JPH01144977A - Novel recombinant plasmid ptpgif2 - Google Patents
Novel recombinant plasmid ptpgif2Info
- Publication number
- JPH01144977A JPH01144977A JP30215487A JP30215487A JPH01144977A JP H01144977 A JPH01144977 A JP H01144977A JP 30215487 A JP30215487 A JP 30215487A JP 30215487 A JP30215487 A JP 30215487A JP H01144977 A JPH01144977 A JP H01144977A
- Authority
- JP
- Japan
- Prior art keywords
- ptpgif2
- coli
- gif
- dhfr
- 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.)
- Granted
Links
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Landscapes
- Peptides Or Proteins (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】
産業上の利用分野
本発明は、成長ホルモン分泌制御因子であるソマトスタ
チン（Ａｌａ−Ｇｌｙ−Ｃｙｓ−Ｌｙｓ−Ａｓｎ−Ｐｈ
ｅ−Ｐｈｅ−Ｔｒｐ−Ｌｙｓ−Ｔｈｒ−Ｐｈｅ−Ｔｈｒ
−５ｅｒ−Ｃｙｓの１４個のアミノ酸配列よりなるペプ
チド、以下、ＧＩＦと略す。）を含む融合タンパク質を
生産可能とする新規組換えプラスミドに関するものであ
る。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to somatostatin (Ala-Gly-Cys-Lys-Asn-Ph), which is a growth hormone secretion regulator.
e-Phe-Trp-Lys-Thr-Phe-Thr
A peptide consisting of a 14-amino acid sequence of -5er-Cys, hereinafter abbreviated as GIF. This invention relates to a novel recombinant plasmid that makes it possible to produce a fusion protein containing the following:
ＧＩＦは、視床下部ペプチドの一種であり、成長ホルモ
ンなど下垂体前葉ホルモンおよびインシュリン、グルカ
ゴンなどの消化管で生産される多くのペプチドホルモン
の分泌を抑制する。このよう作用を有することから、Ｇ
ＩＦは、小人症、糖尿病等の治療薬としての利用が期待
されている。GIF is a type of hypothalamic peptide and suppresses the secretion of many peptide hormones produced in the gastrointestinal tract, such as anterior pituitary hormones such as growth hormone, and insulin and glucagon. Because it has such an effect, G
IF is expected to be used as a therapeutic agent for dwarfism, diabetes, and the like.
本発明の新規組換えプラスミドｐＴＰＧＩＦ２は、第１
図に示されるＤＮＡ配列を有する。ｐＴＰＧ ＩＦ２お
よびｐＴＰＧＩＦ２を含有する大腸菌は２発酵工業、医
薬品工業等の分野に好適である。The novel recombinant plasmid pTPGIF2 of the present invention has the first
It has the DNA sequence shown in the figure. pTPG IF2 and Escherichia coli containing pTPGIF2 are suitable for fields such as fermentation industry and pharmaceutical industry.
従来の技術
本発明Ｍ’、’＋ｉ術的背景としては、いわゆる遺伝子
操作技術がある。ＧＩＦ遺伝子を組み込んだプラスミド
およびその大腸菌での発現に関しては、板倉らの成果が
公知である （に、Ｉｔａｋｕｒａ ｅｔ ａｌ。BACKGROUND OF THE INVENTION The technical background of the present invention M','+i is the so-called gene manipulation technology. Regarding the plasmid incorporating the GIF gene and its expression in E. coli, the results of Itakura et al. are known.
５ｃｉｅｎｃｅ、 ｖｏｌ、１９８．ｐ１０５６（１９
７７））。5science, vol, 198. p1056(19
77)).
一般に９分子量１万以下のポリペプチドは、大腸菌など
の宿主中で生産させても菌体中のプロテアーゼなどによ
って分解されるため安定に細胞内に蓄積されない。これ
は２分子として小さいため安定なコンホメーションをと
れないためであると考えられている。従って、遺伝子操
作を利用してＧＩＦなどの短いポリペプチドを生産しよ
うとした場合、融合遺伝子を作成し、融合タンパク質と
して発現させることが必要である。そのため、板倉らは
、ＧＩＦを暗号化する遺伝子を化学合成いこれをβ−ガ
ラクトシダーゼ遺伝子に融合し、多コピープラスミドに
組み込み得られた組換えプラスミドを大腸菌に導入し、
融合タンパク質として発現させた。融合タンパク質は、
β−ガラクトシダーゼとＧＩＦとをメチオニン（Ｍｅｔ
）残基を介して結合させている。タンパク質をブロムシ
アンで処理することにより、タンパク質中のメチオニン
残基のカルボキシ末端側の結合を特異的に切断すること
ができる。この方法を利用することにより。Generally, polypeptides with a molecular weight of 10,000 or less cannot be stably accumulated in cells even if they are produced in a host such as E. coli because they are degraded by proteases in the bacterial cells. This is thought to be because the two molecules are so small that they cannot maintain a stable conformation. Therefore, when attempting to produce a short polypeptide such as GIF using genetic manipulation, it is necessary to create a fusion gene and express it as a fusion protein. Therefore, Itakura et al. chemically synthesized the gene encoding GIF, fused it to the β-galactosidase gene, integrated it into a multicopy plasmid, and introduced the resulting recombinant plasmid into E. coli.
It was expressed as a fusion protein. The fusion protein is
β-galactosidase and GIF are combined with methionine (Met).
) residues. By treating a protein with bromic cyanide, the carboxy-terminal bonds of methionine residues in the protein can be specifically cleaved. By using this method.
β−ガラクトシダーゼ−ＧＩＦの融合タンパク質からＧ
ＩＦを特異的に切り出すことができる。G from β-galactosidase-GIF fusion protein
IF can be specifically excised.
しかしながら、上記の板倉らの方法は、 （１）大腸菌
で発現した融合タンパク質が不溶化していること、 （
２）β−ガラクトシダーゼがＧＩＦと融合することによ
りその酵素活性を失うこと。などから、ｉｉｉ！Ｉ！合
タンパク質の分離精製に関して問題があった。However, the method of Itakura et al. described above requires that (1) the fusion protein expressed in E. coli is insolubilized;
2) β-galactosidase loses its enzymatic activity by fusing with GIF. From etc., iii! I! There was a problem with the separation and purification of the combined protein.
本発明者らは、この問題を解消するために、枯草菌のジ
ヒドロ葉酸還元酵素遺伝子を利用して。In order to solve this problem, the present inventors utilized the dihydrofolate reductase gene of Bacillus subtilis.
融合タンパク質（以下、 Ｄ ＨＦ Ｒｂｓ Ｇ Ｉ
Ｆと略す。）として発現させることに成功した（特願
昭６２−０７９３７８，６２−０９２８８１）。fusion protein (hereinafter referred to as DHF Rbs GI
Abbreviated as F. ) (Japanese Patent Application No. 62-079378, 62-092881).
その結果に従うと２作られるＤＨＦＲｂ−−−ＧＩＦは
、可溶性の状態で大腸菌面体内に生産され、かつＧＩＦ
と融合してもＤＨＦＲの活性が保持され問題点
しかしながら、上記の本発明者らの方法は２作られるＤ
ＨＦＲｂ−−ＧＩＦの菌体内蓄積量が、菌体タンパク質
のせいぜい数パーセントであり、融合タンパク質を多量
に利用しようと考えた場合。According to the results, DHFRb---GIF produced by 2 is produced in the E. coli hedron in a soluble state, and GIF
However, the above method of the present inventors maintains the activity of DHFR even when fused with
When considering that the amount of HFRb--GIF accumulated in the bacterial body is at most a few percent of the bacterial cell protein, and a large amount of the fusion protein is to be utilized.
生産効率上で問題が考えられた。There was a problem with production efficiency.
発明の目的
本発明の目的は、上記の問題点を解決するために、ＧＩ
Ｆの大量生産を可能にする組換えプラスミドを開発する
ことにある。また２本発明は、遺伝子操作の手法を用い
てＧＩＦを大量に生産する方法の開発の一環として行わ
れたものである。OBJECT OF THE INVENTION An object of the present invention is to solve the above problems by
The objective is to develop a recombinant plasmid that enables mass production of F. Furthermore, the present invention was carried out as part of the development of a method for producing GIF in large quantities using genetic engineering techniques.
既に２本発明者らは（１）大腸菌のＤＨＦＲを大量に発
現する発現プラスミドを構築していること（特願 昭６
０−２１０８１３）、（２）大腸菌のＤＨＦＲのカルボ
キシ末端側の配列を変化させても、枯草菌のＤＨＦＲ同
様酵素活性が失われないこと、（３）大腸菌のＤＨＦＲ
のカルボキシ末端側に異種ペプチドを融合させることを
可能とするプラスミドベクターｐＴＰ７０−１を構築し
ていること（特願 昭６２−３１２８３８）、（４）ｐ
ＴＰ７０−１上の改変ＤＨＦＲは、大腸菌で効率良く発
現すること、を明らかにしている。The present inventors have already (1) constructed an expression plasmid that expresses a large amount of DHFR in E. coli (patent application 1983).
0-210813), (2) Even if the carboxy-terminal sequence of E. coli DHFR is changed, the enzymatic activity is not lost like Bacillus subtilis DHFR, (3) E. coli DHFR
A plasmid vector pTP70-1 has been constructed that enables the fusion of a heterologous peptide to the carboxy-terminal side of
It has been revealed that the modified DHFR on TP70-1 is efficiently expressed in E. coli.
このことを利用し、鋭意研究の結果、ｐＴＰ７０−１を
用いて、ＧＩＦ遺伝子をＤＨＦＲと融合させて発現する
ことにより、上記問題点を解消できることを見いだし、
その知見に従って、ｐＴＰ７０−１にＧＩＦ遺伝子を組
み込んだ組換えプラスミドｐＴＰＧＩＦ２を作成し２本
発明を完成させた。Taking advantage of this fact, as a result of intensive research, we discovered that the above problems could be solved by expressing the GIF gene in a fused form with DHFR using pTP70-1.
Based on this knowledge, the present invention was completed by creating a recombinant plasmid pTPGIF2 in which the GIF gene was integrated into pTP70-1.
発明の構成
第１図は２本発明の組換えプラスミドｐＴＰＧＩＦ２の
全塩基配列を示している。本発明のｐＴＰＧＩＦ２は、
４６６０塩基対の大きさであり。Structure of the Invention FIG. 1 shows the entire base sequence of the recombinant plasmid pTPGIF2 of the present invention. The pTPGIF2 of the present invention is
It has a size of 4660 base pairs.
宿主である大腸菌にトリメトプリムおよびアシビシリン
耐性ｍ、’２ｉ与することができる。ｐＴＰＧＩとε−
Ｆ２は、 Ｅ、ｃｏｌｉ Ｃ６００株に導入されて安定
状態に保たれ、ｐＴＰＧＩＦ２を含有するＥ、 ｃｏｌ
ｉ Ｃ６００株は、微工研にＦＥＲＭ８Ｐ−１５７７と
して寄託されている。It can confer trimethoprim and acivicillin resistance m,'2i to host E. coli. pTPGI and ε-F2 were introduced into E. coli strain C600 and kept stable, and E. coli containing pTPGIF2
The i C600 strain has been deposited with the Microtech Institute as FERM8P-1577.
ｐＴＰＧＩＦ２は、ｐＴＰ７０−１のＢａｍＨＩ切断部
位に、ＧＩＦを暗号化する配列を含む５２塩基対のＤ
Ｎ Ａが挿入した構造である。第１図において、５３３
番目から５８４番目迄の配列が挿入された配列であり、
それ以外の配列がｐＴＰ７０−１の配列と全く同一であ
る。第１図の５７番目から５８１番目の配列は、ｐＴＰ
７０−１の改変ＤＨＦＨのカルボキシ末端側にＧｒＦが
メチオニンを介して結合したＤＨＦＲ−ＧＩＦを暗号化
する。pTPGIF2 contains a 52 base pair D containing a sequence encoding GIF at the BamHI cleavage site of pTP70-1.
This is the structure inserted by NA. In Figure 1, 533
The array from the th to the 584th is the inserted array,
The rest of the sequence is completely identical to that of pTP70-1. The sequence from 57th to 581st in Figure 1 is pTP
GrF is bound to the carboxy terminal side of modified DHFH of 70-1 via methionine to encode DHFR-GIF.
第２図は、 ＤＨＦＲ−ＧＩＦを暗号化する部分のＤ
ＮＡ配列とそれから作られると予想されるタンパク質の
アミノ酸配列を示している。ＤＨＦＲ−ＧＩＦは、１７
５アミノ酸よりなるタンパク質であり、このうちアミノ
末端側から数えて、１から１５９番目までの配列が、大
腸菌の野生型ＤＨＦＲに１箇所アミノ酸置換置換が起こ
った（Ｃｙｓ−１５２（ｗｉｌｄ ｔｙｐｅ） ＋ Ｇ
ｌｕ−１５２）配列であり、１６２番目から１７５番目
までがＧＩＦの配列である。ＧＩＦの配列の直前のアミ
ノ酸はメチオニン（Ｎｅｔ）である。このことにより、
ＤＨＰＲ−ＧＩＦをブロムシアン処理することにより、
ＧＩＦを特異的に切り出すことができる。１６０番目の
イソロイシン（ｌｌｅ）は、ｐＴＰ７０−１のＢａｍＨ
Ｉ部位にＧＩＦを暗号化するＤＮＡを導入する際に、遺
伝暗号の読み取り枠を合わせるために生じた配列である
。ｐＴＰ７０−１が作る改変ＤＨＦＲは、１６２個のア
ミノ酸よりなり、第２図のＤＨＦＲ−ＧＩＦのアミノ酸
配列のうち、アミノ末端側から数えて、１から１６０番
目までの配列に、 Ｇｌｎ−１１ｅの２個のアミノ酸配
列が結合した配列をしている。Figure 2 shows the part D that encrypts DHFR-GIF.
The NA sequence and the amino acid sequence of the protein predicted to be produced from it are shown. DHFR-GIF is 17
It is a protein consisting of 5 amino acids, and the sequence from positions 1 to 159 counting from the amino terminal side has one amino acid substitution in the wild type DHFR of E. coli (Cys-152 (wild type) + G
lu-152) array, and the 162nd to 175th are GIF arrays. The amino acid immediately preceding the GIF sequence is methionine (Net). Due to this,
By subjecting DHPR-GIF to Bromcyan processing,
GIF can be specifically excised. Isoleucine (lle) at position 160 is BamH of pTP70-1.
This sequence was created to match the reading frame of the genetic code when introducing DNA encoding GIF into the I site. The modified DHFR produced by pTP70-1 consists of 162 amino acids, and in the amino acid sequence of DHFR-GIF shown in FIG. It has a sequence in which several amino acid sequences are combined.
ＤＨＦＲ−ＧＩＦは、ｐＴＰ７０−１の改変ＤＨＦＲの
カルボキシ末端側に、ＧＩＦが融合した構造をしている
にもかかわらず、ＤＨＦＲ酵素活性を有する。このため
、大腸菌がＤＨＦＲ−（、ＩＦを多量につくると、ＤＨ
ＦＲの阻害剤であり。DHFR-GIF has a structure in which GIF is fused to the carboxy terminal side of the modified DHFR of pTP70-1, but has DHFR enzyme activity. For this reason, when E. coli produces large amounts of DHFR-(, IF, DHFR-(, IF)
It is an inhibitor of FR.
抗細菌剤でゃＪるトリメトプリムに対して、耐性を示す
ようになる。It becomes resistant to the antibacterial drug trimethoprim.
ＤＨＰＲ−ＧＩＦを暗号化する配列の上流には。Upstream of the sequence encoding DHPR-GIF.
ｐＴＰ７０−１の改変ＤＨＦＲ遺伝子の発現を効率良く
行わせる配列が存在する（特願 昭６ｌ−３１２８３６
）。即ち、４３番目から５０番目までの配列がＳＤ配列
と呼ばれるもので、効率の良い翻訳に、また、４６１８
番目から４６４６番目までが、コンセンサス転写プロモ
ーターであり。There is a sequence that allows efficient expression of the modified DHFR gene of pTP70-1 (Patent application No. 61-312836).
). In other words, the sequence from the 43rd to the 50th is called the SD sequence, and is useful for efficient translation.
The sequence from position 4646 to position 4646 is the consensus transcription promoter.
効率の良い転写に貢献する。また、ｐＴＰ７０−１は、
抗菌剤であるアンピシリンに対して耐性を付与する遺伝
子を有しており、その遺伝子の発現は、ｐＴＰ７０−１
のＢａｍＨＩ部位に異種ＤＮＡが挿入されても影響を受
けない。このことから。Contributes to efficient transcription. In addition, pTP70-1 is
It has a gene that confers resistance to the antibacterial agent ampicillin, and the expression of this gene is controlled by pTP70-1.
Even if foreign DNA is inserted into the BamHI site of , it will not be affected. From this.
ｐＴＰ７０−１のＢａｍＨＩ部位に、ＧＩＦを暗号化す
るＤＮＡ配列が挿入した構造をしているｐＴＰＧＩＦ２
は、大腸菌に導入された場合、多量のＤＨＦＲ−ＧＩ
Ｆを作る。作られたＤＨＦＲ−ＧＩＦは、菌体内に可溶
性の状態で、菌体タンパク質の１５〜２０％にいたるま
で蓄積する。このことによって、ｐＴＰＧＩＦ２を有す
る大腸菌はトリメトプリム耐性を示すようになる。また
、ｐＴＰＧＩＦ２は、ｐＴＰ７０−１由来のアンピシリ
ン耐性を付与する遺伝子を有することから、ｐＴＰＧＩ
Ｆ２が導入された大腸菌は、アンピシリン耐性をも示す
。pTPGIF2 has a structure in which a DNA sequence encoding GIF is inserted into the BamHI site of pTP70-1.
When introduced into E. coli, a large amount of DHFR-GI
Make F. The produced DHFR-GIF accumulates in the bacterial body in a soluble state up to 15 to 20% of the bacterial cell protein. As a result, E. coli having pTPGIF2 becomes resistant to trimethoprim. Furthermore, since pTPGIF2 has a gene that confers ampicillin resistance derived from pTP70-1, pTPGI
E. coli into which F2 has been introduced also exhibits resistance to ampicillin.
このような特長を有するｐＴＰＧＩＦ２は、実施例に従
って作成することができるが１組換えプラスミドの作成
方法によって本発明が制限されるものではない。pTPGIF2 having such features can be constructed according to the Examples, but the present invention is not limited to the method for constructing a single recombinant plasmid.
次に本発明の実施例および参考例を示す。Next, examples and reference examples of the present invention will be shown.
実施例 ｐＴＰＧＩＦ２の作成 ＧＩＦを暗号化するＤＮＡとしては。Example Creation of pTPGIF2 As for the DNA that encodes GIF.
１、５’−ＧＡＴＣＡＴＧＧＣＴＧＧＣＴＧＴＡＡＡＡ
ＡＣＴＴＣＴＴＣＴＧＧＡＡＡＡＣＣＴＴＣＡＣＴＴＣ
ＡＴＧＣＴＡＡ−３’
２、５’−ＧＡＴＣＴＴＡＧＣＡＴＧＡＡＧＴＧＡＡＧ
ＧＴＴＴＴＣＣＡＧＡＡＧＡＡＧＴＴＴＴＴＡＣＡＧＣ
ＣＡＧＣＣＡＴ−３’
ホアミダイト法に従って化学合成し、精製後、ポリヌク
レオチドキナーゼを用いて、各ＤＮＡの５′末端をリン
酸化した。 リン酸化したＤＮＡを約０．１ｍ１（約
０．１ｄｇのＤＮＡを含んでいる。）ずつ取り、これを
６０’Ｃでインキュベートすることによって両ＤＮＡを
アニールさせた（これをＤＮＡＩと呼ぶ）。1,5'-GATCATGGCTGGCTGTAAAA
ACTTCTTCTGGAAAACCCTTCACTTC
ATGCTAA-3'2,5'-GATCTTAGCATGAAGTGAAG
GTTTTCCAGAAGAAGTTTTTACAGC
CAGCCAT-3' was chemically synthesized according to the phoamidite method, and after purification, the 5' end of each DNA was phosphorylated using polynucleotide kinase. Approximately 0.1 ml (containing approximately 0.1 dg of DNA) of the phosphorylated DNA was taken and incubated at 60'C to anneal both DNAs (this is referred to as DNAI).
ＧＩＦを暗号化したＤＮＡを組み込むベクターとしては
、ｐＴＰ７０−１を用いた（特願 昭６ｌ−３１２８３
６）。 約１μｇのｐＴＰ７０−１を、ＢａｍＨＩで
切断した後、アルカリホスファターゼ処理をした。アル
カリホスファターゼ処理したＤＮＡをフェノール処理す
ることにより、共存する酵素タンパク質を変性除去し、
その後エタノールでＤＮＡを沈澱させた。沈澱したＤＮ
Ａを７０％エタノールで洗った後、エタノールを除き。pTP70-1 was used as a vector to incorporate the GIF-encoded DNA (patent application Sho 6l-31283).
6). Approximately 1 μg of pTP70-1 was cleaved with BamHI and then treated with alkaline phosphatase. By treating the alkaline phosphatase-treated DNA with phenol, the coexisting enzyme protein is denatured and removed.
The DNA was then precipitated with ethanol. precipitated DN
After washing A with 70% ethanol, remove the ethanol.
減圧下に沈澱を乾燥させた。ＢａｍＨＩによるＤＮＡの
切断、アルカリホスファターゼ処理、フェノール処理、
およびエタノール沈澱の各操作は。The precipitate was dried under reduced pressure. DNA cleavage with BamHI, alkaline phosphatase treatment, phenol treatment,
and each operation of ethanol precipitation.
いずれも、 ”Ｍｏ１ｅｃｕｌａｒ Ｃｌｏｎｉｎｇ
Ａ ＬｏｂｏｒａｔｏｒｙＭａｎｕａ ｌ”（Ｔ、Ｍａ
ｎｉａｔｉｓ、 Ｅ、Ｆ、Ｆｒ１ｔｓｃｈ、Ｊ、Ｓａ
ｍｂｒｏｏｋ。In both cases, “Mo1ecular Cloning”
A Laboratory Manual” (T, Ma
niatis, E.F., Frltsch, J.Sa.
mbrook.
ｅｄｓ、 Ｃｏ１ｄ Ｓｐｒｉｎｇ ）ｌａｒｂｏｒ
Ｌａｂｏｒａｔｏｒｙ （１９８２）、以下２文献１と
呼ぶ。）に記載している方法に従って行った。乾燥させ
たＤＮＡを５０μｍのリガーゼ用反応液（１０ｍＭ Ｔ
ｒｉｓ−ＨｃＩ、ｐＨ７，４，５ｍＭ ＭｇＣｌ２．１
０ｍＭジチオトレイトール、５ ｍＭ ＡＴＰ）に溶解
後、５μｍのＤＮＡＩを加え、これに１ユニツトの７４
−ＤＮＡリガーゼを加えて、１５°Ｃで、４時間ＤＮＡ
の連結反応を行わせた。この反応物を、形質転換法（ｔ
ｒａｎｓｆｏｒｍａｔｉｏｎ ｍｅｔｈｏｄ、上記文献
１に記載）に従って、大腸菌に取り込ませた。この処理
をした菌体を、５０ｍｇ／ｌのアンピシリンナトリウム
および１０ｍｇ／Ｉのトリメトプリムを含む栄養寒天培
地（培地ｌｉ中に、２ｇのグルコース＋ Ｉｇのリン
酸２カリウム、５ｇのイーストエキスｔ ５ ｇのポリ
ペプトン、１５ｇの寒天を含む。）上に塗布し、３７°
Ｃで２４時間培養することにより、約５００のコロニー
を得ることができた。これらのコロニーから適当に８個
選び、１．５ｍｌのＹＴ＋Ａｐ培地（培８ｇのトリプト
ン、５０ｍｇのアンピシリンナトリウムを含む。）で、
３７°Ｃ，１晩、菌体を培養した。培養液を、各々エッ
ペンドルフ遠心管にとり。eds, Co1d Spring) labor
Laboratory (1982), hereinafter referred to as Reference 2. ) according to the method described. The dried DNA was mixed with a 50μm ligase reaction solution (10mM T
ris-HcI, pH 7, 4, 5mM MgCl2.1
After dissolving in 0mM dithiothreitol, 5mM ATP), 5μm of DNAI was added and 1 unit of 74
- Add DNA ligase and incubate the DNA at 15°C for 4 hours.
A ligation reaction was performed. This reaction product was transformed using the transformation method (t
transformation method, described in Document 1 above). The treated bacterial cells were transferred to a nutrient agar medium containing 50 mg/l ampicillin sodium and 10 mg/l trimethoprim (in medium li, 2 g glucose + Ig dipotassium phosphate, 5 g yeast extract, 5 g polypeptone, containing 15 g of agar) and 37°
Approximately 500 colonies could be obtained by culturing in C for 24 hours. Appropriately select 8 colonies from these colonies and incubate them in 1.5 ml of YT+Ap medium (containing 8 g of tryptone and 50 mg of ampicillin sodium).
The bacterial cells were cultured at 37°C overnight. Transfer each culture solution to an Eppendorf centrifuge tube.
１２．０００回転／分で１０分間遠心分離し、菌体を沈
澱として集めた。これに、０．１ｍｌの電気泳動用サン
プル調製液（０，０６２５ＭのＴｒｉｓ−ＨＣＩ、 ｐ
ｔ（６，８，２χのラウリル硫酸ナトリウム（ＳＤＳ）
。The mixture was centrifuged at 12,000 rpm for 10 minutes, and the bacterial cells were collected as a precipitate. To this, add 0.1 ml of electrophoresis sample preparation solution (0,0625 M Tris-HCI, p
t(6,8,2χ Sodium Lauryl Sulfate (SDS)
.
１０％のグリセリン、５χの２−メルカプトエタノール
。10% glycerin, 5x 2-mercaptoethanol.
０．００１％のブロムフェノールブルーを含む。）を加
え２面体を懸濁し、これを沸騰水中に５分間保ち２面体
を溶かした。この処理をしたサンプルを５ＤＳ−ポリア
クリルアミドゲル電気泳動法（Ｕ、Ｋ。Contains 0.001% bromophenol blue. ) was added to suspend the dihedron, and this was kept in boiling water for 5 minutes to dissolve the dihedron. The treated samples were subjected to 5DS-polyacrylamide gel electrophoresis (U, K).
Ｌａｍｍ１ｉ； Ｎａｔｕｒｅ、 ｖｏｌ、２２７．
ｐ、６８０（１９７０））に従って分析した。標準サン
プルとしてｐＴＰ７０−１を含有する大腸菌に同様な処
理をしたもの、および分子量マーカーとしてラクトアル
ブミン（分子量１４．２００）、 ）リブシンインヒ
ビター（分子量２０、ｔｏｏ） 、 ）リブシノーアン
（分子量２４，０００） 、カルボニックアンヒドラー
ゼ（分子ｆｆ１２９，０００） 、グリ七〇アルデヒド
３−リン酸デヒドロゲナーゼ（分子量３６，０００）
、卵アルブミン（分子量４５，０００） 。Lammli; Nature, vol, 227.
p., 680 (1970)). As a standard sample, Escherichia coli containing pTP70-1 was treated in the same way, and as molecular weight markers, lactalbumin (molecular weight 14.200), ) ribsin inhibitor (molecular weight 20, too), ) ribsinoan (molecular weight 24,000) , Carbonic anhydrase (molecular weight 129,000), Glyseptaldehyde 3-phosphate dehydrogenase (molecular weight 36,000)
, egg albumin (molecular weight 45,000).
および牛血清アルブミン（分子ｆｆ１６６．０００）を
含むサンプルをポリアクリルアミド濃度の１０から２０
％濃度勾配ゲルで泳動した。その結果、８個のコロニー
のうち、３個ではｐＴＰ７０−１のＤＨＦＲのバンドが
消失し、それより明らかに分子量が大きくなったタンパ
ク質（分子量的２３ 、０００と推定される。）を新た
に生産していること、残りの５個のコロニーは、ｐＴＰ
７０−１のＤＨＦＲとほぼ同じ大きさのタンパク質を生
産すること、ｐＴＰ７０−１のＤＨＦＲ（分子量１８，
３７９）は、この条件で分子量的２１ 、０００のタン
パク質として泳動することが明らかになった。分子量の
大きい新たなタンパク質を生産するコロニーのうちから
適当に一つ選び、これをＹＴ＋Ａｐ培地で培養し、Ｔａ
ｎａｋａとＷｅｉｓｂｌｕｍの方法（Ｔ、Ｔａｎａｋａ
、 Ｂ、Ｗｅｉｓｂｌｕｍ； Ｊ。and bovine serum albumin (molecule ff 166.000) with a polyacrylamide concentration of 10 to 20.
% gradient gel. As a result, in three of the eight colonies, the DHFR band of pTP70-1 disappeared, and a new protein with a clearly larger molecular weight (estimated to have a molecular weight of 23,000) was produced. The remaining 5 colonies are pTP
DHFR of pTP70-1 (molecular weight 18,
379) was found to migrate as a protein with a molecular weight of 21,000 under these conditions. Select one of the colonies that produce a new protein with a large molecular weight and culture it in YT+Ap medium.
naka and Weisblum's method (T, Tanaka
, B. Weisblum; J.
Ｂａｃｔｅｒｉｏｌｏｇｙ、 ｖｏｌ、１２１．ｐ、３
５４（１９７５））に従って。Bacteriology, vol, 121. p, 3
54 (1975)).
プラスミドを調製した。得られたプラスミドをｐＴＰＧ
ＩＦ２と名づけだ。ｐＴＰＧＩＦ２は、ｐＴ Ｐ ７
０−１籍Ｂ ａｍＨＩ部位に合成りＮＡが挿□□ｉ−
人された構造をしているはずであるので、ｐ’ｒｐＧＩ
Ｆ２をＥｃｏＲＩと５ａｌＩによる切断によって得られ
る約４００ヌクレオチド長のＤＮＡについて９Ｍ１３フ
アージを用いたジデオキシ法（Ｊ。A plasmid was prepared. The obtained plasmid was transformed into pTPG.
It is named IF2. pTPGIF2 is pTP7
Since it should have a structure in which a synthetic NA is inserted at the amHI site, p'rpGI
Dideoxy method using 9M13 phage (J.
Ｍｅｓｓｉｎｇ； Ｍｅｈｔｏｄｓ ｉｎ Ｅｎｚｙｍ
ｏｌｏｇｙ、 ｖｏｌ、１０１．ｐ、２０（１９８３）
）に従って塩基配列を決定した。その結果。Messing; Methods in Enzym
ology, vol, 101. p, 20 (1983)
) The base sequence was determined according to the following method. the result.
第１図に示すｐＴＰＧＩＦ２の全塩基配列の４７１番目
から８８２番目の配列が明らかにされた。The 471st to 882nd sequences of the entire base sequence of pTPGIF2 shown in FIG. 1 were revealed.
ｐＴＰ７０−１の塩基配列は９本発明者らによりて明ら
かにされている（特願 昭６ｌ−３１２８３６）。ｐＴ
ＰＧＩＦ２のＥｃｏＲＩ−ＳａｌＩの配列は、ｐＴＰ７
０−１のＥｃｏＲＩ−ＳａｌＩの配列に間にあるＢａｍ
ＨＩ部位に、５２ヌクレオチドのＤＮＡ（ＧＩＦを暗号
化する配列として設計・合成した配列）が結合した配列
であった。The nucleotide sequence of pTP70-1 has been revealed by the present inventors (Japanese Patent Application No. 61-312836). pT
The EcoRI-SalI sequence of PGIF2 is pTP7
Bam between the EcoRI-SalI sequence of 0-1
It was a sequence in which 52 nucleotides of DNA (a sequence designed and synthesized as a sequence encoding GIF) was bound to the HI site.
また、ｐＴＰＧＩＦ２のＥｃＯＲｌ−５ａ ｌ Ｉ切断
によって得られる約４．２キロ塩基対のＤＮＡは、Ｐｓ
ｔＩ、ＨｉｎｄＩＩＩ、Ｈｐａｌ、ＡａｔＩ Ｉ、 Ｐ
ｖｕ Ｉ Ｉ、 Ｂｇｌ Ｉ Ｉ、およびＣ１ａＩを用
いた制限酵素による切断実験の結果、ｐ’ｒｐ７０−１
のＥｃｏＲＩ−５ａｌｌ切断によって得られる約４．２
キロ塩基対のＤＮＡと全く同一であることが示された。In addition, approximately 4.2 kilobase pairs of DNA obtained by EcORl-5a l I cleavage of pTPGIF2 is Ps
tI, HindIII, Hpal, AatII, P
As a result of restriction enzyme cleavage experiments using vuII, BglII, and C1aI, p'rp70-1
4.2 obtained by EcoRI-5all cleavage of
It was shown to be completely identical to kilobase pair DNA.
以上の結果から、ｐＴＰＧＩＦ２の全塩基配列が第１図
に示した配列であることが明らかである。From the above results, it is clear that the entire base sequence of pTPGIF2 is the sequence shown in FIG.
参考例
ｐＴＰＧＩＦ２を有する大腸菌の作るＤＨＦＲ−ＧＩＦ
融合タンパク質の精製
ｐＴＰＧＩＦ２を有する大腸菌を１．５１のＹＴ＋Ａｐ
培地で３７°Ｃで一晩培養後、菌体を遠心分離により集
めた。湿重量約６ｇの菌体が得られた。Reference example DHFR-GIF produced by E. coli having pTPGIF2
Purification of fusion protein Escherichia coli harboring pTPGIF2 was incubated with 1.51 YT+Ap.
After culturing in medium at 37°C overnight, the bacterial cells were collected by centrifugation. Bacterial cells with a wet weight of about 6 g were obtained.
菌体を２０ｍ１の１ｍＭのジチオトレイトール（ＤＴＴ
）および０．１ｍＭのエチレンジアミン４酢酸２ナトリ
ウム（ＥＤＴＡ）を含む１０ｍＭリン酸緩衝液ｐＨ７，
０（以下、緩衝液１）に懸濁し。The bacterial cells were mixed with 20ml of 1mM dithiothreitol (DTT).
) and 10 mM phosphate buffer pH 7 containing 0.1 mM disodium ethylenediaminetetraacetate (EDTA),
0 (hereinafter referred to as buffer 1).
フレンチプレスを用いて面体を破砕した。菌体破砕液を
、２０，０００回転／分、１時間の遠心分はとんど全て
が上清中に回収された。このことからＤＨＦＲ−ＧＩＦ
は、可溶性の状態で菌体中に蓄積していると考えられる
。得られた上清中のＤＨＦＲ活性は、１５２０ユニツト
であった。上清なあらかじめ緩衝液１で平衡化したＤＥ
ＡＥ−）コパール６５０Ｍカラム（２，５００ｍｍ ｘ
１，５００．ｍｍ、、約７５０ＴＩ＋３）に吸着させ
、Ｏ，ＩＭのＫＣＩを含む緩衝液１で洗った後、緩衝液
１中で、０．１Ｍから０゜３ＭのＫＣＩの濃度勾配をか
け、タンパク質を溶出させた。約６ｍｌずつのフラクシ
ョンを、フラクションコレクターで集め、各フラクショ
ンについて、ＤＨＦＲの酵素活性を調べ、活性を有する
フラクションを集めた。約４０ｍ１の酵素液が得られた
。回収されたＤＨＦＲ酵素活性は、１，０６５ユニツト
（約７０％）であった。回収酵素液を。The face piece was crushed using a French press. Almost all of the cell suspension obtained by centrifugation at 20,000 rpm for 1 hour was recovered in the supernatant. From this, DHFR-GIF
is thought to accumulate in the bacterial cells in a soluble state. The DHFR activity in the obtained supernatant was 1520 units. Supernatant DE equilibrated in advance with buffer 1
AE-) Copal 650M column (2,500mm x
1,500. After washing with buffer 1 containing KCI of O, IM, a concentration gradient of KCI from 0.1 M to 0.3 M was applied in buffer 1 to elute the protein. Ta. Fractions of approximately 6 ml each were collected using a fraction collector, each fraction was examined for DHFR enzyme activity, and fractions with activity were collected. Approximately 40 ml of enzyme solution was obtained. The DHFR enzyme activity recovered was 1,065 units (approximately 70%). recovered enzyme solution.
アミコン限外ろ過装置を用いて約１ｍｌにまで濃縮し、
これをトヨパールＨＷ５５カラムクロマトグラフィーに
より分画した。約２．５ｍｌずつのフラクションを、フ
ラクションコレクターで集め。Concentrate to approximately 1 ml using an Amicon ultrafiltration device,
This was fractionated by Toyopearl HW55 column chromatography. Collect fractions of approximately 2.5 ml each using a fraction collector.
各フラクションについて、ＤＨＦＲの酵素活性と２８０
ｎｍの吸光度を調べた。各フラクションについて、Ｄ
ＨＦＲの酵素活性を２８０ｎｍの吸光度で割った値を計
算し、一定の値（約３０）を示すフラクションを集めた
。４１０ユニツト（約２７％）、約２３ ｍ ｇの酵素
が回収された。For each fraction, the enzymatic activity of DHFR and 280
The absorbance at nm was examined. For each fraction, D
The value of the HFR enzyme activity divided by the absorbance at 280 nm was calculated, and fractions showing a constant value (approximately 30) were collected. 410 units (about 27%), about 23 mg of enzyme were recovered.
得られた酵素タンパク質をＳＤＳ電気泳動法（上記実施
例に記載の方法）により分析したところ。The obtained enzyme protein was analyzed by SDS electrophoresis (method described in the above example).
約２３ 、０００の単一なタンパク質バンドが示され、
得られた酵素標品が均一であることが示された。Approximately 23,000 single protein bands are shown;
It was shown that the obtained enzyme preparation was homogeneous.
精製したＤＨＦＲ活性を示すタンパク質をエンザイムイ
ムノアッセイにより検討したところ、ＧＩＦに対する抗
体と反応することが示された。即ち、精製して得られた
タンパク質は免疫学的にＧＩＦと同等の構造を有するこ
とが明らかとなった。When the purified protein exhibiting DHFR activity was examined by enzyme immunoassay, it was shown to react with an antibody against GIF. That is, it was revealed that the purified protein had a structure immunologically equivalent to GIF.
精製して得られたタンパク質のカルボキシ末端側のアミ
ノ酸配列を明らかにするために、カルボキシペプチダー
ゼＹを、精製タンパク質に時間を変化させて作用させ、
遊離してくるアミノ酸を定量した（カルボキシペプチダ
ーゼ法によるカルボ−−Ｔｒｐ−Ｌｙｓ−Ｔｈｒ−Ｐｈ
ｅ−Ｔｈｒ−５ｅｒ−（カルボキシ末端）であることが
予想された。ＤＨＦＲ−Ｇ ＩＦのカルボキシ末端のア
ミノ酸はＣｙｓ （システィン）であるが、このアミノ
酸は上記方法では検出できない。In order to clarify the amino acid sequence of the carboxy-terminal side of the purified protein, carboxypeptidase Y was applied to the purified protein at varying times.
The released amino acids were quantified (carbo-Trp-Lys-Thr-Ph by carboxypeptidase method).
It was expected to be e-Thr-5er- (carboxy terminal). The carboxy-terminal amino acid of DHFR-G IF is Cys (cystine), but this amino acid cannot be detected by the above method.
Ｅｌｌｍａｎの方法を用いて、５，５ξジチオビス２−
ニトロ安息香酸（ＤＴＮＢ）を用いて、精製タンパク質
中のチオール（ＳＨ基）の含料を測定したところ、２．
６〜２．９残基／酵素分子という値が得られた。ｐＴＰ
７０−ＩＤＨＦＲについて同様に測定したところ、０．
８〜１．０残基／酵素分子という値であった。Using the method of Ellman, 5,5ξdithiobis2-
When the content of thiol (SH group) in purified protein was measured using nitrobenzoic acid (DTNB), 2.
Values of 6-2.9 residues/enzyme molecule were obtained. pTP
70-IDHFR was similarly measured and found to be 0.
The value was 8 to 1.0 residues/enzyme molecule.
以上の結果は、ｐＴＰＧＩＦ２を有する大腸菌から精製
して得られたＤＨＦＲ活性を有するタンパク質が、ＤＨ
ＦＲ−ＧＩＦであることを示している。The above results indicate that the protein with DHFR activity obtained by purification from E. coli harboring pTPGIF2 is
This indicates that it is FR-GIF.
発明の効果
上記のように、新規組換えプラスミドｐＴＰＧＩＦ２は
、ＤＨＦＲ−ＧＩＦを暗号化しており。Effects of the Invention As described above, the novel recombinant plasmid pTPGIF2 encodes DHFR-GIF.
つｐ’ｒｐｃｌＦ２を有する大腸菌は、ＤＨＦＲ−ＧＩ
Ｆを可溶性の状態で大量に蓄積生産する。さらに、生成
したＤＨＦＲ−Ｇ ＩＦは、ＤＨＦＲ酵素活性を示し、
精製を容易に行うことができる。Escherichia coli with p'rpclF2 has DHFR-GI
Accumulates and produces large amounts of F in a soluble state. Furthermore, the generated DHFR-G IF exhibits DHFR enzyme activity,
Purification can be easily performed.
このような性質を有することから２本発明の新規組換え
プラスミドｐＴＰＧＩＦ２およびそれを有する大腸菌は
、ＤＨＦＲ−ＧＩＦの生産、およびそれを利用したＧＩ
Ｆの生産に有益である。Because of these properties, the novel recombinant plasmid pTPGIF2 of the present invention and E. coli containing it are suitable for the production of DHFR-GIF and for GI using it.
It is beneficial for the production of F.
第１図は、ｐＴＰＧＩＦ２の全塩基配列を示した図であ
り、２本鎖ＤＮＡのうち片方のＤＮＡ鎖配列だけを、５
′末端から３′末端の方向に記述している。図中符号は
、核酸塩基を表し、Ａはアデニンを、Ｃはシトシンを、
Ｇはグアニンを、Ｔはチミンを示している。図中番号は
、ｐ’ｒｐｃｒＦ２に２箇所存在する制限酵素Ｃ１ａｌ
切断認識部位のうち制限酵素ＨｉｎｄＩＩＩ切断部位に
近い方のＣ１ａｌ切断認識部位の、５’ −ＡＴＣＧＡ
Ｔ−３’ 、の最初の”Ａ”を１番として数えたＲ−Ｇ
Ｉ Ｆを暗号化する部分の塩基配列およびタンパク質
のアミノ酸配列を示す図である。図中符号は、核酸塩基
およびアミノ酸を表し、Ａはアデニンを、Ｃはシトシン
を、Ｇはグアニンを、Ｔはチミンを、Ａｌａはアラニン
を、Ａｒｇはアルギニンを、Ａｓｎはアスパラギンを、
Ａｓｐはアスパラギン酸を、Ｃｙｓはシスティンを、Ｇ
ｌｎはグルタミンを、Ｇｌｕはグルタミン酸を、Ｇｌｙ
はグリシンを、Ｈｉｓはヒスチジンを、Ｉｌｅはイソロ
イシンを、Ｌｅｕはロイシンを、Ｌｙｓはリジンを、Ｍ
ｅｔはメチオニンを、Ｐｈｅはフェニルアラニンを、Ｐ
ｒｏはプロリンを、Ｓｅｒはセリンを、Ｔｈｒはトレオ
ニンを、Ｔｒｐはトリプトファンを、Ｔｙｒはチロシン
を、Ｖａｌはバリンを示している。図中番号は、１番目
のアミノ酸であるメチオニンを暗号化するＡＴＧコドン
の”Ａ”を１番として数えた番号を示している。
ＡＴＣＧＡＴＧＴＴＡ ＡＣＡＧＡＴＣＴＡＡＡＣＴＴ
ＣＣＡＴＧＡ ＴＣＡＧＴＣＴＧＡＴｌｌｏ
１２０
ＣＡＴＧＧＡＡＡＡＣＧＣＣＡＴＧＣＣＧＴＡＡＣＧＣ
ＡＡＣＡＣＣＴＴＡＡＡＴＡＡＡＴＣＡＡＴＣＧＧＴＣ
ＧＴＣＣＧＴＴＧＣＣＡＣＣＧＧＧＴＡＣＧ ＧＡＣＧ
ＡＴＣＧＣＧＴＣＧＣＧＧＣＧＴＧ ＴＧＧＴＧＡＣＧ
ＴＡＧＴＴＴＡＴＧＡＡＣＡＧＴＴＣＴＴＧＣＣＧＣＴ
ＴＡＡＣＴＡＡ ＣＴＡＡＣＴＣＣＧＧ ＡＡＡＡＧＧ
ＡＧＧＡＴＧＣＧＧＣＧＴＴＡ ＧＣＧＧＴＡＧＡＴＣ
ＧＣＧＴＴＡＴＣＧＧＧＧＡＡＣＣＴＧＣＣＴＧＣＣＧ
ＡＴＣＴＣＧＣＣＴＧＧＴＴＴＡＣＣＣＧＴＧＡＴＴＡ
ＴＧＧＧＣＣＧＣＣＡ ＴＡＣＣＴＧＧＧＡＡＡＧＧ
ＡＣＧＣＡＡＡ ＡＡＴＡＴＴＡＴＣＣＴＣＡＧＣＡＧ
ＴＣＡＣＧＡＣＧＣＡＧＡＡ ＧＴＧＧＡＡＧＧＣＧＡ
ＣＴＧＧＧＡＡＴＣＧＧＴＡＴＴＣＡＧＣＣＡＣＡＧＣ
ＴＡＴＧ ＡＧＴＴＣＧＡＡＡＴＡＡＡＣＴＴＣＴＴＣ
ＴＧＧＡＡＡＡＣＣＴＡＴＧＡＴＣＣＴＣＴ ＡＣＧＣ
ＣＧＧＡＣＧ６６０ ６７Ｃ
ＴＧＣＧＧＴＴＧＣＴ ＧＧＣＧＣＣＴＡＴハフ１０
７２Ｃ
ＣＴＣＧＣＣＡＣＴＴ ＣＧＧＧＣＴＣＡＴ（１第
１ 図 の １
ＣＴＴＧＣＧＧＣＧＧ ＣＧＧＴＧＣＴＣＡＡ ＣＧＧ
ＣＣＴＣＡＡＣＡＡＴＧＣＡＧＧＡＧ ＴＣＧＣＡＴＡ
ＡＧＧ ＧＡＧＡＧＣＧＴＣＧＴＣＡＡＣＣＣＡＧＴ
ＣＡＧＣＴＣＣＴＴＣＣＧＧＴＧＧＧＣＧＣＧＣＡＣＴ
ＴＡＴＧＡ ＣＴＧＴＣＴＴＣＴＴ ＴＡＴＣＡＴＧＣ
ＡＡｌｏｌｏ １０２０ １０３０
ＡＧＣＧＣＴＣＴＧＧ ＧＴＣＡＴＴＴＴＣＧ ＧＣＧ
ＡＧＧＡＣＣＧＴＧＡＴＣＧＧＣＣＴ ＧＴＣＧＣＴＴ
ＧＣＧ ＧＴＡＴＴＣＧＧＡＡｌｌｌｏ １
１２０ １１３０ＧＣＣＴＴＣＧＴＣＡ ＣＴ
ＧＧＴＣＣＣＧＣＣＡＣＣＡＡＡＣＧＴＴＡＴＣＧＣＣ
ＧＧＣＡＴＧＧＣＧＧＣＣＧ ＡＣＧＣＧＣＴＧＧＧＣ
ＴＡＣＴＡＣＴＧＧ ＧＣＴＧＣＴＴＣＣＴＡＣＣＧＡ
ＴＧＣＣＣＴＴＧＡＧＡＧＣＣＴＧＧＧＧＣＡＴＧＡＣ
ＴＡＴＣＧＴＣＧＣＣＣＴＣＧＴＡＧＧＡＣＡＧＧＴＧ
ＣＣＧＧＣＣＴＴＴＣＧＣＴＧＧ ＡＧＣＧＣＧＡＣＧ
ＡＴＣＴＴＧＣＡＣＧＣＣＣＴＣＧＣＴＣＡＡＴＴＣＧ
ＧＣＧＡＧＡ ＡＧＣＡＧＧＣＣＡＴＣＴＡＣＧＴＣＴ
ＴＧ ＣＴＧＧＣＧＴＴＣＧＣＧＡＣＧＣＧＡＧＧ Ｃ
ＴＧＧＡＴＧＧＣＣＴＴＣＣＣＣＡＴＴＡＧＧＣＡＴＣ
ＧＧＧＡ ＴＧＣＣＣＧＣＧＴＴ ＧＣＡＧＧＣＣＡＴ
ＧＣＧＡＣＣＡＴＣＡＧ ＧＧＡＣＡＧＣＴＴＣＡＡＧ
ＧＡＴＣＧＣＴＣＴＴＣＧＡＴＣＡＣＴＧＧＡＣＣＧＣ
ＴＧ ＡＴＣＧＴＣＡＣＧＧＡＧＣＡＣＡＴＧＧＡ Ａ
ＣＧＧＧＴＴＧＧＣＡＴＧＧＡＴＴＧＴＡ １ＣＴＧＣ
ＣＴＣＣＣＣＧＣＧＴＴＧＣＧＴＣＧＣＧＧＴＧＣＡＴ
Ｇ・ＧＡＡＴＧＧＡＡＧＣＣＧＧＣＧＧＣＡＣＣＴＣＧ
ＣＴＡＡＣＧＧ。
第 １ 図
ＴＧＡＴＴＣＴＴＣＴ ＣＧＣＴＴＣＣＧＧＣＣＴＧＴ
ＣＣＡＧＧＣＡＧＧＴＡＧＡＴＧＡＣＧＣＧＧＣＴＣＴ
Ｔ ＡＣＣＡＧＣＣＴＡＡＣＧＡＴＴＴＡＴＧＣＣＧＣ
ＣＴＣＧＧＣＧＧＧＣＧＣＣＧＣＣＣＴＡＴＡＣＣＴＴ
ＧＴＧＡＧＣＣＧＧＧＣＣＡＣＣＴＣＧＡＣＣＴＡＴＴ
ＣＡＣＣＡＣＴ ＣＣＡＡＧＡＡＴＴＧの ２
ＣＡＧＡＡＣＡＴＡＴ ＣＣＡＴＣＧＣＧＴＣＣＧＣＣ
ＡＴＣＴＣＣＣＴＣＧＧＧＣＡＧＣＧＴＴＧＧＧＴＣＣ
Ｔ ＧＧＣＣＡＣＧＧＧＴＣＧＴＴＧＡＧＧＡＣＣＣＧ
ＧＣＴＡＧＧＣＴＧＧＣＧＧＧＧＴＴＧＡＡＴＣＡＣＣ
ＧＡ ＴＡＣＧＣＧＡＧＣＧ ＡＡＣＧＴＧＡＡＧＣＴ
ＧＣＧＡＣＣＴＧＡ ＧＣＡＡＣＡＡＣＡＴ ＧＡＡＴ
ＧＧＴＣＴＴＧＴＣＴＧＧＡＡＡＣＧＣＧＧＡＡＧＴＣ
Ａ ＧＣＧＣＣＣＴＧＣＡＡＴＣＧＣＡＧＧＡＴ ＧＣ
ＴＧＣＴＧＧＣＴ ＡＣＣＣＴＧＴＧＧＡＧＡＡＧＣＧ
ＣＴＧＧ ＣＡＴＴＧＡＣＣＣＴ ＧＡＧＴＧＡＴＴＴ
ＴＡＧＣＡＧＣＣＧＣＡ ＣＧＣＧＧＣＧＣＡＴＧＣＧ
ＣＡＴＧＡＴＣＧＴＧＣＴＣＣＴＧＴＧＣＣＴＴＡＣＴ
ＧＧ ＴＴＡＧＣＡＧＡＡＴＧＡＣＴＧＣＴＧＣＴ Ｇ
ＣＡＡＡＡＣＧＴＣＣＧＧＴＴＴＣＣＧＴ ＧＴＴＴＣ
ＧＴＡＡＡＣＣＡＴＴＡＴＧＴＴ ＣＣＧＧＡＴＣＴＧ
ＣＡＣＡＣＣＴＡＣＡＴ ＣＴＧＴＡＴＴＡＡＣＴＣＴ
ＣＴＧＧＴＣＣＣＧＣＣＧＣＡＴＣＣＣＡＧＧＧＣＧＣ
ＧＴ ＣＡＧＣＧＧＧＴＧＴ・ＧＴＣＡＣＧＴＡＧＣＧ
ＡＴＡＧＣＧＧＡＧＧＣＡＧＡＴＴＧＴＡ ＣＴＧＡＧ
ＡＧＴＧＣＧＣＧＴＡＡＧＧＡＧ ＡＡＡＡＴＡＣＣＧ
ＣＧＡＣＴＣＧＣＴＧＣＧＣＴＣＧＧＴＣＧＴＡＡＡＧ
ＧＣＧＧＴＡ ＡＴＡＣＧＧＴＴＡＴＡＣＡＴＧＴＧＡ
ＧＣＡＡＡＡＧＧＣＣＡＧＴＴＧＣＴＧＧＣＧＴ ＴＴ
ＴＴＣＣＡＴＡＧＴＣＧＡＣＧＣＴＣＡ ＡＧＴＣＡＧ
ＡＧＧＴ １ＴＧＧＣＧＧＧＴＧＴ ＣＧＧＧＧＣＧＣ
ＡＧ ＣＣＡＴＧＡＣＣＣＡＴＧＴＡＴＡＣＴＧＧ Ｃ
ＴＴＡＡＣＴＡＴＧ ＣＧＧＣＡＴＣＡＧＡＡＣＣＡＴ
ＡＴＧＣＧ ＧＴＧＴＧＡＡＡＴＡ ＣＣＧＣＡＣＡＧ
ＡＴＡＴＣＡＧＧＣＧＣＴ ＣＴＴＣＣＧＣＴＴＣＣＴ
ＣＧＣＴＣＡＣＴＴＣＧＧＣＴＧＣＧＧ ＣＧＡＧＣＧ
ＧＴＡＴ ＣＡＧＣＴＣＡＣＴＣＣＣＡＣＡＧＡＡＴＣ
ＡＧＧＧＧＡＴＡＡＣＧＣＡＧＧＡＡＡＧＡＣＡＡＡＡ
ＧＧＣＣＡ ＧＧＡＡＣＣＧＴＡＡ ＡＡＡＧＧＣＣＧ
ＣＧＧＣＴＣＣＧＣＣＣＣＣＣＴＧＡＣＧＡＧＣＡＴＣ
ＡＣＡＡＡＡＡＧＧＣＧＡＡＡＣＣＣＧＡＣＡＧＧＡＣ
ＴＡ ＴＡＡＡＧＡＴＡＣＣすＯｎ＾ つ
Ｏｎ八 〇ｎｎ凸ｔｏｏｕ−−ｔｏｔｕ
ＣＣＧＣＴＴＡＣＣＧ ＧＡＴＡＣＣＴＧＴＣ＋ＴＴＣ
ＴＣＡＡＴＧＣＴＣＡＣＧＣＴＧＴＡ ＋ＣＣＡＡＧＣ
ＴＧＧＧ ＣＴＧＴＧＴＧＣＡＣ＋ＴＴＡＴＣＣＧＧＴ
Ａ ＡＣＴＡＴＣＧＴＣＴ゛ＧＣＣＡＣＴＧＧＣＡ Ｇ
ＣＡＧＣＣＡＣＴＧ １第
乙ＯＯυ 乙ｏ’ｔｕ 乙
ブＶすＣＧＣＣＴＴＴＣＴＣＣＣＴＴＣＧＧＧＡＡ Ｇ
ＣＧＴＧＧＣＧＣＴＧＧＴＡＴＣＴＣＡＧ ＴＴＣＧＧ
ＴＧＴＡＧ ＧＴＣＧＴＴＣＧＣＴＧＡＡＣＣＣＣＣＣ
Ｇ ＴＴＣＡＧＣＣＣＧＡ ＣＣＧＣＴＧＣＧＣＣ丁Ｇ
ＡＧＴＣＣＡＡＣＣＣＧＧＴＡＡＧＡＣＡＣＧＡＣＴＴ
ＡＴＣＧＴＡＡＣＡＧＧＡＴ ＴＡＧＣＡＧＡＧＣＧ
ＡＧＧＴＡＴＧＴＡＧＡＡＧＴＧＧＴＧＧＣＣＴＡＡＣ
ＴＡＣＧＧ ＣＴＡＣＡＣＴＡＧＡｌ 図 の
４
３８６一
ＡＧＧＡＣＡＧＴＡＴ ＴＴＧＧＴＡＴＣＴＧ ＣＧＣ
ＴＣＴＧＣＴＧＡＡＧＡＧＴＴＧＧＴ ＡＧＣＴＣＴＴ
ＧＡＴ ＣＣＧＧＣＡＡＡＣＡＧＴＴＴ丁ＴＴＴＧＴ
ＴＴＧＣＡＡＧＣＡＧ ＣＡＧＡＩ″ＴＡＣＧＣＡ
ＡＧＣＣＡＧＴＴＡ ＣＣＴＴＣＧＧＡＡＡＡＡＣＣＡ
ＣＣＧＣＴ ＧＧＴＡＧＣＧＧＴＧＧＣＡＧＡＡＡＡＡ
Ａ ＡＧＧＡＴＣＴＣＡＡＧＡＣＧＣＴＣＡＧＴ ＧＧ
ＡＡＣＧＡＡＡＡＡＴＣＡＡＡＡＡＧＧ ＡＴＣＴＴＣ
ＡＣＣＴＡＡＴＣＡＡＴＣＴＡ ＡＡＧＴＡＴＡＴＡＴ
ＴＴＡＡＴＣＡＧＴＧ ＡＧＧＣＡＣＣＴＡＴＡＧＴＴ
ＧＣＣＴＧＡ ＣＴＣＣＣＣＧＴＣＧＣＡＴＧＧＴＴＡ
ＴＧ ＧＣＡＧＣＡＣＴＧＣｒＧＡＴＧＣＴＴＴＴＣＴ
ＧＴＧＡＣＴＧＧＴ １ＴＧＴＡＴＧＣＧＧＣＧＡＣＣ
ＧＡＧＴＴＧ ＋ＣＧＣＧＣＣＡＣＡＴ ＡＧＣＡＧＡ
ＡＣＴＴ“ＣＧＧＧＧＣＧＡＡＡ ＡＣＴＣＴＣＡＡＧ
ＧノＴＡＡＣＣＣＡＣＴＣＧＴＧＣＡＣＣＣＡＡ １Ｃ
ＧＴＴＴＣＴＧＧＧ ＴＧＡＧＣＡＡＡＡＡ ＋ＴＡＡ
ＧＧＧＣＧＡＣＡＣＧＧＡＡＡＴＧＴ −ＡＡ１八
ＡＡり八
へＴＡＡＴＴＣＴＣＴ ＴＡＣＴＧＴＣＡＴＧ Ｃ
ＣＡＴＣＣＧＴＡＡＥＡＧＴＡＣＴＣＡＡ ＣＣＡＡＧ
ＴＣＡＴＴ ＣＴＧＡＧＡＡＴＡＧＣＴＣＴＴＧＣＣＣ
Ｇ ＧＣＧＴＣＡＡＣＡＣＧＧＧＡＴＡＡＴＡＣｒＡＡ
ＡＡＧＴＧＣＴ ＣＡＴＣＡＴＴＧＧＡ ＡＡＡＣＧＴ
ＴＣＴＴへＴＣＴＴＡＣＣＧＣＴＧＴＴＧＡＧＡＴＣＣ
ＡＧＴＴＣＧＡＴＧ：ＴＧＡＴＣＴＴＣＡ ＧＣＡＴＣ
ＴＴＴＴＡ ＣＴＴＴＣＡＣＣＡＧ：ＡＧＧＡＡＧＧＣ
Ａ ＡＡＡＴＧＣＣＧＣＡ ＡＡＡＡＡＧＧＧＡＡｒＧ
ＡＡＴＡＣＴＣＡ ＴＡＣＴＣＴＴＣＣＴ ＴＴＴＴＣ
ＡＡＴＡＴＡＡ″）（Ｉ ＡＡＡｒＩ
ＡＡｒ：、ｎ（＋ＬＩト晶％ＪＬ＋
礪・１ト一一二、υＴＡＴＴＧＡＡＧＣＡ ＴＴＴＡＴ
ＣＡＧＧＧ ”ＡＴＧＴＡＴＴＴＡＧ ＡＡＡＡＡＴＡ
ＡＡＣ７ＡＡＧＴＧＣＣＡＣＣＴＧＡＣＧＴＣＴＡＡ
１ＡＡＡＡＡＴＡＧＧＣＧＴＡＴＣＡＣＧＡＧ （ＣＣ
ＧＣＴＣＡＣＡＡ ＴＴＡＡＴＴＣＴＴＧ ７ＴＣＡＴ
ＡＡＧＣＴＴ
第 １Figure 1 is a diagram showing the entire base sequence of pTPGIF2, in which only one DNA strand sequence of the double-stranded DNA is
It is written in the direction from the 'end to the 3' end. The symbols in the figure represent nucleic acid bases, A is adenine, C is cytosine,
G represents guanine and T represents thymine. The numbers in the figure indicate restriction enzyme C1al, which exists in two places in p'rpcrF2.
5'-ATCGA of the C1al cleavage recognition site that is closer to the restriction enzyme HindIII cleavage recognition site.
T-3', R-G counting the first "A" as number 1
FIG. 2 is a diagram showing the base sequence of the portion encoding IF and the amino acid sequence of the protein. The symbols in the figure represent nucleobases and amino acids, A for adenine, C for cytosine, G for guanine, T for thymine, Ala for alanine, Arg for arginine, Asn for asparagine,
Asp stands for aspartic acid, Cys stands for cysteine, G
ln is glutamine, Glu is glutamic acid, Gly
is glycine, His is histidine, Ile is isoleucine, Leu is leucine, Lys is lysine, M
et is methionine, Phe is phenylalanine, P
ro represents proline, Ser represents serine, Thr represents threonine, Trp represents tryptophan, Tyr represents tyrosine, and Val represents valine. The numbers in the figure indicate the numbers starting from "A" of the ATG codon that encodes the first amino acid, methionine. ATCGATGTTA ACAGATCTAAAACTT
CCATGA TCAGTCTGATllo
120 CATGGAAAACGCCATGCCGTAACGC
AACACCTTAAATAAATCAAATCGGTC
GTCCGTTGCCACCGGGTACG GACG
ATCGCGTCGCGGCGTGTGGTGACG
TAGTTTATGAACAGTTCTTGCCGCT
TAACTAA CTAACTCCGG AAAAGG
AGGATGCGGCGTTA GCGGTAGATC
GCGTTATCGGGGAACCTGCCTGCCG
ATCTCGCCTGGTTTACCCGTGATTA
TGGGCCGCCA TACCTGGGAAAGG
ACGCAAAAATATTATCCTCAGCAG
TCACGACGCAGAA GTGGAAGGGCGA
CTGGGAATCGGTATTCAGCCACAGC
TATG AGTTCGAAATAAACTTC
TGGAAAACCTATGATCCTCTACGC
CGGACG660 67C TGCGGTTGCT GGCGCCTA T Huff 10
72C CTCGCCACTT CGGGCTCAT (1 Fig. 1) 1 CTTGCGGCGG CGGTGCTCAA CGG
CCTCAACAATGCAGGAGTCGCATA
AGG GAGAGCGTCGTCAACCCAGT
CAGCTCCTTCCGGTGGGCGCACT
TATGA CTGTCTTCTT TATCATGC
AAlolo 1020 1030
AGCGCTCTGG GTCATTTTCG GCG
AGGACCGTGATCGGCCT GTCGCTT
GCG GTATTCGGAAlllo 1
120 1130GCCTTCGTCA CT
GGTCCCGCCACCAAACGTTATCGCC
GGCATGGCGGCCG ACGCGCTGGGGC
TACTACTGG GCTGCTTCCTACCGA
TGCCCTTGAGAGCCTGGGGCATGAC
TATCGTCGCCCTCGTAGGACAGGTG
CCGGCCTTTCGCTGG AGCGCGACG
ATCTTGCACGCCCTCGCTCAATTCG
GCGAGA AGCAGGCCATCTACGTCT
TG CTGGCGTTCGCGACGCGAGG C
TGGATGGCCTTCCCCCATTAGGCATC
GGGA TGCCCGCGTT GCAGGCCAT
GCGACCATCAG GGACAGCTTCAAG
GATCGCTCTTCGATCACTGGACCGC
TG ATCGTCACGGAGCACATGGA A
CGGGTTGGCATGGATTGTA 1CTGC
CTCCCCGCGTTGCGTCGCGGTGCAT
G.GAATGGAAGCCGGCGGCACCTCG
CTAACGG. Figure 1 TGATTCTTCT CGCTTCCGGCCTGT
CCAGGCAGGTAGATGACGCGGCTCT
T ACCAGCCTAACGATTTATGCCGC
CTCGGCGGGGCGCCGCCCTATAACCTT
GTGAGCCGGGCCACCTCGACCTATT
CACCACT CCAAGAATTG 2 CAGAACATAT CCATCGCGTCCGCC
ATCTCCCTCGGGCAGCGTTGGGTCC
T GGCCACGGGTCGTTGAGGACCCG
GCTAGGCTGGCGGGGTTGAATCACC
GA TACGCGAGCG AACGTGAAGCT
GCGACCTGA GCAACAACAT GAAT
GGTCTTGTCTGGAAACGCGGAAGTC
A GCGCCCTGCAATCGCAGGAT GC
TGCTGGCT ACCCTGTGGAGAAGCG
CTGG CATTGACCCT GAGTGATTT
TAGCAGCCGCA CGCGGCGCATGCG
CATGATCGTGCTCCTGTGCCTTACT
GG TTAGCAGAATGACTGCTGCT G
CAAAACGTCCGGTTTCCGT GTTTC
GTAAACCATTATGTT CCGGATCTG
CACACCTACAT CTGTATTAACTCT
CTGGTCCCGCCGCATCCCAGGGCGC
GT CAGCGGGTGT・GTCACGTAGCG
ATAGCGGAGGCAGATTGTACTGAG
AGTGCGCGTAAGGAGAAAATACCG
CGACTCGCTGCGCTCGGTCGTAAAG
GCGGTAATACGGTTACATGTGA
GCAAAAAGGCCAGTTGCTGGCGT TT
TTCCATAGTCGACGCTCAAGTCAG
AGGT 1TGGCGGGTGT CGGGGCGC
AG CCATGACCCATGTATACTGG C
TTAACTATG CGGCATCAGAACCAT
ATGCG GTGTGAAAATA CCGCACAG
ATATCAGGCGCTCTTCCGCTTCCT
CGCTCACTTCGGCTGCGG CGAGCG
GTAT CAGCTCACTCCCACAGAATC
AGGGGATAACGCAGGAAAGACAAA
GGCCA GGAACCGTAA AAAGGCCG
CGGCTCCGCCCCCCTGACGAGCATC
ACAAAAAAGGCGAAAACCCGACAGGAC
TA TAAAGATACC On^ Tsu On eight 〇nn convex toou--totu CCGCTTACCG GATACCTGTC+TTC
TCAATGCTCACGCTGTA +CCAAGC
TGGG CTGTGTGCAC+TTATCCGGT
A ACTATCGTCT゛GCCACTGGCA G
CAGCCACTG 1st OOOυ O'tu O'tu VsuCGCCTTTCTCCCTTCGGGAA G
CGTGGCGCTGGTATCTCAG TTCGG
TGTAG GTCGTTCGCTGAACCCCCC
G TTCAGCCCGA CCGCTGCGCC ding G
AGTCCAAACCCGGTAAGACACGACTT
ATCGTAACAGGAT TAGCAGAGCG
AGGTATGTAGAAGTGGTGGCCTAAAC
TACGG CTACACTAGAL Figure
4 386-AGGACAGTAT TTGGTATCTG CGC
TCTGCTGAAGAGTTGGT AGCTCTT
GAT CCGGCAAACAGTTT
TTGCAAGCAG CAGAI″TACGCA
AGCCAGTTTA CCTTCGGAAAAAACCA
CCGCT GGTAGCGGTGGCAGAAAAAA
A AGGATCTCAAAGACGCTCAGTGG
AACGAAAAATCAAAAAGG ATCTTC
ACCTAATCAATCTAAAGTATAATAT
TTAATCAGTGAGGCACCTATAGTT
GCCTGA CTCCCCGTCGCATGGTTA
TG GCAGCACTGCrGATGCTTTTTCT
GTGACTGGT 1TGTATGCGGCGACC
GAGTTG +CGCGCCACAT AGCAGA
ACTT “CGGGGCGAAA ACTCTCAAG
GNOTAACCCACTCGTGCACCCAA 1C
GTTTCTGGG TGAGCAAAAAA+TAA
GGGCGACACGGAAATGT -AA18
AA Rihachi TAATTCTCT TACTGTCATG C
CATCCGTAAEAGTACTCAA CCAAG
TCATT CTGAGAATAGCTCTTGCCC
G GCGTCAAACACGGGATAATACrAA
AAGTGCT CATCATTGGA AAACGT
To TCTTTCTTACCGCTGTTGAGATCC
AGTTCGATG:TGATCTTCA GCATC
TTTTA CTTTCACCAG:AGGAAGGC
AAAATGCCGCAAAAAAAGGGAArG
AATACTCA TACTCTTCCT TTTTC
AATATAA'') (IAAArI
AAr:,n(+LITocrystal%JL+
礪 1to112, υTATTGAAGCA TTTAT
CAGGG ”ATGTATTTAG AAAATA
AAC7AAGTGCACCTGACGTCTAA
1AAAAATAGGCGTATCACGAG (CC
GCTCACAA TTAATTCTTG 7TCAT
AAGCTT 1st
Claims (1)
にトリメトプリム耐性およびアンピシリン耐性を与える
ことができ、トリメトプリム耐性を付与する遺伝子が大
腸菌のジヒドロ葉酸還元酵素遺伝子の３′末端側の配列
が改変されたことによりジヒドロ葉酸還元酵素−ソマト
スタチン融合タンパク質を暗号化し、４６６０塩基対の
大きさを有し、第１図において示されるＤＮＡ配列を有
する新規組換えプラスミドｐＴＰＧＩＦ２。 ２、特許請求範囲第１項記載の新規組換えプラスミドｐ
ＴＰＧＩＦ２を含有するＥ．ｃｏｌｉＣ６００株。[Scope of Claims] 1. The gene that confers trimethoprim resistance and ampicillin resistance can be stably replicated in E. coli and can confer trimethoprim resistance and ampicillin resistance to the host E. coli, and the gene conferring trimethoprim resistance is located at the 3' end of the E. coli dihydrofolate reductase gene. A novel recombinant plasmid pTPGIF2 encodes a dihydrofolate reductase-somatostatin fusion protein by modifying the sequence thereof, has a size of 4660 base pairs, and has the DNA sequence shown in FIG. 2. Novel recombinant plasmid p described in claim 1
E. containing TPGIF2. coli strain C600.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30215487A JPH01144977A (en) | 1987-11-30 | 1987-11-30 | Novel recombinant plasmid ptpgif2 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30215487A JPH01144977A (en) | 1987-11-30 | 1987-11-30 | Novel recombinant plasmid ptpgif2 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01144977A true JPH01144977A (en) | 1989-06-07 |
| JPH0371112B2 JPH0371112B2 (en) | 1991-11-12 |
Family
ID=17905561
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP30215487A Granted JPH01144977A (en) | 1987-11-30 | 1987-11-30 | Novel recombinant plasmid ptpgif2 |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01144977A (en) |
Cited By (8)
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| EP1645631A2 (en) * | 1998-05-01 | 2006-04-12 | Chiron Corporation | Neisseria antigens and compositions |
| US8563006B2 (en) | 2001-10-11 | 2013-10-22 | Wyeth Holdings Corporation | Immunogenic compositions for the prevention and treatment of meningococcal disease |
| US8574597B2 (en) | 2006-12-22 | 2013-11-05 | Wyeth Llc | Immunogenic compositions for the prevention and treatment of meningococcal disease |
| US10183070B2 (en) | 2017-01-31 | 2019-01-22 | Pfizer Inc. | Neisseria meningitidis compositions and methods thereof |
| US10196429B2 (en) | 2012-03-09 | 2019-02-05 | Pfizer Inc. | Neisseria meningitidis composition and methods thereof |
| US10512681B2 (en) | 2010-09-10 | 2019-12-24 | Wyeth Llc | Non-lipidated variants of Neisseria meningitidis ORF2086 antigens |
| US10888611B2 (en) | 2015-02-19 | 2021-01-12 | Pfizer Inc. | Neisseria meningitidis compositions and methods thereof |
| US10899802B2 (en) | 2013-09-08 | 2021-01-26 | Pfizer Inc. | Neisseria meningitidis compositions and methods thereof |
-
1987
- 1987-11-30 JP JP30215487A patent/JPH01144977A/en active Granted
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|---|---|---|---|---|
| US9249198B2 (en) | 1998-05-01 | 2016-02-02 | Glaxosmithkline Biologicals Sa | Neisseria meningitidis antigens and compositions |
| EP1645631B1 (en) * | 1998-05-01 | 2007-10-24 | Novartis Vaccines and Diagnostics, Inc. | Neisseria antigens and compositions |
| US7576176B1 (en) | 1998-05-01 | 2009-08-18 | Novartis Vaccines And Diagnostics, Inc. | Neisseria meningitidis antigens and compositions |
| US7988979B2 (en) | 1998-05-01 | 2011-08-02 | J. Craig Venter Institute, Inc. | Neisseria meningitidis antigens and compositions |
| US8524251B2 (en) | 1998-05-01 | 2013-09-03 | J. Craig Venter Institute, Inc. | Neisseria meningitidis antigens and compositions |
| EP1645631A2 (en) * | 1998-05-01 | 2006-04-12 | Chiron Corporation | Neisseria antigens and compositions |
| US9266929B2 (en) | 1998-05-01 | 2016-02-23 | Glaxosmithkline Biologicals Sa | Neisseria meningitidis antigens and compositions |
| US9249196B2 (en) | 1998-05-01 | 2016-02-02 | Glaxosmithkline Biologicals Sa | Neisseria meningitidis antigens and compositions |
| US9139621B2 (en) | 1998-05-01 | 2015-09-22 | Glaxosmithkline Biologicals Sa | Neisseria meningitidis antigens and compositions |
| US8563006B2 (en) | 2001-10-11 | 2013-10-22 | Wyeth Holdings Corporation | Immunogenic compositions for the prevention and treatment of meningococcal disease |
| US8563007B1 (en) | 2001-10-11 | 2013-10-22 | Wyeth Holdings Corporation | Immunogenic compositions for the prevention and treatment of meningococcal disease |
| US11116829B2 (en) | 2001-10-11 | 2021-09-14 | Wyeth Holdings Llc | Immunogenic compositions for the prevention and treatment of meningococcal disease |
| US10300122B2 (en) | 2001-10-11 | 2019-05-28 | Wyeth Holdings Llc | Immunogenic compositions for the prevention and treatment of meningococcal disease |
| US8574597B2 (en) | 2006-12-22 | 2013-11-05 | Wyeth Llc | Immunogenic compositions for the prevention and treatment of meningococcal disease |
| US11077180B2 (en) | 2010-09-10 | 2021-08-03 | Wyeth Llc | Non-lipidated variants of Neisseria meningitidis ORF2086 antigens |
| US10512681B2 (en) | 2010-09-10 | 2019-12-24 | Wyeth Llc | Non-lipidated variants of Neisseria meningitidis ORF2086 antigens |
| US10829521B2 (en) | 2012-03-09 | 2020-11-10 | Pfizer Inc. | Neisseria meningitidis composition and methods thereof |
| US10550159B2 (en) | 2012-03-09 | 2020-02-04 | Pfizer Inc. | Neisseria meningitidis composition and methods thereof |
| US11472850B2 (en) | 2012-03-09 | 2022-10-18 | Pfizer Inc. | Neisseria meningitidis composition and methods thereof |
| US10196429B2 (en) | 2012-03-09 | 2019-02-05 | Pfizer Inc. | Neisseria meningitidis composition and methods thereof |
| US11680087B2 (en) | 2013-09-08 | 2023-06-20 | Pfizer Inc. | Neisseria meningitidis compositions and methods thereof |
| US10899802B2 (en) | 2013-09-08 | 2021-01-26 | Pfizer Inc. | Neisseria meningitidis compositions and methods thereof |
| US10888611B2 (en) | 2015-02-19 | 2021-01-12 | Pfizer Inc. | Neisseria meningitidis compositions and methods thereof |
| US10183070B2 (en) | 2017-01-31 | 2019-01-22 | Pfizer Inc. | Neisseria meningitidis compositions and methods thereof |
| US10813989B2 (en) | 2017-01-31 | 2020-10-27 | Pfizer Inc. | Neisseria meningitidis compositions and methods thereof |
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| US11730800B2 (en) | 2017-01-31 | 2023-08-22 | Pfizer Inc. | Neisseria meningitidis compositions and methods thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0371112B2 (en) | 1991-11-12 |
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| EXPY | Cancellation because of completion of term |