JPS6232887A - Polypeptide exhibiting interleukin 1 activity and dna coding same - Google Patents
Polypeptide exhibiting interleukin 1 activity and dna coding sameInfo
- Publication number
- JPS6232887A JPS6232887A JP60171493A JP17149385A JPS6232887A JP S6232887 A JPS6232887 A JP S6232887A JP 60171493 A JP60171493 A JP 60171493A JP 17149385 A JP17149385 A JP 17149385A JP S6232887 A JPS6232887 A JP S6232887A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- polypeptide
- acid sequence
- dna
- base sequence
- 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
-
- 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]
- C07K14/545—IL-1
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F02—COMBUSTION ENGINES; HOT-GAS OR COMBUSTION-PRODUCT ENGINE PLANTS
- F02D—CONTROLLING COMBUSTION ENGINES
- F02D41/00—Electrical control of supply of combustible mixture or its constituents
- F02D41/02—Circuit arrangements for generating control signals
- F02D41/14—Introducing closed-loop corrections
- F02D41/1438—Introducing closed-loop corrections using means for determining characteristics of the combustion gases; Sensors therefor
- F02D41/1444—Introducing closed-loop corrections using means for determining characteristics of the combustion gases; Sensors therefor characterised by the characteristics of the combustion gases
- F02D41/1454—Introducing closed-loop corrections using means for determining characteristics of the combustion gases; Sensors therefor characterised by the characteristics of the combustion gases the characteristics being an oxygen content or concentration or the air-fuel ratio
- F02D41/1456—Introducing closed-loop corrections using means for determining characteristics of the combustion gases; Sensors therefor characterised by the characteristics of the combustion gases the characteristics being an oxygen content or concentration or the air-fuel ratio with sensor output signal being linear or quasi-linear with the concentration of oxygen
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
本発明はインターロイキン1活性を示すポリペプチドを
コードするDNA 、 インク−ロイキン1活性を示
すポリペプチド及びそのポリペプチドの誘導体並びにそ
れらの製造法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a DNA encoding a polypeptide exhibiting interleukin-1 activity, a polypeptide exhibiting interleukin-1 activity, derivatives of the polypeptide, and methods for producing them.
ゲリーらはヒト マクロファージの培養土1n中に、マ
イトーゲ/によるマウス胸腺細胞***作用を促進させる
物質を見い出し、これをリンパ球活性化因子(lymp
hocyte acHvating factor。Gerry et al. found a substance in human macrophage culture medium 1N that promotes mitogen-induced mouse thymus cell division, and added this substance to lymphocyte activating factor (lymp).
hocyte acHvating factor.
以下LAFと略記する)と名付けたが、1979年以降
、インターロイキ71(以下、IL−1と略記する)の
名称が用いられている。従って本明細書においてもこの
ような物質をインターロイキンlとして扱う。However, since 1979, the name Interloiki 71 (hereinafter abbreviated as IL-1) has been used. Therefore, in this specification, such a substance is also treated as interleukin I.
IL−1はT細胞やB細胞の増殖分化を促進させ、また
T細胞に作用してリンホカイン、特にインターロイキン
2(T細胞増殖因子)の産生を促進させる効果を仔し、
抗体産生や細胞性免疫のFA節に重要な役割を果たす因
子の一つと考えられている[ 5taruch、M、J
、、 et al、、J、Immunol。IL-1 promotes the proliferation and differentiation of T cells and B cells, and acts on T cells to promote the production of lymphokines, especially interleukin 2 (T cell growth factor).
It is considered to be one of the factors that plays an important role in antibody production and the FA node of cell-mediated immunity [5 Taruch, M, J
,, et al., ,J., Immunol.
130.2191(+983)コ。その他、プロスタグ
ラ/ジンEやコラゲナーゼの産生促進、繊維芽細胞の増
殖促進、又はインターロイキ/2やインターフェロンの
存するNK (ナチュラルキラー)細胞活性化作用を増
強させる効果があると報告されている[: Simon
、P、L、、 et at、、 ” Lymphoki
nes″vo1.o、p、47(+982)^cade
nce I’ress lnc、] aこのようにl−
1は免疫応答のみならず、生体の防御やその修?J1等
にも関与する生体物質であり、免疫不全症に対する治療
蘂や抗腫1g剤としての臨床応用がllI′l待されて
いる。130.2191 (+983). In addition, it is reported to have the effect of promoting the production of Prostagra/gin E and collagenase, promoting the proliferation of fibroblasts, and enhancing the NK (natural killer) cell activation effect of interleukin/2 and interferon [: Simon
, P, L,, et at,, ”Lymphoki
nes″vo1.o, p, 47(+982)^cade
nce I'ress lnc, ] a like this l-
1 is not only the immune response, but also the body's defense and its repair. It is a biological substance that is also involved in J1, etc., and its clinical application as a treatment for immunodeficiency diseases and as an antitumor agent is awaited.
IL−1のこれまでの地組方法は、主としてマクロファ
ージや末哨単核細胞又はマクロファージ様株化細胞(例
えばマ・クスP388D +細胞)や単球性又は弁開性
白血病細胞等を適当な誘導剤の存在下で培養し、その培
養上1?I中より単mするものである。Previous methods for assembling IL-1 have mainly involved the appropriate induction of macrophages, sentinel mononuclear cells, macrophage-like established cell lines (e.g. Ma. 1? It is one m from I medium.
ヒ)IL−1は、ヒト単球性白血病株化細胞であるU9
37細胞及びヒト末梢単核細胞の培養土7i7から分離
精製され、その分子量がI!、300及び15.000
ダルトンであると報告されている[Mizel、S、n
、、 et al、、J、Immunol、131
.1834(1983);Sch++idt、J、A、
、J、Exp、Med、Ioo、772(1984)]
。h) IL-1 is a human monocytic leukemia cell line, U9.
It was isolated and purified from culture medium 7i7 of 37 cells and human peripheral mononuclear cells, and its molecular weight was I! , 300 and 15.000
Dalton [Mizel, S, n
,, et al., ,J.Immunol, 131
.. 1834 (1983); Sch++idt, J.A.
, J. Exp. Med, Ioo, 772 (1984)]
.
最近、マウスP38gD I細胞を用いマウスIL−1
ポリペプチドをコードするc DNAをクローニングし
、IL−1活性を有する100個のアミノ酸からなるポ
リペプチドを大腸閏で生産させることに成功したと報告
されている[Lowedico P4. etal、、
Nature、312,458(+984)] −ま
た、ヒトIL−1に関しては、ヒト単核球或いはヒト
マクロファージからヒトIL−1ポリペプチドをコード
するc DNAがクローニングされ、形質発現に成功し
たことが報告されている[Auron、P、E、ct
at、。Recently, mouse P38gD I cells were used to detect mouse IL-1.
It has been reported that a cDNA encoding a polypeptide was cloned and a polypeptide consisting of 100 amino acids with IL-1 activity was successfully produced in the large intestine [Lowedico P4. etal,,
Nature, 312, 458 (+984)] - Also, regarding human IL-1, human mononuclear cells or human
It has been reported that cDNA encoding human IL-1 polypeptide was cloned from macrophages and successful expression was achieved [Auron, P.E., ct.
at.
Proc、Nat、^cad、sci、Us^81,7
90?(1984)、March、C,Jet al、
、Nature 315,641(1985)]、上8
己のIL−1ポリペプチドをコードするDNAの塩基配
列が同一ではなく、複数のIL−1の存在が示唆されて
いる。Proc, Nat, ^cad, sci, Us^81,7
90? (1984), March, C., Jet al.
, Nature 315, 641 (1985)], supra 8.
The base sequences of the DNAs encoding their own IL-1 polypeptides are not the same, suggesting the existence of multiple IL-1s.
一方、本発明者らは、逍伝子組み換え技術を応用してヒ
トIL−1ポリペプチドを製造すべくv:1.α研究の
結果、ヒ) IIL−Go細胞を用いてヒトIL−1前
駆体ポリペプチドをコードするクローン化c DNAの
単層に成功し、このクローン化cDNAに由来するDN
Aを組み込んだ組み換え体プラスミドで形質転換された
微生物中で、ヒ) IL−1前駆体のC末端側159残
基からなるポリペプチドを生産させ、不純物を実質的に
含有しないヒトIL−1ポリペプチドを単離することに
成功し特許出願を行っている(特開昭GO−11247
4号)6更に研究を重ねた結果、IL−1の生物活性の
うち特に医薬品としての利用価値の高いT細胞の増殖に
及ぼす作用、すなわちLAF活性を発押するには、必ず
しも前記の159残基のアミノ酸配列を必要とせず、そ
れより少ない残基数のアミノ酸配列からなるポリペプチ
ドもLAP活性をイrすることを兄い出し、本発明を完
成した。On the other hand, the present inventors applied v:1 polypeptide to produce human IL-1 polypeptide by applying Shogene recombinant technology. As a result of α research, we succeeded in producing a monolayer of cloned cDNA encoding the human IL-1 precursor polypeptide using human IIL-Go cells, and obtained DNA derived from this cloned cDNA.
In a microorganism transformed with a recombinant plasmid incorporating A), a polypeptide consisting of the C-terminal 159 residues of the human IL-1 precursor is produced, and a human IL-1 polypeptide containing substantially no impurities is produced. We succeeded in isolating the peptide and filed a patent application (Japanese Patent Application Laid-Open No. Sho GO-11247).
No. 4)6 As a result of further research, it was found that among the biological activities of IL-1, activation of LAF activity, which has a particularly high effect on the proliferation of T cells that has high utility value as a pharmaceutical, does not necessarily require the above-mentioned 159 residue. The present invention was completed by discovering that a polypeptide consisting of an amino acid sequence with a smaller number of residues can also induce LAP activity without requiring a basic amino acid sequence.
本発明は、IL−1活性を示すポリペプチドをフードす
るDNA 1すなわち下記式[A]で示されるアミノ酸
配列に対応する塩基配列、或いはそのN末端のアミ/f
i(Set)から17番目C++e)までのアミノ酸が
順次1個づつ除去されたそれぞれのアミノ酸配列に対応
する塩基配列を仔し又は含む新規D N A及びその製
造法に関する。The present invention provides DNA 1 that encodes a polypeptide exhibiting IL-1 activity, that is, a base sequence corresponding to the amino acid sequence represented by the following formula [A], or its N-terminal amino/f
The present invention relates to a novel DNA containing or containing a base sequence corresponding to each amino acid sequence in which amino acids from i (Set) to 17th C++e) are sequentially removed one by one, and a method for producing the same.
Scr Pro Phe Ser Phc
Leu Scr Asn Val LysTy
r Asn Phe Met Arg Ile IIe
Ly’s Tyr GluPhe llc Leu A
sn AsP Ala Leu Asn Gln 5e
r11e lie Arg Ali Asn Asp
Gln Tyr Leu ThrAla Ala Al
a Leu His Asn Leu AspGlu
AlaVat Lys Phe Asp Met Gl
y Ala Tyr Lys 5erScr Lys
Asp Asp Ala Lys IICThr Va
l l1eLeu Arg Ile Ser Lys
Thr Gin Leu Tyr ValThr Al
a Gln Asp Glu Asp Gln Pro
Val LcuLeu Lys Glu Met P
ro Glu llc Pro Lys Thrllc
Thr Gly Ser Glu Thr Asn
Leu Lcu PhcPhe Trp Glu Th
r l1is Gly Thr Lys Asn Ty
rPhe Thr Ser Val Ala l1is
Pro Asn Leu Phe+1e Ala T
hr Lys Gln Asp Tyr Trp Va
t CysLau Ala Gly Gly Pro
Pro Ser Ile Thr AspPhe Gl
n Ile Leu Glu Asn Gin Ala
[A]上言己ポリペプチド[A]自体をコードする
DNAの典型的な塩基配列は次に示す通りである。Scr Pro Phe Ser Phc
Leu Scr Asn Val LysTy
r Asn Phe Met Arg Ile IIe
Ly's Tyr GluPhe llc Leu A
sn AsP Ala Leu Asn Gln 5e
r11e lie Arg Ali Asn Asp
Gln Tyr Leu Thr Ala Ala Al
a Leu His Asn Leu AspGlu
AlaVat Lys Phe Asp Met Gl
y Ala Tyr Lys 5erScr Lys
Asp Asp Ala Lys IICThr Va
l l1eLeu Arg Ile Ser Lys
Thr Gin Leu Tyr Val Thr Al
a Gln Asp Glu Asp Gln Pro
Val LcuLeu Lys Glu Met P
ro Glu llc Pro Lys Thrllc
Thr Gly Ser Glu Thr Asn
Leu Lcu PhcPhe Trp Glu Th
r l1is Gly Thr Lys Asn Ty
rPhe Thr Ser Val Ala l1is
Pro Asn Leu Phe+1e Ala T
hr Lys Gln Asp Tyr Trp Va
t CysLau Ala Gly Gly Pro
Pro Ser Ile Thr AspPhe Gl
n Ile Leu Glu Asn Gin Ala
[A] A typical base sequence of the DNA encoding the self-polypeptide [A] itself is as shown below.
(5’)TCA CCT TTT AGCTTCCTG
AGCAAT GTG AAATACAACTTT
r〜TG AGG ATCΔTCAAA TACGAA
TTCATCCTG AAT GACGCCCTCAA
T CAA AGTATA ATT CGA GCCA
AT GAT CACTACCTCACGGCT GC
T GCA TTA CAT AAT CTG GAT
GAA GCAGTG AAA TTT GA
CATG GGT GCT TAT AAG
TCATCA AAG GAT GAT GCT A
AA ATT ACCGTG ATTCTA AGA
ATCTCA AAA ACT CAA TTG TA
T GTGACT GCCCAA GAT GAA G
ACCAA CCA GTG CTGCTG AAG
GAG ATG CCT GAに ATA
CCCAAA ACCATCAcA GGT A
GT GAG ACCAACCTCCTCTTCTTC
TGG GAA ACT CACGGCACT
AAG AACTATTTCACA TCA GT
T GCCCAT CCA AACTTG TTTAT
T GCCACA AAG CAA GACT
ACTGG GTG TGCTTG GCA
GGG GGG CCA CCCTCT AT
CACT GACTTT CAG ATA C
TG GAA AACCAG GCG(3’)[
I3コ本発明はまた。IL−1活性を示すポリペプチド
、すなわち前記式[A]で示されるアミノ酸配列をイf
するか、或いはそのN末端のアミノa(Set)から1
7番目(Ile)までのアミノ酸が順次1個づつ除去さ
れたそれぞれのアミノ酸配列を存する新規ポリペプチド
又はその誘4体及びその製造法にも関する。(5')TCA CCT TTT AGCTTCCTG
AGCAAT GTG AAATACAACTTT
r〜TG AGG ATCΔTCAAA TACGAA
TTCATCCTG AAT GACGCCCTCAA
T CAA AGTATA ATT CGA GCCA
AT GAT CACTACCTCACGGCT GC
T GCA TTA CAT AAT CTG GAT
GAA GCAGTG AAA TTT GA
CATG GGT GCT TAT AAG
TCATCA AAG GAT GAT GCT A
AA ATT ACCGTG ATTCTA AGA
ATCTCA AAA ACT CAA TTG TA
T GTGACT GCCCAA GAT GAA G
ACCAA CCA GTG CTGCTG AAG
GAG ATG CCT GA to ATA
CCCAAA ACCATCAcA GGT A
GT GAG ACCAAACCTCCTCTTCTT
TGG GAA ACT CACGGCACT
AAG AACTATTTCACA TCA GT
T GCCCAT CCA AACTTG TTTAT
T GCCACA AAG CAA GACT
ACTGG GTG TGCTTG GCA
GGG GGG CCA CCCTCT AT
CACT GACTTT CAG ATA C
TG GAA AACCAG GCG (3') [
The present invention also relates to I3. If a polypeptide exhibiting IL-1 activity, that is, an amino acid sequence represented by the above formula [A],
or from its N-terminal amino a (Set) to 1
The present invention also relates to novel polypeptides having respective amino acid sequences in which amino acids up to the seventh position (Ile) are sequentially removed one by one, or derivatives thereof, and methods for producing the same.
上記ポリペプチドの誘導体とは、該ポリペプチドの項生
の側鎖官能基、N末端のアミノ基又はC末端のカルボキ
シル基を利用して形成される誘導体を意味し、例えばカ
ルボキシル基と脂肪族アルコールとのエステル類、第−
或いは第二アミンとの酸アミド類、アミ7基のN−アシ
ル誘導体又はヒドロキシル基の0−アシル誘導体、さら
には該ポリペプチドのカルボキシル基又はアミ7基等と
で形成される塩、例えば水酸化ナトリウム、水酸化カリ
ウム、アルギニ/、カフェイン、プロカイン、塩酸、グ
ルコン酸等との塩が挙げられる。The above-mentioned derivative of the polypeptide means a derivative formed by utilizing the natural side chain functional group, the amino group at the N-terminus, or the carboxyl group at the C-terminus of the polypeptide, such as a carboxyl group and an aliphatic alcohol. Esters with, No.-
Alternatively, acid amides with secondary amines, N-acyl derivatives of amide 7 groups or O-acyl derivatives of hydroxyl groups, and salts formed with carboxyl groups or amide 7 groups of the polypeptide, such as hydroxylation. Examples include salts with sodium, potassium hydroxide, arginine, caffeine, procaine, hydrochloric acid, gluconic acid, and the like.
本発明のポリペプチドは、会合体として存在し得る場合
もあり、このような会合体も本発明のポリペプチドに包
含される。The polypeptide of the present invention may exist as an aggregate, and such aggregates are also included in the polypeptide of the present invention.
以下に本発明のDNA及びポリペプチドについてその作
製並びに製造法について述べる。The preparation and manufacturing method of the DNA and polypeptide of the present invention will be described below.
ヒ) IL−1前駆体ポリペプチドをコードする塩基配
列を任するDNAは、例えば後記参考例1に示した方法
により単離することができる。h) DNA containing the base sequence encoding the IL-1 precursor polypeptide can be isolated, for example, by the method shown in Reference Example 1 below.
このDNAを適当な制限酵素で切断したのち、これと必
要に応じて常法により合成したDNAアダプターとを結
合することにより、本発明のポリペプチドをコードする
DNA塩基配列の5′末端に開始コドンATG 、 3
’末端に終止コドンを存する塩基配列を含むDNA断片
を作製することができる。After cutting this DNA with an appropriate restriction enzyme, this DNA is ligated with a DNA adapter synthesized by a conventional method as necessary, thereby creating a start codon at the 5' end of the DNA base sequence encoding the polypeptide of the present invention. ATG, 3
A DNA fragment containing a base sequence having a stop codon at its end can be produced.
これを適当なプロモーター及びシャイン・ダルガー7(
SD)配列に続いて結合させ、ベクターに組み込むこと
により、本発明のポリペプチド生産用の形質発現ベクタ
ーを得ることができる。A suitable promoter and Shine Dalgar 7 (
By subsequently ligating the SD) sequence and incorporating it into a vector, an expression vector for producing the polypeptide of the present invention can be obtained.
プロモーターとしては、例えばIac、 trp、 t
ac。Examples of promoters include Iac, trp, t
ac.
phoS、 pho^、 pt、、 SV40初期
プロモーター等が挙げられる。ベクターとしては、形質
転換させる宿主中で増殖するものはすべて用いることが
できる。Examples include phoS, pho^, pt, SV40 early promoter, and the like. Any vector that can be used to propagate in the host to be transformed can be used.
例えば、プラスミド(pnR322等)、ファージ(λ
)7一ジ誘導体等)、ウィルス(SV40等)が挙げら
れる。また、う/ナラエイ プラスミドも有用である。For example, plasmids (pnR322 etc.), phages (λ
)7-di derivatives, etc.), and viruses (SV40, etc.). Also useful are carp/nala ray plasmids.
これらの形質発現ベクターを適当な宿主、例えば大腸苗
などに、例えばコーエ/らの方法[Proc。These expression vectors are transferred to suitable hosts such as large intestine seedlings, for example, by the method of Koe et al. [Proc.
Nat、八cad、sci、UsA、69.2110(
+972)]により導入することにより形質転換体を得
、次いで該形質転換体を培養することにより本発明のポ
リペプチド或いはそのN末端にメチオニンが付加したポ
リペプチドを産生させることができる。該生産物は使用
したプロモーターと形質発現ベクターの構築法により、
宿主中の細胞質内又は細胞質外に蓄積させることができ
る。細胞質外に分泌させるには、例えばアルカリホスフ
ァターゼの構造逍伝子(pho^)やリン酸結合蛋白の
構造逍伝子(phoS )を用い、それらのシグナルペ
プチドをコードする領域に続いてポリペプチドをコード
するDNAを結合させた形質発現ベクターを構築すれば
よい。Nat, 8cad, sci, UsA, 69.2110 (
+972)] to obtain a transformant, and then by culturing the transformant, the polypeptide of the present invention or a polypeptide having methionine added to its N-terminus can be produced. The product depends on the promoter used and the construction method of the expression vector.
It can be accumulated intracytoplasmically or extracytoplasmically in the host. For secretion outside the cytoplasm, for example, the structural gene for alkaline phosphatase (pho^) or the structural gene for phosphate binding protein (phoS) is used, and the polypeptide is inserted following the region encoding the signal peptide. What is necessary is to construct a gene expression vector in which the encoding DNA is linked.
このようにして得られた形質転換体を、それぞれの形質
転換体に応じた適当な培養条件下で、目的のポリペプチ
ドが十分に産生されるまで培養したのち、培養物からポ
リペプチドを抽出する。産生したポリペプチドが細胞質
中に蓄積される場合は、例えば、リゾチーム消化と凍結
融解や超音波破砕、フレンチプレス等により宿主細胞を
破壊したのち、遠心性m又は濾過にて抽出液を集める。The transformants thus obtained are cultured under appropriate culture conditions depending on each transformant until the desired polypeptide is sufficiently produced, and then the polypeptide is extracted from the culture. . If the produced polypeptide is accumulated in the cytoplasm, the host cells are destroyed by, for example, lysozyme digestion, freeze-thawing, ultrasonic disruption, French press, etc., and then the extract is collected by centrifugal m or filtration.
また、ペリプラスムに蓄積される場合は、例えばウィル
スキーらの方法[J、11acteNo1..127,
595(1970)]に従って抽出することができる。In addition, when it is accumulated in the periplasm, for example, the method of Wilsky et al. [J, 11acte No. 1. .. 127,
595 (1970)].
このようにして得られた粗製のポリペプチドは蛋白質の
一般的な精製法(限外d過、透析、イオン交換クロマト
グラフィー、ゲル濾過、電気泳動。The crude polypeptide thus obtained can be purified using general protein purification methods (ultrafiltration, dialysis, ion exchange chromatography, gel filtration, electrophoresis).
アフィニティクロマトグラフィー?)に従い精製するこ
とにより、目的とするポリペプチドを実質的に純品とし
て得ることができる。Affinity chromatography? ), the polypeptide of interest can be obtained as a substantially pure product.
本発明のポリペプチドのうち、前記式[A]で示される
アミノ酸配列のN末端側の1個、9個又は14個のアミ
ノ酸が除去されたアミノ酸配列を佇するポリペプチドが
好ましいものとして挙げることができる。Among the polypeptides of the present invention, polypeptides having an amino acid sequence in which 1, 9, or 14 amino acids on the N-terminal side of the amino acid sequence represented by formula [A] are removed are preferred. I can do it.
本発明のポリペプチドの誘導体は、上記のようにして得
られたポリペプチドを原料として、これに脂肪層アルコ
ールを作用させ該ポリペプチドのエステル体を、カルボ
ン酸の反応性誘導体を作用させ該ポリペプチドのN−ア
シル又は0−アシル体を、第1アミン又は第2アミンを
作用させ該ポリペプチドのアミド体を、それぞれ形成せ
しめることができる。これらの反応は常法に従い行なわ
れる。さらには、本発明のポリペプチドに有機酸、無機
酸、塩基を常法により加えることにより本発明のポリペ
プチドの酸又はアルカリ塩を形成せしめることができる
。The polypeptide derivative of the present invention can be obtained by using the polypeptide obtained as described above as a raw material, treating it with fatty alcohol to form an ester form of the polypeptide, and treating it with a reactive derivative of carboxylic acid to form the polypeptide. The N-acyl or O-acyl form of a peptide can be reacted with a primary amine or a secondary amine to form an amide form of the polypeptide, respectively. These reactions are carried out according to conventional methods. Furthermore, an acid or alkali salt of the polypeptide of the present invention can be formed by adding an organic acid, an inorganic acid, or a base to the polypeptide of the present invention using a conventional method.
(以下余白)
次に、本発明のポリペプチドの特性について以下に記述
する。(The following is a blank space) Next, the characteristics of the polypeptide of the present invention will be described below.
分子Q、N末端アミノM、C末端アミノ酸及びIL−1
活性の測定方法は次の通りである。Molecule Q, N-terminal amino M, C-terminal amino acid and IL-1
The method for measuring activity is as follows.
(1) 分子ffi測定
分子量はドデシル硫酸ナトリウム(SDS )存在下に
おけるポリアクリルアミドゲル(ゲル0度12.5%)
電気泳動分析により測定した。(1) The molecular weight measured by molecular ffi was measured using polyacrylamide gel (gel 0 degrees 12.5%) in the presence of sodium dodecyl sulfate (SDS).
Measured by electrophoretic analysis.
実施例1〜3で得たそれぞれのポリペプチド溶液を等容
量の4%SDS 、10%2−メルカプトエタノール、
20%グリセロール及び0.02%ブロムフェノールブ
ルーを含む0.+25M T r i s −11CI
B樹液(plIO,8>と混合し、30分間室at’
放置後、5DS−ポリアクリルアミドゲル電気泳動分析
に付した。Each polypeptide solution obtained in Examples 1 to 3 was mixed with equal volumes of 4% SDS, 10% 2-mercaptoethanol,
0.01 containing 20% glycerol and 0.02% bromophenol blue. +25M Tris -11CI
Mix with B sap (plIO,8> and chamber at' for 30 min.
After standing, it was subjected to 5DS-polyacrylamide gel electrophoresis analysis.
泳動用溶媒として0.1%SDSを含む25mMTri
s−0,2Mグリシン液を用い、200J?ルトで3時
間泳動させた。泳動終了後、クマシーブリリアントブル
ー6250を用いる染色で蛋白質の泳動位置を、また別
途にゲルを1■巾で切り出し各ゲル片を1%重炭酸ア/
モ二ウつ水溶Zに浸漬し、ゲル中のfJf白質を抽出し
た。各ゲル片から得たそれぞれの抽出液について後記す
る方法に従ってTL−1活性を測定し、精製ポリペプチ
ドを得た。25mMTri containing 0.1% SDS as a running solvent
Using s-0.2M glycine solution, 200J? The electrophoresis was carried out for 3 hours at a normal temperature. After the electrophoresis is completed, the electrophoresis position of the protein is determined by staining with Coomassie Brilliant Blue 6250.Separately, the gel is cut out into 1-inch width and each gel piece is soaked in 1% bicarbonate/water.
The fJf white matter in the gel was extracted by immersion in Monitsu aqueous Z. The TL-1 activity of each extract obtained from each gel piece was measured according to the method described later, and purified polypeptide was obtained.
分子量既知の標窄蛋臼賀としてホスフJリラーゼb (
MW94,000) 、 ウシ血清アルブミン(MW
07.000> 、オボアルプミy MW 43,00
0) 、炭酸脱水酵素(MW 30,000) 、大豆
トリプシン インヒビター(MW 20,100)及び
α−ラクトアルブミン(MW 14,400)を用いた
。Phosph J-lylase b (
MW94,000), bovine serum albumin (MW
07.000>, Oboalpumiy MW 43,00
0), carbonic anhydrase (MW 30,000), soybean trypsin inhibitor (MW 20,100) and α-lactalbumin (MW 14,400).
C) N末端アミノ酸分析
(1)項に記載の方法により得られた精製ポリペプチド
について、ダンシル化法[Gray、W、R,Meth
odsin Enzymol、volXI 、pL19
(19B?)]により、N末rJアミノ酸を同定した。C) N-terminal amino acid analysis The purified polypeptide obtained by the method described in (1) was analyzed using the dansylation method [Gray, W, R, Meth
odsin Enzymol, volXI, pL19
(19B?)], the N-terminal rJ amino acid was identified.
精製ポリペプチド溶液の100μlに、IO%SDSの
lOμ1.N−エチルモルフオリ/の100μ!及びダ
ンシルクロリド(アセトン中5 mg/ ml)の50
μlを添加し、37°Cで1時間加温した。更に、21
1のアセトンを加え、析出した沈殿を遠心分層にて集め
、これを80%アセトンにて洗浄した。沈殿を真空乾燥
させたのち、f’E N l−lClの100μpに6
解し、真空密封ガラス口中で105°C,5〜18時間
加熱した。To 100 μl of purified polypeptide solution, add 10 μl of IO% SDS. 100μ of N-ethylmorpholy/! and 50% of dansyl chloride (5 mg/ml in acetone)
μl was added and warmed at 37°C for 1 hour. Furthermore, 21
1 of acetone was added, and the precipitate precipitated was collected by centrifugation and washed with 80% acetone. After vacuum drying the precipitate, 6
The mixture was dissolved and heated at 105° C. for 5 to 18 hours in a vacuum-sealed glass cap.
真空乾燥にて乾燥させたのち、水飽和酢酸エチルで抽出
し、ダンシル化されたアミノ酸をポリアミン シート(
Chang−Chin Trading Co、Tai
wan)を用いる二次元薄層クロマトグラフィー[Wo
ods、に、Roand Wang、に、T、、11i
ochim、l1liophys、^cta 、 13
3 、309(1907>]により同定した。After drying in vacuum, the dansylated amino acids were extracted with water-saturated ethyl acetate and transferred to a polyamine sheet (
Chang-Chin Trading Co.,Tai
Two-dimensional thin layer chromatography [Wo
ods, to, Roand Wang, to, T,, 11i
ochim, l1liophys, ^cta, 13
3, 309 (1907>).
(3)C末端アミノ酸分析
精製ポリペプチドIfJ液の20μ!に、100mM酢
Mす) IJ ウ1Jli& (p1]5.4) ノ+
8μL 1 %SDSの4μl及びカルボキシペプチ
ダーゼY (0,1B/1)の2μ!を添加し、25°
Cで5〜15分静置した。この反応液の10μaに10
0mMff1炭酸ナトリウム緩衝液(pH9,3)の5
μ!及びダンシルクロリド(アセトン中5 mg/■1
)の10μlを添加し、37℃で1時間加温した。更に
、酢酸2μ!及び酢酸エチルの20μ!を加え、激しく
振丑したのち酢酸エチル層に回収されたダンシル化アミ
ノ酸を、ポリアミン シートを用いる二次元Fg層クロ
マトグラフィーにより同定した。(3) C-terminal amino acid analysis 20μ of purified polypeptide IfJ solution! 100mM vinegar Msu) IJ U1Jli& (p1]5.4) No
8 μl 4 μl of 1% SDS and 2 μl of carboxypeptidase Y (0,1B/1)! and 25°
The mixture was left standing at C for 5 to 15 minutes. 10μa of this reaction solution
5 of 0mMff1 sodium carbonate buffer (pH 9,3)
μ! and dansyl chloride (5 mg/■1 in acetone
) was added and heated at 37°C for 1 hour. Furthermore, acetic acid 2μ! and 20μ of ethyl acetate! was added and shaken vigorously, and the dansylated amino acid recovered in the ethyl acetate layer was identified by two-dimensional Fg layer chromatography using a polyamine sheet.
+41 1L−1活性
IL−1活性は、マイトーダンによるマウス胸腺細胞分
裂作用を促進させる生物活性、すなわちり73球活性化
因子(LAF )活性により評価した。+41 1L-1 activity IL-1 activity was evaluated based on the biological activity of mitodan to promote mouse thymocyte division, ie, ly73 cell activating factor (LAF) activity.
精製ポリペプチド溶液を5%牛脂児血1nを含む組織培
養用培地RPM+−1(+40にて希釈した。各希釈液
の50ulを96穴平底型プレート(Flow Lab
s、)に入れ、それぞれに25μg/■10度のフィト
ヘマグルチニン−p (Difco社)の50μ!を添
加し、更にC311/ lleマウスより採取した胸腺
細胞(IXIO7個/1)溶液の100μ!を加え、3
7°Cで5%炭酸ガス含有空気中、湿度90〜100%
で2日間培養した。The purified polypeptide solution was diluted in tissue culture medium RPM+-1 (+40) containing 1 n of 5% tallow blood. 50 ul of each dilution was added to a 96-well flat bottom plate (Flow Lab
) and 50 μg of phytohemagglutinin-p (Difco) at 25 μg/■10 degrees each. and 100 μl of a thymocyte (7 IXIO cells/1) solution collected from C311/lle mice. Add 3
At 7°C, in air containing 5% carbon dioxide, humidity 90-100%
The cells were cultured for 2 days.
次いで、引−チミジンの1μCiを加え、更に18時間
培養したのち、細胞内にとりこまれた刊−チミジン量を
計測することにより、 LAF活性を測定した。Next, 1 μCi of thymidine was added, and after further culturing for 18 hours, LAF activity was measured by measuring the amount of thymidine incorporated into the cells.
その結果を下表1に示す。The results are shown in Table 1 below.
表 1
表中において、前記式[A]で示されるポリペプチドの
N末端のSetが除去されたポリペプチドを、IL−1
(+57) 、同ポリペプチド[AコのN末端側の5e
r−Pro−Phe−3er−Phe−Leu−Ser
−Asn−Vatが除去されたポリペプチドを、IL−
1(149) 、同ポリペプチド[A]のN末端側の5
er−Pro−Phe−3er−Phe−Leu−Se
r−Asn−Val−Lys−Tyr−A++n−Ph
e−Mctが除去されたポリペプチドをIL−1(14
4)と略記した。Table 1 In the table, the polypeptide represented by the formula [A] from which the N-terminal Set was removed was compared to IL-1
(+57), the same polypeptide [5e on the N-terminal side of A
r-Pro-Phe-3er-Phe-Leu-Ser
-Asn-Vat removed polypeptide is IL-
1 (149), 5 on the N-terminal side of the same polypeptide [A]
er-Pro-Phe-3er-Phe-Leu-Se
r-Asn-Val-Lys-Tyr-A++n-Ph
The e-Mct-depleted polypeptide was isolated from IL-1 (14
4).
本発明のポリペプチドおよびその誘導体は、免疫不全治
療剤又は抗kR瘍剤として用いられうる。The polypeptide of the present invention and its derivatives can be used as an immunodeficiency therapeutic agent or an anti-kR tumor agent.
本発明のポリペプチドおよびその誘4体の製剤化にあた
っては、溶液及びi*結乾燥品のいずれでも良いが、長
期安定性の点から凍結乾操品が望ましい。そして試形剤
や安定化剤をrFx加するのが好ましい。安定化剤とし
ては、例えばアルブミン、グロブリフ、ゲラチン、プロ
タミノ、プロクミン塩。In formulating the polypeptide of the present invention and its derivative, either a solution or an i*lyophilized product may be used, but a freeze-dried product is preferable from the viewpoint of long-term stability. Then, it is preferable to add rFx to the sample or stabilizer. Stabilizers include, for example, albumin, globrif, gelatin, protamino, procumin salts.
グルコース、ガラクトース、キシロース、マンニット、
グルクロ/酸、トレハロース、デキストラン、ヒドロキ
シエチルデンプン、非イオン界面活性剤(ポリオキシエ
チレン脂肪酸エステル、ポリオキシエチレンアルキルエ
ーテル
レンアルキルフェニルエーテル、ポリオキシエチレンン
ルビタン脂肪酸エステル、ポリオキシエチレングリセリ
ン脂肪酸エステル、ポリオキシエチレ7 12 化ヒマ
シ油.ポリオキシエチレンヒマシ油。glucose, galactose, xylose, mannitol,
Glucuro/acid, trehalose, dextran, hydroxyethyl starch, nonionic surfactants (polyoxyethylene fatty acid ester, polyoxyethylene alkyl etherene alkylphenyl ether, polyoxyethylene rubitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, Oxyethylene 7 12 castor oil.Polyoxyethylene castor oil.
ポリオキシエチレンポリオキシプロピレンアルキルエー
テル、ポリオキシエチレンポリオキシプロピレンブロッ
クポリマー,ンルビタ/脂肪酸エステル、シ9糖脂肪酸
エステル、グリセリ/脂肪酸エステル)等が挙げられる
。これらの製剤は常法に従い製造することができる。Examples include polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene polyoxypropylene block polymer, Nrubita/fatty acid ester, ci-9 sugar fatty acid ester, glycerin/fatty acid ester), and the like. These preparations can be manufactured according to conventional methods.
本明細書では2柾の簡略化のために以下の略号を使用す
る。In this specification, the following abbreviations are used for the sake of brevity.
A アブ二/
C シトシン
G グアニン
T チミン
Ala アラニン
八rg アルギニン
Asn アスパラギン
Asl) アスパラギン酸
Cys システィア
Gln グルタミン
Glu グルタミン酸
Gly グリシ/
II i s ヒスヂクン
+1e インロイシ/
Leu ロイ7ン
Lysリジ/
Met メチオニン
Phe フェニルアラニン
Pro プロリ/
Setセリ/
T)+r スレオニン
Trp)リプトファ/
Tyr チロシン
Val バリン
DNA デオキシリボ核酸
c DNA 相補DNA
s s c DNA 単鎖c DNAdscDNA
二重Sr1cDNARNAリボ核酸
mRNA 伝令RNA
ポリ(^)mRNAポリアデニル酸含有伝令RNAdA
TP デオキシアデノシン三リン酸dcTP
デオキシシチジン三リン酸dGTP
デオキシグアノシン三リン酸dTTP デオキ
シチミジン三リン酸オリゴ(dC) オリゴデオキ
シシチジル酸オリゴ(dG) オリゴデオキシグア
ニル酸オリゴ(dT) オリゴデオキシチミジル酸
ポリ(A) ポリアデニル酸
ポリ(U) ポリウリジル酸
ポリ(dA) ポリデオキシアデニル酸ポリ(d
C) ポリデオキシシチジル酸ポリ(dG)
ポリデオキシグアニル酸ポリ(dT) ポリ
デオキシチミジル酸ATP アデノシフ三す
/i!lIEDTA エチレンジアミ/四酢酸
kb キロ塩基
kbp キロ塩基対
bp 塩基対
以下に実施例及び参考例を挙げて本発明を更に具体的に
説明するが、本発明はこれらの実施例に限定されるもの
ではない。A Abdi/ C Cytosine G Guanine T Thymine Ala Alanine 8rg Arginine Asn Asparagine Asl) Aspartic acid Cys Cystia Gln Glutamine Glu Glutamic acid Gly Glycine/ II is Hisadikun+1e Inleuci/ Leu Roy7in Lys Rizi/ Met Methionine Phe Phenyl Alanine Pro Proli / Set Seri / T) + r Threonine Trp) Liptopha / Tyr Tyrosine Val Valine DNA Deoxyribonucleic acid c DNA Complementary DNA s s c DNA Single strand c DNA Adsc DNA
Double Sr1 cDNA RNA Ribonucleic acid mRNA Messenger RNA Poly(^)mRNA Polyadenylic acid-containing messenger RNA AdA
TP deoxyadenosine triphosphate dcTP
Deoxycytidine triphosphate dGTP
Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate oligo(dC) Oligodeoxycytidylate oligo(dG) Oligodeoxyguanylate oligo(dT) Oligodeoxythymidylate poly(A) Polyadenylate poly(U) Polyuridylate poly( dA) polydeoxyadenylate poly(d
C) poly(dG) polydeoxycytidylate
Polydeoxyguanylate poly(dT) Polydeoxythymidylate ATP Adenosyph Sans/i! lIEDTA ethylenediami/tetraacetic acid kb kilobase kbp kilobase pair bp base pair The present invention will be explained in more detail by giving examples and reference examples below, but the present invention is not limited to these examples. .
また下記の実施例の理解を容易にするため第1、2図を
示した。Further, FIGS. 1 and 2 are shown to facilitate understanding of the following embodiments.
(以下余白)
実施例I
IL−1活性を示すポリペプチド[I L−1(+57
)]の″IA造
第2表のアミノ酸番号115〜271の157残基から
なるアミノ酸配列を有するポリペプチド生産用形質発現
プラスミド(pHLP384)を、参考例2に示しり方
法ニ従って*築した。ただし、参考例2で用いた合成オ
リゴヌクレオチド アダプター[I[11にかえて、次
式で示される合成アダプターを用いた。(Left below) Example I Polypeptide showing IL-1 activity [IL-1(+57
An expression plasmid (pHLP384) for producing a polypeptide having an amino acid sequence consisting of 157 residues of amino acid numbers 115 to 271 in Table 2 was constructed according to the method shown in Reference Example 2. However, instead of the synthetic oligonucleotide adapter [I[11] used in Reference Example 2, a synthetic adapter represented by the following formula was used.
得られた形質発現プラスミドを参考例2に示した方法に
従って、E、collll[1I01に導入し形質転換
体を得た。The obtained expression plasmid was introduced into E. collll [1I01] according to the method shown in Reference Example 2 to obtain a transformant.
この形質転換体をLI3ブロス中37°Cで一夜振盪培
養した。その菌体懸濁液のIOm+を11の改良M9培
地(組成:1.5%Na2HPOa・l2H20,0,
3%KH2POa。The transformants were cultured overnight at 37°C with shaking in LI3 broth. The IOm+ of the bacterial cell suspension was transferred to 11 modified M9 medium (composition: 1.5% Na2HPOa, 12H20,0,
3%KH2POa.
0.05%NaC1,0,1%NHaC1、2mg/
1ビタミンIl+。0.05% NaCl, 0.1% NHaCl, 2 mg/
1 vitamin Il+.
0.5%カザミノ酸+ 2 mM M g S Oa
+ 0 、1 mM C’aCl 210.5%ブドウ
糖)に接種し、37℃で1時間培養し、次いでインドー
ル−3−アクリル酸を終濃度20μg/11になるよう
に加え、更に24時間培養を継続した後、遠心分離によ
り菌体を集めた。菌体を100177)0.1%リゾチ
ーム及び30mM NaC+を含む50 mMTris
−11CI (PI(8,0) 緩衝液に再懸濁し、0
°Cで30分間静置した後、ドライアイス/エタノール
浴での凍結と37℃での融解を繰り返した後、21の1
0%ポリエチレンイミンを加え静置した。次いで、遠心
分離により菌体残渣を除き、T?′I澄な抽出液を得た
。0.5% Casamino Acids + 2mM MgS Oa
+ 0, 1 mM C'aCl (210.5% glucose) and cultured at 37°C for 1 hour, then indole-3-acrylic acid was added to a final concentration of 20 μg/11, and the culture was continued for an additional 24 hours. After continuing, the bacterial cells were collected by centrifugation. 100177) 50mM Tris containing 0.1% lysozyme and 30mM NaC+
-11CI (PI(8,0)) resuspended in buffer and 0
After standing for 30 minutes at °C, freezing in a dry ice/ethanol bath and thawing at 37 °C was repeated.
0% polyethyleneimine was added and left to stand. Next, bacterial cell residue was removed by centrifugation, and T? A clear extract was obtained.
この抽出液に等容量の飽和硫酸アンモニウム水溶液を加
え静置したのち、遠心分離にて沈殿画分を集めた。この
沈殿画分を約1001の20mM Tr i s−I
C1緩8T液(pH8,0)に溶解し、同11衝液に対
して透析したのち、予め同Ii液にて平衡化されたDE
AE−セファロースCL−Onカラムに負荷した。An equal volume of saturated ammonium sulfate aqueous solution was added to this extract, and the mixture was allowed to stand, and then centrifuged to collect a precipitate fraction. This precipitate fraction was mixed with approximately 1001 20mM Tri s-I
DE was dissolved in C1 mild 8T solution (pH 8,0) and dialyzed against the same 11 buffer solution, and then equilibrated in advance with the same solution Ii.
Loaded onto an AE-Sepharose CL-On column.
同緩衝液にて該カラムを充分洗浄したのち、NaCl濃
度0〜0.5Mの濃度勾配にて溶出した。IL−1活性
を存する溶出画分を集め、限外濾過にてQ縮したのち、
セファクリルS−200によるゲル濾過に付し、IL−
1活性を存する両分を集めた。After thoroughly washing the column with the same buffer, it was eluted with a NaCl concentration gradient of 0 to 0.5M. Elution fractions containing IL-1 activity were collected, and after Q-condensation by ultrafiltration,
After gel filtration with Sephacryl S-200, IL-
Both fractions containing 1 activity were collected.
この溶出液のIL−1活性測定では、1×104倍希釈
液で45 、394cp■の刹−チミジンの取り込みを
認め、IL−1活性を確認した。When measuring the IL-1 activity of this eluate, incorporation of 45,394 cp of thymidine was observed in a 1x104 diluted solution, confirming IL-1 activity.
実施例2
1L−1活性を示すポリペプチド[I L−DI49)
]の製造
第2表のアミノ酸番号123〜271の149残基から
なるアミノ酸配列を存するポリペプチド生産用形質発現
プラスミドを第1図に示すように構築した。Example 2 Polypeptide showing 1L-1 activity [IL-DI49]
An expression plasmid for producing a polypeptide having an amino acid sequence consisting of 149 residues of amino acid numbers 123 to 271 in Table 2 was constructed as shown in FIG.
参考例2で得た組み換え体プラスミドpHLP383か
ら制限酵素EcoRIとl1indIffにて、第2表
に示した塩基配列の第398番目から下流側の約422
bPのDNA断片を得た。このDNA断片に、常法によ
り合成した次式
で示されるオリゴヌクレオチド アダプターをT4DN
Aリガーゼを用いて結合させた。From the recombinant plasmid pHLP383 obtained in Reference Example 2, about 422 downstream from the 398th nucleotide sequence shown in Table 2 was extracted using the restriction enzymes EcoRI and l1indIff.
A DNA fragment of bP was obtained. To this DNA fragment, an oligonucleotide adapter represented by the following formula synthesized by a conventional method was added to T4DN.
The ligation was performed using A ligase.
別途に、プラスミドpHLP383から制限酵素C1a
Iと旧ndI[1にて切断し、トリプトファ/ プロモ
ーター頭載の一部及びアンピシリン耐性遺伝子とテトラ
サイクリン耐性逍伝子を含む大きなりNA fr片を得
た。Separately, from plasmid pHLP383, restriction enzyme C1a
By cutting with I and old ndI [1, a large NAfr fragment containing part of the tryptopha promoter head, an ampicillin resistance gene, and a tetracycline resistance gene was obtained.
このDNA断片に、先にN製したDNA断片をT4DN
Aリガーゼを用いて結合させることにより、149残基
のアミノ酸よりなるポリペプチド生産用形質発現プラス
ミド(pHLP385)を構築した。To this DNA fragment, add the previously prepared DNA fragment to T4DN.
By ligation using A ligase, a polypeptide-producing expression plasmid (pHLP385) consisting of 149 amino acid residues was constructed.
このプラスミドpHLr’385を用い、実施例1に示
したと同様の方法で形質転換体(E、colillll
lQI/pHLP385)を得、この形質転換体を培養
し、更にその菌体抽出液から目的とするポリペプチドを
+11した。Using this plasmid pHLr'385, a transformant (E, colillll.
lQI/pHLP385) was obtained, this transformant was cultured, and the target polypeptide was obtained from the cell extract.
このポリペプチド液のIL−1活性測定では、lX10
4倍希釈液で27 、700cpmの引−チミジ/の取
り込みを認め、IL−1活性を確認した。In the IL-1 activity measurement of this polypeptide solution, lX10
In the 4-fold diluted solution, uptake of 27,700 cpm of thymidine was observed, confirming IL-1 activity.
実施例3
の製造
第2表のアミノ酸番号128〜271の144残基から
なるアミノ酸配列を存するポリペプチド生産用形質発現
プラスミドを、実施例2に示した方法に従ってvl築し
た。ただし、実施例2で用いた合成オリゴヌクレオチド
アダプター[I]にかえて、次式
で示される合成アダプターを用いることにより、!44
残基のアミノ酸よりなるポリペプチド生産用形質発現プ
ラスミド(pHLPa80)を構築した。Production of Example 3 An expression plasmid for producing a polypeptide having an amino acid sequence consisting of 144 residues of amino acid numbers 128 to 271 in Table 2 was constructed according to the method shown in Example 2. However, by using a synthetic adapter represented by the following formula in place of the synthetic oligonucleotide adapter [I] used in Example 2,! 44
An expression plasmid (pHLPa80) for producing a polypeptide consisting of amino acid residues was constructed.
このプラスミドpHLP38Bを用い、実施例1に示し
たと同様の方法で形質転換体を得、この形質転換体を培
養し、更にその菌体抽出液から目的とするポリペプチド
を単離した。Using this plasmid pHLP38B, a transformant was obtained in the same manner as shown in Example 1, this transformant was cultured, and the target polypeptide was isolated from the bacterial cell extract.
このポリペプチドのIL−1活性測定では、1×103
倍希釈液で15,092cp−の’l(−チミジンの取
り込みを認め、IL−1活性を確認した。In the IL-1 activity measurement of this polypeptide, 1×103
Incorporation of 15,092 cp-'l(-thymidine) was observed in the diluted solution, confirming IL-1 activity.
実施例4
143残基からなるポリペプチドの製造第2表のアミノ
酸番号129〜271の143残基からなるアミノ酸配
列を有するポリペプチド生産角形MQ現プラスミド(p
utp387)を、実施例2に示した方法に従って構築
した。ただし、実施例2で用いた合成オリゴヌクレオチ
ドアダプター[I]にかえて、次式
%式%
で示される合成アダプターを用いた。Example 4 Production of polypeptide consisting of 143 residues Polypeptide production having an amino acid sequence consisting of 143 residues with amino acid numbers 129 to 271 in Table 2. Square MQ current plasmid (p
utp387) was constructed according to the method described in Example 2. However, instead of the synthetic oligonucleotide adapter [I] used in Example 2, a synthetic adapter represented by the following formula % was used.
このプラスミドpHLP387を用い、実施例1に示し
たと同様の方法で形質転換体を得、この形質転換体を培
養し、更にそのΔ体抽出液から目的とするポリペプチド
を単離した。Using this plasmid pHLP387, a transformant was obtained in the same manner as shown in Example 1, this transformant was cultured, and the target polypeptide was isolated from the Δ body extract.
このポリペプチド液のIL−1活性測定では、10倍希
釈液で12,700cp■の上−チミジンの取り込みを
認めた。In IL-1 activity measurement of this polypeptide solution, uptake of 12,700 cp of supra-thymidine was observed in a 10-fold diluted solution.
(以下余白)
参考例1
ヒトIL−1前駆体ポリペプチドをコードするc DN
Aのクローニング及び塩基配列の決定HL−E30細胞
をペトリディフシュ(直径80鳳)にI X 107個
/ l0m1/ dishの条件で播いた。培養液には
IO%牛脂児血1n含仔のRPMI−1840培地を用
い、分化誘導剤としてホルボール−12−ミリステート
−13−アセテートとビタミンAMをいずれも最終濃度
として500ng/ mlになるように添加した。37
℃で5%炭酸ガス含存空気中、湿度90〜100%で2
日間培養したのち、培養液と浮遊細胞を吸引除去した。(Left below) Reference Example 1 c DN encoding human IL-1 precursor polypeptide
Cloning of A and Determination of Base Sequence HL-E30 cells were seeded in a petri dish (diameter: 80 mm) under the condition of I x 107 cells/10 ml/dish. The culture medium used was RPMI-1840 medium containing 1N of IO% tallow baby blood, and phorbol-12-myristate-13-acetate and vitamin AM were used as differentiation inducers at a final concentration of 500 ng/ml. Added. 37
℃ in air containing 5% carbon dioxide, humidity 90-100%
After culturing for one day, the culture solution and floating cells were removed by suction.
分化した細胞が伸行したディツシュにIO%牛脂児血清
含仔Rr’M11640培地に誘導剤としてエンドトキ
シン(大腸菌由来のりボポリサブカライド)をIOμg
/閣1濃度に、蛋白合成阻害剤としてシクロへキシミド
を1Mg/■1濃度に添加した培地の101を加え、更
に5時間培養した。培養終了後、培養液を吸引除去し、
ディツシュ上に残った分化細胞を0.5%ラウロイルサ
ルコシン酸ナトリウム、5mMクエン酸ナトリウム及び
0.1M 2−メルカプトエタノールを含む6Mグアニ
ジルチオシアネート液で溶解し、ホモジナイズした。こ
のホモジネートをO,IM EDTA含を5.7M塩化
セシウム水溶液上に重層し、超遠心分離機(RPS27
−20一ター1日立工機)を用い26,500rpmで
20時間遠心し全RNA画分をベレフトとして得た。こ
れを0.35M NaCl、 20mM Tris及
び20mM EDTAを含む7M尿素液の少量に溶解し
、エタノール沈殿として回収した。Add IO μg of endotoxin (Noribopolysubcalide derived from Escherichia coli) as an inducer to the dish in which the differentiated cells have grown into Rr'M11640 medium containing IO% beef tallow serum.
A medium 101 containing cycloheximide as a protein synthesis inhibitor added to a concentration of 1 Mg/1 was added to the culture medium at a concentration of 1 Mg/1 and cultured for an additional 5 hours. After culturing, remove the culture solution by suction,
The differentiated cells remaining on the dish were dissolved in a 6M guanidyl thiocyanate solution containing 0.5% sodium lauroyl sarcosinate, 5mM sodium citrate and 0.1M 2-mercaptoethanol, and homogenized. This homogenate was layered on a 5.7M cesium chloride aqueous solution containing O, IM EDTA, and placed in an ultracentrifuge (RPS27
The whole RNA fraction was centrifuged at 26,500 rpm for 20 hours using a Hitachi Koki (Hitachi Koki) to obtain a total RNA fraction. This was dissolved in a small amount of 7M urea solution containing 0.35M NaCl, 20mM Tris and 20mM EDTA, and collected as an ethanol precipitate.
この全RNA画分を1 mM EDTAを含むIOmM
Tris−T−ICI緩衝液(f)87.4) (以
下TE液という)21に溶解し、65℃で5分間加熱し
た。これにNaC+溶液を0.5Mとなるように加えた
後、あらかじめ0.5M NaC+を含むTE液で平衡
化したオリゴ(dT)セルロース男ラムに付し、吸わし
たポリ(A) mRNAをTE液で溶出した。This total RNA fraction was dissolved in IOmM containing 1mM EDTA.
It was dissolved in Tris-T-ICI buffer (f) 87.4) (hereinafter referred to as TE solution) 21 and heated at 65°C for 5 minutes. After adding NaC+ solution to a concentration of 0.5M, it was applied to an oligo(dT) cellulose membrane equilibrated in advance with a TE solution containing 0.5M NaC+, and the absorbed poly(A) mRNA was added to the TE solution. It was eluted.
ここで得られたポリ(A) mRNAを以下の実験に用
いた。The poly(A) mRNA obtained here was used in the following experiment.
■ c DNAの合成
(1)項で得られたポリ(A) mRNAを鋳型として
グプラーとホフマンの方法[Gene25,203(1
983)]に準じてc DNAを合成した。該ポリ(A
) 、 mRNA(6μg)を6μlの蒸留水に溶解さ
せ、これに0.6μl0100mM水酸化メチル水銀水
溶液を添加し室温で10分間放置した1次いで、20単
位のRNA分解酵素阻害剤(RNas+n■、 Pro
megaLotec社製品)を含む500mM 2−メ
ルカプトエタノール液の1,7μlを添加した。室温で
5分間放置したのち、更に10mM MgCl2.1.
25mM dGTP、 1.25mM dAT+’。■ Synthesis of c DNA Using the poly(A) mRNA obtained in section (1) as a template, the method of Gupler and Hoffmann [Gene 25, 203 (1)
cDNA was synthesized according to [983)]. The poly(A
), mRNA (6 μg) was dissolved in 6 μl of distilled water, 0.6 μl of 100 mM methylmercury hydroxide aqueous solution was added thereto, and the mixture was left at room temperature for 10 minutes.
1.7 μl of a 500 mM 2-mercaptoethanol solution containing 2-mercaptoethanol (product of megaLotec) was added. After standing at room temperature for 5 minutes, 10mM MgCl2.1.
25mM dGTP, 1.25mM dAT+'.
1.25mM dTTP、 0.5mM dCTP、O
,+7μM a−”P−dCTP (比活性、 750
Ci/ m5ole) 、 4 ttgオリゴ(dT)
12〜+a、 120単位トリ骨髄、性白血病ウィ
ルス由来逆転写酵素を含む32ulの50mM Tri
s−HCI(pH8,3)緩衝液を添加し、42℃で6
0分間反応させた後、 EDTAを加えて反応を停止さ
せた。フェノール/クロロホルム混液(1:1)で抽出
し、その水腹に酢酸アンモニウムを終濃度2.5Mにな
ルヨうに加え、エタノールにより反応生成物(sscD
NA−mRNA複合体)を沈殿させた。コ(QsscD
N八−mRNA複合体を下記組成の反応緩衝液100μ
!に溶解した。1.25mM dTTP, 0.5mM dCTP, O
, +7 μM a-”P-dCTP (specific activity, 750
Ci/m5ole), 4ttg oligo(dT)
12~+a, 120 units avian bone marrow, 32ul of 50mM Tri containing leukemia virus-derived reverse transcriptase
Add s-HCI (pH 8,3) buffer and incubate at 42°C for 6 hours.
After reacting for 0 minutes, EDTA was added to stop the reaction. Extract with phenol/chloroform mixture (1:1), add ammonium acetate to a final concentration of 2.5M, and extract the reaction product (sscD) with ethanol.
NA-mRNA complex) was precipitated. Ko (QsscD
The N8-mRNA complex was mixed with 100μ of a reaction buffer having the following composition.
! dissolved in.
反応緩衝液組成:
5 mM MgCI2. l0mM (NHa) 2s
O4,100mMKC1,0,15mMβ−ニコチンア
ミド アデニン ジヌクレオチド、40μM dGTI
’、 40μMdATP、 40μMdTTP、 40
μM dCTP、及び5μgウシ血1nアルブミン、
1.25単位大腸菌リボヌクレアーゼI夏、21位大腸
ΔD N Aポリメラーゼ■を含む20mM Tris
−HCI (PH7,5) B衝/IIi′。Reaction buffer composition: 5mM MgCI2. 10mM (NHa) 2s
O4, 100mM KC1, 0,15mM β-nicotinamide adenine dinucleotide, 40μM dGTI
', 40μMdATP, 40μMdTTP, 40
μM dCTP, and 5 μg bovine blood 1n albumin,
20mM Tris containing 1.25 units E. coli ribonuclease I summer, 21st colon ΔDNA polymerase■
-HCI (PH7,5) B/IIi'.
該溶解液を12゛Cで60分間反応させ、これに2,5
単位の大腸菌DNAリガーゼを添加し、更に22°Cで
60分間反応させた。 EDTAを加えて反応を停止さ
せた後、上記と同様に7エノール/クロロホルム混液で
抽出し、エタノールにより反応生成物(dscDNA
)を沈殿させ、回収した。The solution was reacted at 12°C for 60 minutes, and then 2,5
One unit of E. coli DNA ligase was added, and the mixture was further reacted at 22°C for 60 minutes. After stopping the reaction by adding EDTA, extraction was performed with 7 enol/chloroform mixture in the same manner as above, and the reaction product (dscDNA) was extracted with ethanol.
) was precipitated and collected.
(3) dCテール付付加 DNAの調製2項で得ら
れたdscDNAを下記組成の反応緩衝液100μlに
溶解させ、37℃で30分間反応させ、d s c D
NAにdCテールを付加させた。(3) Addition of dC tail DNA preparation The dscDNA obtained in Section 2 was dissolved in 100 μl of reaction buffer with the following composition, reacted at 37°C for 30 minutes, and dscD
A dC tail was added to NA.
反応緩衝液組成:
2 mM COCl2.0.2mMジチオスL/イトー
ル。Reaction buffer composition: 2mM COCl2.0.2mM dithios L/itol.
0.1mMα−″P−dCTP (比活性I C!/
m5ole)及びIO単単位ターミナルデオキシヌクレ
オリジルトランスフェラーゼ含有する100mMカコジ
ル酸ナトリウム(pH7,2)。0.1mM α-″P-dCTP (specific activity I C!/
m5ole) and 100 mM sodium cacodylate (pH 7.2) containing IO single unit terminal deoxynucleolysyl transferase.
反応はEDTA水溶液を添加して停止させ、フェノール
/クロロホルム混液フ抽出し、dCテール付加dscD
NAをエタノールにより沈殿させ回収した。これを1
mM EDTA及びl00mM NaC+を含む10m
MTris−)菖CI (PH7,4)Inn液液て、
2μg/mlの濃度に溶解させた。The reaction was stopped by adding an aqueous EDTA solution, extracted with a phenol/chloroform mixture, and the dC-tailed dscD
NA was precipitated and collected with ethanol. This is 1
10m containing mM EDTA and 100mM NaC+
MTris-) Iris CI (PH7,4) Inn liquid,
It was dissolved at a concentration of 2 μg/ml.
(4) 組み換え体プラスミドの作製
dGデテール加PnR322(Bethesda Rc
s、Labs。(4) Preparation of recombinant plasmid dG detail addition PnR322 (Bethesda Rc
s, Labs.
Inc、製)と(3)項で得られたdCテール付付加
s cDNAを1.5mlのl mM EDTA及び1
00mM NaC1を含む10mM Tris−HCI
(pI−17,4) 13衝液中、それぞれ1.5μ
g及び0.09μg含むように溶解混合させた後、65
℃で10分間、57°Cで2時間、さらに45℃で2時
間加温しアニーリングを行い、組み換え体プラスミド溶
液を調製した。Inc.) and the dC tailed addition obtained in section (3).
s cDNA in 1.5 ml of lmM EDTA and 1
10mM Tris-HCI with 00mM NaCl
(pI-17,4) 1.5μ each in 13 buffers
After dissolving and mixing to contain 65 g and 0.09 μg,
Annealing was performed by heating at 57°C for 10 minutes, 2 hours at 57°C, and 2 hours at 45°C to prepare a recombinant plasmid solution.
■ 形質転換体のi!!択
(4)項で得られた組み換え体プラスミド溶液を用い、
E、col+χ1776株を形質転換させた。即ち、E
、coliχ1776株を、ジアミノピメリ7m100
μg/ml及びチミジ/4θμg/mlを補ったし一グ
ロス(組成=II当りトリプト710g、酵母エキス5
g 、NaC15g、 ブドウ糖1 g 、 P
)17.2) 20m1中、37℃で吸光度(GOOn
m)が0.5となるまで培養し、菌体を遠心分離し、5
0mM CaC+2含有10mMTris−HCI緩衝
液(pH7,3) 10ulにて洗浄した。■ Transformant i! ! Using the recombinant plasmid solution obtained in option (4),
E, col+χ1776 strain was transformed. That is, E
, coli χ1776 strain, Diaminopimeli 7m100
Supplemented with μg/ml and timidium/4θ μg/ml (composition = 710 g of tryptope per II, 5 yeast extract
g, NaC 15g, glucose 1g, P
)17.2) Absorbance (GOOn) at 37℃ in 20ml
Culture until m) becomes 0.5, centrifuge the bacterial cells, and
It was washed with 10ul of 10mM Tris-HCI buffer (pH 7,3) containing 0mM CaC+2.
集めた菌体を同じ緩衝液21に懸濁させ、0℃で5分間
0置した。この懸濁液0.21に上記組み換え体プラス
ミド溶液0.11を添加混合し、0℃で15分間り置し
、更に42℃で2分間保持した後、上記の培養で用いた
のと同一組成のし−ブロス0.51を加えて1時間振W
培養を行った。この培養液の一部を取り、上記組成に加
えてテトラサイタリン(15μg/l)が添加されたし
一グロス寒天平板に広げ37°Cで約12時間培養し、
テトラサイクリン耐性菌を進択してcD、NAライブラ
リーを作製した。The collected bacterial cells were suspended in the same buffer solution 21 and incubated at 0°C for 5 minutes. Add and mix 0.11 of the above recombinant plasmid solution to 0.21 of this suspension, leave at 0°C for 15 minutes, and then hold at 42°C for 2 minutes. Add Noshi Broth 0.51 and shake for 1 hour.
Culture was performed. A portion of this culture solution was taken, spread on a gloss agar plate to which tetracytalin (15 μg/l) was added in addition to the above composition, and cultured at 37°C for about 12 hours.
A cD and NA library was created by selecting tetracycline-resistant bacteria.
(6) クローニング
(5)項で得られたcDNAう(ブラリーから、参考例
3で得た組み換え体プラスミドpRL+5からウサギI
L−1をコードするクローン化c DNAの断片をプロ
ーブとして用いたコロニー ハイプリダイゼーシクン試
験及びハイブリダイゼーシ117トランスレーシヨン試
験CManiatis、T、、et al、。(6) Cloning The cDNA obtained in section (5) (from library, rabbit I from the recombinant plasmid pRL+5 obtained in Reference Example 3)
Colony hybridization test and hybridization translation test using a fragment of cloned cDNA encoding L-1 as a probe CManiatis, T., et al.
“Mo1ecularCloniB”329(+980
) Co1d Springllarbor Lab、
]によりヒトIL−1ポリペプチドをコードするc D
NAを含むプラスミドを存する形質転換体を選び出した
。“Mo1ecularCloniB” 329 (+980
) Cold Springllarbor Lab,
] c D encoding human IL-1 polypeptide
Transformants containing a plasmid containing NA were selected.
この組み換え体プラスミドをp II L 4と名づけ
た。This recombinant plasmid was named pIIL4.
(2) クローン化c DNAの塩基配列の決定クロー
ン化c DNAの塩基配列はM13フ1−ジを用いるジ
テオキン法にて決定した。MI3mp18及びM 13
ap19(Pharmacia p−を旧acheli
ca +slJ:製)をクローニングベクターとし、旧
3ンークエノンングキフト(Amersham Int
ernaNonal plc社製)を用い、“MI3ク
ロー二/グ及びシークエ/シ7グハ7ドブコク″ (A
mersham Internationalplct
t製)に従って実施した。(2) Determination of the base sequence of the cloned c DNA The base sequence of the cloned c DNA was determined by the ditheokine method using M13 fluoride. MI3mp18 and M13
ap19 (Pharmacia p- old acheli
ca + slJ:) was used as a cloning vector, and the former 3rd generation company (Amersham Int.
ernaNonal plc) and "MI3 Croni/G and Sequence/Shi7guha7 Dobukoku" (A
mersham internationalplct
It was carried out according to the method (manufactured by T.T.).
その塩基配列及びその塩基配列から推測されるアミノ酸
は下記第2表に示すとおりであり、ヒトIL−1前駆体
ポリペプチドをコードしている。The base sequence and the amino acids deduced from the base sequence are shown in Table 2 below, and encode the human IL-1 precursor polypeptide.
第1〜3番の塩基が開始コドンATGであり、第814
〜816番の塩基は終止コドンTAGである。The 1st to 3rd bases are the start codon ATG, and the 814th base is the start codon ATG.
The base at positions 816 to 816 is the stop codon TAG.
(以下余白)
第2表
CAAGCTGCCAGCCAGAGAGGGAGTC
ATTTCATTGGCGTTTGAGTCAGCAA
AGAAGTCAAG(1バυ。(Left below) Table 2 CAAGCTGCCAGCCAGAGGGAGTC
ATTTCATTGGCGTTTGAGTCAGCAA
AGAAGTCAAG (1 bar υ.
[註]※×※は終止コド/を示す。[Note] *×* indicates the stop code /.
()中の数字はアミノl!1番号を示す。The numbers in parentheses are amino l! 1 number is shown.
(以下余白)
参考例2
ヒ) IL−1(+59残基)ポリペプチド生産用形質
転換体の作製
第2表のアミノ酸番号113〜27+のアミノ酸配列を
有する+59残基からなるヒトIL−1ポリペプチド生
産用形質発現プラスミド(pH[、P2S5)を第2図
に示すように構笑した。(Leaving space below) Reference Example 2 H) Preparation of a transformant for producing IL-1 (+59 residues) polypeptide A human IL-1 polypeptide consisting of +59 residues having an amino acid sequence of amino acid numbers 113 to 27+ in Table 2. A peptide-producing expression plasmid (pH[, P2S5) was constructed as shown in FIG.
すなわち、参考例1− (61で得た組み換え体プラス
ミドp II L 4から制限酵素Pst Iによりク
ローン化cDNA部分を切り出し、更に制限酵素Alu
Iを作用させ、第2表に示した塩基配列の第351番目
から下流側の約533bPのDNA断片を得、更にこれ
に制限酵素BstN Iを作用させ、第2表の塩基配列
の第351番から第808番までに和光するDNA断片
を単離した。このDNA断片に、常法により合成した次
式
%式%[]
及び次式
5′−AGGCGTGATGA
[IVコ3′−CCGCAC
TACTTCGA
で示されるオリゴヌクレオチド アダプターを順次T4
DNAリガーゼを用いて結合させることにより、上記の
+59残基のアミノ酸からなるヒトIL−1ポリペプチ
ドをコードする塩基配列の5′末端に開始コドンATG
を付加し、更に終止コドンT G A T G Aを付
加したDNA断片を得た。このDNA断片をIIIL−
1断片という。That is, from the recombinant plasmid p II L 4 obtained in Reference Example 1-(61), the cloned cDNA portion was excised with the restriction enzyme Pst I, and further with the restriction enzyme Alu
A DNA fragment of approximately 533 bP downstream from the 351st position of the base sequence shown in Table 2 was obtained by reacting with BstN I, and this was further reacted with the restriction enzyme BstN I to obtain a DNA fragment of about 533 bP downstream from the 351st position of the base sequence shown in Table 2. A DNA fragment corresponding to Wako from No. 808 was isolated. To this DNA fragment, the following formula % formula % [] and the following formula 5'-AGGCGTGATGA were synthesized by a conventional method.
[IV co3'-CCGCAC
TACTTCGA oligonucleotide adapters are sequentially added to T4.
By linking using DNA ligase, a start codon ATG is attached to the 5' end of the base sequence encoding the human IL-1 polypeptide consisting of the above +59 amino acid residues.
A DNA fragment was obtained in which a termination codon TGA TGA was added. This DNA fragment was
It is called one fragment.
一方、プラスミドpCT−1[1kehara、M、e
t al、。On the other hand, plasmid pCT-1 [1kehara, M, e
tal,.
Proc、Nat、^cad、sci、UsA 81,
595(i(1984)]に制限酵酵素1paIとυ止
■を作用させ里プロモーター領域の一部を含む約380
bpのDNA断片を切り出し、このDNA断片に、常法
により合成した次式5式%(1
で示されるオリゴヌクレオチド アダプターをT4DN
Aリガーゼを用いて結合させた。Proc, Nat, ^cad, sci, UsA 81,
595 (i (1984))] was treated with restriction enzyme 1paI and υstop■ to create approximately 380 cells containing part of the Sato promoter region.
A bp DNA fragment was excised, and an oligonucleotide adapter expressed by the following formula 5 (1) synthesized by a conventional method was added to the DNA fragment with T4DN.
The ligation was performed using A ligase.
とcD DNA IIi 片ニ、先ニFA製したIII
L−1断片をT4DNΔリガーゼを用いて結合させ、D
NA断片を得た。このDNA断片をプロモーター+11
L−1断片という。and cD DNA IIi.
The L-1 fragment was ligated using T4DNA ligase and D
An NA fragment was obtained. This DNA fragment is attached to the promoter +11
It is called L-1 fragment.
別aに、プラスミドpBR322に制限酵素Ava I
とPvu Uを作用させ、大きなりNA断片(約3.7
kbp)を0.7%アガロースゲル電気泳動により分離
した。Separately, restriction enzyme Ava I was added to plasmid pBR322.
A large NA fragment (approximately 3.7
kbp) were separated by 0.7% agarose gel electrophoresis.
このDNA断片の両端をDNAポリメラーゼ■(クレノ
ー フラグメント)及びdGTP、 dATP、 dC
TP。Both ends of this DNA fragment were treated with DNA polymerase (Klenow fragment) and dGTP, dATP, dC.
T.P.
dTTPを用い平滑末端とし、その両端をT4 DNA
リガーゼを用いて結合させた。このプラスミドベクター
をP[1R5Oという。さらに、このpIIRsoベク
ターに制限酵素AatIIと1lindlllを作用さ
せ、大きなりNA断片(約3.8kbp)を単離精製し
た。Make blunt ends using dTTP, and connect both ends with T4 DNA.
The conjugation was performed using ligase. This plasmid vector is called P[1R5O. Furthermore, this pIIRso vector was treated with restriction enzymes AatII and 1lindll to isolate and purify a large NA fragment (approximately 3.8 kbp).
このDNA断片に先に調整したプロモーター+11L−
1断片をTa DNAリガーゼを用いて結合させること
により、159残基からなるヒトIL−1ポリペプチド
生産用形質発現プラスミドを構来した。この形質発現プ
ラスミドをPIILP383と名づけた。The promoter +11L- that was previously adjusted to this DNA fragment
By ligating the two fragments using Ta DNA ligase, an expression plasmid for producing human IL-1 polypeptide consisting of 159 residues was constructed. This expression plasmid was named PIILP383.
この形質発現プラスミド(pHLP383)を下記の方
法によりE、coli 118101に導入し形質転換
体を得た。This expression plasmid (pHLP383) was introduced into E. coli 118101 by the method described below to obtain a transformant.
すなわち、E、coli 11BIOIをLブロス(組
成:11当たり、トリプトンIOg、酵母エキス5g、
NaC15g、 ブドウII! 1 g ; pH7
,2)の51に接種し、37°Cで一夜培養した。その
菌体懸濁液の1mlを100菖lのLブロスに接種し、
濁度(吸光度650n鳳)がO,Gになるまで37°C
で培養した。氷水中で30分間静置後、菌体を遠心分離
により集め、これを501の50mM CaC+ 2に
懸濁し、0°Cで60分間静置した。次いで、遠心性m
により菌体を集め、20%グリセリンを含む50mM
CaC+2のl0w1に再懸濁した。That is, E. coli 11 BIOI was added to L broth (composition: per 11, tryptone IOg, yeast extract 5g,
15g of NaC, Grape II! 1 g; pH7
, 2) and cultured overnight at 37°C. 1 ml of the bacterial cell suspension was inoculated into 100 liters of L broth,
37°C until turbidity (absorbance 650n) reaches O, G
It was cultured in After standing in ice water for 30 minutes, the bacterial cells were collected by centrifugation, suspended in 50mM CaC+ 2 of 501, and left standing at 0°C for 60 minutes. Then, efferent m
Collect the bacterial cells and add 50mM containing 20% glycerin.
Resuspended in l0w1 of CaC+2.
この懸濁液に上記の形質発現ベクターpHLP383を
添加し、これを氷水中で20分間、42°Cで1分間。The above expression vector pHLP383 was added to this suspension, and the mixture was incubated in ice water for 20 minutes and at 42°C for 1 minute.
室温で10分間インキュベートした後、LBブロス(組
成;11当たり、トリプト710g 、酵母エキス5g
及びNaCl IOg、 pl−17,5)を加え、
37°Cで60分間振盪した。その菌体懸濁液の一部を
25μg/−lアンピシリンを含むLI3寒天平板に播
き、37°Cで一夜培養した後、ア/ピンリ/耐性クロ
ーンを選択して形質転換体を得た。After incubating for 10 minutes at room temperature, LB broth (composition; 710 g of trypto, 5 g of yeast extract per 11
and NaCl IOg, pl-17,5),
Shake at 37°C for 60 minutes. A portion of the bacterial cell suspension was plated on an LI3 agar plate containing 25 μg/-l ampicillin, cultured overnight at 37°C, and A/pinli/resistant clones were selected to obtain transformants.
(以下余白)
参考例3
ウサギIL−1cDNAのyA製
(1) ウサギIL−1mRNAの調製ウサギにプロピ
オニバクテリウム アクネス死菌体を1別当たり+00
■gの投与量で静脈内に注入し、8日後に屠殺した。直
ちに開胸気管切開し、気管内に挿入したチューブを介し
てリン酸rA衝化生理食塩液を用い肺洗浄を操り返し、
肺胞マクロファージを採取した。この肺胞マクロファー
ジをlθ%牛脂児血清含をのRPMI−1640培地に
懸濁させてベトリディッシュ(直径80曹)に1枚当た
りlXl0’個となるように播き、37℃で5%炭酸ガ
ス含仔空気中、湿度90〜100%で前培養した。1時
間の前培養の後、エンドトキシン(大腸菌由来のりボポ
リサブカライド) 、 TI’A(ホルボール−12−
ミリステート−13−アセテート)及びシクロへキシミ
ドをそれぞれ最終0度がIOμg/ ml、 500n
g/ ml及びIμg/mlとなるように添加混和し、
更に培養を継続した。(Leaving space below) Reference Example 3 Rabbit IL-1 cDNA manufactured by yA (1) Preparation of rabbit IL-1 mRNA Injecting killed Propionibacterium acnes cells into rabbits at +000% per portion.
The mice were injected intravenously at a dose of 1 g and sacrificed 8 days later. Immediately, a thoracic tracheotomy was performed, and lung lavage was repeated using phosphate rA buffered saline through a tube inserted into the trachea.
Alveolar macrophages were collected. These alveolar macrophages were suspended in RPMI-1640 medium containing lθ% tallow serum, plated on Vetri dishes (diameter 80 Ca) at lXl0' per plate, and heated at 37°C under 5% carbon dioxide gas. Preculture was performed in air containing larvae at a humidity of 90 to 100%. After 1 hour of preincubation, endotoxin (Noribopolysubcharide derived from Escherichia coli), TI'A (phorbol-12-
myristate-13-acetate) and cycloheximide, each with a final concentration of IO μg/ml, 500n
Add and mix to give g/ml and Iμg/ml,
Further culture was continued.
4時間後に培養液を吸引除去し、ディツシュ上に残った
マクロファージから参考例1−(1)項に示した方法に
従ってポリ(^)mRNAを得た。After 4 hours, the culture solution was removed by suction, and poly(^) mRNA was obtained from the macrophages remaining on the dish according to the method shown in Reference Example 1-(1).
ここで得たポリ(A) mRNAをアガロースゲル電気
泳動(ゲル濃度1%、6M尿素存在下、 pH4)に
付し、2.6〜3.7kbの分子サイズに相当する泳動
位置からポリ(A) mRNAを回収した。The poly(A) mRNA obtained here was subjected to agarose gel electrophoresis (gel concentration 1%, in the presence of 6M urea, pH 4), and poly(A) ) mRNA was collected.
■ c DNAライブラリーの作製
(1)項で得られたポリ(A) mRNAを鋳型として
、参考例1−(2)から6)に示した方法に準じて、
c DNAライブラリーを作製した。■ Preparation of c DNA library Using the poly(A) mRNA obtained in section (1) as a template, according to the method shown in Reference Example 1-(2) to 6),
A cDNA library was constructed.
(3) クローニング
上J己のc DNAライブラリーについて、ウサギIL
−1をコードするcDNAを含むプラスミドを持つ形質
転換体をスクリーニングするため32 p標識c DN
Aプローブを用いるコロニー・ハイブリダイゼーンgノ
試験をハナへンらの方法[Gene。(3) Cloning of own cDNA library using rabbit IL
To screen for transformants carrying a plasmid containing cDNA encoding 32p-labeled cDNA
The colony hybridization assay using the A probe was performed according to the method of Hanahen et al. [Gene.
10.03(+980)]に従って行った。エアトドキ
シン。10.03 (+980)]. Airtodoxin.
TPA及びシクロへキシミドと共に培養[上3i! (
1)項参照コした肺胞マクロファージ及びこれらのiJ
Ω操作を省略した肺胞マクロファージからそれぞれ上記
(1)項の方法で得たポリ(A) mRNAを鋳型とし
て、参考例1−(2)項の方法で合成し、ffff p
で標識したc DNAをそれぞれ誘導プラス及びHaマ
イナスプローブとした。この試験により誘導プラスのプ
ローブと結合し、誘導マイナスのプローブとはハイブリ
ダイズしない塩基配列を含む組み換え体プラスミドを有
する形質転換体を選別した。Culture with TPA and cycloheximide [Top 3i! (
See section 1) Alveolar macrophages and their iJ
Poly(A) mRNA obtained from alveolar macrophages by the method described in section (1) above was used as a template and synthesized by the method described in Reference Example 1-(2), and ffff p
cDNA labeled with Ha was used as induction plus and Ha minus probes, respectively. Through this test, transformants were selected that had a recombinant plasmid containing a base sequence that bound to the induction-plus probe but did not hybridize to the induction-minus probe.
次イテ、これらの遭択されたクローンについてハイブリ
ダイゼーシヨ/・トランスレージg7試験を上記(鳳)
項で得たポリ(^)mRNAを用い、ウサギIL−1m
RNAと強くハイブリダイズするクローンを選び出した
。Next, perform the hybridization/transrage G7 test on these selected clones (Otori).
Using the poly(^) mRNA obtained in Section 1, rabbit IL-1m
Clones that strongly hybridized with RNA were selected.
このクローンの有する組み換え体プラスミドをpRL+
5と名づけた。The recombinant plasmid of this clone is pRL+
I named it 5.
第1図は形質発現プラスミドPHLP385の構築工程
を示す。尚、図中の式[11は実施例2で示したオリゴ
ヌクレオチド アダプターを音味する。
第2図は形質発現ベクターpHLP383の構築工程を
示す。尚、図中の式[I[11、[TV] 、 [V
]は参考例2で示したそれぞれのオリゴヌクレオチド
アダプターを特徴する
特許出願人 大日本製薬株式会社
代 理 人 坪 井 存 四 部第1−
JLulFIG. 1 shows the steps for constructing the expression plasmid PHLP385. In addition, the formula [11 in the figure represents the oligonucleotide adapter shown in Example 2. FIG. 2 shows the construction steps of the expression vector pHLP383. In addition, the formula [I[11, [TV], [V
] is each oligonucleotide shown in Reference Example 2
Patent applicant featuring an adapter: Dainippon Pharmaceutical Co., Ltd. Representative: Tsuboi Tsuboi Part 4, Part 1 - JLul
Claims (25)
その誘導体。(1) A polypeptide or a derivative thereof that exhibits interleukin-1 activity.
、或いはそのN末端から17番目までのアミノ酸が順次
1個づつ除去されたそれぞれのアミノ酸配列を有する特
許請求の範囲第(1)項記載のポリペプチド又はその誘
導体。 【アミノ酸配列があります】[A](2) Claim (1) which has an amino acid sequence represented by the following formula [A], or has each amino acid sequence in which the amino acids from the N-terminus to the 17th amino acid are sequentially removed one by one. The described polypeptide or derivative thereof. [There is an amino acid sequence] [A]
の範囲第(1)項記載のポリペプチド。 【アミノ酸配列があります】(3) The polypeptide according to claim (1), which has an amino acid sequence represented by the following formula. [There is an amino acid sequence]
の範囲第(1)項記載のポリペプチド。 【アミノ酸配列があります】(4) The polypeptide according to claim (1), which has an amino acid sequence represented by the following formula. [There is an amino acid sequence]
はそのN末端のArgが除去されたアミノ酸配列を有す
る特許請求の範囲第(1)項記載のポリペプチド。 【アミノ酸配列があります】(5) The polypeptide according to claim (1), which has an amino acid sequence represented by the following formula, or has an amino acid sequence in which Arg at the N-terminus has been removed. [There is an amino acid sequence]
かのポリペプチドのN末端にMetが結合してなる特許
請求の範囲第(1)項記載のポリペプチド。(6) The polypeptide according to claim (1), wherein Met is bound to the N-terminus of any of the polypeptides according to claims (3) to (5).
ードする塩基配列を有し又は含むDNA。(7) DNA having or containing a base sequence encoding a polypeptide exhibiting interleukin-1 activity.
塩基配列、或いはそのN末端から17番目までのアミノ
酸が順次1個づつ除去されたそれぞれのアミノ酸配列に
対応する塩基配列を有し又は含む特許請求の範囲第(7
)項記載のDNA。 【アミノ酸配列があります】(8) It has a base sequence corresponding to the amino acid sequence represented by the following formula [A], or a base sequence corresponding to each amino acid sequence in which the amino acids from the N-terminus to the 17th amino acid are sequentially removed one by one, or Claim No. 7 (7)
) DNA described in section. [There is an amino acid sequence]
列を有し又は含む特許請求の範囲第(7)項記載のDN
A。 【アミノ酸配列があります】(9) DN according to claim (7), which has or contains a base sequence corresponding to the amino acid sequence represented by the following formula:
A. [There is an amino acid sequence]
配列を有し又は含む特許請求の範囲第(7)項記載のD
NA。 【アミノ酸配列があります】(10) D according to claim (7), which has or contains a base sequence corresponding to the amino acid sequence represented by the following formula:
N.A. [There is an amino acid sequence]
末端のArgが除去されたアミノ酸配列に対応する塩基
配列を有し又は含む特許請求の範囲第(7)項記載のD
NA。 【アミノ酸配列があります】(11) Amino acid sequence represented by the following formula, or its N
D according to claim (7), which has or contains a base sequence corresponding to the amino acid sequence from which Arg at the terminal has been removed.
N.A. [There is an amino acid sequence]
請求の範囲第(7)項記載のDNA。 【塩基配列があります】(12) The DNA according to claim (7), which has or contains a base sequence represented by the following formula. [There is a base sequence]
求の範囲第(7)項記載のDNA。 【塩基配列があります】(13) The DNA according to claim (7), which has or contains a base sequence represented by the following formula. [There is a base sequence]
請求の範囲第(7)項記載のDNA。 【塩基配列があります】(14) The DNA according to claim (7), which has or contains a base sequence represented by the following formula. [There is a base sequence]
端のコドン(AGG)が欠失した塩基配列を有し又は含
む特許請求の範囲第(7)項記載のDNA。 【塩基配列があります】(15) The DNA according to claim (7), which has or contains a base sequence represented by the following formula, or a base sequence in which a codon (AGG) at the 5' end thereof is deleted. [There is a base sequence]
ずれかの塩基配列の5′末端に開始コドン及び/又は3
′末端に終止コドンが結合した塩基配列を有し又は含む
特許請求の範囲第(7)項記載のDNA。(16) A start codon and/or 3
The DNA according to claim (7), which has or contains a base sequence with a stop codon attached to its end.
ずれかのDNAを組み込んだベクター。(17) A vector incorporating the DNA according to any one of claims (7) to (16).
範囲第(17)項記載のベクター。(18) The vector according to claim (17), wherein the vector is a gene expression vector.
る特許請求の範囲第(17)項又は第(18)項記載の
ベクター。(19) The vector according to claim (17) or (18), which is a vector that can propagate in E. coli.
範囲第(17)〜(19)項記載のいずれかのベクター
。(20) The vector according to any one of claims (17) to (19), wherein the vector is an E. coli plasmid.
pHLP386又はpHLP387である特許請求の範
囲第(20)項記載のベクター。(21) The vector is pHLP384, pHLP385,
The vector according to claim (20), which is pHLP386 or pHLP387.
いずれかのベクターで形質転換された宿主。(22) A host transformed with the vector according to any one of claims (17) to (21).
範囲第(22)項記載の宿主。(23) The host according to claim (22), wherein the transformed host is a microorganism.
範囲第(22)項又は第(23)項記載の宿主。(24) The host according to claim (22) or (23), wherein the transformed host is E. coli.
ずれかのDNAを形質発現ベクターに組み込み、これに
より形質転換された宿主を培養し、産生されるインター
ロイキン1活性を示すポリペプチドを採取することを特
徴とする該ポリペプチドの製造法。(25) The DNA according to any one of claims (7) to (16) is incorporated into a gene expression vector, a host transformed with the vector is cultured, and a polypeptide exhibiting interleukin-1 activity is produced. A method for producing the polypeptide, which comprises collecting the peptide.
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60171493A JPS6232887A (en) | 1985-08-02 | 1985-08-02 | Polypeptide exhibiting interleukin 1 activity and dna coding same |
EP85309453A EP0188920B1 (en) | 1984-12-25 | 1985-12-23 | Interleukin 1 and its derivative |
DE85309453T DE3587605T2 (en) | 1984-12-25 | 1985-12-23 | Interleukin-1 and its derivative. |
KR1019850009774A KR950000712B1 (en) | 1984-12-25 | 1985-12-24 | Mehtod for preparation of interieukin-1 |
US07/954,418 US5376639A (en) | 1984-12-25 | 1992-09-30 | Treatment of sarcoma with interleukin 1α polypeptides |
US08/252,826 US5494663A (en) | 1984-12-25 | 1994-06-02 | Treatment of microbial infection with interleukin 1 polypeptides |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60171493A JPS6232887A (en) | 1985-08-02 | 1985-08-02 | Polypeptide exhibiting interleukin 1 activity and dna coding same |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS6232887A true JPS6232887A (en) | 1987-02-12 |
Family
ID=15924118
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP60171493A Pending JPS6232887A (en) | 1984-12-25 | 1985-08-02 | Polypeptide exhibiting interleukin 1 activity and dna coding same |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6232887A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989003671A1 (en) * | 1987-10-29 | 1989-05-05 | Dainippon Pharmaceutical Co., Ltd. | Sustained-release preparation |
JPH02167298A (en) * | 1988-07-29 | 1990-06-27 | Otsuka Pharmaceut Co Ltd | Interleukin-1alpha derivative |
-
1985
- 1985-08-02 JP JP60171493A patent/JPS6232887A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989003671A1 (en) * | 1987-10-29 | 1989-05-05 | Dainippon Pharmaceutical Co., Ltd. | Sustained-release preparation |
JPH02167298A (en) * | 1988-07-29 | 1990-06-27 | Otsuka Pharmaceut Co Ltd | Interleukin-1alpha derivative |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5464939A (en) | Highly purified protein, production and use thereof | |
JP2515308B2 (en) | Human immune interferon | |
AU648670B2 (en) | A-C-B Proinsulin, method of manufacturing and using same, and intermediates in insulin production | |
EP0155549B1 (en) | Dna encoding human tumor necrosis factor and human tumor necrosis factor polypeptide | |
IE63991B1 (en) | Novel fusion proteins and their purification | |
JPH0789934B2 (en) | Manufacture and expression of structural genes | |
HU209152B (en) | Method for producing human interferon protein of high purity and pharmaceutical preparative containing it | |
JP2525023B2 (en) | Purification of recombinant interleukin-1 | |
JP2002509691A (en) | Production and use of recombinant protein multimers with altered biological activity | |
JP2653061B2 (en) | Novel polypeptide and method for producing the same | |
SK43097A3 (en) | Method for purifying keratinocyte growth factors | |
PT93956A (en) | PROTEIN PURIFICATION METHOD USING CTAP-III FUSES | |
CN107556376B (en) | A kind of Interferon Alpha-2b mutant and its preparation method and application | |
JPS6232887A (en) | Polypeptide exhibiting interleukin 1 activity and dna coding same | |
CN109776653B (en) | Human serum albumin adhesion peptide and application thereof | |
EP0205038A1 (en) | Polypeptide, process for preparing it, microorganism and pharmaceutical use | |
JP2698976B2 (en) | Novel polypeptide and DNA encoding the same | |
JP2515975B2 (en) | Polypeptide having antitumor effect | |
CN113845599B (en) | Recombinant porcine interferon fusion protein and application thereof | |
JPH0560480B2 (en) | ||
JP2579747B2 (en) | Novel DNA encoding human interleukin 1 | |
JPS6323898A (en) | Novel polypeptide, dna coding said polypeptide and production thereof | |
JP2577304B2 (en) | Polypeptide having human interleukin 1 activity | |
JP2584206B2 (en) | Human cancer necrosis factor | |
JPS6261585A (en) | Polypeptide exhibiting interleukin 1 activity and dna coding said polypeptide |