JPH01257492A - Method for enhancing production of paraprotein - Google Patents
Method for enhancing production of paraproteinInfo
- Publication number
- JPH01257492A JPH01257492A JP62048935A JP4893587A JPH01257492A JP H01257492 A JPH01257492 A JP H01257492A JP 62048935 A JP62048935 A JP 62048935A JP 4893587 A JP4893587 A JP 4893587A JP H01257492 A JPH01257492 A JP H01257492A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- dna
- medium
- psv
- paraprotein
- 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
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- 108010064255 Paraproteins Proteins 0.000 title abstract 5
- 210000004102 animal cell Anatomy 0.000 claims abstract description 24
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims abstract description 14
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
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- 102100023702 C-C motif chemokine 13 Human genes 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
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- MKXKFYHWDHIYRV-UHFFFAOYSA-N flutamide Chemical compound CC(C)C(=O)NC1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 MKXKFYHWDHIYRV-UHFFFAOYSA-N 0.000 description 1
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- PZLXYMQOCNYUIO-UHFFFAOYSA-N lithium;hydrochloride Chemical compound [Li].Cl PZLXYMQOCNYUIO-UHFFFAOYSA-N 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
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- 229910052700 potassium Inorganic materials 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
イ、利用分野
本発明は、組換え動物細胞の培養方法に関する詳細には
、組換え動物細胞の培養時に異種蛋白質の産生を増強さ
せる方法に関する。DETAILED DESCRIPTION OF THE INVENTION A. Field of Application The present invention relates in detail to a method for culturing recombinant animal cells, and more particularly to a method for enhancing the production of a heterologous protein during culturing of recombinant animal cells.
口、従来技術
遺伝子組換え技術によって目的の異種蛋白質を生産する
宿主としては、当初大腸菌を中心に研究が進められ、さ
らに酵母、枯草菌等の微生物が、その培養の簡便さ及び
宿主−ベクター系の研究の進展等から繁用されていた。Previously, as a host for producing a target heterologous protein using genetic recombination technology, research initially focused on Escherichia coli, and further microorganisms such as yeast and Bacillus subtilis were used due to their ease of cultivation and the host-vector system. It was frequently used due to the progress of research on.
(Goeddel、 D、 v、IItakura+
K、、 et al、 Proc、 Nath、 Ac
ad、 Sci。(Goeddel, D, v, IItakura+
K., et al., Proc., Nath, Ac.
ad, Sci.
u、s、A、、 76、106 (1979)およびN
agata、 S、。u, s, A, 76, 106 (1979) and N
agata, S.
Ta1ra、 S、 H,and Weisssann
、仁、 Nature、 284+1316 (191
30)参照〕。Ta1ra, S., H., and Weisssann.
, Jin, Nature, 284+1316 (191
See 30)].
しかし、近年高分子糖蛋白質を天然型に近い形で、しか
も可溶な型で遺伝子組換え技術による物質生産をおこな
うために宿主として、動物細胞を用いる発現系に焦点が
移っている。However, in recent years, the focus has shifted to expression systems that use animal cells as hosts in order to produce high-molecular glycoproteins in a form close to their natural form and in a soluble form using genetic recombination technology.
既知の動物細胞としては、 C1(O−K。Known animal cells include: C1 (OK.
(チャイニーズハムスター卵巣細胞;
ATCCCCL61)
BHK (新生仔ハムスター腎細胞;ATCCCCL
IO)
CO5−7
(CLI Origin、 5V−40細胞ニー A
TCCCRL1651)
Vero (アフリカミドリザル腎細胞:^TCC
CCL−81)
等がある。(Chinese hamster ovary cells; ATCCCCL61) BHK (Newborn hamster kidney cells; ATCCCCL
IO) CO5-7 (CLI Origin, 5V-40 cell knee A
TCCCRL1651) Vero (African green monkey kidney cells: ^TCC
CCL-81) etc.
ハ0本発明が解決しようとする問題点
そこで、次の関心事は異種蛋白質の産生をいかに増強さ
せるかということである。Problems to be Solved by the Present Invention Therefore, the next concern is how to enhance the production of heterologous proteins.
今回、本発明者らは、組換え動物細胞の壇養時に、培地
に酪酸またはその塩を添加することにより異種蛋白質の
産生を増強できることを見出し、本発明を完成した。The present inventors have now completed the present invention by discovering that production of a heterologous protein can be enhanced by adding butyric acid or its salt to the culture medium when culturing recombinant animal cells.
二0問題点を解決するための手段
本発明は、異種蛋白質をコードするDNA配列を組み込
んだベクターを用いて形質転換した動物細胞を培養して
異種蛋白質を産生ずる際に、培地に酪酸またはその塩を
添加することにより異種蛋白質の産生を増強させる方法
に関する。Means for Solving the 20 Problems The present invention provides for the production of a heterologous protein by adding butyric acid or its The present invention relates to a method for enhancing the production of a heterologous protein by adding salt.
(1)組換え動物細胞の調製
組換え動物細胞は、遺伝子組換え技術により目的とする
異種蛋白質をコードするDNA配列を組み込んだベクタ
ーを宿主である動物細胞に導入して当該細胞を形質転換
させることにより得られる。(1) Preparation of recombinant animal cells Recombinant animal cells are obtained by introducing a vector into which a DNA sequence encoding the desired heterologous protein is introduced into host animal cells using gene recombination technology to transform the cells. It can be obtained by
異種蛋白質は、高分子蛋白質または高分子糖蛋白質であ
れば特に限定されない。The heterologous protein is not particularly limited as long as it is a high molecular weight protein or high molecular weight glycoprotein.
このような異種蛋白質としては、ウロキナーゼ(UK)
、プロウロキナーゼ(ProUK)、組織プラスミノー
ゲンアクチベーター(t−PA)、B型肝炎表面抗原(
HBsAg)、プレS 8N域を含むII B s A
g(PreS−HBsAg)、インターフェロン−r
(IFN−T)、コロニー形成刺激因子(C3F)など
が挙げられる。Such heterologous proteins include urokinase (UK)
, prourokinase (ProUK), tissue plasminogen activator (t-PA), hepatitis B surface antigen (
HBsAg), II BsA including pre-S 8N area
g (PreS-HBsAg), interferon-r
(IFN-T), colony formation stimulating factor (C3F), and the like.
また、これらの活性断片(ドメイン)または誘導体であ
ってもよい。In addition, active fragments (domains) or derivatives thereof may be used.
これらの生理活性物質をコードするDNA配列およびそ
の調製方法は、
ProUK (特開昭6O−180591)Protl
K ドメイン(特願昭6l−156936)t−PA
(特開昭59−183693. 同59−42321
)tlBs八8 へ(同 559−36698)Pr
ey−HBsA (同56−63995)IFN−r
(同6l−108397)C3F (同6l−
199787)などで開示されている。The DNA sequences encoding these physiologically active substances and their preparation methods are described in ProUK (Japanese Unexamined Patent Publication No. 60-180591) Protl.
K domain (patent application Sho 6l-156936) t-PA
(Unexamined Japanese Patent Publication No. 59-183693. No. 59-42321
) to tlBs 88 (same 559-36698) Pr
ey-HBsA (56-63995) IFN-r
(6l-108397) C3F (6l-108397)
199787) and others.
異種蛋白質発現用ベクターは、発現系遺伝子として例え
ばSV −40に由来するエンハンサ−1初期領域プロ
モーター、ポリアブニレ−シランシグナル及び有用な異
種蛋白質をコードする遺伝子から構成される。このよう
な発現系ベクターとしては、p S V −Gt(特開
昭6l−177987)、psV−Gt(特願昭6l−
79086)などが例示される。又他の動物ウィルスや
動物細胞に由来するプロモーターなども使用できる。A vector for expressing a heterologous protein is composed of, for example, an enhancer-1 early region promoter derived from SV-40, a polyabnylene silane signal, and a gene encoding a useful heterologous protein as expression system genes. Examples of such expression system vectors include pSV-Gt (Japanese Unexamined Patent Publication No. 61-177987), psV-Gt (Japanese Patent Application No. 61-177987),
79086), etc. Furthermore, promoters derived from other animal viruses and animal cells can also be used.
動物細胞としては、CHO−Kl 、BHK、COS
−7、Vero、ヒト腎由来株細胞(特願昭60−29
0325参照)、チャン肝細胞(ATCC磁CCL13
)、チンパンジー肝細胞(^TCC1tccL6311
)などが挙げられる。特にC)10−に、が好ましい。Animal cells include CHO-Kl, BHK, COS
-7, Vero, human kidney-derived cell line (patent application 1986-29)
0325), Chang hepatocytes (ATCC magnetic CCL13)
), chimpanzee hepatocytes (^TCC1tccL6311
), etc. Particularly preferred is C) 10-.
かくして、異種蛋白質発現用ベクターをこの宿主細胞の
核内に挿入することによって形質転換がおこなわれる。Transformation is thus performed by inserting the heterologous protein expression vector into the nucleus of this host cell.
組換えDNAの細胞への導入方法は、リン酸カルシウム
沈澱法(ストレインら、Bioches+、J、+21
8+475−482.1984)などが挙げられる。こ
の方法は、組換えDNAを含む、リン酸緩衝液に塩化カ
ルシウム溶液を滴下することによって、D N A I
Jン酸カルシウムの微小沈澱を形成させ、その微小沈澱
を細胞に吸着させ、細胞内にとりこませる方法である。The method for introducing recombinant DNA into cells is the calcium phosphate precipitation method (Strain et al., Bioches+, J, +21
8+475-482.1984). This method involves adding a calcium chloride solution dropwise to a phosphate buffer containing recombinant DNA.
This is a method of forming microprecipitates of calcium J phosphate, adsorbing the microprecipitates to cells, and incorporating them into the cells.
形質転換は、シャーレに約1 = 10 X 10’c
el Is10ml/100mdishの割合で宿主細
胞を調製し、これに異種蛋白質発現用ベクターをDNA
量として2〜4pgを加える。DNAを宿主細胞にとり
こませた後、培養を行い形質転換細胞の選別をおこなう
。Transformation is carried out in a Petri dish with approximately 1 = 10 x 10'c
Prepare host cells at a ratio of el Is10ml/100mdish, and add the heterologous protein expression vector to the DNA.
Add 2 to 4 pg as an amount. After the DNA is introduced into host cells, they are cultured and transformed cells are selected.
外来遺伝子が発現している細胞を選択するためには、同
時に薬剤耐性遺伝子を導入して、薬剤耐性株を選択する
方法が用いられる(スブラマニら、Aral、Bioc
hem、、 135.1−15+ 1983)
e例えば、動物細胞内でG−418に耐性を示すネオマ
イシン耐性遺伝子:a+sinoglycostde
3’ −phospho−transferase I
Iを用いる。薬剤耐性遺伝子は先述の発現用ベクター中
に直接挿入するかまたはコトランスフェクション((:
o−transfection)により導入される。ネ
オマイシン耐性遺伝子発現プラスミドとしては、5V4
Qのプロモーター、スブライシングジャンクシッン、ポ
リA付加シグナルを有するベクターにネオマイシン耐性
遺伝子を接続したp 5V−G、−Ne oあるいはp
SV Gt Neoなどを用いることができる。In order to select cells in which a foreign gene is expressed, a method is used that simultaneously introduces a drug-resistant gene and selects a drug-resistant strain (Subramani et al., Aral, Bioc.
hem,, 135.1-15+ 1983)
For example, a neomycin resistance gene that shows resistance to G-418 in animal cells: a+sinolycostde
3'-phospho-transferase I
Use I. The drug resistance gene can be directly inserted into the expression vector described above or co-transfected ((:
o-transfection). As a neomycin resistance gene expression plasmid, 5V4
p5V-G, -Ne o or p, in which a neomycin resistance gene is connected to a vector having a Q promoter, a splicing junkin, and a polyA addition signal.
SV Gt Neo etc. can be used.
(2)前培養 組換え動物細胞を予め、増殖・継代培養しておく 。(2) Preculture Recombinant animal cells are propagated and subcultured in advance.
培地としては、WaBouth培地、ダルベツコ変法イ
ーグル培地、F−12培地、RITC−80−1培地(
J、 Ce11. Phygiol、、111,155
−162(1982))などが挙げられ、好適にはF−
12培地、RITC−80−7などが用いられる。As a medium, WaBouth medium, Dulbecco's modified Eagle medium, F-12 medium, RITC-80-1 medium (
J, Ce11. Phygiol, 111,155
-162 (1982)), preferably F-
12 medium, RITC-80-7, etc. are used.
また、10〜20v/v%程度のウシ胎児血清を添加す
ることがより好ましい。Moreover, it is more preferable to add about 10 to 20% v/v of fetal bovine serum.
さらに、プロリン10〜100μg/請1を添加しても
よい。Furthermore, 10 to 100 μg/kg of proline may be added.
組換え動物細胞を1〜20万cel ls/mlとなる
ように培地中に植え込み、2〜3日間培養を続けた後、
必要ならば培地を交換して継代培養し、細胞数を10〜
200万celLs/mlに調製する。Recombinant animal cells were implanted in a medium at a concentration of 1 to 200,000 cells/ml, and after continued culture for 2 to 3 days,
If necessary, change the medium and subculture to increase the number of cells to 10~
Adjust to 2 million cells/ml.
(3) 本培養
基本培地としては、Way+*outh培地、ダルベツ
コ変法イーグル培地、F−12培地、RITC−80−
7培地などが挙げられ、好適にはF−12培地、RIT
C−80−7培地などが用いられる。(3) Main culture basic medium includes Way+*outh medium, Dulbecco's modified Eagle medium, F-12 medium, RITC-80-
7 medium, etc., preferably F-12 medium, RIT medium, etc.
C-80-7 medium etc. are used.
本工程は、好適には無血清壇地中で行う。This step is preferably carried out in a serum-free platform.
これに、本発明の特徴である酪酸またはその塩(例えば
、ナトリウム塩、カリウム塩など)が添加される。Butyric acid or a salt thereof (eg, sodium salt, potassium salt, etc.), which is a feature of the present invention, is added to this.
添加量としては、0.1〜105M程度、好適には1〜
5mMが例示される。The amount added is about 0.1 to 105M, preferably 1 to 105M.
An example is 5mM.
培地にはその他うクトアルブミン氷解吻、トランスフェ
リン、各種アミノ酸、各種脂肪酸、インシュリン等のホ
ルモンなどを添加してもよい。Other substances such as uctoalbumin, transferrin, various amino acids, various fatty acids, and hormones such as insulin may be added to the medium.
この培地中には空気(Air 95%、CO!5%)を
適宜導入(流速:10〜500+wl/分)することが
好ましく、温度は20〜37℃が好ましい、培養液は2
〜3日程度ごとに、交換する。It is preferable to appropriately introduce air (Air 95%, CO! 5%) into this medium (flow rate: 10-500+wl/min), the temperature is preferably 20-37°C, and the culture solution is
Replace it every ~3 days.
かくして、培地中または、細胞質中に異種蛋白質が産生
される。Thus, a heterologous protein is produced in the medium or in the cytoplasm.
培地からの産生物の回収は、例えば、当該培地を遠心分
離、減圧濃縮、塩析分画、ゲル濾過、濃縮、イオン交換
クロマトグラフィー、アフィニティークロマトグラフィ
ー等を二適宜組み合わせることによって行なわれる。The product is recovered from the medium, for example, by centrifugation of the medium, concentration under reduced pressure, salting out fractionation, gel filtration, concentration, ion exchange chromatography, affinity chromatography, etc. in an appropriate combination.
細胞質からの回収は、機械的破壊または界面活性剤など
で細胞を破壊した後に得られた細胞抽出液を上記の培養
上清と同様に処理する。For recovery from the cytoplasm, the cell extract obtained after disrupting cells mechanically or with a surfactant or the like is treated in the same manner as the culture supernatant described above.
ホ、効果
本発明の方法により、組換え動物細胞における異種蛋白
質の産生が2〜3倍程度増強される。E. Effect The method of the present invention enhances the production of heterologous proteins in recombinant animal cells by about 2 to 3 times.
従って、本発明の方法は遺伝子工学分野における動物細
胞を用いての異種蛋白質の大量生産に極めて有用な方法
と考えられる。Therefore, the method of the present invention is considered to be an extremely useful method for mass production of heterologous proteins using animal cells in the field of genetic engineering.
へ、実施例・実験例
本発明をより詳細に説明するために、実施例・実験例を
挙げるが、本発明はこれらにより限定されるものではな
い。Examples and Experimental Examples In order to explain the present invention in more detail, Examples and Experimental Examples are given, but the present invention is not limited by these.
実施例1
参考例1により得られたProUに産生形質転換株(C
−68−53,C−68−61)およびCHOOK+細
胞、各々20万cellsを10v/v%牛脂児血清含
有F−12培地4m+1(60■dish)中に植え込
み、37°Cで3日間培養した。培地を除去して維持用
培地(F−12またはRITC−80−7)4甑lで培
養皿を洗った後に、維持用培地または5@M酪酸ナトリ
ウム含有維持用培地を加え、37゛Cで24時間培養し
た。培養上清のUK活性をフィブリン平板法により測定
した。Example 1 ProU production transformant strain (C
-68-53, C-68-61) and CHOOK+ cells, 200,000 cells each, were implanted in 4 m+1 (60 dishes) of F-12 medium containing 10 v/v% tallow serum and cultured at 37°C for 3 days. . After removing the medium and washing the culture dish with 4 liters of maintenance medium (F-12 or RITC-80-7), add maintenance medium or maintenance medium containing 5@M sodium butyrate and incubate at 37°C. Cultured for 24 hours. UK activity of the culture supernatant was measured by fibrin plate method.
実施例2
参考例2により得られたPrey−HBsAg産生形質
転換株を用いて実施例1に準じて培養を行った(培地は
F−12)。Example 2 The Prey-HBsAg producing transformant obtained in Reference Example 2 was cultured according to Example 1 (medium was F-12).
PreS−HBsAgの定量はELISA法で行った。Quantification of PreS-HBsAg was performed by ELISA method.
第 2 表
実施例3
参考例3により得られたTPA産生形質転換株を用いて
実施例1に準じて培養を行った。培地はF−12または
35μg/mlプロリン含有ダルベツコ変法イーグル培
地(DME)を用いた。t−PAの定量はフィブリン平
板法を用いた。標準試料としてUKを用い、活性はUK
の国際単位に換算した第 3 表
実験例1(濃度)
参考例1により得られたProUK産生形質転換株(C
−68−53株) 20万cellsを10v/v%牛
脂児血清含有F−12培地4 ml (60mdish
)に植え込み、37℃で3日間培養したところ、細胞数
が2..8X10’に達した。培地を取り除き、培養皿
を4+ilのF−12で洗った後、酪酸ナトリウム(最
終濃度O〜20sM)含有F−12を4m+1ずつ分注
した。24時間培養後、培養上清のUK活性をフィブリ
ン平板法により測定した。またUK活性測定用の試料を
採取後、トリプシン処理を行い、細胞を培養皿か・らは
がし、コールタ−カウンターを用いて細胞数を測定した
。Table 2 Example 3 The TPA-producing transformant obtained in Reference Example 3 was cultured according to Example 1. The medium used was F-12 or Dulbecco's modified Eagle's medium (DME) containing 35 μg/ml proline. The fibrin plate method was used to quantify t-PA. UK was used as a standard sample, and the activity was determined by UK.
Table 3 Experimental example 1 (concentration) converted to international units of ProUK producing transformant strain (C
-68-53 strain) 200,000 cells in 4 ml of F-12 medium containing 10 v/v% beef tallow serum (60 m dish
) and cultured at 37°C for 3 days, the number of cells was 2. .. It reached 8X10'. After removing the medium and washing the culture dish with 4+il of F-12, 4ml+1 of F-12 containing sodium butyrate (final concentration O~20 sM) was dispensed. After culturing for 24 hours, the UK activity of the culture supernatant was measured by fibrin plate method. After collecting a sample for UK activity measurement, it was treated with trypsin, the cells were peeled off from the culture dish, and the number of cells was measured using a Coulter counter.
第 4 表
実験例2(種類)
参考例1により得られたProUK産生形質転換株(C
−68−53株)20万cellsをIQv/v%牛脂
児血・涜含有F−12培地4 ml (60mdish
)に植え込み、37℃で3日間培養したところ、細胞数
が2.8 X 10’に達した。培地を取り除き、4■
lのF−12で洗った後、各種添加剤含有F−12培地
を4+ilずつ分注した。37℃で24時間培養後、培
養上清のUK活性をフィブリン平板法により測定した。Table 4 Experimental Example 2 (Type) ProUK producing transformant strain (C
-68-53 strain) 200,000 cells in 4 ml (60 mdish) of F-12 medium containing IQv/v% beef tallow blood and
) and cultured at 37°C for 3 days, the number of cells reached 2.8 x 10'. Remove the medium and incubate 4
After washing with 1 ml of F-12, 4+ il of F-12 medium containing various additives were dispensed. After culturing at 37°C for 24 hours, the UK activity of the culture supernatant was measured by fibrin plate method.
第 5 表
実験例3(培養時間)
参考例1により得られたPro[IK産生形質転換株(
C−68−53株)20万cellsを10v/v%牛
脂児血清含有F−12培地4m1(60閣dish)に
植え込み、37°Cで3日間培養したところ、細胞数が
2.8 X 10’に達した。培地を取り除き、41の
F−12で洗った後、5IIM酪酸ナトリウム含有F−
12培地を4+11ずつ分注した。37℃で6〜24時
間培養後、培養上清のUK活性をフィブリン平板法で測
定した。F−12培地のみで培養した場合の活性を対照
として、各時間における酪酸ナトリウムのProUK産
生の増強効果を百分率で示した。Table 5 Experimental Example 3 (Culture time) The Pro[IK-producing transformed strain obtained in Reference Example 1 (
C-68-53 strain) 200,000 cells were implanted into 4 ml of F-12 medium (60-kaku dish) containing 10 v/v% beef tallow serum and cultured at 37°C for 3 days, resulting in a cell count of 2.8 x 10. ' has been reached. After removing the medium and washing with 41 F-12, F-1 containing 5IIM sodium butyrate was added.
12 media were dispensed into 4+11 portions. After culturing at 37°C for 6 to 24 hours, the UK activity of the culture supernatant was measured by fibrin plate method. The enhancing effect of sodium butyrate on ProUK production at each time point is shown as a percentage, with the activity when cultured only in F-12 medium being used as a control.
第 6 表
実験例4
酪酸ナトリウムによるProUK産生の活性化のメカニ
ズムについて調べるために、UK産生株の培養上清と細
胞抽出液を調製してUK活性と蛋白含量を測定した。Table 6 Experimental Example 4 To investigate the mechanism of activation of ProUK production by sodium butyrate, culture supernatants and cell extracts of UK producing strains were prepared and UK activity and protein content were measured.
参考例1により得られたProUK産生形質転換株(C
−68−53) 66万cellsを10v/v%牛脂
児血清含存F−12培地10ml(110m1(100
)に植え込み、3日間培養後、101のF−12で洗っ
た。5n+M酪酸ナトリウム含有F−12培地またはF
−12培地を各々10m1ずつ分注した。37°Cで2
4時間培養後に細胞抽出液と培養上清のUK活性と蛋白
含量を各々、フィブリン平板法とローリ−法で測定した
。ProUK producing transformant strain (C
-68-53) 660,000 cells were grown in 10ml (110ml) of F-12 medium containing 10v/v% tallow serum.
), and after culturing for 3 days, it was washed with 101 F-12. F-12 medium containing 5n+M sodium butyrate or F
-12 medium was dispensed in 10 ml portions. 2 at 37°C
After 4 hours of culture, the UK activity and protein content of the cell extract and culture supernatant were measured by the fibrin plate method and the Lowry method, respectively.
細胞抽出液の調製方法:単層状となっている細胞(細胞
数は約500万)をトリプシン処理で集め、10m1の
等張リン酸緩衝液で2度洗った後に、0.5mlの等張
リン酸緩衝液に細胞を懸濁し、0.5mlの0.5v八
%トリトンX−100溶液を加えて撹拌した後、37°
Cで5分間装置することにより得られる。Preparation method for cell extract: Collect monolayered cells (approximately 5 million cells) by trypsin treatment, wash twice with 10 ml of isotonic phosphate buffer, and add 0.5 ml of isotonic phosphate buffer. Suspend the cells in acid buffer, add 0.5ml of 0.5v 8% Triton X-100 solution, stir, and incubate at 37°
Obtained by incubating at C for 5 minutes.
第7表中、抽出画分での結果は、酪酸ナトリウム処理に
より選択的に細胞に導入した異種の蛋白合成が促進され
ることを証明している。In Table 7, the results for the extracted fraction prove that sodium butyrate treatment promotes the synthesis of a heterologous protein selectively introduced into cells.
また、非分泌性産物をコードしているプラスミドを用い
た場合にも、酪酸処理が適用できることを示唆するもの
である。This also suggests that butyric acid treatment can be applied even when a plasmid encoding a non-secreted product is used.
参考例1
proLIK産生形質転換株は特開昭61−17798
7に開示された方法により調製される。Reference Example 1 The proLIK-producing transformed strain is JP-A-61-17798
7.
すなわち、当該株はネオマイシン耐性遺伝子とProU
に遺伝子を共導入(コトランスフェクション)したCH
OK+細胞に由来する。That is, the strain contains the neomycin resistance gene and ProU.
CH in which genes were co-introduced (co-transfection) into
Derived from OK+ cells.
(1)cDNA
UK′@産生する大賢由来株化細胞から抽出されたmR
NAからc DNAを調製する。(1) mR extracted from Daiken-derived cell line producing cDNA UK'@
Prepare cDNA from NA.
解析された全塩基配列を以下に表わす。The entire base sequence analyzed is shown below.
このようなProUKの全塩基配列を含むプラスミドと
して常法により、pUK33を調製する。pUK33 is prepared by a conventional method as a plasmid containing the entire base sequence of ProUK.
(2) ベクターのウロキナーゼDNA配列の挿入(
1)で得たp IJ K33 (全長約2.2Kbpの
ウロキナーゼcDNAを含む)をPst Iで部分消
化し、1.7Kbp断片を単離してpSV−G、に挿入
した。この際の反応は、反応混合液(pUK331川μ
g、10XPst Ibuffer 50 p 1.
Pst I 10 tt 1. (60U )
、 dH!Oを加え500μe> を、37゛Cに保ち
、反応開始後10分、15分および20分で、それぞれ
約160μrサンプリングし、65’C,5分間加熱処
理後、それぞれの、反応液を混合して、1%アガロース
ゲルにて電気泳動にかけ、1.7Kbp断片を約10μ
g回収した。回収された約1.7Kbp断片の約5μg
を用い、T4−DNAポリメラーゼ処理で付着末端を平
滑末端にかえKpn Iリンカ−を付加した。その後
Kpn Iで消化したpsV−G、 と連結して、
大腸菌JIB
101を形質転換して、pSV−G、−proUK(第
1図)を得た。(2) Insertion of the urokinase DNA sequence into the vector (
pIJ K33 (containing urokinase cDNA with a total length of approximately 2.2 Kbp) obtained in 1) was partially digested with Pst I, and a 1.7 Kbp fragment was isolated and inserted into pSV-G. The reaction at this time was carried out using the reaction mixture (pUK331 Kawaμ
g, 10XPst Ibuffer 50 p 1.
Pst I 10 tt 1. (60U)
, dH! Add O and maintain the temperature at 37°C, sample approximately 160 μr each at 10, 15, and 20 minutes after the start of the reaction, heat treat at 65°C for 5 minutes, and mix each reaction solution. electrophoresed on a 1% agarose gel, and the 1.7Kbp fragment was separated into approximately 10μ
g was collected. Approximately 5 μg of the approximately 1.7 Kbp fragment recovered
Using T4-DNA polymerase treatment, sticky ends were changed to blunt ends and Kpn I linkers were added. It was then ligated with psV-G digested with Kpn I,
E. coli JIB 101 was transformed to obtain pSV-G, -proUK (Figure 1).
pSV −Cz−proUKは、5V−40のmRNA
転写調節域の下流にproUKcDNAの5′−非コー
ド化領域、シグナル配列コード化領域、proUKコー
ド化領域及び3″−非コード化領域が挿入されており、
適当な受容細胞へDNAを導入することでヒト−Pro
LJKの産生を行いうる(3) ドミナント選択マー
カーを含むプラスミドDNAの調製
上記pSV−G、を用いて培養細胞を形質転換する際の
ドミナント選択マーカーとして用いるプラスミドである
psV7G、−Neo’ は、以下の様にして作製した
。pSV-Cz-proUK is 5V-40 mRNA
The 5'-non-coding region, signal sequence-coding region, proUK-coding region and 3''-non-coding region of proUK cDNA are inserted downstream of the transcriptional regulatory region,
By introducing DNA into suitable recipient cells, human-Pro
(3) Preparation of plasmid DNA containing a dominant selection marker psV7G, -Neo', a plasmid used as a dominant selection marker when transforming cultured cells using the above pSV-G, is as follows: It was made as follows.
Tn5由来のネオマイシン耐性遺伝子がクローニングさ
れているプラスミドp N E O(Southern
。Plasmid pNEO (Southern
.
P、J、 and P、 Berg、 J、 Mo1e
c、 and AppliedGenet、、 1.3
27−341 (1982) をBamHIと旧nd
lllで切断後、エタノール沈澱してDNA断片を回収
した。回収したDNAは4dNTP存在下でE。P, J, and P, Berg, J, Mo1e.
c, and AppliedGenet, 1.3
27-341 (1982) as BamHI and former nd
After cutting with lll, DNA fragments were recovered by ethanol precipitation. The recovered DNA was transformed into E in the presence of 4dNTPs.
coliDNA−ポリメラーゼI、クレノー断片で付着
末端を平滑末端に変更した。 1.5KbpD N A
断片はアガロースゲル電気泳動で分離回収し、次にT、
−DNAリガーゼによる反応でその末端に前述の様にし
てにpn Iリンカ−を付加した。The sticky ends were changed to blunt ends with E.coli DNA-polymerase I, Klenow fragment. 1.5Kbp DNA
The fragments were separated and recovered by agarose gel electrophoresis, then T.
- A pn I linker was added to the end by a reaction using DNA ligase as described above.
にpn 1リンカ−の付加後、Xpn16UでDNA
断片を完全に消化し、リンカ−の付加された目的の約1
.5KbpのDNA断片を回収した。After addition of pn1 linker to
Completely digest the fragment and add linkers to approximately 1
.. A 5 Kbp DNA fragment was recovered.
回収したDNA断片は、そのうち15ngを前述の様に
してKpn Iで切断したp S V G+ 20
ngと連結し、大腸菌HBIOIを形質転換して目的の
プラスミドpsV−G、 −Neo’を得た(第2図
)。Of the recovered DNA fragments, 15 ng of them were digested with Kpn I as described above.
ng and transformed E. coli HBIOI to obtain the target plasmid psV-G, -Neo' (Fig. 2).
(4) 受容細胞(recipient cell)
の調製受容細胞として、チャイニーズハムスター卵巣細
胞(CHO−に、、^TCCCCL61(Flow−L
abo、 Inc。(4) Recipient cell
Chinese hamster ovary cells (CHO-, ^TCCCCL61 (Flow-L
abo, Inc.
より購入))を用い、細胞の培養を10シ/V%ウシ胎
児血清(Boehringer Manheim社製)
を含むハムのF −12(Gibco社製)で行った。Cells were cultured using 10%/V% fetal bovine serum (manufactured by Boehringer Manheim).
The test was carried out using ham F-12 (manufactured by Gibco) containing
細胞はファルコン3028フラスコで継代維持した。形
質転換に用いる細胞は、接種後2日の単層なものを用い
た。Cells were maintained for passage in Falcon 3028 flasks. The cells used for transformation were monolayer cells 2 days after inoculation.
トリプシン処理で細胞を集め、前記培養液に懸濁後、フ
ァルコン3003(100m)培養シャーレに5×10
Sce11s/10@lで植え込んだ。24時間5%の
C08−95%airのCO,−インキュベータで培養
後、形質転換に用いた。この時シャーレあたりの細胞数
は、?、0xlO’ cellsであった。Collect cells by trypsin treatment, suspend them in the above culture medium, and place them in a Falcon 3003 (100 m) culture dish at 5 x 10 cells.
It was implanted as Sce11s/10@l. After culturing in a 5% CO8-95% air CO incubator for 24 hours, it was used for transformation. At this time, what is the number of cells per petri dish? , 0xlO' cells.
(5) 形質転換
長田等の方法(蛋白質核酸・酵素、 28 (14)
。(5) Transformation method of Nagata et al. (Protein nucleic acid/enzyme, 28 (14)
.
1569〜1581.1983)に準じて調製したプラ
スミドDNA (pSV−G+ −proUK及びp
SV−G、−Neo’ (混合比率は100;1)〕
をDNA量として2.0μg/mlおよびキャリアDN
Aとしてサケ***D N A (PL−Biochem
ical)を20 p g/+wl含有するDNA−C
aPO,微小沈澱液を(4)で調製したシャーレあたり
11づつ加えた。シャーレを十字に動かして静置し、D
N A Ca P Oa微小沈澱液を細胞に吸着さ
せた後、再びインキュベータで培養を開始した。18時
間後壇地交換を行い、さらに48時間以上壇培養た後に
、形質転換細胞の選別を開始した。1569-1581.1983) prepared according to plasmid DNA (pSV-G+-proUK and pSV-G+-proUK).
SV-G, -Neo' (mixing ratio is 100; 1)]
2.0 μg/ml as DNA amount and carrier DN
Salmon sperm DNA (PL-Biochem
DNA-C containing 20 pg/+wl of
11 pieces of aPO and microprecipitate were added to each petri dish prepared in (4). Move the petri dish crosswise and let it stand still, D
After the N A Ca P Oa microprecipitate was adsorbed onto the cells, culturing was started again in the incubator. After 18 hours, the plates were exchanged, and after culturing the plates for 48 hours or more, selection of transformed cells was started.
(6) 形質転換細胞の選別
真核細胞において、G−418耐性を与えるTn5由来
のネオマイシン耐性遺伝子を
ドミナントマーカーとして用い(前述のpSV−GIN
e o’ ) 5outhern等の方法(J、 M
o1ec、 and Applied Genet、、
1.327−341.1982)に準じて細胞をG−
418含有培地(400μg/l)で培養することによ
ってトランスフエクシゴンした細胞の選別を行った。な
お、G−418はGtbco社製(Geneticin
、 Gibco、 lot No、75 K6043
及び74N8040)を用いた。(6) Selection of transformed cells In eukaryotic cells, the neomycin resistance gene derived from Tn5, which confers G-418 resistance, was used as a dominant marker (pSV-GIN
e o' ) 5other et al.'s method (J, M
o1ec, and Applied Genet,
1.327-341.1982).
Transfected cells were selected by culturing in 418-containing medium (400 μg/l). Note that G-418 is manufactured by Gtbco (Geneticin
, Gibco, lot No. 75 K6043
and 74N8040) were used.
4日おきに培地を交換し、G−418耐性コロニーを順
次クローニングして成育させ、10日回定83クローン
を得た。得られたクローンをヒト−ウロキナーゼRPI
(A試薬(ミドリ十字社製)及びフィブリン平板法でウ
ロキナーゼの踵体を調べた。RPHA試薬で、第一次の
スクリーニングを行った結果、G−418耐性株のうち
約25%で凝集がみられ、ヒト−ウロキナーゼ様物質が
産生されていると推定できた。The medium was replaced every 4 days, and G-418-resistant colonies were successively cloned and grown, yielding 83 clones in a 10-day cycle. The obtained clone was subjected to human-urokinase RPI
(The heel of urokinase was examined using A reagent (manufactured by Midori Juji Co., Ltd.) and fibrin plate method. As a result of the first screening using RPHA reagent, agglutination was observed in approximately 25% of the G-418 resistant strains. It was assumed that a human urokinase-like substance was produced.
凝集のみられた15株をフィブリン平板法によってプラ
スミノーゲンアクキベーター活性を調べたところ培養上
清に活性が認められ、しかもこの活性は、ウロキナーゼ
抗原カラムで精製した抗ヒト−ウロキナーゼ抗体で中和
された。スクリーニングによって得たウロキナーゼ様物
質の産生量の高いと思われる形質転換体C−68を選び
、低密度培養によるクローニングを行って、C−68−
53とC−68−61の2株を得た。When the plasminogen activator activity of the 15 aggregated strains was examined by the fibrin plate method, activity was observed in the culture supernatant, and this activity was neutralized by anti-human urokinase antibody purified with a urokinase antigen column. Ta. The transformant C-68, which was obtained by screening and seemed to have a high production amount of urokinase-like substance, was selected and cloned by low-density culture to transform C-68-
Two strains, 53 and C-68-61, were obtained.
これらのクローンの培養上清を用いて、合成・分泌され
たヒトーUK様蛋白の性状を検討した。Using the culture supernatants of these clones, the properties of the synthesized and secreted human-UK-like protein were investigated.
なお、C−68−53とC−68−61の両クローンは
、100日以上にわたって安定にさらに構成的に培地中
にヒトーUK様蛋白を分泌した。そして、形質転換細胞
から、分子量が54.000ダルトンの糖鎖の付加され
たp r o−UKの産生が確認された。In addition, both clones C-68-53 and C-68-61 secreted human-UK-like protein into the medium stably and constitutively for over 100 days. Production of pro-UK to which a sugar chain with a molecular weight of 54,000 Daltons was added was confirmed from the transformed cells.
参考例2
PreS−HBsAg産生形質転換株として、SV40
エンハンサ−および初期プロモーターの下流にPreS
−HRsAg遺伝子を接続して導入したCHO−に、細
胞を用いた。Reference Example 2 As a transformed strain producing PreS-HBsAg, SV40
PreS downstream of enhancer and early promoter
Cells were used for CHO- into which the -HRsAg gene was connected and introduced.
(1) P S V −3r d組換えDNAの作製
(第3図)
Pre S−HBsAg構造遺伝子全体がpBR322
のHindl[[部位に挿入されているプラスミドpP
s411から、Pre S jl域の3番目のATGか
らHB’sA’g遺伝子領域までを含んだ1.2Kbp
断片(3rd Pre S−HBsAg)をMst I
L Hindlにより切り出し、動物細胞発現ベクター
p S V G x E c oのEcoR1部位
に正方向に挿入したプラスミドpsV−3rdを作製す
る。(1) Preparation of PSV-3rd recombinant DNA (Figure 3) The entire Pre S-HBsAg structural gene is pBR322.
The plasmid pP inserted at the Hindl [[ site
1.2 Kbp including from s411 to the third ATG of Pre S jl region to HB'sA'g gene region
The fragment (3rd Pre S-HBsAg) was converted into MstI
Plasmid psV-3rd is prepared by cutting out the plasmid using L Hindl and inserting it into the EcoR1 site of the animal cell expression vector pSV G x Eco in the forward direction.
pP5411 10μgをMst n 12U、
Hindll112Uで37°C5時間反応させ、フェ
ノール抽出を2回行い、エタノール沈澱によりDNAを
回収する。pP5411 10μg Mst n 12U,
React with Hindll 112U at 37°C for 5 hours, perform phenol extraction twice, and collect DNA by ethanol precipitation.
このDNAは40閣Hトリス塩酸緩衝液(pH7,2)
、8s+M硫酸マグネシウム、80μMジチオスレイト
ール、2s+MdNTP存在下、DNAポリメラーゼ!
クレノー断片10Uにより室温で30分反応させ、付着
末端を平滑末端にし、さらに50IIMトリス塩酸緩衝
液(pH8,2)で牛小腸由来アルカリホスファターゼ
IUにより37℃1時間反応し、5′末端を脱リン酸化
したのち、クロロホルム、フェノール抽出を2回行いエ
タノール沈澱によりDNAを回収する。このDNA4μ
gに66mM )リス塩酸緩衝液(pH7,6) 6.
6 sM塩化マグネシウム、10mMジチオスレイトー
ル、1.OsM A T P 、 EcoRIリンカ
−(9GGAATTCC) 2 # g存在下、T4D
NAリガーゼ5.6Uを16℃−晩反応させ、EcoR
Iリンカ−を接続する。このDNAをエタノール沈澱に
より回収し、I!coRI 50 Uで37°C5時間
反応させ、0,8%低融点アガロースゲル電気泳動を行
い、1.2Kbp断片をゲルから切り出し、フェノール
抽出を2回行い、エタノール沈澱によりDNAを回収す
る。This DNA is 40% H Tris-HCl buffer (pH 7.2)
, 8s+M magnesium sulfate, 80 μM dithiothreitol, 2s+MdNTP, DNA polymerase!
The sticky ends were made blunt by reacting with 10 U of Klenow fragment at room temperature for 30 minutes, and then reacted with IU of bovine small intestine alkaline phosphatase in 50 IIM Tris-HCl buffer (pH 8,2) at 37°C for 1 hour to dephosphorylate the 5' end. After oxidation, the DNA is extracted twice with chloroform and phenol, and then precipitated with ethanol to recover the DNA. This DNA 4μ
66mM) Lis-HCl buffer (pH 7,6) 6.
6 sM magnesium chloride, 10mM dithiothreitol, 1. OsM ATP , T4D in the presence of EcoRI linker (9GGAATTCC) 2 #g
5.6 U of NA ligase was reacted at 16°C overnight, and EcoR
Connect the I linker. This DNA was recovered by ethanol precipitation and I! React with 50 U of coRI at 37°C for 5 hours, perform 0.8% low melting point agarose gel electrophoresis, cut out a 1.2 Kbp fragment from the gel, perform phenol extraction twice, and recover DNA by ethanol precipitation.
SV40のエンハンサ−、プロモーター、スプライシン
グジャンクション、ポリアデニル化シグナルを有する動
物細胞発現ベクターpsVGt EcoのEcoR1
部位に、I!coRIリンカ−の接続したPre 5−
HBsAg 1.2Kbp断片を挿入する。psVG。EcoR1 of the animal cell expression vector psVGt Eco that has the enhancer, promoter, splicing junction, and polyadenylation signals of SV40
In the part, I! Pre 5- connected with coRI linker
Insert the HBsAg 1.2 Kbp fragment. psVG.
−Eco3IjgをEcoRI 10 Uで37℃6時
間反応させ、さらに50mM )リス塩酸(9118,
2)緩衝液中で、牛小腸由来アルカ・リフオスファター
ゼ20Uで37°C1時間反応させ、5′末端を脱リン
酸化した後、0.8%低融点アガロースゲル電気泳動を
行う。-Eco3Ijg was reacted with 10 U of EcoRI at 37°C for 6 hours, and then 50 mM) lithium-hydrochloric acid (9118,
2) In a buffer solution, react with 20 U of bovine small intestine-derived alkaline phosphatase at 37°C for 1 hour to dephosphorylate the 5' end, and then perform 0.8% low melting point agarose gel electrophoresis.
泳動後、断片を切り出し、フェノール抽出2回、エタノ
ール沈澱によりDNAを回収する。EcoR1ン肖 化
pSVGz−Ecol μ g 11!: Pr
eS−HBsAg 1゜’2Kbp断片0.5μgを
66n+M )リス塩酸緩衝液(pH7,6) 、6.
6 mM塩化マグネシウム、10mMジチオスレイトー
ル、1.0m門A T P反応液中で、T4DNAリガ
ーゼ8.4Uにより16°C−晩反応させ、大腸菌HB
l 01に形質転換し、アンピシリン耐性形質転換体
を得る。形質転換体のなかから、EcoRI 、 Ba
l1+Hl 、 Sal I +Xba Iの切断パ
ターンを分析することにより、Pre S−HBsAg
断片が正方向に入ったpSV−3rdを得る。After electrophoresis, the fragments are cut out, and the DNA is recovered by phenol extraction twice and ethanol precipitation. EcoR1 expression pSVGz-Ecol μg 11! : Pr
0.5 μg of eS-HBsAg 1°'2 Kbp fragment in 66n+M) Lis-HCl buffer (pH 7,6), 6.
E. coli HB was incubated overnight at 16°C with 8.4 U of T4 DNA ligase in a 6 mM magnesium chloride, 10 mM dithiothreitol, 1.0 mATP reaction solution.
l 01 to obtain ampicillin-resistant transformants. Among the transformants, EcoRI, Ba
Pre S-HBsAg
pSV-3rd containing the fragment in the forward direction is obtained.
(2) 動物細胞の形質転換
CHO−に、細胞の形質転換は、リン酸カルシウム沈澱
法により行う。2倍濃度のヘペス緩衝液(2XHBS)
(ヘペス(N−(2−ヒドロキシエチル)ピペラジン−
N′−2−エタンスルホン酸) 10g / i、Na
Ce 6g71.pH7,1に調節) 2.5ml、7
0mM NaHzPO4と70IIIM Na2HPO
4の水?容液50μffi。(2) Transformation of animal cells Transformation of CHO- cells is performed by calcium phosphate precipitation method. 2x Hepes buffer (2XHBS)
(Hepes(N-(2-hydroxyethyl)piperazine-
N'-2-ethanesulfonic acid) 10g/i, Na
Ce 6g71. (adjusted to pH 7.1) 2.5ml, 7
0mM NaHzPO4 and 70mM Na2HPO
4 water? Volume of liquid: 50μffi.
lμg/+nlサケ***DNA100μffi、組み換
えDNA[pSV−3rd 104g、 pSV
Ct+ NeO′ (図2) 0.1μgl
100μffiをチューブに加え、滅菌水で全量4
.7mlにしたB液を調製する。このB液にA液(2M
CaCj! z) 300IIlを加える、B液に空
気を送りながら撹拌し、A液をゆっくり滴下する。A液
を加え終った後、1〜2分空気を送りつづけ、均質な沈
澱を形成させ、10分間静置する。CHOK+ 細胞は
、トランスフェクションの前日に、100 mシャーレ
に5XIO’個まき、24時間CO□インキュベーター
で培養する。培養液は10v/v%胎仔牛血清を含むハ
ムのF−12培地を用いる。この培養シャーレに、微小
沈澱液1mlを滴下し、均一に拡散させる。10分間の
静置により、DNA−リン酸カルシウム微小沈澱を細胞
に吸着させた後、再びCO□インキュベーターで培養す
る。トランスフェクションの18時間後に、培地交換を
行い、さらに48時間後G−418含有培地と交換する
。 G−418の濃度は400gg/mlである。lμg/+nl salmon sperm DNA 100μffi, recombinant DNA [pSV-3rd 104g, pSV
Ct+ NeO' (Figure 2) 0.1μgl
Add 100 μffi to the tube and dilute the total volume with sterile water
.. Prepare solution B to 7 ml. Add this B solution to A solution (2M
CaCj! z) Add 300 IIl, stir while blowing air into liquid B, and slowly drop liquid A. After adding Solution A, continue blowing air for 1 to 2 minutes to form a homogeneous precipitate, and let stand for 10 minutes. The day before transfection, 5XIO' CHOK+ cells are seeded in a 100 m petri dish and cultured in a CO□ incubator for 24 hours. The culture medium used is Ham's F-12 medium containing 10% v/v fetal calf serum. 1 ml of the microprecipitation solution is dropped into this culture dish and dispersed uniformly. After allowing the DNA-calcium phosphate microprecipitate to adsorb to the cells by standing for 10 minutes, the cells are cultured again in the CO□ incubator. 18 hours after transfection, the medium is replaced, and after another 48 hours, the medium is replaced with G-418-containing medium. The concentration of G-418 is 400 gg/ml.
G−418含有培地で成育したコロニーをシリンダー法
で分離し、G−418耐性形質転換細胞を得る。Colonies grown in the G-418-containing medium are separated by the cylinder method to obtain G-418-resistant transformed cells.
(3) Pre S−HBsAgの生産上記のG−4
18耐性細胞の培養液中のHBsAg活性をRPHA法
(アンティヘプセル:ミドリ十字!り及びEIA法(オ
ースザイム■、グイナボット社製)により検討した。G
−418耐性株のなかから、HB s A g陽性株が
高頻度に得られる。さらにポリアルブミン(pH3A)
結合活性をポリアルブミン(pH3A)のPHA法(レ
ンケイら、Immunol。(3) Production of Pre S-HBsAg G-4 above
The HBsAg activity in the culture medium of 18-resistant cells was examined by the RPHA method (Antihepsel: Midori Jujiri!) and the EIA method (Auszyme ■, manufactured by Guinabot).
Among the -418-resistant strains, HBsAg-positive strains are frequently obtained. Furthermore, polyalbumin (pH3A)
The binding activity was determined by the PHA method of polyalbumin (pH 3A) (Renkei et al., Immunol.
Methods、 16.23−30.1977)によ
り検討し、HBsAg陽性細胞上清にpH3A結合活性
が認められ、Pre S 85域が発現していることを
、確認した。この上清を塩化セシウム密度勾配遠心分離
法を用いて精製した。この精製サンプルをSO3PAG
Eによって分子量を求めたところ約35,000、約2
7,000、約24 、000の3本のバンドを確認し
た。このことにより形質転換した細胞がPre S−H
BsAgペプタイドを産生じていることを確認した。な
お本発明で得られたPre S−HBsAgの粒子を電
子顕微鏡で観察したところ直径的22nmの球形粒子で
あった。Methods, 16.23-30.1977), pH3A binding activity was observed in the HBsAg positive cell supernatant, and it was confirmed that the Pre S 85 region was expressed. This supernatant was purified using cesium chloride density gradient centrifugation. This purified sample was added to SO3PAG.
The molecular weight was determined by E and was approximately 35,000, approximately 2
Three bands of 7,000 and approximately 24,000 were confirmed. This allows the transformed cells to become Pre S-H
It was confirmed that BsAg peptide was produced. When the particles of Pre S-HBsAg obtained in the present invention were observed with an electron microscope, they were found to be spherical particles with a diameter of 22 nm.
参考例3
t−PA産生形質転換株として、5V−40エンハンサ
−および初期プロモーターの下流にt−PA遺伝子を接
続して導入したCHO(O−Kl細胞を用いた。Reference Example 3 As a t-PA producing transformant, CHO (O-Kl cells) into which the t-PA gene was introduced downstream of the 5V-40 enhancer and early promoter were used.
(1) 発現ベクターの構築
メラノーマ細胞G−361株由来t−PAcDNAを有
するプラスミドpTPA−2より、t−PAの全コード
化領域を含む断片を取り出し、動物細胞用発現ベクター
p S V G tの発現調節領域の間に挿入してt
−PA発現ベクターpDX−2を構築する。(1) Construction of expression vector A fragment containing the entire coding region of t-PA was extracted from plasmid pTPA-2 containing t-PA cDNA derived from melanoma cell line G-361, and the expression vector for animal cells pSV Gt was extracted. Insert between the expression regulatory regions and t
-Construct PA expression vector pDX-2.
(1−i)pXK−2の作製(第4図)プラスミドpT
PA−2をHindl[I及びXmn1で消化し、アガ
ロースゲル電気泳動によりt−PAcDNAを含む2.
9KbのDNAバンドを切り取り、エレクトロエル−シ
ランにてDNAを回収した。(1-i) Construction of pXK-2 (Figure 4) Plasmid pT
2. PA-2 was digested with Hindl[I and Xmn1 and t-PAcDNA was analyzed by agarose gel electrophoresis.
A 9 Kb DNA band was cut out, and the DNA was recovered using Electrol-Silane.
この2.9Kb Hind m−Xmn■断片の両末端
をフィルイン(fill−in) シ、Kpn I
リンカ−を付加した。Fill-in both ends of this 2.9 Kb Hind m-Xmn fragment with Kpn I
A linker was added.
Kpn Iリンカ−の結合した断片は、さらにKpn
1を用いて消化し、アガロースゲル電気泳動により
2.9Kb付近のバンドを切り取りエレクトロエル−シ
コンにてDNAを回収した。The Kpn I linker-bound fragment is further linked to the Kpn I linker.
1, a band around 2.9 Kb was excised by agarose gel electrophoresis, and the DNA was recovered using electrophoresis.
一方、動物細胞用発現ベクターpSV GxをKpn
I消化し、さらにCHOP (牛小腸由来アルカリ
ホスファターゼ)処理により5′末端のリン酸基を除い
た0次にこの3.2Kb p S V −Gt −Kp
ni断片と先に調製した2、9Kb t −P A
c D N Aを含む断片を連結した0反応後二の溶液
を用いて大腸菌HBIOIを形質転換した。On the other hand, the expression vector pSV Gx for animal cells was used as Kpn
This 3.2Kb pSV-Gt-Kp was digested with I and further treated with CHOP (alkaline phosphatase derived from bovine small intestine) to remove the 5'-terminal phosphate group.
ni fragment and the previously prepared 2,9Kb t-PA
After the 0 reaction in which the fragments containing cDNA were ligated, E. coli HBIOI was transformed using the second solution.
形質転換体より抽出したプラスミドDNAから、目的と
するプラスミドPXK−2を選択した。The target plasmid PXK-2 was selected from plasmid DNA extracted from the transformant.
(1−ii)pDD−2プラスミドの作製(第5図)プ
ラスミドpTPA−2をBbe Iと旧ndlllで
消化し、このDNAをアガロースゲル電気泳動にかけゲ
ルよりG−Cテイル及びt−PAcDNAの1部を含む
1.IKbのDNAバンドを切り取り、エレクトロエル
−ジョンによりDNAを回収した。(1-ii) Preparation of pDD-2 plasmid (Figure 5) Plasmid pTPA-2 was digested with Bbe I and old ndlll, and this DNA was subjected to agarose gel electrophoresis to extract the G-C tail and t-PA cDNA. Including parts 1. The IKb DNA band was excised, and the DNA was recovered by electrolysis.
そしてこのHind m −Bbe n断片をOde
Iで消化し、次に5′側の突出末端をフィルイン(
fill−in) シた後ポリアクリルアミドゲルにか
け270bp断片のバンドをゲルより切り出しエレクト
ロエル−シランにてDNAを回収した。このG−Cテイ
ルが除かれた270bp Ode 1断片の両末端に
Kpn Iリンカ−を付加した。この断片を一旦Kp
n I消化した後ポリアクリルアミドゲル電気泳動に
かけ、リンカ−DNA由来のDNA切断片を除いた。泳
動後ゲルより270bp付近のバンドを切り取り、エレ
クトロエル−ジョンにてDNAを回収した。Then, this Hind m -Bbe n fragment was
Digest with I, then fill in the 5' overhanging end (
After filling-in), the DNA was run on a polyacrylamide gel, a 270 bp fragment band was cut out from the gel, and the DNA was recovered using Electrol-Silane. Kpn I linkers were added to both ends of this 270 bp Ode 1 fragment from which the G-C tail had been removed. Kp this fragment once.
After digestion with n I, the resulting product was subjected to polyacrylamide gel electrophoresis to remove DNA fragments derived from the linker DNA. After electrophoresis, a band around 270 bp was cut out from the gel, and the DNA was recovered by electrolysis.
(1−i)で調製した[pn I処理pSV Gt
と先に調製した270bp Kpn n断片を連結し
た。反応後この液を用い大腸菌DHIを形質転換した。[pn I-treated pSV Gt prepared in (1-i)
and the previously prepared 270 bp Kpn n fragment were ligated. After the reaction, this solution was used to transform Escherichia coli DHI.
形質転換体より抽出したプラスミドDNAから目的とす
るプラスミドpDD−2を選択した。The target plasmid pDD-2 was selected from plasmid DNA extracted from the transformant.
(1−’iii D X −2プラスミドの ’
(6−’)(1−i)で作製したプラスミドpXK−2
をRan m (C1a Iのisoschizom
er )消化し、さらにBgl uを用い部分消化し
、t−PAのN末付近に位置するBgl 11部位だ
けを切断しアガロースゲル電気泳動にてゲルより4.8
Kb Ran m −Bgl In断片のDNAバンド
を切り取り、エレクトロエル−ジョンにてDNAを回収
した後この断片をBAP (バクテリア由来アルカリホ
スファターゼ)処理した一方、(1−ii )で作製し
たpDD−2をRan■とBgl IIを用い消化し
た。これをアガロースゲルにかけ、ゲルよりSV40初
期プロモーターを含む730bpのDNAバンドを切り
取り、エレクトロエル−シランにてDNAを回収した。(1-'iii DX-2 plasmid'
(6-') Plasmid pXK-2 prepared in (1-i)
Ran m (C1a I isoschizom
er), and then partially digested with Bglu to cut only the Bgl 11 site located near the N-terminus of t-PA, and analyzed by agarose gel electrophoresis.
The DNA band of the Kb Ran m -Bgl In fragment was cut out, and the DNA was recovered by electrolysis. This fragment was treated with BAP (bacterial alkaline phosphatase), while the pDD-2 prepared in (1-ii) was Digestion was performed using Ran■ and Bgl II. This was applied to an agarose gel, a 730 bp DNA band containing the SV40 early promoter was excised from the gel, and the DNA was recovered using Electrol-Silane.
この730bp Ran I −Bgl n断片と先に
調製した4、8KbRan In −Bgl n断片を
連結し、この反応液を用い大腸菌DHIを形質転換した
。This 730 bp Ran I-Bgl n fragment was ligated with the previously prepared 4.8 Kb Ran In-Bgl n fragment, and E. coli DHI was transformed using this reaction solution.
形質転換体より抽出したプラスミドDNAから目的とす
るプラスミドpDX−2を選択した。The target plasmid pDX-2 was selected from plasmid DNA extracted from the transformant.
(2)t−PA産生形質転換株の調製
(1)で得られたプラスミドpDX−2を用い、参考例
1または2の方法に準じて、p S V −G +−N
eo’ と共に、DNA−リン酸カルシウム沈澱法によ
り、CHOK11in胞をコトランスフェクションした
。(2) Preparation of t-PA producing transformant strain pSV -G + -N using plasmid pDX-2 obtained in (1) and according to the method of Reference Example 1 or 2.
CHOK11in cells were cotransfected with eo' by DNA-calcium phosphate precipitation.
導入効率は1.8 X 10−’〜4.6 X 10−
’コロニー/pg DNA/dish程度であった。Introduction efficiency is 1.8 x 10-' to 4.6 x 10-'
'Colony/pg DNA/dish.
この中から、目的とするt−PA産生形質転換株(R−
72−3)を選別した。From among these, the desired t-PA producing transformant strain (R-
72-3) was selected.
第1図はp UK33 (p r o UKのcDNA
を担持)とp S V G+(S V40初期プロモ
ーターを担持)からp S V −G r p r
o U K (両者を担持)を構築する手順を示す。
第2図はpNEO(ネオマイシン耐性遺伝子を担持)
トp S V G t(S V40初期プロモーター
を担持)からpSV−G、Neo’ (両者を担持)
を構築する手順を示す。
第3図はpPs411 (preS−HBsAgのcD
NAを担持)とpsV−Gt−Eco (SV40初
期プロモーターを担持)からpSV−3rd(両者を担
持)を構築する手順を示す。
第4図はpTPA−2(t−PAのcDNAを(U持)
(!: p S V Gz(S V40初朋プロモ
ーターを1u持)からpXK−2を構築する手順を示す
。
第5図はpTPA−2とpSV−G、からp[)D−2
を構築する手順を示す。
第6図はpXK−2とpDD−2からpDX−2(TP
Aのc DNAとSV40初朋プ初子プロモーターを担
持)を構築する手順を示す。
回申、ニー囚はSV40エンハンサ−
※燕μsはSV40初朋プロモーター
mはネオマイシン耐性遺伝子
一一一は外来DNA
口二二はSV40ポリアデニル化シグナルAP’ はア
ンピシリン耐性
Tc’ はテトラサイクリン耐性
□ はpBR322由来
を表わす。
また、制限酵素部位は以下のように表わされる。
Ac:Acc I、 B :Bamtll、 Ba
:Bal I+Bb:BbeI、C:Cff1aI、D
:DdeI。
GI:Bgl21. GII :Bgl I[、H:
H4ndI[I。
tip:I(pa I、 K :Kpn I、
P :Pst IPv:Pvu II、
S :Sal !+ Sc:Sca I+Sa
:Sph l、 T :Tth IV、 X
:Xho I。
(ばか3名)
第 4 図
第 5 図
1F篇“;。、−□、6
第 6 図
手続補正力
昭和62年5り/日Figure 1 shows pUK33 (p r o UK cDNA
) and pSVG+ (carrying the SV40 early promoter) to pSV-Grpr
The procedure for constructing o U K (carrying both) is shown. Figure 2 shows pNEO (carrying neomycin resistance gene)
Top pSV G t (carrying the SV40 early promoter) to pSV-G, Neo' (carrying both)
Here are the steps to build the . Figure 3 shows the cD of pPs411 (preS-HBsAg).
The procedure for constructing pSV-3rd (carrying both NA) and psV-Gt-Eco (carrying the SV40 early promoter) is shown. Figure 4 shows pTPA-2 (t-PA cDNA (containing U)).
(!: Shows the procedure for constructing pXK-2 from pSV Gz (containing 1 u of SV40 Hatsutomo promoter). Figure 5 shows the construction of p[)D-2 from pTPA-2 and pSV-G.
Here are the steps to build the . Figure 6 shows pXK-2 and pDD-2 to pDX-2 (TP
The procedure for constructing the A cDNA and the SV40 Hatsuko promoter (carrying the Hatsuko promoter) is shown. ※Tsubame μs is SV40 hatsatomo promoter m is neomycin resistance gene 111 is foreign DNA Mouth 22 is SV40 polyadenylation signal AP' is ampicillin resistance Tc' is tetracycline resistance □ is derived from pBR322 represents. In addition, restriction enzyme sites are expressed as follows. Ac: Acc I, B: Bamtll, Ba
:Bal I+Bb:BbeI, C:Cff1aI, D
:DdeI. GI:Bgl21. GII:Bgl I[,H:
H4ndI[I. tip: I(pa I, K: Kpn I,
P:Pst IPv:Pvu II,
S: Sal! + Sc:Sca I+Sa
:Sph l, T :Tth IV, X
:Xho I. (Three idiots) Figure 4 Figure 5 Figure 1F section “;., -□, 6 Figure 6 Procedure correction power 5/day 1985
Claims (3)
ベクターを用いて形質転換した動物細胞を培養して異種
蛋白質を産生する際に、培地に酪酸またはその塩を添加
することを特徴とする異種蛋白質の産生増強方法。(1) A heterologous protein characterized in that butyric acid or its salt is added to the medium when producing the heterologous protein by culturing animal cells transformed using a vector incorporating a DNA sequence encoding the heterologous protein. A method for enhancing the production of.
第(1)項記載の方法。(2) The method according to claim (1), wherein the animal cell is CHO-K_1.
請求の範囲第(1)項記載の方法。(3) The method according to claim (1), wherein the vector contains the SV40 early promoter.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62048935A JPH01257492A (en) | 1987-03-05 | 1987-03-05 | Method for enhancing production of paraprotein |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62048935A JPH01257492A (en) | 1987-03-05 | 1987-03-05 | Method for enhancing production of paraprotein |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH01257492A true JPH01257492A (en) | 1989-10-13 |
Family
ID=12817118
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
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Country | Link |
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JP (1) | JPH01257492A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0649900A3 (en) * | 1993-09-08 | 1996-07-03 | Suntory Ltd | Process for production of protein. |
US5705364A (en) * | 1995-06-06 | 1998-01-06 | Genentech, Inc. | Mammalian cell culture process |
US5721121A (en) * | 1995-06-06 | 1998-02-24 | Genentech, Inc. | Mammalian cell culture process for producing a tumor necrosis factor receptor immunoglobulin chimeric protein |
US6656466B1 (en) | 1995-06-06 | 2003-12-02 | Genetech, Inc. | Human tumor necrosis factor—immunoglobulin(TNFR1-IgG1) chimera composition |
-
1987
- 1987-03-05 JP JP62048935A patent/JPH01257492A/en active Pending
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0649900A3 (en) * | 1993-09-08 | 1996-07-03 | Suntory Ltd | Process for production of protein. |
US5834249A (en) * | 1993-09-08 | 1998-11-10 | Suntory Limited | Process for production of protein |
US5705364A (en) * | 1995-06-06 | 1998-01-06 | Genentech, Inc. | Mammalian cell culture process |
US5721121A (en) * | 1995-06-06 | 1998-02-24 | Genentech, Inc. | Mammalian cell culture process for producing a tumor necrosis factor receptor immunoglobulin chimeric protein |
US6656466B1 (en) | 1995-06-06 | 2003-12-02 | Genetech, Inc. | Human tumor necrosis factor—immunoglobulin(TNFR1-IgG1) chimera composition |
US7666416B2 (en) | 1995-06-06 | 2010-02-23 | Genentech, Inc. | Mammalian cell culture process |
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