JPH01137976A - Novel dna fragment - Google Patents
Novel dna fragmentInfo
- Publication number
- JPH01137976A JPH01137976A JP29184187A JP29184187A JPH01137976A JP H01137976 A JPH01137976 A JP H01137976A JP 29184187 A JP29184187 A JP 29184187A JP 29184187 A JP29184187 A JP 29184187A JP H01137976 A JPH01137976 A JP H01137976A
- Authority
- JP
- Japan
- Prior art keywords
- gene
- tnf
- mouse
- dna
- fragment
- 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
- 239000012634 fragment Substances 0.000 title claims abstract description 24
- 101100046526 Mus musculus Tnf gene Proteins 0.000 claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 230000002759 chromosomal effect Effects 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 abstract description 22
- 238000000034 method Methods 0.000 abstract description 10
- 239000002299 complementary DNA Substances 0.000 abstract description 9
- 239000000126 substance Substances 0.000 abstract description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 abstract description 5
- 101150033527 TNF gene Proteins 0.000 abstract description 4
- 239000000523 sample Substances 0.000 abstract description 4
- 241000701959 Escherichia virus Lambda Species 0.000 abstract description 3
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 abstract description 3
- 108091026890 Coding region Proteins 0.000 abstract description 2
- 238000010367 cloning Methods 0.000 abstract description 2
- 238000009396 hybridization Methods 0.000 abstract description 2
- 150000007523 nucleic acids Chemical class 0.000 abstract description 2
- 230000006798 recombination Effects 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- 108020004707 nucleic acids Proteins 0.000 abstract 1
- 102000039446 nucleic acids Human genes 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 238000005215 recombination Methods 0.000 abstract 1
- 230000014509 gene expression Effects 0.000 description 16
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 15
- 102000003390 tumor necrosis factor Human genes 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 9
- 239000002158 endotoxin Substances 0.000 description 8
- 229920006008 lipopolysaccharide Polymers 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 210000004102 animal cell Anatomy 0.000 description 6
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 101150074155 DHFR gene Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108700026226 TATA Box Proteins 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 101150034814 F gene Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 101000658545 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) Type I restriction enyme HindI endonuclease subunit Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101000666411 Mus musculus Tenascin Proteins 0.000 description 1
- 101000626128 Mus musculus Tetranectin Proteins 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 102000057041 human TNF Human genes 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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
- C07K14/525—Tumour necrosis factor [TNF]
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明はマウスＴＮＦをコードする染色体遺伝子を含む
新規ＤＮＡフラグメントに関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel DNA fragment containing a chromosomal gene encoding mouse TNF.
腫瘍壊死因子（ＴＮＦ）は、ＢＣＧ感作したマウスに一
定期間後リポ多糖を静脈投与して得られるもので、血清
中に存在し、Ｍｅｔｈ Ａ肉腫を皮下移植したマウスに
投与すると、肉腫組織の出血性壊死をひきおこす物質に
対して与えられた名称である。ＴＮＦは、ある種の腫瘍
細胞を直接傷害し、正常細胞には影響を与えにくい為、
制ガン剤として開発されている。一方、ＴＮＦは種特異
性がない為、ヒト以外の動物由来のＴＮＦも制ガン剤と
して極めて有用である。このＴＮＦをコードする染色体
遺伝子の構造及び編成を解明すれば、その発現のメカニ
ズム、異常発現の疾病に於ける役割、制ガンメカニズム
等の究明に多大な寄与をもたらす事は必定である。たと
えば発現のメカニズムの解明により、ガン患者の体内に
ＴＮＦを産生させ、治療する事も可能になる。さらにプ
ロモータ部分だけをとり出す事により、ＴＮＦ遺伝子の
発現を促進する物質のスクリーニングにも応用でき、そ
の解明が待たれていた。Tumor necrosis factor (TNF) is obtained by intravenously administering lipopolysaccharide to BCG-sensitized mice after a certain period of time, and is present in the serum. This is the name given to substances that cause hemorrhagic necrosis. TNF directly damages certain tumor cells and has little effect on normal cells, so
It is being developed as an anti-cancer drug. On the other hand, since TNF has no species specificity, TNF derived from animals other than humans is also extremely useful as an anticancer agent. Elucidation of the structure and organization of the chromosomal gene that encodes TNF will undoubtedly make a significant contribution to the investigation of its expression mechanism, the role of abnormal expression in diseases, and the anticancer mechanism. For example, by elucidating the mechanism of expression, it will become possible to produce TNF within the body of cancer patients and treat them. Furthermore, by extracting only the promoter portion, this method can be applied to screening for substances that promote TNF gene expression, and clarification has been awaited.
本発明は、上記に示した如き現状に鑑みなされたもので
、その目的はＴＮＦの遺伝子の構造及び編成を解明する
事により、その発現のメカニズム、疾病における役割、
制ガンの機構等を究明するべく、マウスＴＮＦ遺伝子を
含むＤＮＡフラグメントを提供する事にある。さらに、
このＤＮＡフラグメントを動物細胞に導入する事により
、制ガン剤としてのマウスＴＮＦを効率良く得ることも
可能となる。さらに、このＤＮＦフラグメントのプロモ
ータ一部分をとり出し、このプロモータ一部分に生理活
性物質をコードするＤＮＡ断片を接続して、動物細胞に
導入する事により、該生理活性物質を効率良く得ること
も可能となる。The present invention was made in view of the current situation as described above, and its purpose is to elucidate the structure and organization of the TNF gene, thereby understanding the mechanism of its expression, its role in diseases, and the like.
The purpose of this project is to provide a DNA fragment containing the mouse TNF gene in order to investigate the mechanism of cancer control. moreover,
By introducing this DNA fragment into animal cells, it becomes possible to efficiently obtain mouse TNF as an anticancer agent. Furthermore, by extracting a portion of the promoter of this DNF fragment, connecting a DNA fragment encoding a physiologically active substance to this promoter portion, and introducing the DNA fragment into animal cells, it becomes possible to efficiently obtain the physiologically active substance. .
本発明者らは、遺伝子組換えの手法を用いて、鋭意ＴＮ
Ｆ遺伝子の構造について研究を重ねてきたが、このたび
マウスのＴＮＦ遺伝子をクローニングする事に成功し、
本発明を完成するに到った。The present inventors have earnestly developed TN using genetic recombination techniques.
After much research into the structure of the F gene, we have now succeeded in cloning the mouse TNF gene.
The present invention has now been completed.
本発明は、エクソン及びイントロン、並びに該エクソン
に隣接した５′末端約８００個の核酸塩基配列を含んで
成るマウスＴＮＦ遺伝子を含むＤＮＡフラグメントの発
明である。The present invention is a DNA fragment comprising the mouse TNF gene, which comprises an exon and an intron, and a 5'-terminal approximately 800 nucleobase sequence adjacent to the exon.
本発明に係るマウスＴＮＦ遺伝子は４個のエクソンと３
個のイントロンを含む。また５′末端側ＴＡＴＡボツク
スを含む、第２図または第３図の４３３−６２３の塩基
配列は、ヒトと８６％のホモロジーを示し、発現に重要
な塩基配列である。The mouse TNF gene according to the present invention has 4 exons and 3 exons.
Contains several introns. Furthermore, the nucleotide sequence 433-623 in FIG. 2 or 3, which includes the 5' terminal TATA box, shows 86% homology with human and is an important nucleotide sequence for expression.
本発明のマウスＴＮＦ遺伝子を含むＤＮＡフラグメント
は、概路次のようにして得られる。The DNA fragment containing the mouse TNF gene of the present invention can be obtained generally as follows.
即ち、マウスのＤＮＡを５ａｕ３ＡＩで部分分解したも
のをλファージに組み込み、マウスの遺伝子ライブラリ
ーを作製する。次いで既に得られているウサギＴＮＦ
ｃＤＮＡ （デイ−・エヌ・ニー（ＤＮＡ） 、Ｖｏｌ
５．ｐ１４９ （１９８６））のコーディング領域を
含むＰｓｔＩフラグメントをプローブとして、プラーク
ハイブリダイゼーションを行ないスクリーニングすると
、４個のポジイティブなりローンが単離される。このう
ちの１つを選びＤＮＡを単離し、ＥｃｏＲＩで分解して
、ウサギＴＮＦｃＤＮＡとハイブリダイズする約２．８
ｋｂ及び３′側をコードする０、８ ｋｂのフラグメン
トの全塩基配列をサンガー （Ｓａｎｇｅｒ）法により
決定する。That is, mouse DNA partially digested with 5au3AI is incorporated into λ phage to create a mouse gene library. Next, the already obtained rabbit TNF
cDNA (DNA), Vol.
5. Plaque hybridization was performed and screened using a PstI fragment containing the coding region of p149 (1986) as a probe, and four positive clones were isolated. Select one of these, isolate the DNA, digest with EcoRI, and hybridize with rabbit TNF cDNA. Approximately 2.8
The entire base sequence of the 0.8 kb fragment encoding the 3' side and the 3' side is determined by the Sanger method.
本発明のマウスＴＮＦ遺伝子を含むＤＮＡフラグメント
は、遺伝子発現メカニズムや、制ガンの機構解明に必要
不可欠なものである。The DNA fragment containing the mouse TNF gene of the present invention is essential for elucidating gene expression mechanisms and anticancer mechanisms.
更に本発明によりマウスＴＮＦ遺伝子のＤＮＡ塩基配列
が決定されたことから、発現に重要な領域の存在が判り
、今後の研究に寄与するところが大きい。この部分は、
リポポリサッカライド（ＬＰＳ）、丁ＰＡ（１２−０−
テトラデカノイルホルボール−１３−アセテート）等に
よるＴＮＦ誘導に関与する部分であり、このプロモータ
ー領域だけをとり出し下流に発現させたい物質をコード
する遺伝子を連結すれば、誘導可能な発現ベクターとな
る。Furthermore, since the DNA sequence of the mouse TNF gene has been determined according to the present invention, the existence of an important region for expression has been determined, which will greatly contribute to future research. This part is
Lipopolysaccharide (LPS), Ding PA (12-0-
This is the part involved in TNF induction by eg (tetradecanoylphorbol-13-acetate), etc., and if only this promoter region is removed and a gene encoding the substance to be expressed downstream is linked, it becomes an inducible expression vector. .
つまり、動物細胞での発現において細胞自身が、発現を
狙っている物質によって成長がおさえられたり、毒性を
与えられたりする時、まず細胞を増殖させた後、ＬＰＳ
等で誘導をかければ目的とする物質を大量に得ることが
できるのである。In other words, in the case of expression in animal cells, when the cells themselves are inhibited from growth or rendered toxic by the substance they are aiming to express, the cells are first grown, then LPS
By applying induction using methods such as these, it is possible to obtain a large amount of the desired substance.
又、動物細胞でＴＮＦを発現させると、イントロン３（
第２図の１９７８−１９８５の塩基配列）にウィルスの
エンハンサ−配列が存在し、さらにイントロンｌ （第
２図の１２２７−１２３４及び１３６９−１３７６の塩
基配列）、イントロン２（第２図の１６６４−１６７１
及−５＝
び１６７３−１６８０の塩基配列）、３′末端（第２図
の３０３１−３０３８の塩基配列）にも存在することか
ら、ＴＮＦのｃＤＮＡを組込んだものよりも高い発現を
得ることができる。Furthermore, when TNF is expressed in animal cells, intron 3 (
There is a viral enhancer sequence in the base sequence 1978-1985 in Figure 2), intron 1 (base sequence 1227-1234 and 1369-1376 in Figure 2), and intron 2 (base sequence 1664-1364 in Figure 2). 1671
-5= and nucleotide sequence 1673-1680) and at the 3' end (nucleotide sequence 3031-3038 in Figure 2), it is possible to obtain higher expression than when incorporating TNF cDNA. Can be done.
〔実施例１〕
マウス遺伝子ライブラリーは、マウスＤＮＡをＳａｕ３
Ａ Ｉで部分的に切断し、λファージ（Ｃｈａｒｏｎ２
８）に組込んだ物を用いた。マウスＴＮＦ遺伝子クロー
ニングの為のプローブは、本発明者らが先に分離したウ
サギＴＮＦｃＤＮＡのＰｓｔＩ断片をラベル化して作製
した。６Ｘ１０’個のプラークを４０％のホルムアミド
存在下ベントン（Ｂｅｎｔｏｎ）とデーヴイス（Ｄａｖ
ｉｓ）の方法（サイエンス（Ｓｃｉｅｎｃｅ）Ｕ四１８
０−１８２（１９７７））に従ってプラークハイブリダ
イゼーションを行ない、４つのホジティブクローンを得
た。このうちの１つからＤＮＡを抽出し、ＥｃｏＲＩで
切断した。上記のプローブを用いハイブリダイズした２
．８ｋｂのフラグメント及びマウスＴＮＦｃＤＮＡの３
′非翻訳領域と相補的なオリゴヌクレオチド、５ ′−
ＡＧＧＡＡＴＧＧＡＣＡＴＴＣＧＡＧＧＣＴ−３′とハ
イブリダイズする０、８ ｋｂをサブクローニングし、
塩基配列を決定した。その結果を第１図に制限酵素地図
と塩基配列決定手法、第２図にＤＮＡ塩基配列を示した
。この塩基配列とベニ力（Ｐｅｎｎｉｃａ）らの報告（
プロシーデイングズ・オブ・ザ・ナショナル・アカデミ
−・オブ・サイエンシーズ・ニー・ニス・ニーＰｒｏｃ
Ｎａｔｌ、Ａｃａｄ、Ｓｃｉ、 ＵＳＡ ） ８２．
６０６０−６０６４（１９８４））から、第２図の８１
４−９９９．１５１９−１５７３．１７５２−１７９９
゜２０９３−２５０８の塩基配列が構造遺伝子の部分で
あると同定できる。５′非翻訳領域には、３５６−３８
３の位置にＺ型のコンホメーションをとるＡＣの（り返
し配列（第２図の点線部分） 、Ｓｐ １結合部位（５
９Ｂ−６０３） 、ＴＡＴＡ配列は６１Ｂ−６２３（第
２図の下線部分）の位置に確認された。３′非翻訳領域
には、ポリアゾニレ−ジョン暗号ＡＡＴＡＡＡが３２６
０−３２６５ （第２図下線部分）に存在する。さらに
第３図に示す様に、５′非翻訳領域にヒ）ＴＮＦ遺伝子
と高いホモロジーを示す領域が存在し、特に４３３−６
２３は８６％と、遺伝子発現に重要な役割を果しており
、ＬＰＳ等による誘導に関与する。[Example 1] Mouse gene library was created by converting mouse DNA into Sau3
Partially digested with A I, λ phage (Charon2
8) was used. A probe for mouse TNF gene cloning was prepared by labeling the PstI fragment of rabbit TNF cDNA that the present inventors had previously isolated. 6 x 10' plaques were prepared using Benton and Dav in the presence of 40% formamide.
is) method (Science) U418
0-182 (1977)) and four positive clones were obtained. DNA was extracted from one of these and cut with EcoRI. 2 hybridized using the above probe
．． 8 kb fragment and 3 of mouse TNF cDNA
'Oligonucleotide complementary to untranslated region, 5'-
0.8 kb hybridizing with AGGAATGGACATTCGAGGCT-3' was subcloned,
The base sequence was determined. The results are shown in Fig. 1 and the restriction enzyme map and nucleotide sequence determination method, and Fig. 2 shows the DNA nucleotide sequence. This base sequence and the report by Pennica et al.
Proceedings of the National Academy of Sciences
Natl, Acad, Sci, USA) 82.
6060-6064 (1984)), 81 in Figure 2
4-999.1519-1573.1752-1799
The base sequence of ゜2093-2508 can be identified as part of the structural gene. The 5' untranslated region contains 356-38
AC has a Z-shaped conformation at position 3 (repeated sequence (dotted line in Figure 2)
9B-603), the TATA sequence was confirmed at position 61B-623 (underlined portion in Figure 2). In the 3' untranslated region, the polyazonylation code AATAAA is 326
0-3265 (underlined part in Figure 2). Furthermore, as shown in Figure 3, there is a region in the 5' untranslated region that shows high homology with the human) TNF gene, especially 433-6.
23 plays an important role in gene expression, accounting for 86%, and is involved in induction by LPS and the like.
〔実施例２〕
プラスミドｐＳＶ２−ｄｈｆｒ （Ａ Ｔ ＣＣ３７
１４６）をＨｉｎｄＩ［Ｉ及びＢｇｌ ＩＩで完全消化
したベクターを用意する。このベクターはＳＶ４０の初
期プロモーター及びＳＶ４０のターミネータ−を持って
いる。次に第２図に示すＤＮＡをＮａｒ Ｉ 、 Ｅｃ
ｏＲＩで完全消化したのち、ＤＮＡポリメラーゼ（クレ
ノー断片）で平滑末端とし約２００ｂｐを分離する。上
記ベクターも同様に平滑末端とした後、Ｔ４ＤＮＡリガ
ーゼでつなぎ合せ、本発明物質の発現用プラスミドｐＳ
Ｖ２−１’１ＴＮＦを得る。この工程を第４図に示す。[Example 2] Plasmid pSV2-dhfr (AT CC37
A vector is prepared by completely digesting 146) with HindI[I and Bgl II. This vector has an SV40 early promoter and an SV40 terminator. Next, the DNA shown in Figure 2 was transformed into Nar I, Ec
After complete digestion with oRI, about 200 bp are separated into blunt ends using DNA polymerase (Klenow fragment). After making the above vector blunt-ended in the same way, it was ligated with T4 DNA ligase to create a plasmid pS for expressing the substance of the present invention.
V2-1'1 TNF is obtained. This process is shown in FIG.
〔実施例３〕
プラスミドｐＳＶ２−ｄｈｆｒをＰｖｕ ＩＩ及びＢｇ
ｌｌＩで完全消化したベクターを用意する。次に第２図
に示すＤＮＡをＮｃｏ Ｉ 、 Ｎａｒ Ｉで完全消化
した後、ＤＮＡポリメラーゼ（クレノー断片）で平滑末
端とし、約５６０ｂｐの断片をとり出す（第２図の２２
８−７８５）。[Example 3] Plasmid pSV2-dhfr was transformed into Pvu II and Bg
Prepare a vector completely digested with llI. Next, the DNA shown in Figure 2 was completely digested with Nco I and Nar I, and the ends were made blunt with DNA polymerase (Klenow fragment), and a fragment of about 560 bp was taken out (22 in Figure 2).
8-785).
これにγ−ＩＦＮの遺伝子を含むプラスミドｐＧ ｔａ
ｃｌｌｌｏ （ネイチャー（Ｎａｔｕｒｅ）３１３ 、
８０３ （１９８５））をＢｇｌ Ｉ［、Ｓａｌ Ｉで
完全消化し、平滑末端とする。これらをＴ４ＤＮＡリガ
ーゼでつなき合せ、ｐＳＶ２−ＴＰＮとする。この工程
を第５図に示す。Plasmid pGta containing the γ-IFN gene
clllo (Nature 313,
803 (1985)) was completely digested with Bgl I and Sal I to make blunt ends. These are ligated together using T4 DNA ligase to form pSV2-TPN. This process is shown in FIG.
〔実施例４〕
Ｃ０８−１１胞（ＡＴＣＣＣＲＬ １６５０）をダＪレ
ベッコのＭＥＭ培地（フロー・ラボラトリイズ（Ｆｌｏ
ｗＬａｂｏｒａｔｏｒｉｅｓ）社製）を用いて、３７℃
、５％ｃＯ□インキュベータ中で対数増殖期になる迄培
養し、０．１％トリプシン及び０．０２％ＥＤＴＡを用
いて細胞をはがし、ハンクス平衡塩（フロー・ラボラト
リイズ（Ｆｌｏｗ Ｌａｂｏｒａｔｏｒｉｅｓ）社製）
に約１×１０７ｃｅｌｌｓ／ ｍβの濃度になる様に懸
濁する。５μｌの１ ｍＭ ）リス−塩酸緩衝液（ｐＨ
８，０）に約１０μｇの実施例２で作製したｐＳＶ２−
ＭＴＮＦを加えた溶液を１．５ｍｌのエツペンドルフ型
試験管にとり、さらに上述のＣＯ３−１細胞の懸濁液を
約２００μｌ加える。０℃で１０分管放置した後、デイ
−イーピー・システム（ＤＩｌｉＰ ５ＹＳＴＢ旧社製
細胞融合装置ＦＰＨ１００１型のキュベツトに懸濁液を
移し、１．２ＫＶで４０℃秒の条件で２回処理する。こ
の処理後、０℃で５分間放置する。次いでダルベツコの
ＭＥＭ培地を１０ｍ１加え、３７℃、５％ＣＯ□インキ
ューベーター中で培養する。２４時間後に培地を交換し
、さらに２４時間培養を続ける。この培養液のし細胞障
害活性を測定したところ以下の様であった。すなわちｃ
ＤＮＡを組込んだものより、本発明で得られたＤＮＡフ
ラグメントを組込んだものの方が発現量が多いことがわ
かった。[Example 4] C08-11 cells (ATCC CRL 1650) were grown in Da J Rebecco's MEM medium (Flo Laboratories (Flo Laboratories)).
w Laboratories) at 37°C.
The cells were cultured in a 5% cO
Suspend at a concentration of approximately 1 x 107 cells/mβ. 5 μl of 1 mM) Lis-HCl buffer (pH
About 10 μg of pSV2- produced in Example 2
The solution containing MTNF is placed in a 1.5 ml Eppendorf test tube, and approximately 200 μl of the above-mentioned suspension of CO3-1 cells is added thereto. After leaving the tube at 0°C for 10 minutes, the suspension was transferred to a cuvette of DIliP System (formerly DIliP 5YSTB cell fusion device FPH1001 model) and treated twice at 1.2 KV and 40°C for seconds. After treatment, leave at 0°C for 5 minutes. Next, add 10ml of Dulbecco's MEM medium and culture in a 5% CO□ incubator at 37°C. After 24 hours, replace the medium and continue culturing for an additional 24 hours. When the cytotoxic activity of this culture solution was measured, it was as follows: c.
It was found that the expression level was higher in the DNA fragments obtained by the present invention than in the DNA fragments.
Ｌ細胞障害活性
ｐＳＶ２− ｄｈｆｒ ２０ｕ／ｎｕ以下
ｐＳＶ２− ＭＴＮＦ ４ Ｘ １０’ｕ
／ｍ １２ｐＳＶ２− ＭｃＤＮＡ
８ Ｘ１０”ｕ／ｍＩ！（マウスＴＮＦの
ｃＤＮＡ）
〔実施例５〕
実施例４と同様に行なうが、ＣＯ５−１細胞のかわり１
．：ｃ）ｔｏ細胞、ｐＳＶ２−１’１ＴＮＦ（７）かわ
りにｐＳＶ２−ＴＰＮを加え、処理する。２４時間後に
培地を交換した後、ＬＰＳを１０℃ｇ／ｍ １又はＴＰ
ＡをＬｏｎｇ／ｍ Ｉ！の濃度になる様に加え、さらに
２４時間培養を続ける。この培養液をとりインターフェ
ロン活性を測定したところ以下の様であった。すなわち
ＬＰＳで誘導する事により、高発現の系として使える事
がわかった。L cytotoxic activity pSV2- dhfr 20 u/nu or less pSV2- MTNF 4 X 10'u
/m 12pSV2- McDNA
8 X 10”u/mI! (cDNA of mouse TNF) [Example 5] Perform the same as Example 4, but use 1 instead of CO5-1 cells.
．． :c) To cells, add pSV2-TPN instead of pSV2-1'1 TNF (7) and treat. After changing the medium after 24 hours, add LPS at 10°C g/m 1 or TP.
A Long/m I! The culture was continued for an additional 24 hours. When this culture solution was taken and the interferon activity was measured, the results were as follows. In other words, it was found that it can be used as a high expression system by inducing it with LPS.
ＲＩＡ（γ−ＩＦＮ）
ｐＳＶ２− ｄｈｆｒ １０ｕ／ｍｊ！以
下ｐＳＶ２− ＴＰＮ １００ｕ／＋＋
１ｐＳＶ２− ＴＰＮ（＋ＬＰＳ） ６００ｕ／
ｍ＃〔発明の効果〕
以上述べた如く、本発明はマウスＴＮＦ遺伝子を含むＤ
ＮＡフラグメントを初めて提供するものであり、特に５
′非翻訳領域は遺伝子発現に重要であり、その範囲を限
定できた。又、このプロモーターを動物細胞での発現ベ
クターとして使用できた。さらにマウスは実験系が確立
しており、今後の研究に極めて有用である。RIA (γ-IFN) pSV2- dhfr 10u/mj! Below pSV2- TPN 100u/++
1pSV2- TPN (+LPS) 600u/
m# [Effects of the Invention] As described above, the present invention provides D
It is the first to provide NA fragments, especially 5
'Untranslated regions are important for gene expression, and we were able to limit their range. Additionally, this promoter could be used as an expression vector in animal cells. Additionally, mice have an established experimental system and will be extremely useful for future research.
さらに、動物細胞での発現において、ｃ ＤＮＡを組み
込むより（ｐＳＶ２−ＭｃＤＮＡ） 、本発明で得られ
たマウスＴＮＦ遺伝子を含むＤＮＡフラグメント組み込
んだ方が（ｐＳＶ２−ＭＴＮＦ）、高い発現を得る事が
でき、産業上極めて有用である。Furthermore, in expression in animal cells, higher expression can be obtained by integrating the DNA fragment containing the mouse TNF gene obtained in the present invention (pSV2-MTNF) than by integrating cDNA (pSV2-McDNA). , is extremely useful industrially.
第１図は本発明に係るマウスＴＮＦ遺伝子の構造地図で
あり、制限酵素地図である。白で囲った部分は構造遺伝
子を示す。矢印は塩基配列決定の方向を示す。
第２図は本発明に係るマウスＴＮＦ遺伝子の核酸配列で
あり、ＡＣくり返し配列には点線、ＴＡＴＡボックス及
びポリアゾニレ−ジョン暗号には下線を施した。
第３図は本発明に係るマウスＴＮＦ５′非翻訳領域（第
２図の３２８−８２８の塩基配列、上段）とヒトＴＮＦ
５′非翻訳領域のホモロジー（下段）を示したものであ
る。同じ塩基には＊印が施しである。
第４図はマウスＴ Ｎ’Ｆ発現ベクターｐＳＶ２−ＭＴ
ＮＦの構築を示した。
第５図はマウスＴＮＦ遺伝子のプロモータ領域にγ−Ｉ
ＦＮ遺伝子を接続したプラスミド構築を示したものであ
る。
特許出願人 旭化成工業株式会社
第１図FIG. 1 is a structural map and restriction enzyme map of the mouse TNF gene according to the present invention. The area surrounded by white indicates the structural gene. Arrows indicate the direction of base sequencing. FIG. 2 shows the nucleic acid sequence of the mouse TNF gene according to the present invention, in which the AC repeating sequence is marked with a dotted line, and the TATA box and polyazoni region code are marked with an underline. Figure 3 shows the mouse TNF 5' untranslated region (nucleotide sequence 328-828 in Figure 2, upper row) and human TNF according to the present invention.
The homology of the 5' untranslated region (bottom row) is shown. The same bases are marked with *. Figure 4 shows the mouse TN'F expression vector pSV2-MT.
The construction of NF was demonstrated. Figure 5 shows γ-I in the promoter region of the mouse TNF gene.
This figure shows the construction of a plasmid connecting the FN gene. Patent applicant: Asahi Kasei Industries, Ltd. Figure 1
Claims (2)
接した５’末端約８００個の核酸塩基配列を含んで成る
マウスＴＮＦをコードする染色体遺伝子を含むＤＮＡフ
ラグメント(1) A DNA fragment containing a chromosomal gene encoding mouse TNF, comprising an exon, an intron, and a 5'-terminus sequence of about 800 nucleobases adjacent to the exon.
許請求の範囲第（１）項記載のＤＮＡフラグメント(2) The DNA fragment according to claim (1), which has the base sequence shown in FIG.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29184187A JPH01137976A (en) | 1987-11-20 | 1987-11-20 | Novel dna fragment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29184187A JPH01137976A (en) | 1987-11-20 | 1987-11-20 | Novel dna fragment |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01137976A true JPH01137976A (en) | 1989-05-30 |
Family
ID=17774113
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP29184187A Pending JPH01137976A (en) | 1987-11-20 | 1987-11-20 | Novel dna fragment |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01137976A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996010647A1 (en) * | 1994-09-30 | 1996-04-11 | Fuso Pharmaceutical Industries, Ltd. | Probe for diagnosing infectious diseases |
| WO1999043840A1 (en) * | 1998-02-27 | 1999-09-02 | Boehringer Ingelheim Pharmaceuticals, Inc. | Self-regulated apoptosis of inflammatory cells by gene therapy |
| EP0989853A1 (en) * | 1997-02-28 | 2000-04-05 | Boehringer Ingelheim Pharmaceuticals Inc. | Self-regulated apoptosis of inflammatory cells by gene therapy |
-
1987
- 1987-11-20 JP JP29184187A patent/JPH01137976A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996010647A1 (en) * | 1994-09-30 | 1996-04-11 | Fuso Pharmaceutical Industries, Ltd. | Probe for diagnosing infectious diseases |
| EP0989853A1 (en) * | 1997-02-28 | 2000-04-05 | Boehringer Ingelheim Pharmaceuticals Inc. | Self-regulated apoptosis of inflammatory cells by gene therapy |
| EP0989853A4 (en) * | 1997-02-28 | 2004-03-17 | Boehringer Ingelheim Pharma | Self-regulated apoptosis of inflammatory cells by gene therapy |
| WO1999043840A1 (en) * | 1998-02-27 | 1999-09-02 | Boehringer Ingelheim Pharmaceuticals, Inc. | Self-regulated apoptosis of inflammatory cells by gene therapy |
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