて、E.coli DH5α
MCR (Bethesda Research Lab. 社製) をカルシウム法に
より形質転換した。この形質転換されたE.coli DH5αMC
R を含む液をアンピシリン50μg/ml含有プレートに播い
て形質転換株を選抜した。このE.coli形質転換株を10ml
培養して上記と同じ方法でプラスミドｐＡＱＥ１を10μ
g 得た。 ４) E.coliのｓＧＨ発現ベクターｐｓＧＨＩＢ−２ (Pr
oc. Natl. Acad. Sci. USA. 82, 4306-4310 (1985)) 10
μg に DraＩの20ユニットとBgl II 3ユニットを加え消
化して、PtrpとｓＧＨ遺伝子、及びリポプロテインター
ミネーターの入ったＤＮＡセグメントを得た。一方先の
ｐＡＱＥ１ 10μｇを同様に処理してPvu IIとBam HIの
セグメントを得た。両者を同様な方法でライゲーション
し、E.coliの形質転換を行って増殖し、その組み換えプ
ラスミドｐＱＳＧ１, 10μg を得た。 ５) Synechococcus sp. PCC7002 株をＯＤ550 0.5-1.0
になるまで生育させる。この培養液１mlにプラスミドｐ
ＱＳＧ１を５μg/mlの濃度になるように加え、37℃、20
00lxの蛍光燈下で、１％CO2 雰囲気に90分置いた。この
培養液を１％寒天-medium A プレートに塗布し、減光
下、30℃ 48時間培養した。その後アンピシリン (2.５
μg/ml) を含む0.６％寒天を重層して37℃ 2000lx の蛍
光燈下で培養し、コロニーの形成で形質転換株を得た。EXAMPLE According to FIG.Synecococcus sp. of PCC7002
SGH expression plasmid using the existing plasmid pAQ1
Shows the construction of the vector. This strain is Pasteur Institute Cal
Saved as PCC7002 in the Char Collection. (1)Synecococcus A strain of sp. PCC7002 was added to medium A (J. Ph.
ysiol.,9, 427 (1973)) In addition to 300 ml, 500 ml A
1% CO in a Renmeier flask under fluorescent lighting 2sky of
OD while feeling550Cultured to 2.0. (2) The obtained culture solution is centrifuged to collect the cells, and the bacterial cells are reduced to 0.
2g was obtained. From 0.2 g of these cells, the alkaline method (Muramatsu Masami)
Volume: Lab Manual Genetic Engineering, Maruzen (1988))
30 μg of rasmid was isolated. This plasmid solution is
Rose electrophoresis and cut out the smallest size band
PAQ after purification with Gene Clean (Bio-Rad)
13 μg was obtained. (3)E.coliPlasmid pBR322 (Pharmacia)
5 μg of restriction enzyme HindIII (manufactured by Takara Shuzo) 10 units
And digest the plasmid by adding
Dephosphorylation with phosphatase (Calf in stestine Takara)
Chemical treatment was carried out, followed by purification with Gene Clean. Also p
Cut 3 μg of AQ1 with 5 units of HindIII
Purified with lean. T4 DNA ligase (both
(Sake Brewing Co.) Add 10 units, process and connect both
It was Using this ligation mixture,E.coli DH5α
MCR (Bethesda Research Lab.) Is used for calcium method
More transformed. This transformedE.coli DH5αMC
Plate the solution containing R on a plate containing 50 μg / ml ampicillin.
The transformants were selected by 10 ml of this E. coli transformant
Incubate and p10Q plasmid pAQE1 in the same manner as above.
g got. 4)E.coliSGH expression vector psGHIB-2 (Pr
oc. Natl. Acad. Sci. USA.82, 4306-4310 (1985)) 10
Add 20 units of DraI and 3 units of Bgl II to μg
And Ptrp and sGH genes, and lipoprotein
A DNA segment containing a minator was obtained. One way ahead
Similarly, 10 μg of pAQE1 was treated with Pvu II and Bam HI.
Got a segment. Ligate both in the same way
The E. coli strain is transformed, grown, and the recombinant
10 μg of rasmid pQSG1 was obtained. 5)Synechococcussp. PCC7002 strain OD5500.5-1.0
Grow until. Plasmid p was added to 1 ml of this culture.
Add QSG1 to a concentration of 5 μg / ml, and keep at 37 ℃ for 20
1% CO under fluorescent light of 00lx2Leave it in the atmosphere for 90 minutes. this
Apply the culture solution to 1% agar-medium A plate and dimm
The cells were cultured at 30 ° C. for 48 hours under the conditions. Then Ampicillin (2.5
μg / ml) containing 0.6% agar and layered at 37 ℃ 2000lx
The cells were cultured under a light lamp and colonies were formed to obtain a transformant strain.
【００１４】このSynechococcus sp. PCC7002 の形質転
換株はSynechococcus sp. PCC7002(sQSG1) として工業
技術院微生物工業技術研究所に寄託し、その寄託番号は
微工研菌寄第12356号である。 （６) こうして得たSynechococcus sp. PCC7002 の形質
転換株はまず、アンピシリンを含まない培地A で一日間
培養し、次に、アンピシリン0.５μg/ml含む培地A 培地
を重層し増殖させた。[0014] The Synechococcus sp. PCC7002 transformed strain of is deposited with the Synechococcus sp. Agency Fermentation Research Institute as PCC7002 (sQSG1), the accession number is FERM No. 12356. (6) The thus obtained transformant strain of Synechococcus sp. PCC7002 was first cultured in the medium A containing no ampicillin for one day, and then the medium A containing 0.5 μg / ml of ampicillin was overlaid and grown.
【００１５】この形質転換株の菌体中に蓄積されたｓＧ
Ｈの検出はイムノブロッテイン法により、次のように行
った。Synechoccocus sp. PCC7002 の形質転換株の培養
液３00mlをＯＤ550 1.0 で集菌し、５mlの緩衝液Ａに懸
濁し、３00W の超音波ホモジナイザーで10分間処理し
た。これを15000rpm、10分間遠心分離し、沈澱を2.5ml
緩衝液Ａに懸濁し、10μｌをＳＤＳ電気泳動にかけた。
次にゲル上のタンパクをＰＶＤＦメンブレン (バイオッ
ド) に電気泳動的に転写した。このメンブレンを薄いポ
リエチレン袋に入れて、３％ゼラチン溶液を加えて１時
間インキュベートした後、まずｓＧＨのモノクローナル
抗体 (マウス) の１％ゼラチン溶液を加えて２時間イン
キュベートし、次にパーオキシダーゼでラベルした抗マ
ウス・ヤギ抗体 (バイオラッド) を加えて更に60分間イ
ンキュベートした。発色はナフトール誘導体 (コニカイ
ムノステインＨＲＰ、コニカ)を用いて行った。ＳＤＳ
電気泳動でコントロールとして流した市販のｓＧＨ(協
和発酵) と比べると、同じ位置である、約25KDa にバン
ドが検出された。SG accumulated in the cells of this transformant
The detection of H was performed by the immunoblottein method as follows. 300 ml of a culture solution of a transformant of Synechoccocus sp. PCC7002 was collected at OD 550 1.0, suspended in 5 ml of buffer solution A, and treated with an ultrasonic homogenizer at 300 W for 10 minutes. This is centrifuged at 15000 rpm for 10 minutes, and the precipitate is 2.5 ml.
Suspended in buffer A, 10 μl was subjected to SDS electrophoresis.
Next, the protein on the gel was electrophoretically transferred to a PVDF membrane (Biod). Put this membrane in a thin polyethylene bag, add 3% gelatin solution and incubate for 1 hour, then add 1% gelatin solution of sGH monoclonal antibody (mouse) and incubate for 2 hours, then label with peroxidase. Anti-mouse / goat antibody (Bio-Rad) was added and incubated for another 60 minutes. Color was developed using a naphthol derivative (Konica Immunostain HRP, Konica). SDS
As compared with the commercially available sGH (Kyowa Hakko) used as a control by electrophoresis, a band was detected at the same position, about 25 KDa.
【図１】ｓＧＨの発現のためのシヤトルベクターの構築
図。FIG. 1 is a schematic drawing of a shuttle vector for expressing sGH.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ａ２３Ｋ 1/165 Ｂ 7110−2Ｂ Ｃ１２Ｎ 15/18 (Ｃ１２Ｎ 15/74 Ｃ１２Ｒ 1:19 1:89） (Ｃ１２Ｐ 21/02 Ｃ１２Ｒ 1:89） (72)発明者 小嶋 洋之 大阪府池田市伏尾台２―９―１，１―506 (72)発明者 山野 尚子 大阪府大阪市平野区平野西３―９―１， 803 (72)発明者 伊藤 菁莪 東京都千代田区大手町一丁目６番１号 協 和醗酵工業株式会社内 (72)発明者 杉本 整治 東京都千代田区大手町一丁目６番１号 協 和醗酵工業株式会社内─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Internal reference number FI Technical display area A23K 1/165 B 7110-2B C12N 15/18 (C12N 15/74 C12R 1:19 1:89) (C12P 21/02 C12R 1:89) (72) Inventor Hiroyuki Kojima 2-9-1, 1-506 Fushiodai, Ikeda City, Osaka Prefecture (72) Naoko Yamano 3-9 Hirano Nishi, Hirano-ku, Osaka City, Osaka Prefecture ―1, 803 (72) Inventor Satoshi Ito 1-6-1, Otemachi, Chiyoda-ku, Tokyo Kyowa Fermentation Industry Co., Ltd. (72) Inventor Keiji Sugimoto 1-6-1, Otemachi, Chiyoda-ku, Tokyo Kyo Inside Wako Fermentation Co., Ltd.
Claims (4)
にE.coliのトリプトファンプロモーターとサケ成長ホル
モン遺伝子を導入した組み換えプラスミド1. A recombinant plasmid obtained by introducing the tryptophan promoter of E. coli and the salmon growth hormone gene into a plasmid derived from the cyanobacteria Synechococcus.
質転換されたラン藻シネココッカスの形質転換体2. A transformant of the cyanobacterium Synechococcus transformed with the recombinant plasmid according to claim 1.
ネココッカスを培地に生育せしめて該ラン藻シネココッ
カスの菌体中においてサケ成長ホルモンを蓄積せしめる
ことを特徴とするサケ成長ホルモンの製造方法。3. A method for producing salmon growth hormone, which comprises growing the transformed cyanobacterial synechococcus according to claim 2 in a medium to accumulate salmon growth hormone in the cells of the cyanobacterial synechococcus.
ホルモンを含むシネココッカスの菌体を魚類に投与する
ことを特徴とするサケ成長ホルモンの投与方法。4. A method for administering salmon growth hormone, which comprises administering to a fish a bacterium of Synechococcus containing salmon growth hormone obtained by the method according to claim 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3232069A JPH0568572A (en) | 1991-09-11 | 1991-09-11 | Blue-green alga synechococcus capable of producing salmon growth hormone |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3232069A JPH0568572A (en) | 1991-09-11 | 1991-09-11 | Blue-green alga synechococcus capable of producing salmon growth hormone |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0568572A true JPH0568572A (en) | 1993-03-23 |
Family
ID=16933503
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3232069A Pending JPH0568572A (en) | 1991-09-11 | 1991-09-11 | Blue-green alga synechococcus capable of producing salmon growth hormone |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0568572A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998042748A1 (en) * | 1997-03-24 | 1998-10-01 | Hih.Biocenter Inc. | New synthetic polypeptide |
| KR100443843B1 (en) * | 1999-05-28 | 2004-08-09 | 주식회사 알진텍 | Biosynthesis of foreign proteins using transformed microalgae |
| JP2008528027A (en) * | 2005-01-26 | 2008-07-31 | ユニヴェルシテイト レイデン | Means and methods for improving the development and maturation of fish eggs and / or fish sperm using hormones produced by transplanted cells |
| CN114736842A (en) * | 2022-05-06 | 2022-07-12 | 宁波大学 | Method for detecting bioavailability of nutritive salt in water body by using promoter of synechococcus gene |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58124799A (en) * | 1982-01-19 | 1983-07-25 | Mitsui Toatsu Chem Inc | Preparing method of blue-green alga plasmid pba1 and preparation thereof |
| JPS6115699A (en) * | 1984-06-29 | 1986-01-23 | Kyowa Hakko Kogyo Co Ltd | Growth hormone of fish |
| JPH0376583A (en) * | 1989-08-16 | 1991-04-02 | Hagiwara Yoshihide | Manifestation of human protein in blue-green algae |
-
1991
- 1991-09-11 JP JP3232069A patent/JPH0568572A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58124799A (en) * | 1982-01-19 | 1983-07-25 | Mitsui Toatsu Chem Inc | Preparing method of blue-green alga plasmid pba1 and preparation thereof |
| JPS6115699A (en) * | 1984-06-29 | 1986-01-23 | Kyowa Hakko Kogyo Co Ltd | Growth hormone of fish |
| JPH0376583A (en) * | 1989-08-16 | 1991-04-02 | Hagiwara Yoshihide | Manifestation of human protein in blue-green algae |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998042748A1 (en) * | 1997-03-24 | 1998-10-01 | Hih.Biocenter Inc. | New synthetic polypeptide |
| KR100443843B1 (en) * | 1999-05-28 | 2004-08-09 | 주식회사 알진텍 | Biosynthesis of foreign proteins using transformed microalgae |
| JP2008528027A (en) * | 2005-01-26 | 2008-07-31 | ユニヴェルシテイト レイデン | Means and methods for improving the development and maturation of fish eggs and / or fish sperm using hormones produced by transplanted cells |
| CN114736842A (en) * | 2022-05-06 | 2022-07-12 | 宁波大学 | Method for detecting bioavailability of nutritive salt in water body by using promoter of synechococcus gene |
| CN114736842B (en) * | 2022-05-06 | 2023-12-22 | 宁波大学 | Method for detecting bioavailability of nutrient salts in water body by using promoter of Synechococcus gene |
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