JPS623796A - Production of interleukin 2 - Google Patents
Production of interleukin 2Info
- Publication number
- JPS623796A JPS623796A JP60143675A JP14367585A JPS623796A JP S623796 A JPS623796 A JP S623796A JP 60143675 A JP60143675 A JP 60143675A JP 14367585 A JP14367585 A JP 14367585A JP S623796 A JPS623796 A JP S623796A
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- Prior art keywords
- dna
- interleukin
- plasmid
- gene
- bacillus
- Prior art date
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
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- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
この発明はインターロイキン2(以下「IL2Jと記す
)の製造法に関する。IL2は免疫不全あるいは、腫瘍
等の治療薬として使用できる可能性が大きい。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to a method for producing interleukin 2 (hereinafter referred to as "IL2J").IL2 has great potential to be used as a therapeutic agent for immunodeficiency, tumors, etc.
従来の技術
IL2の製造法の一つとして、IL2をコードする遺伝
子が挿入されているプラスミドを有する大腸菌を培養す
る方法が知られている(欧州特許出願公開筒00915
39号)。しかし従来の組換えDNA法によシ育種され
た微生物は、いずれも細胞内にIL2は生成蓄積され、
IL2を回収するためには、細胞破砕と、細胞質蛋白の
IL2の分離といり繁雑な工程を必要としていた。BACKGROUND ART As one method for producing IL2, there is a known method of culturing E. coli containing a plasmid into which a gene encoding IL2 has been inserted (European Patent Application Publication No. 00915
No. 39). However, in all microorganisms bred using conventional recombinant DNA methods, IL2 is produced and accumulated within the cells.
In order to recover IL2, complicated steps were required, including cell disruption and separation of IL2 from cytoplasmic protein.
発明が解決しようとする問題点
従ってこの発明の目的は、微生物Ivlifl胞外にI
L2を生成せしめることによるIL−2の製造法を開発
することにある。Problems to be Solved by the Invention Therefore, it is an object of the present invention to
The purpose of this invention is to develop a method for producing IL-2 by producing L2.
問題点を解決するだめの手段
上述の問題点に対し、本発明者らはシグナル41グチド
のDNAと同DNAに接続されているIL2遺伝子を有
するプラスミドが導入されているバチルス属の細菌を培
養し、同細菌の細胞外にIL2を生成蓄積せしめること
よシなるIL2の製造法を見出した。Means to Solve the Problems In order to solve the above-mentioned problems, the present inventors cultured bacteria of the genus Bacillus into which a plasmid having the DNA of signal 41 and the IL2 gene connected to the same DNA was introduced. discovered a method for producing IL2 that does not involve producing and accumulating IL2 outside the cells of the same bacterium.
バチルス属の細胞内で機能する分泌ベクターDNAとし
ては、α−アミラーゼのシグナル配列を有するpTUB
226 (J、 Biochem、 、 95 、8
7−93(1984)参照) 、 pTUB285(G
ene 、34 、1−3(1985)参照)などがあ
る。これらのベクターへのIL2遺伝子の挿入箇所はそ
れぞれ上記文献に記載されている。As a secretion vector DNA that functions in cells of the genus Bacillus, pTUB having a signal sequence of α-amylase is used.
226 (J.Biochem, 95, 8
7-93 (1984)), pTUB285 (G
ene, 34, 1-3 (1985)). The insertion sites of the IL2 gene into these vectors are described in the above-mentioned documents.
IL2遺伝子として、欧州特許出願公開第009153
9号に記載されているpIL2−50Aなどすべての遺
伝子が使用できる。As IL2 gene, European Patent Application Publication No. 009153
All genes such as pIL2-50A described in No. 9 can be used.
上記ベクターDNAとIL2遺伝子を接続して、組換え
DNAを調製する方法は、例えば第1図に示すように合
成りNA リンカ−を用いて接続する方法、その他通常
の方法が使用できる。A method for preparing recombinant DNA by connecting the above vector DNA and the IL2 gene can be, for example, the method of connecting using a synthetic NA linker as shown in FIG. 1, or other conventional methods.
得られた組換えDNAを用いてバチルス属細菌を形質転
換する方法も又、通常の方法である。即ち宿主として用
いるバチルス属細菌トしては、バチルス・ズブチリス、
マーパーグ株、バチヤス、メガテリウム1ノゞチルス0
プミリス、パチルスーアミロリキファシエンス、バチル
ス・リフェニフォルミス、バチルス・プレビス、バチル
ス−ステアロサーモフィラム等の中で修飾、制限系を有
しない宿主として利用可能な菌株が考えられる。The method of transforming Bacillus bacteria using the obtained recombinant DNA is also a conventional method. In other words, the Bacillus bacteria used as hosts include Bacillus subtilis, Bacillus subtilis,
Marperg strain, Batyas, Megatherium 1 Nochirus 0
Bacterial strains that can be used as hosts without modification or restriction systems are considered, such as Bacillus pumilis, Patylsus amyloliquefaciens, Bacillus rifeniformis, Bacillus plevis, and Bacillus stearothermophilum.
このようなバチルス属細菌宿主を上記組換えDNAによ
り形質転換する方法は、Cohenらのプロトプラスト
形質転換法(Chang + S、 + and Co
hen +S、 N、 、 Mo1. Gen、 Ge
netics 、 168 、111 (1979)参
照)やDuncanらのコンピテントセル法(Dunc
an +C,H,* Wilaon lc、A、 +−
and Young lF、E、 eGene 、 1
、153 (1977)参照)がある。A method for transforming such a Bacillus bacterial host with the above recombinant DNA is the protoplast transformation method of Cohen et al. (Chang + S, + and Co
hen +S, N, , Mo1. Gen, Ge
netics, 168, 111 (1979)) and the competent cell method of Duncan et al.
an +C,H,* Wilaon lc,A, +-
and Young IF, E, eGene, 1
, 153 (1977)).
これらの方法を使ってIL2発現プラスミドを形質転換
し、ベクターDNA上の選択マーカー形質、及びIL2
産生能の有無によシ形質転換株を選択できる。IL2の
定性及び定量分析は英国特許出願公開第91539号に
記載された方法によシ行うことができる。These methods are used to transform the IL2 expression plasmid, and the selectable marker trait on the vector DNA and the IL2 expression plasmid are transformed.
Transformants can be selected based on the presence or absence of production ability. Qualitative and quantitative analysis of IL2 can be carried out by the method described in GB 91539.
得られた形質転換株であるIL2生産能を有するバチル
ス属細菌を用いてIL2を生産する方法は、IL2生産
能を有する微生物を炭素源、窒素源、及び無機イオン、
更に必要によシビタミン、アミノ酸等の有機微量栄養素
を含有する通常の培地を用いて培養すればよい。培養方
法は好気条件下で通常30℃から40℃の範囲で行う。A method for producing IL2 using the obtained transformed strain, a Bacillus bacterium capable of producing IL2, involves using a microorganism capable of producing IL2 as a carbon source, a nitrogen source, and an inorganic ion.
Furthermore, if necessary, the culture may be carried out using an ordinary medium containing organic micronutrients such as vitamins and amino acids. The culturing method is usually carried out under aerobic conditions at a temperature in the range of 30°C to 40°C.
得られた培養液よシIL2yll被グチkを採取する方
法は遠心によシ菌体を除去した後、培養上清を硫安分画
等によシ濃縮し、英国特許出願公開第91539号に記
載されている方法で行うことができる。The method for collecting IL2yl cells from the obtained culture solution is to remove the bacterial cells by centrifugation, and then concentrate the culture supernatant by ammonium sulfate fractionation, etc., as described in British Patent Application Publication No. 91539. It can be done using the method described.
実施例
(1)IL2遺伝子のpTUB285への組込みは第1
図に示すように行った。pIL2−5OAを制限酵素H
g iAI及びDraIにょシ切断して生じる約450
bpの断片を5%ポリアクリルアミドゲル電気泳動によ
シ分離、精製した。次にこの断片を33mMTris−
acetate (P[(7,9) 、66 mM酢酸
カリヮム、10 mM h 1lf2マグネシウム、0
.5 mMジチオスレイトール、100μ々驚牛血清ア
ルブミン、0.2mMdATP 、 dcTP 、 d
GTP 、 dTTP存在下で4単位の74 DNAポ
リメラーゼ(全酒造iR)と37℃で15分間反応させ
、平滑末端化した。これと別にCCAAGCTTGGな
るHlnd mリンカ−(全酒造製)を66 mMTr
ls−HO2(pH7,6) 、10 mMMg(t2
.1 mM ATP、1mMスペルミジン塩酸塩、15
mMジチオスレイトール、200 Aii’7匂牛血清
アルブミン反応液中でT4ポリヌクレオチドキナーゼ(
全酒造製)3単位と37℃で1時間反応させてリン酸化
した。続いて同じ゛反応液中で上記の平滑末端化した断
片へのリン酸化したリンカ−の付加を、T 4 DNA
リガーゼ(全酒造製)1.4単位と4℃で一晩反応させ
ることによシ行った。反応終了後等量の水飽和フェノー
ルと混和し、水層を回収した。水層は等量のクロロホル
ムで一回抽出し、水層よJ DNAをエタノール沈澱に
より回収した。Example (1) Integration of IL2 gene into pTUB285 in the first step
This was done as shown in the figure. pIL2-5OA with restriction enzyme H
About 450 g produced by iAI and DraI cleavage
The bp fragments were separated and purified by 5% polyacrylamide gel electrophoresis. Next, this fragment was added to 33mM Tris-
acetate (P[(7,9), 66 mM potassium acetate, 10 mM h1lf2 magnesium, 0
.. 5mM dithiothreitol, 100μ dithiothreitol, 0.2mM dATP, dcTP, d
This was reacted with 4 units of 74 DNA polymerase (Zen Shuzo iR) at 37°C for 15 minutes in the presence of GTP and dTTP to form blunt ends. Separately, a Hlnd m linker called CCAAGCTTGG (manufactured by Zenshuzo Co., Ltd.) was added to 66 mMTr.
ls-HO2 (pH 7,6), 10 mM Mg (t2
.. 1mM ATP, 1mM spermidine hydrochloride, 15
T4 polynucleotide kinase (
It was phosphorylated by reacting with 3 units (manufactured by Zenshuzo Co., Ltd.) at 37°C for 1 hour. Subsequently, a phosphorylated linker was added to the blunt-ended fragment in the same reaction solution, and T4 DNA was added to the blunt-ended fragment.
This was carried out by reacting with 1.4 units of ligase (manufactured by Zenshuzo Co., Ltd.) at 4°C overnight. After the reaction was completed, the mixture was mixed with an equal amount of water-saturated phenol, and the aqueous layer was collected. The aqueous layer was extracted once with an equal volume of chloroform, and J DNA was recovered from the aqueous layer by ethanol precipitation.
DNAは10 mM Tris−HCl(p)17.5
) 、7mM MgCl2.60 mM NaC4に
溶解し、50単位のHIndI[[を加え37℃で4時
間反応させ、HIndIIIの付着末端を生じせしめた
。反応終了、後、フェノール抽出、クロロホルム抽出、
エタノール沈澱の後、5%のz +7アクリルアミドグ
ル電気泳動によりvンカーの除去を行った。DNA in 10 mM Tris-HCl (p) 17.5
) was dissolved in 7mM MgCl2.60mM NaC4, and 50 units of HIndI [[ was added thereto and reacted at 37°C for 4 hours to generate a cohesive end of HIndIII. After completion of reaction, phenol extraction, chloroform extraction,
After ethanol precipitation, v-linker removal was performed by 5% z +7 acrylamide gel electrophoresis.
ベクターDNAは以下のように調製した。pTUB28
5をHInd mで切断後生じる大きい方の断片(5,
3kbp )を1.0%アガロース電気泳動によシ分離
・精製した。Vector DNA was prepared as follows. pTUB28
The larger fragment generated after cutting 5 with HInd m (5,
3kbp) was separated and purified by 1.0% agarose gel electrophoresis.
このベクターDNAと上記のリンカ−を付加したIL2
遺伝子断片との結合は、20μ!の66mMTris−
HCt(pH7,6) 、6 mM MgCl2.1
mM ATP 。This vector DNA and IL2 with the above linker added
The binding with the gene fragment is 20μ! 66mM Tris-
HCt (pH 7,6), 6 mM MgCl2.1
mM ATP.
10mMジチオスレイトールの反応液中で1.4単位の
T 4 DNA リガーゼを加え、4℃で一晩反応させ
ることによシ行った。This was carried out by adding 1.4 units of T 4 DNA ligase to a reaction solution of 10 mM dithiothreitol and reacting overnight at 4°C.
(2)組換えプラスミドのバチルス・ズプチ藻ス207
−25株への導入はS、 Chang r S、N、
Cohenらのプロトゲラスト形質転換法(Mo1e、
Gen。(2) Recombinant plasmid Bacillus sputum 207
-25 strain was introduced with S, Chang r S, N,
Cohen et al.'s protogellast transformation method (Mo1e,
Gen.
Genet4cm 、 168 、111 (1979
)参照)によった。パーツ
f ルス−x”ブチ、pス207−25株を100rr
L/!のペンアッセイ培地中で37℃で2時間培養した
後5JMしlQm/の10■のリゾチームを含む0.5
Mシェークロース、2.0rnMマレイン酸、20m
MMgCt2(以下「S八ツ」と記す)に懸濁し、37
℃で1時間保温することによシプロトプラスト化した。Genet4cm, 168, 111 (1979
)). Part f Rusu-x” Buchi, ps 207-25 stock 100rr
L/! After incubation for 2 hours at 37°C in pen assay medium containing 5JM and 10μ of lysozyme/0.5
M Shake Rose, 2.0rnM Maleic Acid, 20m
Suspended in MMgCt2 (hereinafter referred to as "S Yatsu"), 37
Cyprotoplasts were formed by incubating at ℃ for 1 hour.
上記の連結されたDNAを含む溶液20μlに2倍濃度
の8MM溶液20μlとこのプロトゲラスト溶液0.5
dを加え、更に40%PKG6000 1.5−を加え
てよく混合し、2分間室温に放置後、。Add 20 μl of the above ligated DNA-containing solution to 20 μl of the 2x 8MM solution and 0.5 μl of this protogellast solution.
Add d, and then add 40% PKG6000 1.5-, mix well, and leave at room temperature for 2 minutes.
SMM 5 mを加え、低速遠心でプロトプラストを集
めた。そして、集められたプロトプラストは標準濃度の
インアッセイ培地を含むSMMに懸濁した。5 m of SMM was added and protoplasts were collected by low speed centrifugation. The collected protoplasts were then suspended in SMM containing a standard concentration of in-assay medium.
なお、40チPEG 6000溶液とは40?のPEG
6000を5WLl!の2倍濃度−のSMMにあわせ蒸
留水で100−としたものである。プロトプラストを上
記培地中で37℃で3時間振とり培養した後、100μ
に徳のカナマイシンを含むDI(3培地のプレート(0
,8%寒天、0.5Mコハク酸Na 2.0.5%カザ
ミノ酸、0.5%イーストエキストラクト、0.35%
に2HPO4,0,15%藷、、PO4,0,5%りk
:ff −ス、0.02 MMgCt2.0.01%
牛血清アルブミン)上に敷き、37℃で72時間培養し
、再生させた。pTUB285にはカナマイシン耐性を
示す遺伝子があるので、プラスミドを保持する宿主細菌
のみがコロニーを再生する。再生コロニーはランダムに
選択し保持するプラスミドを分離・精製し制限酵素によ
る切断試験を行いIL2遺伝子が挿入されたプラスミド
pYT−amy IL2B42を保持するクローンを同
定した。By the way, 40% PEG 6000 solution is 40? PEG of
5WLLl for 6000! The concentration of SMM was adjusted to 100 with distilled water. After culturing protoplasts in the above medium at 37°C for 3 hours with shaking, 100μ
A plate of DI (3 medium) containing Kanamycin (0
, 8% agar, 0.5M Na succinate 2.0.5% Casamino acids, 0.5% yeast extract, 0.35%
Add 2HPO4,0,15%, PO4,0,5%
:ff-su, 0.02 MMgCt2.0.01%
The cells were spread on bovine serum albumin) and cultured at 37°C for 72 hours to regenerate. Since pTUB285 contains a gene that exhibits kanamycin resistance, only host bacteria that carry the plasmid will reproduce colonies. Regenerated colonies were randomly selected, the retained plasmids were isolated and purified, and a restriction enzyme cleavage test was performed to identify clones retaining the plasmid pYT-amy IL2B42 into which the IL2 gene had been inserted.
(3) pYT−amy IL2B42を保持する形
質転換株を0.5チイーストエキストラクト、1.0%
ポリ4グトン、0、54 NaCL 、 0.2%グル
コース、s ttt/mlカナマイシン、pH7,5の
培地100mA’中で37℃で振とり培養し、菌体を遠
心分離によシ除去した培養上清をIL2活性アッセイ用
サンプルとした。その結果10,000〜15,000
単位/ mlのIL2活性が認められた。(3) Transformed strain harboring pYT-amy IL2B42 was treated with 0.5% yeast extract, 1.0%
The culture medium was shaken and cultured at 37°C in 100 mA' of poly4gtone, 0.54 NaCL, 0.2% glucose, sttt/ml kanamycin, pH 7.5, and the bacterial cells were removed by centrifugation. The supernatant was used as a sample for IL2 activity assay. Result: 10,000-15,000
Units/ml of IL2 activity was observed.
pTUB 28 s (第2図参照)は以下の手順で構
築された。pTUB 4はpUBlloのB amHI
部分にα−アミラーゼの全遺伝子を含んだ2.3kbの
DNA断片が組込まれたプラスミドである。この組込ま
れたDNA断片の全塩基配列はすでに決定されている(
文献J、 Bacteriol 156 、327 (
1983)参照)。pTUB28s (see Figure 2) was constructed using the following procedure. pTUB 4 is B amHI of pUBllo
This is a plasmid in which a 2.3 kb DNA fragment containing the entire α-amylase gene is integrated. The entire base sequence of this integrated DNA fragment has already been determined (
Reference J, Bacteriol 156, 327 (
(1983)).
またpUB 110はバチルス属細菌内で増殖可能なプ
ラスミドでクローニング・ベクターとして広く用い
−られている。pTUB4をEcoRIで切断すると、
4.9kbと1.9 kbの断片にわかれる。4.9k
bの断片について大腸菌DNA Jリメラーゼ■のクレ
ノー7ラグメントによシ平滑末端とした後、H1ndl
llリンカ−を付加したのち、T 4 DNA ’)ガ
ーゼによりffl状化させて得られるプラスミドがpT
UB201である。pUB110 is a plasmid that can be propagated within Bacillus bacteria and is widely used as a cloning vector.
- is being given. When pTUB4 is cut with EcoRI,
It is divided into 4.9 kb and 1.9 kb fragments. 4.9k
After making the fragment b blunt-ended with the Klenow 7 fragment of E. coli DNA J limerase, H1ndl
After adding a ll linker, the plasmid obtained by converting the T4 DNA' into ffl form with gauze is called pT.
It is UB201.
次K 1.9 kbの断片をAluIによシ切断し、α
−アミラーゼのプロモーター、及びシグナル配列を含ん
だ424 bpの断片を分離・精製し、これにHl n
d mリンカ−を付加して上記のpTUB201のH1
ndu部位に組込んで得られるプラスミドがpTU82
18である。pTUB 218をHlndII[で部分
分解した後、大腸菌DNAポリメラーゼIクレノー フ
ラグメントによシ干滑末端化しT 4 DNA IJガ
ーゼにより環状化し、さらにこれをHlnd mで切断
した後、pKTH74よ、りH1ndIIIで切シ出さ
れる1、4 kbの断片ヲ組込ミ、バチルス・サチルス
・マーパーク株に導入すると、β−ラクタマーゼ産生能
を付与するプラスミドがpTUB 285である。pK
TH74はpBR322にコードされるβ−ラクタマー
ゼ遺伝子のうち、そのシグナル配列を欠いた部分がGC
AAGCTGCなるHlndl[リンカ−を介して、p
BR322のHlnd ll[部分に組込まれたもので
ある(文献Proc、Natl。The next K 1.9 kb fragment was cut with AluI and α
- Isolate and purify a 424 bp fragment containing the amylase promoter and signal sequence, and add it to Hl n
H1 of the above pTUB201 by adding a d m linker
The plasmid obtained by integrating into the ndu site is pTU82.
It is 18. pTUB 218 was partially digested with HlndII, then blunt-ended with E. coli DNA polymerase I Klenow fragment, circularized with T4 DNA IJ gauze, further cut with Hlndm, and then cut with HlndIII from pKTH74. pTUB 285 is a plasmid that confers the ability to produce β-lactamase when the 1 to 4 kb fragment is introduced into a Bacillus subtilis marpark strain. pK
TH74 is the part of the β-lactamase gene encoded by pBR322 that lacks the signal sequence.
AAGCTGC Hlndl [via linker, p
It was incorporated into the Hlnd ll [portion of BR322 (Reference Proc, Natl.
Acad、 Sci 、 79 、5582 (198
2)参照)。Acad, Sci, 79, 5582 (198
2)).
以上のようにpTUB 285はpUBlloのBam
HI部位に、その塩基配列がすべて決定されている2、
3kbの外来DNA断片が挿入された・ぐチルス属細菌
内で増殖可能なプラスミドである。As mentioned above, pTUB 285 is pUBllo's Bam
All base sequences have been determined at the HI site2.
This is a plasmid into which a 3 kb foreign DNA fragment has been inserted, and which can proliferate in bacteria belonging to the genus Gutilus.
第1図は、本発明のインターロイキン2遺伝子を有する
プラスミドの造成経過説明図である。
第2図は、本発明において使用されたプラスミドベクタ
ーpTUB 285の説明図である。
特許用゛願大 味の素株式会社
第1図
IL2 ζON^
i H91AI/ Ora+FIG. 1 is an explanatory diagram of the construction process of a plasmid having the interleukin 2 gene of the present invention. FIG. 2 is an explanatory diagram of the plasmid vector pTUB 285 used in the present invention. Patent application Ajinomoto Co., Ltd. Figure 1 IL2 ζON^ i H91AI/ Ora+
Claims (2)
ているインターロイキン2遺伝子を有するプラスミドが
導入されているバチルス属の細菌を培養し、同細菌の細
胞外にインターロイキン2を生成蓄積せしめることを特
徴とするインターロイキン2の製造法。(1) Cultivating a Bacillus bacterium into which a plasmid containing the interleukin 2 gene connected to the signal peptide DNA is introduced, and producing and accumulating interleukin 2 outside the cells of the bacterium. Characteristic manufacturing method of interleukin 2.
ものである特許請求の範囲第1項記載の製造法。 【アミノ酸配列があります】(2) The production method according to claim 1, wherein the signal peptide has the following amino acid sequence. [There is an amino acid sequence]
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60143675A JPS623796A (en) | 1985-06-28 | 1985-06-28 | Production of interleukin 2 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60143675A JPS623796A (en) | 1985-06-28 | 1985-06-28 | Production of interleukin 2 |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS623796A true JPS623796A (en) | 1987-01-09 |
Family
ID=15344319
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP60143675A Pending JPS623796A (en) | 1985-06-28 | 1985-06-28 | Production of interleukin 2 |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS623796A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05105542A (en) * | 1991-10-14 | 1993-04-27 | Akechi Ceramics Kk | Air-passable refractory |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57132895A (en) * | 1980-12-31 | 1982-08-17 | Parubua Irutsuka | Production of rearranged dna molecule and protein |
-
1985
- 1985-06-28 JP JP60143675A patent/JPS623796A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57132895A (en) * | 1980-12-31 | 1982-08-17 | Parubua Irutsuka | Production of rearranged dna molecule and protein |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05105542A (en) * | 1991-10-14 | 1993-04-27 | Akechi Ceramics Kk | Air-passable refractory |
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