JPS63237795A - Production of apo lipoprotein - Google Patents
Production of apo lipoproteinInfo
- Publication number
- JPS63237795A JPS63237795A JP7114687A JP7114687A JPS63237795A JP S63237795 A JPS63237795 A JP S63237795A JP 7114687 A JP7114687 A JP 7114687A JP 7114687 A JP7114687 A JP 7114687A JP S63237795 A JPS63237795 A JP S63237795A
- Authority
- JP
- Japan
- Prior art keywords
- gene
- dna
- apoa
- vector
- plasmid
- 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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- WPWRNMZBLPVTAW-UHFFFAOYSA-N dichloromethane;2,2,2-trichloroacetic acid Chemical compound ClCCl.OC(=O)C(Cl)(Cl)Cl WPWRNMZBLPVTAW-UHFFFAOYSA-N 0.000 description 1
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- 238000013508 migration Methods 0.000 description 1
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- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- CCVKPWUMYBYHCD-UHFFFAOYSA-N oxolane;pyridine Chemical compound C1CCOC1.C1=CC=NC=C1 CCVKPWUMYBYHCD-UHFFFAOYSA-N 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
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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
- C07K14/775—Apolipopeptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は遺伝子工学的手法によるアポリポタンパク質、
殊にじトアポＡ−ｎの製造法に関しており、より詳しく
は化学合成したアポリポタンパク貿遺伝子を挿入したプ
ラスミド組換体（アポリポタンパク質発現ベクター）で
形質転換された形質転換体の培養による上記タンパク質
の製造方法に関している。[Detailed Description of the Invention] Industrial Application Field The present invention relates to apolipoproteins produced by genetic engineering techniques.
In particular, it relates to a method for producing ditoapoA-n, and more specifically, the production of the above protein by culturing a transformant transformed with a plasmid recombinant (apolipoprotein expression vector) into which a chemically synthesized apolipoprotein trade gene has been inserted. It's about the method.
従来の挟装
アポリポタンパク質とは、血漿リポタンパク質より脂質
部分を除いたタンパク質部分で必って、リポタンパク質
の構造と機能を支配する重要な構成タンパク質である。Conventional sandwiched apolipoproteins are the protein parts of plasma lipoproteins with the lipid part removed, and are important constituent proteins that control the structure and function of lipoproteins.
該アポリポタンパク質は脂質を結合して可溶性を保ち、
また脂質の運搬に役立つものでおり、これには例えばＡ
−Ｉ、△−■、Ｂ、Ｃ−Ｉ、Ｃ−■、Ｃ−■、Ｄ、Ｅ等
が知られている。之等のうちで特にヒトアポＡ−ｎは、
ヒトの肝臓、小腸等で合成され、これは下式（Ａ＞に示
す一次構造のアミノ酸配列（Ｎ末端がピロリドンカルボ
ン酸で、Ｃ末端がグルタミンであるアミノ酸残基数７７
個、分子量約８７００＞を有するポリペプチド鎖が、そ
の６位のシスティンでジスルフィド結合した二量体の構
造でおることが知られている。The apolipoprotein binds lipids to remain soluble,
It also helps transport lipids, such as A
-I, Δ-■, B, C-I, C-■, C-■, D, E, etc. are known. Among these, human apoAn is particularly
It is synthesized in the human liver, small intestine, etc., and has the amino acid sequence of the primary structure shown in the following formula (A> (the number of amino acid residues is 77, with the N-terminus being pyrrolidone carboxylic acid and the C-terminus being glutamine).
It is known that a polypeptide chain having a molecular weight of about 8,700 is in the form of a dimeric structure in which the cysteine at position 6 is disulfide bonded.
ＰＣＡ−Ａｌａ−ＩＪｓ−Ｇｌｕ−Ｐｒｏ−Ｃｙｓ−Ｖ
ａｌ−Ｇｌｕ−３ｅｒ−Ｌｅｕ Ｖａｔ ３ｅｒ Ｇｌ
ｎ Ｔｙｒ Ｐｈｅ Ｇｌｎ Ｔｈｒ Ｖａｌ−Ｔｈｒ
−Ａｓｏ−Ｔｙｒ−Ｇｌｙ−Ｌｙｓ−Ａｓｐ−Ｌｅｕ−
Ｍｅｔ−Ｇｌｕ−ＬＶＳ Ｖａｔ ＬＶＳ Ｓｅｒ Ｐ
ｒｏ ＧＩＩＪ−ＬｅＬＪ Ｇｉｎ ’△ｌａ−（３１
ｕ−Ａｌａ（ｙｓ−３ｅｒ−Ｔｙｒ−ｐｈｅ−Ｑｌｕ−
１ｙｓ−３ｅｒ−ｌ−ｙｓ−Ｇｌｕ−Ｇｌｎ−１−ｅｕ
−Ｔｈｒ−Ｐｒｏ−１ｅｕ−１１ｅ−ＬＶＳ−ＩＪＳ−
Ａｌａ−Ｇｌｙ−Ｔｈｒ−（３ｔｕ −１−ｅｕ−Ｖａ
ド△５ｎ−ｐｈｅ−Ｌｅｕ−３ｅｒ−Ｔｙｒ−Ｐｈｅ−
Ｖａｌ−Ｇｌｕ−Ｌ ｅｕ−Ｇｌｙ−Ｔｈｒ−Ｇｌｎ−
Ｐｒｏ−Ａｌａ−Ｔｈｒ−Ｇｉｎ−ＣＯＯＨ（Ａ＞上記
式（Ａ＞に示すポリペプチド鎖を有するアポＡ−ＩＩは
、血漿中高比重りボタンバク質（ＨＤＬ）の主要構成タ
ンパクの一つで、アポＡ−工と共にＨＤＬ中に存在する
ことは知られているが、その特有の生理的機能の詳細は
いまだ解明されていない。しかして、該アポＡ−ｎにつ
いては、高脂血症患者における血中濃度の増加傾向や、
肝疾患（例えば急性肝炎、慢性肝炎、肝硬変、肝ガン等
）患者における有意な血中濃度低下が認められる旨の報
告〔臨床病理ＸＸＩＸ：２，１３５〜１３８ （１９８
１））があり、またインスリン作用促進活性を有し、イ
ンスリン作用増強剤、ひいては抗糖尿病剤として有用で
あるとの報告（特開昭６１−５３２２２号公報〕もおり
、之等の病態等に対して何らかの生理的機能を果してい
る。従って、上記アポＡ−ｍを容易に高純度で且つ大量
に生産する技術の確立は、その生理的機能の研究、解明
等を初めとして、上記各種病態の研究、その治療法の確
立等、基礎研究分野、医療分野等の各種分野で非常に意
義がある。PCA-Ala-IJs-Glu-Pro-Cys-V
al-Glu-3er-Leu Vat 3er Gl
n Tyr Phe Gln Thr Val-Thr
-Aso-Tyr-Gly-Lys-Asp-Leu-
Met-Glu-LVS Vat LVS Ser P
ro GIIJ-LeLJ Gin'△la-(31
u-Ala(ys-3er-Tyr-phe-Qlu-
1ys-3er-l-ys-Glu-Gln-1-eu
-Thr-Pro-1eu-11e-LVS-IJS-
Ala-Gly-Thr-(3tu-1-eu-Va
Do△5n-phe-Leu-3er-Tyr-Phe-
Val-Glu-L eu-Gly-Thr-Gln-
Pro-Ala-Thr-Gin-COOH (A> ApoA-II, which has a polypeptide chain shown in the above formula (A>), is one of the main constituent proteins of plasma high-density protein (HDL). Although it is known that apoA-n exists in HDL together with ApoA-n, the details of its specific physiological function have not yet been elucidated. There is an increasing trend in concentration,
Report that a significant decrease in blood concentration is observed in patients with liver diseases (e.g. acute hepatitis, chronic hepatitis, cirrhosis, liver cancer, etc.) [Clinical Pathology XXIX: 2,135-138 (198
1)), and there is also a report (Japanese Unexamined Patent Publication No. 61-53222) that it has insulin action-promoting activity and is useful as an insulin action enhancer and even as an antidiabetic agent. Therefore, the establishment of a technology to easily produce the above-mentioned apoA-m in high purity and in large quantities is necessary, starting with the research and elucidation of its physiological functions, as well as the prevention of the various pathological conditions mentioned above. It is of great significance in various fields such as research, establishment of treatment methods, basic research fields, medical fields, etc.
発明が解決しようとする隔題点
しかして、従来ヒトアポＡ−ｎは、ヒト血清より通常の
タンパクの分離、精製手段、例えばヒト血清よりＨＤＬ
画分を超遠心分離法等により採取した後、脱脂し、ゲル
濾過し、イオン交換クロマトグラフィー等を行なって得
られているが、かかる方法は、ヒト血清の入手自体困難
となりつつある現在、決して実用的に満足なものとはい
えず、これに代る新しい方法、特に遺伝子工学的手法を
利用して微生物により産生させる方法の確立が、斯界で
要望されている。Problems to be Solved by the Invention However, conventionally, human apoA-n can be obtained from human serum using conventional protein separation and purification means, such as HDL from human serum.
Fractions are collected by ultracentrifugation, etc., then delipidated, gel-filtered, and subjected to ion-exchange chromatography, etc., but such methods are difficult to obtain these days, as it is becoming increasingly difficult to obtain human serum. It cannot be said that it is practically satisfactory, and there is a demand in the art for the establishment of a new alternative method, especially a method for producing it by microorganisms using genetic engineering techniques.
問題点を解決するための手段
本発明者らは、上記現状に鑑み、鋭意研究を重ねた結果
、上記ヒトアポＡ−ｎをコードする遺伝子を新たに設計
し、化学合成することに成功すると共に、かくして得ら
れる遺伝子を適当なプラスミドベクターに組込んで遺伝
子組換体（発現ベクター）を構築し、これを利用して微
生物を形質転換し、該形質転換体を培養して目的とする
アポＡ−ｍの発現を確認するに成功し、ここに上記斯界
の要望に合致する技術を提供できる本発明を完成するに
至った。Means for Solving the Problems In view of the above-mentioned current situation, the present inventors have conducted extensive research, and as a result, have succeeded in designing a new gene encoding the above-mentioned human apoA-n and chemically synthesizing it. The gene thus obtained is inserted into an appropriate plasmid vector to construct a genetic recombinant (expression vector), which is used to transform microorganisms, and the transformant is cultured to produce the desired apoA-m. We have succeeded in confirming the expression of this problem, and have now completed the present invention, which can provide a technology that meets the needs of the above-mentioned industry.
本発明によれば、下記式（１）で表わされる塩基配列を
含有するアポリポタンパク質遺伝子をプラスミドベクタ
ーに挿入してアポリポタンパク貿遺伝子発現プラスミド
組換体を作成し、該組換体を宿主細胞に形質転換させて
形質転換体を構築し、これを培養して発現されるアポリ
ポタンパク質を採取することを特徴とするアポリポタン
パク質の製造法が提供される。According to the present invention, an apolipoprotein gene containing the base sequence represented by the following formula (1) is inserted into a plasmid vector to create an apolipoprotein trade gene expression plasmid recombinant, and the recombinant is transformed into a host cell. A method for producing an apolipoprotein is provided, which comprises constructing a transformant, culturing the transformant, and collecting the expressed apolipoprotein.
しＩ（ｊ （ｊ、Ａｔ； ＩＡＵ （、；ＩＩ
ＩＩＩ ＣＡＡ 王Ｔ下本明細書において、塩基
配列、アミノ酸配列、之等を構成する各核酸塩基、アミ
ノ酸乃至その残基等の表示は、ＩＵＰＡＣ−ＩＵＢの規
定乃至当該分野において慣用される略号によるものとす
る。I(j (j, At; IAU (,; II
III CAA Under T. Wang In this specification, each nucleobase, amino acid, residue, etc. constituting a base sequence, amino acid sequence, etc. will be indicated according to the IUPAC-IUB regulations or abbreviations commonly used in the field. shall be.
以下、本発明のアポリポタンパク質の製造技術につき、
これに利用するアポリポタンパク質遺伝子（以下単に「
本発明遺伝子」という〉の設計及び合成、該遺伝子を含
む発現ベクターの構築、該ベクターの導入による形質転
換体の製造、該形質転換体の培養の順で詳述する。Below, regarding the production technology of apolipoprotein of the present invention,
The apolipoprotein gene used for this purpose (hereinafter simply "
The following will be detailed in the following order: design and synthesis of the gene of the present invention, construction of an expression vector containing the gene, production of a transformant by introducing the vector, and cultivation of the transformant.
本発明遺伝子の塩基配列の設計に当たっては、以下の基
準を採用した。In designing the base sequence of the gene of the present invention, the following criteria were adopted.
（１）宿主細胞として用いる、例えば大腸菌での使用頻
度の高いトリヌクレオチドコドンを選択する。(1) Select trinucleotide codons that are frequently used in, for example, E. coli, which is used as a host cell.
（２）遺伝子内及びその両端に特定の制限酵素認識部位
を持たせ、任意にその部位を操作して、他の遺伝子との
連結、プラスミドベクターへの挿入を行ない得るように
する。(2) A specific restriction enzyme recognition site is provided within the gene and at both ends thereof, and the site is arbitrarily manipulated to enable ligation with other genes and insertion into a plasmid vector.
（３）化学合成した遺伝子断片を集合、連結させる場合
、目的とする連結状態とは異なる遺伝子の連結が起こら
ないか、または最小限に留め得るようにする。(3) When assembling and ligating chemically synthesized gene fragments, ensure that ligation of genes different from the desired ligation state does not occur or can be kept to a minimum.
（４）前記式（Ａ）に示すアポＡ−ＩＩ−次構造のＮ末
端ピロリドンカルボン酸に対応するトリヌクレオチドコ
ドンとしては、シャープら（Ｃ，Ｒ，５ｈａｒｐｅ ｅ
ｔ ａｌ、＞ ！、：Ｊニッチ決メラれた遺伝子配列（
Ｎｕｃｌｅｉｃ ＡＣｉｄＳＲｅｓｅａｒｃｈ、 １
２ （９） 、 ３９１７−３９３２（１９８４））に
習って、グルタミン（Ｇ Ｉｎ）を示すコドンを選択す
る。(4) As the trinucleotide codon corresponding to the N-terminal pyrrolidone carboxylic acid of the apoA-II-order structure shown in formula (A), Sharp et al.
tal,>! , :J niche determined gene sequence (
Nucleic ACidSRearch, 1
2 (9), 3917-3932 (1984)), a codon representing glutamine (GIn) is selected.
上記基準より設計された本発明遺伝子構成部分の好まし
い塩基配列の一興体例は、前記式（１）に示す通りであ
り、これはヒトアポＡ−ＩＩのアミノ酸配列に対応する
もの、即ち該アミノ酸配列をコードするものである。し
かして本発明遺伝子は、ヒトアポＡ−ＩＩのアミノ酸配
列をコードし、）Ｗ ｂｉ子組換え技術により該ヒトア
ポＡ−ｎを発現、製造できる限り、上記式（１）の塩基
配列に限定されるものではなく、これを基礎としてその
塩基配列の若干の変更、削除、付加等の改変乃至氷飾が
可能である。かかる改変、修飾等の行なわれた塩基配列
もまた、これが上記式（１〉の塩基配列と同−のアポＡ
−ｎ遺伝情報を有する限り、本発明遺伝子に包含される
。A preferred example of the base sequence of the gene component of the present invention designed based on the above criteria is as shown in the above formula (1), which corresponds to the amino acid sequence of human apoA-II, that is, the amino acid sequence It is something to code. Therefore, the gene of the present invention encodes the amino acid sequence of human apoA-II, and is limited to the base sequence of formula (1) above, as long as the human apoA-n can be expressed and produced by W bi recombinant technology. Based on this, modifications or decorations such as slight changes, deletions, and additions to the base sequence are possible. The base sequence that has been subjected to such alterations, modifications, etc. also has the same apoA base sequence as the base sequence of the above formula (1>).
-n genetic information is included in the genes of the present invention.
上記塩基配列の改変乃至修飾は、当業界で知られている
。その具体例は、上記（１）の塩基配列によりコードさ
れるアミノ酸配列と同一のアミノ酸配列をコードする遺
伝暗号（ｇｅｎｅｊＩＣＣ０ｄＯｎ ）の採用にある。Alteration or modification of the base sequence described above is known in the art. A specific example thereof is the adoption of a genetic code (genejICC0dOn) that encodes the same amino acid sequence as the amino acid sequence encoded by the base sequence in (1) above.
また、上記塩基配列の修飾（付加）には、これにより得
られる塩基配列を実際に適当なベクターに挿入し、微生
物で発現させるために必要なプロモーター等の各種調節
因子との連結を行なうための各種制限酵素認識部位の付
与が包含される。即ち、本発明遺伝子は、これを利用し
て遺伝子工学的手法により目的のヒトアポＡ−Ｉ［発現
ベクターを構築するに当たって、アポＡＩ［遺伝子に更
に、プロモーター、シャイン・ダルガノ配列（Ｓｈｉｎ
ｅ−［） ａ１ｇａｒｎｏ配列、ＳＤ配列）、タンハク
合成ノ開始コドン、終止コドン等の各種調節因子を適宜
連結させる必要がおるが、之等各塩基配列の切断、結合
等の操作はいずれも制限酵素の利用により行なわれるた
め、上記遺伝子には、その前後に適当な制限酵素認識部
位の付与が必須となる。In addition, the modification (addition) of the base sequence described above involves actually inserting the resulting base sequence into an appropriate vector and linking it with various regulatory factors such as promoters necessary for expression in microorganisms. This includes the provision of various restriction enzyme recognition sites. That is, the gene of the present invention can be used to construct the target human apoA-I [expression vector by genetic engineering techniques.
It is necessary to appropriately link various regulatory factors such as e-[) a1garno sequence, SD sequence), start codon for tanhaku synthesis, and stop codon, but operations such as cutting and joining of each base sequence are performed using restriction enzymes. Therefore, it is essential to provide appropriate restriction enzyme recognition sites before and after the gene.
かかる適当な制限酵素認識部位の付与された遺伝子もま
た、本発・明遺伝子に包含される。その−具体例として
は、下記式（２）で表わされるものを例示できる。これ
は、前記式（１）で表わされる塩基配列の前後に、その
発現に必要なプロモーター等の各種調節因子との連結の
ための特定の制限酵素認識部位を付加したものであり、
引続く当該遺伝子発現ベクターの構築に特に好適でおる
。Genes provided with such suitable restriction enzyme recognition sites are also included in the present invention. As a specific example thereof, one represented by the following formula (2) can be exemplified. This is obtained by adding specific restriction enzyme recognition sites before and after the base sequence represented by the above formula (1) for linking with various regulatory factors such as promoters necessary for its expression,
It is particularly suitable for the subsequent construction of the gene expression vector.
下記式（２）には、該塩基配列中の制限酵素認識部位及
び該塩基配列でコードされるアミノ酸配列をも併記する
。The following formula (2) also includes the restriction enzyme recognition site in the base sequence and the amino acid sequence encoded by the base sequence.
尚、本発明遺伝子中に存在させるべき制限酵素認識部位
は、上記式（２）に示すものに限定されることなく、構
築すべきアポＡ−［発現ベクターの種類に応じて、従来
より公知の各種のものを適宜選択することができる。Note that the restriction enzyme recognition site to be present in the gene of the present invention is not limited to that shown in the above formula (2), and may be any of the conventionally known restriction enzyme recognition sites depending on the type of the expression vector to be constructed. Various types can be selected as appropriate.
上記式（１）及び式（２〉で表わされる特定の塩基配列
を有する遺伝子を代表として、本発明遺伝子は、之等の
塩基配列に従い、各核酸を順次反応させることにより合
成できる。この反応は通常の方法、例えば同相リン酸ト
リエステル法（Ｎａｔｕｒｅ 、３１０，１０５ （１
９８４））等により行ない得る。また、得られる各塩基
配列の単離精製は、例えば高速液体クロマトグラフィー
等の常法に従うことができ、精製された各塩基配列の確
認は、例えばホモクロマトグラフィーによる二次元展開
法（Ｅ、 Ｊａｙ、 Ｒ，Ａ、 Ｂａｍｂａｒａ、 Ｒ
５Ｐａｄｍａｎｂｈａｎ ａｎｄ Ｒ，Ｗｕ 、 Ｎｕ
ｃｌｅｉｃ Ａｃ１ｄｓＲｅｓ、、１，３３１ （１
９７４））やマキサム−ギルバート法（Ａ、 Ｍ、 Ｍ
ａＸａｍ ａｎｄ Ｗ、 Ｇ１１ｂｅｒｔ。The gene of the present invention can be synthesized by sequentially reacting each nucleic acid according to the base sequences represented by the above formulas (1) and (2>). Conventional methods, such as the homophase phosphotriester method (Nature, 310, 105 (1
984)) etc. Further, the isolation and purification of each obtained base sequence can be carried out according to a conventional method such as high performance liquid chromatography, and the confirmation of each purified base sequence can be carried out, for example, by a two-dimensional development method using homochromatography (E, Jay , R.A., Bambara, R.
5 Padmanbhan and R, Wu, Nu
cleic Ac1dsRes, 1,331 (1
974)) and the Maxam-Gilbert method (A, M, M
aXam and W, G11bert.
Ｐｒｏｃ、Ｎａｔｌ、Ａｃａｄ、Ｓｃｉ、、ＵＳＡ、
７４． ５６０（１９７７） ： Ａ、 Ｍ、 Ｍ
ａｘａｌＩｌａｎｄＷ、 Ｇ１１ｂｅｒｔ。Proc, Natl, Acad, Sci, USA,
74. 560 (1977): A, M, M
axalIlandW, G11bert.
Ｍｅｔｈｏｄｓ ｉｎ Ｅ ｎｚｙｍｏｌ、、Ｖｏｌ、
６５ 、 ｐｐ４９９ 。Methods in Enzymol, Vol.
65, pp499.
Ａｃａｄ、 Ｐｒｅｓｓ （１９８０） ）等により、
それぞれ行なうことができる。Acad, Press (1980), etc.
You can do each.
本発明遺伝子の合成は、特に好ましくは前記式（１）又
は式（２）に示される塩基配列の中間に位置するｐｓｔ
Ｉ制限酵制限酵素部２識を前半部（サブユニットＡ）と
後半部（サブユニットＢ）の２つに分け、之等を別々に
構築することにより実施される。この方法につき、以下
に詳述する。The synthesis of the gene of the present invention is particularly preferably carried out using pst
This is carried out by dividing the I restriction enzyme restriction enzyme section into two parts, the first half (subunit A) and the second half (subunit B), and constructing these parts separately. This method will be explained in detail below.
この方法においては、まずサブユニツ１〜Ａを合成する
ために、塩基数１６〜１９個のオリゴヌクレオチド断片
Ａ−１〜△−１５を合成する。またサブユニットＢの合
成のために、塩基数１４〜１９個のオリゴヌクレオチド
断片Ｂ−１〜Ｂ−１５を合成する。之等各オリゴヌクレ
オチド断片の塩基数及び塩基配列は下記第１表に示す通
りである。In this method, first, in order to synthesize subunits 1 to A, oligonucleotide fragments A-1 to Δ-15 having 16 to 19 bases are synthesized. Furthermore, for the synthesis of subunit B, oligonucleotide fragments B-1 to B-15 having 14 to 19 bases are synthesized. The number of bases and base sequences of each oligonucleotide fragment are shown in Table 1 below.
第 １ 表
次いで、上記各オリゴヌクレオチド断片の各５個ずつを
下記式（３）〜（８）に示す通りに集合、連結させて、
ブロック１〜ブロツク６をそれぞれ合成する。Table 1 Next, five of each of the above oligonucleotide fragments were assembled and linked as shown in the following formulas (3) to (8),
Blocks 1 to 6 are synthesized, respectively.
得られるブロック１〜ブロツク３を集合、連結させるこ
とにより、所望のサブユニットＡが収得される。同様に
ブロック４〜ブロツク６の集合、連結により所望のサブ
ユニツｉ−８が収得される。A desired subunit A is obtained by assembling and connecting the obtained blocks 1 to 3. Similarly, a desired subunit i-8 is obtained by aggregating and concatenating blocks 4 to 6.
かくして得られるサブユニットＡ及びサブユニットＢの
各塩基配列は、次式（９）及び（１０）にそれぞれ示す
通りである。The base sequences of subunit A and subunit B thus obtained are as shown in the following formulas (9) and (10), respectively.
サブユニットＡは、その両末端にＥＣ０ＲＩ、ＰＳｔ工
制御！艮酵素認識部位をそれぞれ有しており、またサブ
ユニットＢは、その両末端にＰＳｔＩ、ＭＩＵＩ制限酵
素認識部位をそれぞれ有している。Subunit A has EC0RI and PSt engineering control at both ends! Subunit B has PStI and MIUI restriction enzyme recognition sites at both ends.
ブロック １：
（うＡＣ； ＩＡＧ ＩＩＣＩＩＩ Ｃす１（β
（Ｘ；Ａ ”’ｌ’ＧＧかくして構築されたサブユ
ニットＡは、これを例えばプラスミドベクターｐＢＲ３
２２のＥ Ｃ０ＲＩ−ｐｓｔＩ制限酵素切断サイトに容
易に組込むことができる。このサブユニットＡを組込ん
で構築したプラスミド組換体の具体例としては、後記実
施例に示すプラスミドｐＡＰＯ１を例示できる。Block 1: (AC; IAG IICIII C1 (β
(X;A ”'l'GGThe thus constructed subunit A can be transferred to the plasmid vector pBR3, for example.
It can be easily integrated into the ECORI-pstI restriction enzyme cleavage site of 22. A specific example of a plasmid recombinant constructed by incorporating this subunit A is plasmid pAPO1 shown in Examples below.
また、サブユニットＢは、例えばプラスミドｐＢＲ３２
２のＥＣ０ＲＩ切断部位に予めＭＩｕＩリンカ−（化学
合成オリゴヌクレオチド：ＧＴＣＧＡＣＧＣＧＴＣＧＡ
Ｃ）を挿入したベクターｐＢＲ３２２−ＭｌｕのＰｓｔ
Ｉ−ＭＩｕ工制限酵素切断サイトに、上記サブユニット
Ａと同様にして容易に組込むことができる。かくして得
られる組換体の具体例としては、後記実施例に示すプラ
スミドＤＡＰ○２を例示できる。In addition, subunit B is, for example, plasmid pBR32
A MIuI linker (chemically synthesized oligonucleotide: GTCGACGCGTCGA
C) Pst of vector pBR322-Mlu inserted with
It can be easily incorporated into the I-MIu restriction enzyme cleavage site in the same manner as subunit A above. A specific example of the recombinant thus obtained is plasmid DAP○2 shown in Examples below.
上記各プラスミドは、各々カルシウム法（Ｅ。Each of the above plasmids was prepared using the calcium method (E).
Ｌ ｅｄｅｒｂｅｒｇ、 Ｓ、 Ｃｏｈｅｎ、 Ｊ 、
Ｂａｃｔｅｒｉｏｌ、、 １１９．１０７２ （１９
７４））等の通常の方法に従い、之等を大腸菌に形質転
換させることができ、この方法によりそれぞれ保存、増
幅させることができる。Lederberg, S., Cohen, J.
Bacteriol,, 119.1072 (19
These can be transformed into Escherichia coli according to a conventional method such as 74)), and each can be stored and amplified by this method.
本発明遺伝子を保有するベクターは、上記プラスミドｐ
ＡＰＯＩ及びｐＡＰＯ２より、それらの各々保有する各
サブユニットを切出し、連結させ、この連結物をプラス
ミドベクター１：）ＢＲ３２２等の適当なベクターに組
込むことにより得られる。The vector carrying the gene of the present invention is the plasmid p
It can be obtained by cutting out the respective subunits possessed by APOI and pAPO2, ligating them together, and incorporating the ligated product into an appropriate vector such as plasmid vector 1:) BR322.
その具体例は後記実施例に示す通りであり、以下、かく
して得られるプラスミド組換体を、Ｉ）ＡＰＯＡ−ＩＩ
という。このプラスミドｐＡＰＯＡ−ＩＩもまた上記Ｄ
ＡＰＯＩ及びｐＡＰＯ２”と同様にこれを例えば大腸菌
等の適当な宿主細胞に形質転換させることができ、該微
生物内で安定に保存、増幅させ得る。Specific examples thereof are shown in Examples below, and hereinafter, the plasmid recombinants thus obtained will be described as I) APOA-II
That's what it means. This plasmid pAPOA-II is also
Like APOI and pAPO2'', it can be transformed into a suitable host cell such as E. coli, and can be stably stored and amplified within the microorganism.
上記のごとくして得られる各プラスミドベクターが、宿
主細胞中に存在することの確認は、通常の方法、例えば
アルカリ−３ＤＳ抽出法（３ｉｒｎｂｏｉｍ、Ｈ，Ｃ，
ａｎｄ Ｄｏｌｙ、Ｊ、、ＮｕｃｌｅｉｃＡｃｉｄｓ
Ｒｅｓ、、７．１５１３ （１９７９））等に従って
、プラスミドＤＮＡを分取後、種々の制限酵素で処理し
、２等制限酵素の認識部位の存在の有無乃至は生成りＮ
Ａ断片の長さの検討を行なうことにより判断できる。The presence of each plasmid vector obtained in the above manner in the host cell can be confirmed by conventional methods, such as the alkaline-3DS extraction method (3irnboim, H, C,
and Doly, J., Nucleic Acids.
Res., 7.1513 (1979), etc., the plasmid DNA is separated and treated with various restriction enzymes to determine the presence or absence of recognition sites for secondary restriction enzymes or the production of N.
This can be determined by examining the length of the A fragment.
上記本発明遺伝子を保有するベクターは、これを導入し
て得られる形質転換体が、目的とするアポＡ−ｎを発現
するためには、本発明遺伝子と共に、その発現のための
各種調節因子、例えばプロモーター、ＳＤ配列、翻訳停
止シグナル、転写終結信号等を保有する必要がおる。之
等を保有するベクターにおける本発明遺伝子の発現様式
及びそのために用いられる各種調節因子は、特に限定は
なく、例えば大腸菌を宿主細胞として利用する場合、そ
の菌体内に直接発現させる系、ペリプラズム層に分泌発
現させる系等を任意に採用できる。In order for the transformant obtained by introducing the above-mentioned gene of the present invention to express the desired apoA-n, the vector carrying the gene of the present invention must contain various regulatory factors for its expression, as well as the gene of the present invention. For example, it is necessary to have a promoter, SD sequence, translation stop signal, transcription termination signal, etc. There are no particular limitations on the expression mode of the gene of the present invention in the vector carrying these and the various regulatory factors used for this purpose. Any system for secretion expression can be adopted.
上記各発現系の構築等の際に用いられる遺伝子工学的手
法は、いずれも常法に従うことができ、各種制限酵素に
よるＤＮＡの切断処理、Ｓ１ヌクレアーゼ、Ｔ４ＤＮＡ
リガーゼ等を用いたＤＮＡの連結処理、アガロースゲル
電気泳動法、ポリアクリルアミドゲル電気泳動法等によ
るＤＮＡの単離、精製、フェノール抽出法によるＤＮＡ
の回収、精製等を包含する。また得られる各発現系の確
認も常法に従い、例えば遺伝子の塩基配列を直接マキサ
ム−ギルバート法で解析するか、ミニプレバレージョン
やマツピング法により遺伝子の挿入やその方向を確認す
る方法（Ｈ，Ｃ，Ｂｉｒｎｂｏｉｍ ｅｔａｌ、、
Ｎｕｃｌｅｉｃ Ａｃ１ｄｓ Ｒｅ５ｅａｒｃｈ
、 ７゜１５１３−１５２３ （１９７９））等によ
ることができる。之等各操作の具体例は、後記実施例に
詳述する。Genetic engineering methods used in constructing each of the above expression systems can be carried out using conventional methods, such as cutting DNA with various restriction enzymes, S1 nuclease, T4 DNA, etc.
DNA ligation using ligase etc., DNA isolation and purification by agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc., DNA by phenol extraction method.
This includes recovery, purification, etc. Confirmation of each expression system obtained also follows conventional methods, for example, by directly analyzing the gene base sequence using the Maxam-Gilbert method, or by confirming the insertion and direction of the gene by mini-prevalence or mapping methods (H, C , Birnboim et al.
Nucleic Ac1ds Re5earch
, 7°1513-1523 (1979)). Specific examples of each of these operations will be described in detail in Examples below.
上記大腸菌ペリプラズム層への分泌発現系につき詳）ホ
ずれば、この弁環系は、例えば大腸菌β−ラクタマーゼ
のシグナルペプチドを利用して構築されるものであり、
この系では、目的タンパク貿はシグナルペプチドとの融
合タンパクとして大腸菌内で発現され、該シグナルペプ
チドの作用により内膜まで移行され、該内股内で融合タ
ンパクよりシグナルペプチドがシグナルペプチド−げに
より酵素的に切断され、かくして目的タンパクのみが大
腸菌ペリプラズム層に選択的に蓄積される。Regarding the secretion expression system to the E. coli periplasm layer (details), this valve ring system is constructed using, for example, the signal peptide of E. coli β-lactamase,
In this system, the target protein is expressed in E. coli as a fusion protein with a signal peptide, and transported to the inner membrane by the action of the signal peptide. In this way, only the target protein is selectively accumulated in the periplasmic layer of E. coli.
従って、この系の利用によれば、大腸菌菌体内にあける
プロテアーゼによる目的タンパク貿の分解の危険性を回
避できる利点があると共に、目的タンパク質はペリプラ
ズム層に局在するため、その分離、精製が容易となる利
点があり、更に内膜に移行した融合タンパクはシグナル
ペプチドの直後、即ち目的タンパクの直前でシグナルペ
プチダーゼにより選択的に切断され、結果として他のい
かなる不要アミノ酸配列をも含まない目的タンパクのみ
がペリプラズム層に分泌、蓄積される利点もめる。Therefore, the use of this system has the advantage of avoiding the risk of the target protein being degraded by proteases in the E. coli cells, and since the target protein is localized in the periplasmic layer, it is easy to isolate and purify it. Furthermore, the fusion protein transferred to the inner membrane is selectively cleaved by signal peptidase immediately after the signal peptide, that is, just before the target protein, resulting in only the target protein containing no other unnecessary amino acid sequences. The benefits of secretion and accumulation in the periplasmic layer are also highlighted.
上記β−ラクタマーゼ（ｂｌａ ）のシグナルペプチド
を利用した分泌発現系の具体例としては、後記実施例に
示したプラスミドｐＳＡＰ−０を例示できる。これは、
ｔａＣプロモーター、ＳＤ配列及びｂｌａシグナルペプ
チド（２３１固のアミノ酸よりなる）をコードする塩基
配列が、同一方向に並んだ塩基配列を有するプラスミド
ベクターｐＵＧＴ１５０（特願昭６１−１５３７８３号
）を起源として、まずそのｂｌａシグナルペプチドＣ末
端末端化学合成リンカ−［ＣＣＧＧＣＣＧＧ］を挿入し
て上記ｂｌａシグナルペプチドのＣ末端アミノ酸（２３
番目のＡｌａ）の第３コドンの直後にＮａｅ工制限酵素
認識部位を設けたベクターｐＫＴＮを（構築し、該ベク
ターのＥＣＯＲＩ−Ｎａｅ工制限酵素切断断片、ｃｌＧ
Ｔｌ ５０３ （特願昭６１−３１４１５号）のＥｃｏ
ＲニーＸｈｏ工制限酵素切断断片及びＩ）Ａ ＰＯＡ−
Ｉ［由来のプロアポＡ ＩＩＪ伝子を含む塩基配列を
連結させることにより構築される。かくして得られる発
現ベクター１）ＳＡ Ｐ−○は、ｔａｃプロモーター、
ＳＤ配列、ｂａａシグナルペプチドーアボＡ−ＩＩ融合
タンパクをコードする遺伝子が同一方向に並んだ塩基配
列を有している。殊に該塩基配列中の融合タンパクをコ
ードする遺伝子・は、ｂｌａシグナルペプチドをコード
する塩基の後にアポＡ−ｎ遺伝子の第１番目のアミノ酸
をコードする第１コドンが直結されているため、読取り
フレームがずれるおそれもなく、ｔａｃプロモーターの
働きにより目的とする融合タンパクが大腸菌菌体内で発
現され、内膜を通過後、ペリプラズム層に所望のアポＡ
−ＩＩのみが分泌、蓄積される。A specific example of a secretory expression system using the signal peptide of β-lactamase (bla) is plasmid pSAP-0 shown in Examples below. this is,
Originating from the plasmid vector pUGT150 (Japanese Patent Application No. 153783/1983), which has the base sequences encoding the taC promoter, SD sequence, and bla signal peptide (consisting of 231 unique amino acids) aligned in the same direction, The C-terminal amino acid (23
A vector pKTN (constructed) in which a Nae restriction enzyme recognition site was provided immediately after the third codon of the
Eco of Tl 503 (Patent Application No. 61-31415)
Rnie Xho restriction enzyme cleavage fragment and I) A POA-
It is constructed by linking the base sequences containing the proapoA IIJ gene derived from A. The expression vector thus obtained 1) SAP-○ is a tac promoter,
It has a base sequence in which the SD sequence and the gene encoding the baa signal peptide-AvoA-II fusion protein are aligned in the same direction. In particular, in the gene encoding the fusion protein in the base sequence, the first codon encoding the first amino acid of the apoAn gene is directly linked after the base encoding the bla signal peptide, making it difficult to read. The desired fusion protein is expressed in E. coli by the action of the tac promoter without fear of frame shift, and after passing through the inner membrane, the desired apoA is delivered to the periplasmic layer.
Only -II is secreted and accumulated.
上記発現ベクターで形質転換される宿主細胞としては、
例えば大腸菌等のダラム陰性菌、枯草菌等のダラム陽性
菌、放線菌等の原核生物細胞及び酵母等の真核生物細胞
を例示できる。之等の内では特に大腸菌が好適である。The host cells transformed with the above expression vector include:
Examples include Durham-negative bacteria such as Escherichia coli, Durham-positive bacteria such as Bacillus subtilis, prokaryotic cells such as actinomycetes, and eukaryotic cells such as yeast. Among these, Escherichia coli is particularly preferred.
その具体例としては、例えば大腸菌に１２株由来のＨＢ
ｌＣ）１株（Ｈ。As a specific example, for example, HB derived from 12 strains of E. coli.
1C) 1 strain (H.
Ｗ、 １３ｏｙｅｒ ａｎｄ Ｏ，Ｒｏｕｌｌａｎｄ
−Ｄｕｓｓｏｉｘ、。W, 13oyer and O, Roulland
-Dussoix,.
Ｊ、Ｍｏ１．Ｂｉｏｌ、、４１．４５９−４７２（’１
９６９））及びＪＭ１０３株（Ｊ、 Ｍｅｓｓｉｎｇｅ
ｔ ａｌ、、Ｎｕｃｌｅｉｃ Ａｃ１ｄｓ Ｒｅｓ
、、９．３０９（１９８１））等を例示できる。J, Mo1. Biol,,41.459-472('1
969)) and JM103 strain (J, Messinge
tal, Nucleic Ac1ds Res
, 9.309 (1981)).
上記により得られる弁用ベクターで形質転換された宿主
細胞の培養は、通常の細胞培養用培地を用いて行なうこ
とができる。上記培地としては、例えばＬ−ｂｒｏｔｈ
培地のほか、Ｅ培地、Ｍ９培地、Ｍ６３培地等の各種の
ものを利用できる。之等の培地には、更に通常知られて
いる各種の栄養を添加することもできる。培養条件とし
ては、微生物の生育に適したｐＨ，温度、通気、撹拌条
件等を適宜選択して採用できる。例えば大腸菌の場合に
は、ｐＨ約５〜８の範囲、特に約７が適当でおり、約２
０〜４３°Ｃの温度で、通気撹拌培養すればよい。この
培養により、目的タンパク質はペリプラズム層に生産、
蓄積される。Host cells transformed with the valve vector obtained above can be cultured using a normal cell culture medium. As the above-mentioned medium, for example, L-broth
In addition to culture media, various media such as E culture medium, M9 culture medium, and M63 culture medium can be used. Various commonly known nutrients can also be added to these media. As culture conditions, pH, temperature, aeration, stirring conditions, etc. suitable for the growth of microorganisms can be appropriately selected and employed. For example, in the case of Escherichia coli, a pH range of about 5 to 8, particularly about 7, is suitable, and about 2.
Culture may be carried out with aeration and stirring at a temperature of 0 to 43°C. Through this culture, the target protein is produced in the periplasmic layer.
Accumulated.
かくして生産された目的タンパク質は、これを常法に従
い分離、精製できる。この分離、精製操作としては、例
えば培養上澄、浸透圧ショック法等により調製したペリ
プラズム画分、超音波破砕等により調製した細胞内画分
等の各々について、それぞれゲル濾過、吸着クロマトグ
ラフィー、゛イオン交換クロマトグラフィー、高速液体
クロマトグラフィー等が単独で又は適宜組合せて採用で
きる。The target protein thus produced can be separated and purified using conventional methods. The separation and purification operations include gel filtration, adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography, etc. can be employed alone or in appropriate combination.
また精製された目的タンパク質の確認は、高速液体クロ
マトグラフィーによる単一ピークの出現、ポリアクリル
アミドゲル電気泳動法による単一ハンドの出現等により
行ない得る。更に目的タンパク質の同定は、通常のタン
パク質乃至ポリペプチドの構造解析手段と同様にして、
例えば５ＤＳ−ＰＡＧＥによる分子足の分析、アミノ酸
分析器によるアミノ酸組成の測定、アミノ酸シークエン
サーによるアミノ酸配列の解析等により行なうことがで
きる。Further, the purified target protein can be confirmed by the appearance of a single peak by high-performance liquid chromatography, the appearance of a single hand by polyacrylamide gel electrophoresis, and the like. Furthermore, the target protein can be identified using the same method as conventional protein or polypeptide structural analysis methods.
For example, this can be carried out by analyzing molecular legs using 5DS-PAGE, measuring amino acid composition using an amino acid analyzer, and analyzing amino acid sequences using an amino acid sequencer.
実 施 例
以下、本発明を更に詳しく説明するため参考例及び実施
例を挙げる。尚、各側において用いられる各方法及び操
作は、特に明記しない限り、以下の通り行なわれたもの
である。EXAMPLES Reference examples and examples are given below to explain the present invention in more detail. It should be noted that each method and operation used on each side was performed as follows, unless otherwise specified.
１、制限酵素によるＤＮＡの切断操作
使用した制限酵素は、宝酒造社製又はＮＥＢ（Ｎｅｗ
Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ）社製のものであ
り、反応溶液としては同各社が指定する組成のものを用
いた。反応温度は３７°Ｃとし、温浴中で３時間静置し
て反応させた。制限酵素の標準的使用量は、ＤＮＡ１μ
Ｑに対して１ユニツトであり、最終反応液量は１００μ
Ｑ以上となるようにした。また反応容器としては滅菌済
みの１．５ｍ（２容エツペンドルフチユーブを用いた。1. Cutting DNA with restriction enzymes The restriction enzymes used were those manufactured by Takara Shuzo Co., Ltd. or NEB (New
(England Biolabs), and the reaction solution used was one with a composition specified by each company. The reaction temperature was 37°C, and the mixture was left standing in a hot bath for 3 hours to react. The standard amount of restriction enzyme used is 1μ of DNA.
1 unit for Q, and the final reaction volume is 100μ
It was set to be Q or higher. A sterilized 1.5 m (2 volume) Eppendorf tube was used as the reaction vessel.
２、フェノール抽出法
酵素反応の終了後、酵素を失活させ反応を停止させるた
めにこのフェノール抽出法を行なった。2. Phenol extraction method After the enzymatic reaction was completed, this phenol extraction method was performed to deactivate the enzyme and stop the reaction.
即ち、反応液に、その液量の半量となるＴＥ緩衝液飽和
フェノール［１ｍＭ ＥＤＴＡを含む１０ｍＭトリス
塩酸（ｐＨ８，０＞緩衝液をフェノールに飽和させたち
の］を加えて充分振盪撹拌し、次いで遠心分離（１２０
００回転／分、５分間）してＤＮＡの含まれる水層を採
取し、更に同量のエーテルを加え、同様に振盪撹拌後、
遠心分離して水層を採取した。この操作を２〜３回繰返
した。That is, half of the volume of TE buffer-saturated phenol [10 mM Tris-HCl containing 1 mM EDTA (pH 8,0> buffer was saturated with phenol)] was added to the reaction solution, and the mixture was thoroughly shaken and stirred. Centrifugation (120
00 rotations/min for 5 minutes) to collect the aqueous layer containing DNA, add the same amount of ether, and after shaking and stirring in the same manner,
The aqueous layer was collected by centrifugation. This operation was repeated 2 to 3 times.
採取された水層に０．１倍容量の３Ｍ＃酸ナトナトリウ
ム緩衝液Ｈ４，８＞と２．５倍容量の冷エタノールを加
え、振盪撹拌し、−８０℃で３０分以上放置後、遠心分
離（１２０００回転／分回転弁間）することによりＤＮ
Ａを沈澱させて回収した。To the collected aqueous layer, add 0.1 times volume of 3M #acid sodium buffer H4,8> and 2.5 times volume of cold ethanol, shake and stir, leave at -80°C for 30 minutes or more, and then centrifuge. DN by separating (12,000 revolutions/minute between rotating valves)
A was precipitated and collected.
３、ＤＮＡのプラントエンド化方法
（１）Ｔ４ＤＮＡポリメラーゼによる方法６７ｍＭトリ
ス塩酸（１）Ｈ８，８）、６．７ｍＭ塩化マグネシウム
、１０ｍＭ ２−メルカプトエタノール、１６．６ｍ
Ｍ硫１アンモニウム、６．７μＭ ＥＤＴＡ及び各１
ｍＭのｄＡＴＰ。3. DNA plant-end modification method (1) Method using T4 DNA polymerase 67mM Tris-HCl (1) H8,8), 6.7mM magnesium chloride, 10mM 2-mercaptoethanol, 16.6mM
1 ammonium sulfate, 6.7 μM EDTA and 1 each
dATP in mM.
ｄＣＴＰ、ｄＧＴＰ及びｄＴＴＰを含む水溶液中にＤＮ
Ａを溶かし、ＤＮＡ１μｑに対して１ユニツトとなる量
のＴ４ＤＮＡポリメラーゼ（宝酒造社製）を加え、３７
℃で１時間反応させた。最終反応液量は１００ｔＩＱと
した。反応終了後、前記フェノール抽出法にてＤＮＡを
回収した。DN in an aqueous solution containing dCTP, dGTP and dTTP.
Dissolve A, add T4 DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) in an amount of 1 unit per 1 μq of DNA, and add 37
The reaction was carried out at ℃ for 1 hour. The final reaction liquid volume was 100 tIQ. After the reaction was completed, DNA was collected using the phenol extraction method described above.
（２）３１ヌクレアーゼによる方法
３０ｍＭ＃ｌす［・リウム（ｐＨ４，６＞、５０ｍＭ塩
化ナトリウム及び１ｍＭ硫酸亜鉛を含む水溶液にＤＮＡ
を溶かし、ＤＮＡ１μｑに対して３ユニツトとなる量の
８１ヌクレアーゼ（ＢＲＬ社製）を加えて、３７°Ｃで
１０分間反応させた。最終反応液量は１００μＱとした
。次いで０．５ＭＥＤ丁Ａ２μＱ及び１Ｍトリス塩酸（
ｐＨ８，０＞１μＱを加えて反応を終了させ、その後、
前記フェノール抽出法にてＤＮＡを回収した。(2) Method using 31 nuclease DNA was added to an aqueous solution containing 30mM #l[.
81 nuclease (manufactured by BRL) was added in an amount of 3 units per μq of DNA, and the mixture was reacted at 37°C for 10 minutes. The final reaction solution volume was 100 μQ. Then 2μQ of 0.5MED A and 1M Tris-HCl (
The reaction was terminated by adding pH 8,0 > 1 μQ, and then
DNA was recovered using the phenol extraction method described above.
４、Ｔ４ＤＮＡリガーゼによるＤＮＡ断片の結合操作
６７ｍＭトリスＦＡ酸（［）Ｈ７，６＞、６．７ｍＭ塩
化マグネシウム、１０ｍＭジチオスレイトール及び１ｍ
〜１ △ＴＰを含む水溶液にＤＮＡを溶かし、ＤＮＡ１
μＱに対して１ユニツトとなる量のＴ４ＤＮＡリガーゼ
（宝酒造社製）を加え、１２°Ｃで５時間以上又は４°
Ｃで一晩以上反応させることによりＤＮＡを結合させた
。最終反応液量は１００μＱとした。反応終了後、前記
フェノール抽出法にてＤＮＡを回収した。4. Linking of DNA fragments using T4 DNA ligase 67mM TrisFA acid ([)H7,6>, 6.7mM magnesium chloride, 10mM dithiothreitol and 1mM
~1 Dissolve DNA in an aqueous solution containing △TP, and
Add T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) in an amount equal to 1 unit per μQ, and incubate at 12°C for 5 hours or more or at 4°C.
The DNA was bound by reacting at C for at least one night. The final reaction solution volume was 100 μQ. After the reaction was completed, DNA was collected using the phenol extraction method described above.
５゜形質転換法
宿主細胞として、大腸菌Ｈ８１０１株又はＪ Ｍ２Ｏ３
株を用いた。5゜transformation method As the host cell, E. coli H8101 strain or JM2O3
A stock was used.
宿主細胞株を、Ｌ−ｂｒｏｔｈ培地（１％バクト］〜リ
プトン、０．５％バクトイ−ストエキストラクト、０．
５％塩化ナトリウム〉で、３７°Ｃ下、６１０ｎｍの吸
光度が０．２５になるまで撮盪培養して増殖させた。こ
の培養液’＋ｏｍＱを遠心分離（７０００回転／分、５
分間）して国体を回収し、水冷した。これを０．１Ｍ塩
化マグネシウム５ＴｎＱに懸濁させて洗浄し、続いて遠
心分１ （７０００回転／分、１分間）により菌体を回
収し、氷冷したＯ、１Ｍ塩化カルシウム及び０．０５Ｍ
塩化マグネシウム混合溶液５ｍ１２に懸濁させた。これ
を水中で３０分以上放置した後、遠心分１１ｉ１１ （
７０００回転／分、１分間）して菌体を回収し、再度同
溶液０．５ｍ１２に懸濁させた。この懸濁液０．２ｍＱ
にＴ４ＤＮＡリカーゼを用いて結合させたＤＮＡの反応
組成液を加え、３０分間氷冷した。次いで、４２．５°
Ｃの温浴で３０秒間加温し、ｌ −ｂｒｏｔｈ培地１．
０ｒｒＩ！Ｑを加え、これを３７°Ｃの温浴中で１時間
静置した。The host cell line was grown in L-broth medium (1% Bacto) to Lipton, 0.5% Bacto yeast extract, 0.
5% Sodium Chloride> at 37°C with shaking culture until the absorbance at 610 nm reached 0.25. This culture solution'+omQ was centrifuged (7000 rpm, 5
(min), the national polity was collected and cooled in water. This was suspended in 0.1M magnesium chloride 5TnQ and washed, and then the bacterial cells were collected by centrifugation 1 (7000 revolutions/min, 1 minute).
It was suspended in 5 ml of a magnesium chloride mixed solution. After leaving this in water for more than 30 minutes, centrifuge 11i11 (
7,000 revolutions/minute for 1 minute), the bacterial cells were collected and suspended again in 0.5 ml of the same solution. This suspension 0.2mQ
A reaction composition of DNA bound using T4 DNA licase was added to the mixture, and the mixture was cooled on ice for 30 minutes. Then 42.5°
Warm for 30 seconds in a warm bath of l-broth medium 1.
0rrI! Q was added and the mixture was left standing in a 37°C hot bath for 1 hour.
かくして、得られる形質転換株を以下の抗生物質耐性を
指標として選択した。即ち、１．５％寒天を含む１−ｂ
ｒｏｔ１１培地にアンピシリン５０μｑ／ｍＱ又はテト
ラサイクリン２０ｕＱ／ｍＱを添加して調製した平板培
地に、上記で得た反応組成液の溶液各０．２ｍＱずつを
拡げ、これを３７°Ｃで一晩静置培養し・、生育するコ
ロニーを分離した。The resulting transformed strain was selected using the following antibiotic resistance as an indicator. i.e. 1-b containing 1.5% agar
Spread 0.2 mQ each of the reaction composition solutions obtained above onto a plate medium prepared by adding 50 μq/mQ of ampicillin or 20 uQ/mQ of tetracycline to rot11 medium, and culture this overnight at 37°C. Then, growing colonies were isolated.
６、プラスミドの単離、精製
プラスミドを保有する菌株を、アンピシリン５０μＣｌ
／ｍＱ又はテトラサイクリン２０μｇ ／／ ｍＱを
添加したＬ−ｂｒｏｔｈ培地４００ ｍＱ中で、３７°
Ｃで１２〜１６時間振盪培養した。これを遠心分前（６
０００回転／分、１０分間）して菌体を集め、これに溶
液Ｉ［５０ｍＭグルコース、１０ｍＭＥＤＴＡ、２５ｍ
Ｍトリス塩酸（１）Ｈ８，０＞及び２Ｉ！Ｉｇ／Ｉｒ１
Ｑリゾチーム、滅菌後便用］の１４前を加えて懸濁させ
、水中に３０分間放置１ノだ。更にこれに溶液ＩＩ ［
０，２Ｎ水酸化ナトリウム及び１％ソジウムドデシル硫
酸］の２８ｍ（２を加えて撹拌し、水中で５分間放置し
た後、溶液ＩＪＩ ［３Ｍ＃酸ナトリウム（ｐＨ４，８
）、滅菌後便用］の２１ｒａＱを加えて、水中で６０分
以上放置した。遠心分離（８０００回転／分、１０分間
）して、十清を集め、２．５倍容量（１５０ＩＩ′ＩＱ
〉の冷エタノールを加え、〜８０℃で３０分間放置し、
更に遠心分離（８０００回転／分、１０分間）して沈渣
を得た。これに溶液ＩＶ［０，１’Ｍ酢酸ナトリウム及
び０．０５Ｍトリス塩酸（［Ｈ８，Ｏ＞ ］の１１１Ｔ
ＩＱを加えて溶解させ、更に２．５倍容量（２７，５＋
ｎＱ）の冷エタノールを加え、−８０°Ｃで３０分間放
置した。もう一度遠心分離（１２０００回転／分、１５
分間）して沈渣を集めた。6. Plasmid isolation and purification The plasmid-carrying strain was treated with ampicillin 50 μCl.
/mQ or 20 μg of tetracycline // 37° in L-broth medium 400 mQ supplemented with mQ
The cells were cultured with shaking at C for 12 to 16 hours. This was centrifuged for 6 minutes before centrifugation.
000 revolutions/min for 10 minutes) to collect the bacterial cells, and add solution I [50mM glucose, 10mM EDTA, 25mM
M Tris-HCl (1) H8,0> and 2I! Ig/Ir1
Add 14 minutes of Q-lysozyme (for sterilized stool), suspend it, and leave it in water for 30 minutes. Furthermore, solution II [
After adding 28 m (2.
), 21raQ (for fecal use after sterilization) was added and left in water for 60 minutes or more. Centrifuge (8000 rpm, 10 minutes), collect the supernatant, and dilute to 2.5 times the volume (150 II'IQ
> Add cold ethanol and leave at ~80℃ for 30 minutes.
Further centrifugation (8000 rpm, 10 minutes) was performed to obtain a precipitate. This was added with solution IV [0.1'M sodium acetate and 0.05M Tris-HCl ([H8,O>] of 111T
Add IQ to dissolve, and then dilute to 2.5 times the volume (27,5+
nQ) cold ethanol was added and left at -80°C for 30 minutes. Centrifuge again (12000 rpm, 15
minutes) and collected the precipitate.
次に、得られた沈渣にＴＥ緩衝液［１０ｍＭトリス塩酸
（ｐＨ７，５＞及びｉｍＭ ＥＤ下Ａ（７）溶液］を
４−加えて溶解させ、これに塩化セシウム４．６２ｇを
加えて撹拌溶解させ、更にエチジウムブロマイド５ｍＭ
ｍ１２溶液を０．４２ｍ１２加えた。Next, TE buffer [10mM Tris-HCl (pH 7.5> and imM ED A(7) solution]) was added to the resulting precipitate to dissolve it, and 4.62g of cesium chloride was added thereto and dissolved with stirring. and further add 5mM ethidium bromide.
0.42 m12 of m12 solution was added.
得られた溶液を遠心分離（３０００回転／分、１０分）
して浮遊物を除き、溶液を超遠心分離（５００００回転
／分、１５時間）した。Centrifuge the obtained solution (3000 rpm, 10 minutes)
The suspended matter was removed, and the solution was subjected to ultracentrifugation (50,000 rpm, 15 hours).
上記超遠心分離終了後、紫外線照射により螢光を発する
プラスミドＤＮＡ部分を採取した。これを５Ｍ塩化ナト
リウム溶液で飽和したイソプロパツールで５〜６回抽出
し、これからエチジウムブロマイドを除去した。最後に
バイオゲルＡ−５０（Ｂｉｏｇｅｌ Ａ−５０）カラム
クロマトグラフィー［２，５Ｃｍ直径×１５〜２０Ｃｍ
カラムサイズ、溶出溶媒−ＴＥ緩衝液＋０．５Ｍ塩化ナ
トリウム溶液、Ｕ Ｖ ２５４ｎｍにより検出コにより
塩化セシウム及び混入しているＲＮＡ等を除去し、前記
フェノール抽出法によりプラスミドＤＮＡを回収した。After the ultracentrifugation was completed, the plasmid DNA portion that emitted fluorescence upon irradiation with ultraviolet rays was collected. This was extracted 5-6 times with isopropanol saturated with 5M sodium chloride solution to remove ethidium bromide. Finally, Biogel A-50 column chromatography [2.5 cm diameter x 15-20 cm
Column size, elution solvent - TE buffer + 0.5M sodium chloride solution, detection using UV 254 nm to remove cesium chloride and contaminating RNA, etc., and plasmid DNA was recovered by the phenol extraction method described above.
得られた精製プラスミドＤＮＡ量は、ＯＤ２６０ｎｍ測
定による０Ｄ２６ｏ＝０．０２２を１μｇ１ｍＱＤＮＡ
量と換算して、算出した。The amount of purified plasmid DNA obtained was 0D26o=0.022 measured by OD260nm, 1μg1mQDNA.
It was calculated by converting it into the amount.
７、オリゴヌクレオチドの合成
オリゴヌクレオチドの合成は、同相リン酸トリエステル
法により行なった（Ｈ，Ｉ ｔｏ ｅｔ ａｌ、。7. Synthesis of oligonucleotides Oligonucleotides were synthesized by the in-phase phosphotriester method (H, I to et al.
Ｎｅｃｌｅｉｃ Ａｃ１ｄｓ Ｒｅ５ｅａｒｃｈ、
１０．１７５５−１７６９ （１９８２））。Necleic Ac1ds Research,
10.1755-1769 (1982)).
即ち、まず１％架橋ポリスチレン樹脂５−Ｘｌ（２００
〜４００メツシユ、バイオラボラトリーズ社製）をアミ
ノメチル化したものと、５’ −０−ジメトキシトリチ
ルヌクレオシドのモノコハク酸エステルとを反応させて
、ヌクレオシド担持樹脂を得た。次に、バーチエム社製
ＤＮＡ合成装置を用いて以下の操作を行なった。That is, first, 1% crosslinked polystyrene resin 5-Xl (200
A nucleoside-supported resin was obtained by reacting aminomethylated 400 mesh (manufactured by Bio Laboratories) with a monosuccinic acid ester of 5'-0-dimethoxytrityl nucleoside. Next, the following operations were performed using a DNA synthesizer manufactured by Birchiem.
上記樹脂２５ｍ０を同装置の反応管に入れ、２％トリク
ロロ酢酸−ジクロロメタン溶液を加えて、５′位のジメ
トキシトリチル基を脱離させた。この操作は溶液に橙色
の着色が消えるまで数回繰返した。更にピリジンで２回
、アセトニトリルで２回それぞれ洗浄後、窒素ガスを通
して樹脂を乾燥させた。次に完全に保護されたジヌクレ
オチド又はモノヌクレオチド（Ｃ，Ｂｒｏｋａ ｅｔ
ａｌ。25 m0 of the above resin was placed in a reaction tube of the same apparatus, and a 2% trichloroacetic acid-dichloromethane solution was added to remove the dimethoxytrityl group at the 5' position. This operation was repeated several times until the orange coloring of the solution disappeared. Furthermore, after washing twice with pyridine and twice with acetonitrile, the resin was dried by passing nitrogen gas through it. Next, a fully protected dinucleotide or mononucleotide (C, Broka et al.
al.
Ｎｕｃｌｅｉｃ Ａｃ１ｄｓ Ｒｅ５ｅａｒｃｈ、
旦、５４６１−５４７１ （１９８０）の方法により調
製した）のトリエチルアンモニウム塩２０ｍｐを加え、
縮合剤（メシチレンスルホニル−５−ニトロトリアゾー
ルのピリジン溶液）を用いて、４５°Ｃで２５分間反応
させて縮合させた。反応終了後、反応液を除き、ピリジ
ンで洗浄し、キャツピング剤（ジメチルアミノピリジン
のテトラヒドロフラン−ピリジン溶液）と無水酢酸とを
加え、室温で５分間反応させ、未反応の水ＩＪ３をマス
クさせた。最後に、樹脂をピリジンとアセトニトリルと
で洗浄し、同相合成法の１サイクルを終了させた。Nucleic Ac1ds Research,
5461-5471 (1980)) was added,
Using a condensing agent (a pyridine solution of mesitylenesulfonyl-5-nitrotriazole), the mixture was reacted at 45°C for 25 minutes for condensation. After the reaction was completed, the reaction solution was removed, washed with pyridine, a capping agent (tetrahydrofuran-pyridine solution of dimethylaminopyridine) and acetic anhydride were added, and the mixture was allowed to react at room temperature for 5 minutes to mask unreacted water IJ3. Finally, the resin was washed with pyridine and acetonitrile to complete one cycle of the in-phase synthesis method.
以上の操作を繰返して、順次鎖長をのばして、目的の保
護基を有するオリゴヌクレオチドを担持した樹脂を得た
。The above operations were repeated to sequentially increase the chain length to obtain a resin carrying an oligonucleotide having the desired protecting group.
得られた樹脂に、切断剤［テトラメチルグアニ 。A cutting agent [tetramethylguani] is added to the resulting resin.
ジウムー２−ピリジンアルドキシメートのピリジン：水
（９０：１０）溶液コを加え４０℃で１５時間放置した
。次に脱脂綿を充填したパスツールピペットを用いて濾
過し、切離された樹脂を除去した後、炉液を減圧下で濃
縮した。この残渣に２８％アンモニア水２．５ｍ（！を
加え、ガラス管（内径８ｍｍｘ１０ｃｍ＞に移し、封管
後、５０〜６０″Ｃの温浴中に４時間放置して反応させ
た。反応終了後、アンモニアを除去し、０．０１Ｍトリ
エチルアンモニウムー二炭酸塩水溶液２ｍｆ２を加えて
溶解し、エーテルで洗浄した。ここまでの操作により、
目的のオリゴヌクレオチドは、樹脂から切断され、５′
末端水酸基の保護基（ジメトキシトリチル基）以外のす
べての保護基が除去された状態となった。A solution of dium-2-pyridine aldoximate in pyridine:water (90:10) was added, and the mixture was left at 40°C for 15 hours. Next, the mixture was filtered using a Pasteur pipette filled with absorbent cotton to remove the separated resin, and the filtrate solution was concentrated under reduced pressure. To this residue was added 2.5 m of 28% ammonia water, transferred to a glass tube (inner diameter 8 mm x 10 cm), and after sealing the tube, it was left to react in a hot bath at 50 to 60"C for 4 hours. After the reaction was completed, Ammonia was removed, 2 mf2 of 0.01 M triethylammonium dicarbonate aqueous solution was added to dissolve, and the mixture was washed with ether.
The oligonucleotide of interest is cleaved from the resin and the 5′
All the protecting groups other than the terminal hydroxyl protecting group (dimethoxytrityl group) were removed.
次に、副反応生成物、遊離した保護基、脱保惑剤等を、
目的とするオリゴヌクレオチドから効率よく除くために
、逆相Ｃ１８シリカゲル（ウォーターズ社製）を充填し
たカラム（バイオラッド社製、エコツカラム、内径１ｃ
ｍｘ ３ Ｑｃｍ）を用いて、粗精製を以下の通り行な
った。Next, remove side reaction products, liberated protecting groups, deabsorbing agents, etc.
In order to efficiently remove the oligonucleotide from the target oligonucleotide, a column packed with reversed phase C18 silica gel (manufactured by Waters) (manufactured by Bio-Rad, Ecotu column, inner diameter 1c) was used.
Crude purification was performed as follows using mx 3 Qcm).
このカラムクロマトグラフィーは、５％アセトニトリル
の０．１Ｍ酢酸トリエチルアンモニウム水溶液から３０
％アセトニトリルの同水溶液の勾配溶出溶媒系を用いて
、２５４ｎＩ！′！での吸光度測定により検出し、目的
とするフラクションを単離した。This column chromatography was performed from 0.1M aqueous triethylammonium acetate solution in 5% acetonitrile to 30% acetonitrile.
Using the same gradient elution solvent system of % acetonitrile in water, 254 nI! ′! The target fraction was isolated by absorbance measurement.
かくして集めたフラクションを、減圧下に濃縮し、残渣
を高速液体クロマトグラフィー（ポンプ；ウォーターズ
社製モデル６０００Ａ型及び同社製Ｍ−４５型、グラジ
ェンター；同社製モデル６６０型ソルベントプログラマ
−１検出器：同社製／４４０型デイテクター）で単一ピ
ークとなるまで分取、精製した。ここで用いたカラムは
逆相Ｃ１８ＹＭＣＣ１３Ｙ ０ＤＳ−Ａ−３１２（山村
化学研究所社製、Ｑ、６ｃｍ直径ｘ１５ｃｍ）であり、
溶出溶媒としては５％→４０％アセトニトリル１０．１
Ｍ酢酸トリエチルアンモニウム水溶液（ｐＨ７，２＞を
用いて勾配溶出させた。The thus collected fractions were concentrated under reduced pressure, and the residue was subjected to high performance liquid chromatography (pump; Waters Model 6000A and Waters Model M-45; gradienter; Waters Model 660 Solvent Programmer-1 detector: The sample was fractionated and purified using a 440 type detector (manufactured by the same company) until a single peak was obtained. The column used here was a reverse phase C18YMCC13Y 0DS-A-312 (manufactured by Yamamura Kagaku Kenkyusho Co., Ltd., Q, 6 cm diameter x 15 cm),
Elution solvent: 5% → 40% acetonitrile 10.1
Gradient elution was performed using M aqueous triethylammonium acetate solution (pH 7.2).
かくして精製されたオリゴヌクレオチドは、その５′末
端がまだジメトキシトリチル基で保護されているので、
これを８０％酢酸水溶液で２０分間反応処理し、脱ジメ
トキシトリチル基化後、再度高速液体クロマトグラフィ
ーにより単一ピークになるまで分取、精製した。このク
ロマトグラフィーは前記と同一の０ＤＳ−Ａ−３１２カ
ラムを用い、５％→１５％アセトニトリル１０．１Ｍ酢
酸トリエチルアンモニウム水溶液（ｐｉ−１７，５＞で
勾配溶出により実施した。The thus purified oligonucleotide is still protected at its 5' end with a dimethoxytrityl group;
This was reacted with an 80% aqueous acetic acid solution for 20 minutes, removed from the dimethoxytrityl group, and then fractionated and purified again by high performance liquid chromatography until a single peak was obtained. This chromatography was carried out using the same 0DS-A-312 column as described above, with gradient elution from 5% to 15% acetonitrile in a 10.1 M aqueous triethylammonium acetate solution (pi-17,5>).
かくして目的の化学合成オリゴヌクレオチド精製物を得
た。尚、オリゴヌクレオチドの固相合成法にあける縮合
反応の収率は、各サイクルで脱離させたジメトキシトリ
チルアルコールの量から換算できる。即ち６０％過塩素
酸−エタノールの溶液中での５００ｎｌｌｌにあける吸
光度を測定することによりその収率を求め得る。In this way, a purified chemically synthesized oligonucleotide of interest was obtained. Note that the yield of the condensation reaction involved in the solid phase synthesis method for oligonucleotides can be calculated from the amount of dimethoxytrityl alcohol eliminated in each cycle. That is, the yield can be determined by measuring the absorbance at 500 nll in a 60% perchloric acid-ethanol solution.
８、オリゴヌクレオチド塩基配列の分析、確認化学合成
したオリゴヌクレオチドの塩基配列の分析は、ホモクロ
マトグラフィーを用いた二次元展開法及びマキサム−ギ
ルバート法により、以下のようにまずオリゴヌクレオチ
ドの５′末端側に３２Ｐの導入を行なって実施した。8. Analysis and confirmation of oligonucleotide base sequence Analysis of the base sequence of chemically synthesized oligonucleotides is carried out using the two-dimensional development method using homochromatography and the Maxam-Gilbert method, as shown below. This was carried out by introducing 32P into the side.
ｉ）５′末端３２ｐの標識化
オリゴヌクレオチド５μｇ（凍結乾燥品）を、蒸留水１
００ｕＱに溶解し、この溶液１４μＱに２５０ｍＭ１−
リス塩！ （＋）Ｈ７，６＞ 、５０ｍＭ塩化マグネシ
ウム、１０ｍＭスペルミン、５０ｍＭジチオスレイトー
ル、５００ｍＭ塩化カリウムの混合液１２μＱを、次い
でγ−３２ｐ−ＡＴＰ水溶液（アマジャム社製、１０μ
Ｃｉ／μＱ）１μＱ及びＴ４ポリヌクレオチドキナーゼ
（宝酒造社製）の各１μＱをそれぞれ加え、更に蒸留水
を加えて全量を６０μＱとした。これを３７℃で１時間
反応させ、５′末端を３２Ｐで標識化した。１００’Ｃ
湿浴中に２分間浸漬して反応を停止させた後、全量が５
μＱ程度になるまで濃縮し、次いで２０Ｘ２０Ｃｍに切
ったＤＥＡＥ−セルロースプレート（マチェレーナーゲ
ル社製、ｒＰｏｌｙｇｒａｍ Ｊ ＣＥＬ３００ＤＥＡ
Ｅ／ＨＲ−２／１５）にスポットし、ホモミックスチャ
−タイプ■［シグマ社製イーストＲＮＡタイプＶ１１０
ＣＩを５ Ｍ ７に酸化カリウム水溶液８ｍｌ及び水４
２ｍＱと共に３７°Ｃで２４時間振りまぜて分解させた
後、１Ｎ塩酸で中和し、尿素を７Ｍとなるように加えて
溶解ざけた後、全量を５００ ｍＱとする。使用時は濾
過して用いる］を用いて７０〜ｓｏ’ｃで展開させた。i) 5 μg of 5′-end 32p labeled oligonucleotide (lyophilized product) was added to 1 ml of distilled water.
00uQ, and 250mM 1- to 14μQ of this solution.
Squirrel salt! (+)H7,6>, 12μQ of a mixture of 50mM magnesium chloride, 10mM spermine, 50mM dithiothreitol, 500mM potassium chloride, and then a γ-32p-ATP aqueous solution (manufactured by Amajam, 10μ
Ci/μQ) 1μQ and T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) 1μQ each were added, and distilled water was further added to make the total amount 60μQ. This was reacted at 37°C for 1 hour, and the 5' end was labeled with 32P. 100'C
After stopping the reaction by immersing it in a wet bath for 2 minutes, the total amount
DEAE-cellulose plate (manufactured by Macherenagel, rPolygram J CEL300DEA) was concentrated to about μQ and then cut into 20×20 cm
E/HR-2/15) and homomixture type ■ [Sigma Yeast RNA Type V110].
CI to 5 M 7 with 8 ml of potassium oxide aqueous solution and 4 ml of water
After decomposition by shaking with 2 mQ at 37°C for 24 hours, neutralize with 1N hydrochloric acid, add urea to 7M to dissolve, and make the total volume 500 mQ. Before use, the solution was filtered before use] and developed at 70 to so'c.
上記展開は、キシレンシアツール、オレンジＧ及び酸性
ツクシンの各１％混合水溶液をマーカーとしてスポット
し、キシレンシアツールの青色マーカーが約１Ｑｃｍ程
度上がるまで行なった。乾燥後、プレートをポリ塩化ビ
ニリデンフィルムで包み、オートラジオグラムをとった
。感光時間は室温で約３０分とした。Ｘ線フィルムを現
像し、３２ｐ−標識されたオリゴヌクレオチドのスポッ
トの位置をトレーシングペーパーに移しとり、これをも
とにして更にＤＥＡＥ−セルロースプレート上に印をつ
けた。印をつけた位置のＤＥＡＥ−セルロース部分をか
き取り、エタノール洗浄後、１Ｍトリエチルアンモニウ
ム・二次酸塩水溶液で溶出させた。濃縮乾固後、カウン
トを測定し、２０００ＣＩ）ｍ／μＱとなるように蒸留
水を添加して溶解させた。The above development was carried out by spotting a 1% mixed aqueous solution of xylene cyan tool, orange G and acidic tsukusin as markers until the blue marker of the xylene cyan tool rose by about 1 Qcm. After drying, the plates were wrapped in polyvinylidene chloride film and autoradiograms were taken. The exposure time was about 30 minutes at room temperature. The X-ray film was developed and the position of the 32p-labeled oligonucleotide spot was transferred to tracing paper, and based on this the spot was further marked on the DEAE-cellulose plate. The DEAE-cellulose portion at the marked position was scraped off, washed with ethanol, and then eluted with a 1M triethylammonium secondary acid salt aqueous solution. After concentrating to dryness, the count was measured, and distilled water was added to dissolve the solution at a concentration of 2000 CI) m/μQ.
ｉｉ）二次元ホモクロマトグラフィー・フィンガープリ
ント法
上記ｉ）で得られた５′末端３２Ｐ標識オリゴヌクレオ
チド溶液を７μＱづつ、０．４ｒｎＱ容工ツペンドルフ
チユーブ各３本に各々採取し、之等に各々２５０ｍＭト
リス塩Ｍ（ｐＨ８，０）及び５０ｍＭ塩化マグネシウム
の水溶液２μＱを加え、更に蛇毒フォスフオシエステラ
ーゼ（べ一すンガーマンハイム山之内社製、１．５Ｕ／
ｍｃｌ、／１７ＩＱ）をそれぞれＯ，”ＩＩＩｇ／ｕＱ
、０．２μｇ／μＱ又は０．５μｑ／μＱ加え、３７°
Ｃで３０分間反応させた。反応の停止は、５ｍＭＥＤＴ
Ａを２μＱ加えた後、１００’Ｃ温浴中に２分間浸漬す
ることにより行なった。ii) Two-dimensional homochromatography/fingerprinting method Collect 7 μQ each of the 5'-end 32P-labeled oligonucleotide solution obtained in i) above into three 0.4rnQ Tuppendorf tubes, and Add 2 μQ of an aqueous solution of 250 mM Tris salt M (pH 8.0) and 50 mM magnesium chloride, and add snake venom phosphoosiesterase (manufactured by Bassinger Mannheim Yamanouchi, 1.5 U/
mcl, /17IQ) respectively O,”IIIg/uQ
, 0.2μg/μQ or 0.5μq/μQ added, 37°
The reaction was carried out at C for 30 minutes. To stop the reaction, add 5mMEDT
After adding 2 μQ of A, the sample was immersed in a 100'C hot bath for 2 minutes.
上記反応液の一部（３μＱ）を取って、ＤＥＡＥ−セル
ロースプレートにスポットし、ホモミックスチャ−タイ
プＶｌ ［シグマ社製イーストＲＮＡタイプＶＩＩＯＣ
Ｉを５Ｍ水酸化カリウム水溶液１０ｍ１２及び水４０ｍ
Ｑと共に３７°Ｃで４８時間振りまぜて分解させた後、
１Ｎ塩酸で中和し、尿素を７Ｍとなるように加えて溶解
させた後、全量を５００ ｍＱとする。使用時は濾過し
て用いる］を用いて７０〜８０’Ｃで展開させた。展開
後、オートラジオグラムをとり、オリゴヌクレオチドの
部分分解の状態を調べた。部分分解が確認された反応液
２μＱを、７Ｍ尿素水溶液９５鵬に酢酸５ ｍＱとピリ
ジン０．５１ｒｌ（２とを加えたもので湿らせた酢酸セ
ルロース膜（力−ルツ１イン社製、２．５Ｘ３６Ｃｍ）
の一端中央部にスポットした。これを酢酸−ピリジン水
溶液中で電気泳動させ、オレンジＧの色素マーカーが’
１５ｃｍ泳動したところで停止させた。ドライヤーで酢
酸セルロース膜を乾燥させ、ＤＥＡＥ−セルロースプレ
ート（２０Ｘ ２０ｃｍ）の一端から１．５ｃｍの位置
に酢酸セルロース膜の下端を合わせて重ねた。更に上か
らは蒸留水で湿らせたワットマン３Ｍペーパー（２，５
Ｘ２０ｃｍ）５〜６枚を載せ、ガラス板と約２ｋＣＩの
重しを載せて約３０分間放置してオリゴヌクレオチド部
分分解物をＤＥＡＥ−セルロースプレート上に移行させ
た。酢酸セルロース膜、ワットマン３Ｍペーパー及び重
しを除去し、ＤＥＡＥ−セルロースプレートを蒸留水で
約１Ｑｃｍ展開させた後、ホモミックスチャ−タイプＶ
ｌを入れた展開槽に移し、７０〜８０’Ｃで約２時間展
開させた。展開後、オートラジオグラムをとり、Ｘ線フ
ィルムを現像し、現われるスポットの泳動パターンより
解析を行なった。A portion (3 μQ) of the above reaction solution was taken, spotted on a DEAE-cellulose plate, and homomixture type Vl [Yeast RNA type VIIOC manufactured by Sigma]
I in 10ml of 5M aqueous potassium hydroxide solution and 40ml of water
After shaking and decomposing it with Q at 37°C for 48 hours,
Neutralize with 1N hydrochloric acid, add and dissolve urea to a concentration of 7M, and then adjust the total volume to 500 mQ. The mixture was developed at 70 to 80'C using a filter (filter before use). After development, an autoradiogram was taken to examine the state of partial degradation of the oligonucleotide. 2 μQ of the reaction solution in which partial decomposition was confirmed was added to a cellulose acetate membrane (manufactured by Ritz Iin Co., Ltd., 2.0 μL) moistened with 95 μL of a 7M urea aqueous solution, 5 mQ of acetic acid, and 0.51 rL of pyridine. 5X36cm)
A spot was spotted in the center of one end. This was electrophoresed in an acetic acid-pyridine aqueous solution, and the orange G dye marker was '
It was stopped after migrating 15 cm. The cellulose acetate membrane was dried with a dryer and stacked on a DEAE-cellulose plate (20×20 cm) with its lower edge aligned 1.5 cm from one edge. Furthermore, from above, Whatman 3M paper (2,5
A glass plate and a weight of about 2 kCI were placed on the plate, and the partially degraded oligonucleotide was transferred onto the DEAE-cellulose plate by leaving it for about 30 minutes. After removing the cellulose acetate membrane, Whatman 3M paper and weight, and developing the DEAE-cellulose plate with distilled water to a depth of about 1 Qcm, homomixture type V
The mixture was transferred to a developing tank containing 100 ml of water and developed at 70 to 80'C for about 2 hours. After development, an autoradiogram was taken, the X-ray film was developed, and the electrophoretic pattern of the spots that appeared was analyzed.
１ｉｉ） マキサム−ギルバート法
この方法は、上記ｉ）で得られた５′末端３２Ｐ標識オ
リゴヌクレオチドを用いて、各塩基をこれに特異的な修
飾反応、切断反応を利用して、化学的に分解させ、ポリ
アクリルアミドゲル電気泳動を利用して、分解物をその
切断断片の鎖長差によって分離し、オートラジオグラム
をとり、その泳動パターンより塩基配列を読取る方法で
ある（Ａ、 Ｍ、 Ｍａｘａｎ＋ ａｎｄ Ｗ、 Ｇ
１１ｂｅｒｔ。1ii) Maxam-Gilbert method This method uses the 5'-end 32P-labeled oligonucleotide obtained in i) above to chemically decompose each base using a specific modification reaction and cleavage reaction. This method uses polyacrylamide gel electrophoresis to separate the degraded products based on the difference in chain length of the cleaved fragments, takes an autoradiogram, and reads the base sequence from the migration pattern (A, M, Maxan+ and W, G
11 bert.
ｐｒｏｃ、Ｎａｔｌ、Ａｃａｄ、Ｓｃｉ、、ＵＳＡ、
７４゜５６０ （１９７７＞）。proc, Natl, Acad, Sci, USA,
74°560 (1977>).
上記方法のための分析用キットは市販されており、本方
法でもニューイングランドヌクレアー社製（Ｎｅｗ
Ｅｎｇｌａｎｄ Ｎｕｃｌｅａｒ＞のマキサム−ギル
バート分析キットを用いた。Analytical kits for the above method are commercially available, and this method also uses a kit manufactured by New England Nuclear Co.
A Maxam-Gilbert analysis kit from England Nuclear was used.
化学分解は、同キットのマニュアルを参考にして実施し
た。電気泳動は２０Ｘ６０Ｃｍの２０％ポリアクリルア
ミドゲル（７Ｍ尿素含有）を用いて行なった。泳動後は
、ゲルをポリ塩化ビニリデンフィルムで包み、オートラ
ジオグラムをとった（−８０’Ｃ１−晩感光〉。Chemical decomposition was carried out with reference to the kit's manual. Electrophoresis was performed using a 20×60 cm 20% polyacrylamide gel (containing 7M urea). After electrophoresis, the gel was wrapped in polyvinylidene chloride film and an autoradiogram was taken (-80'C1-night exposure).
９．７ガロースゲル電気泳動 アガロースゲル濃度は、０．９％又は１．６％とした。9.7 Garose gel electrophoresis The agarose gel concentration was 0.9% or 1.6%.
アガロース■（同口化学研究所製）を上記各濃度となる
ように秤量し、ＴＢＥ緩衝液［０，０８９Ｍトリスホウ
酸、０．００２ＭＥＤＴＡを含む〕を加えて、加熱溶解
させてゲルを作成した。泳動は、ミニゲル電気泳動シス
テム ミューピッド２（大昔石油社製）を用い、ＴＢＥ
緩衝液を泳動用緩衝液として用いて行なった。泳動後、
０．５μｇ／ＩＱエチジウムブロマイド溶液にゲルを浸
漬し、紫外線照射により蛍光を発するＤＮＡ断片を確認
した。ゲルからのＤＮＡの溶出は、目的のバンド部分の
ゲルをナイフ等で切りとり、透析チューブ（３５００Ｍ
Ｗカット）にいれ、ＴＥ緩衝液を満たし、同ミニゲル電
気泳動装置で３０分間泳動させることにより行なった。Agarose ■ (manufactured by Doguchi Kagaku Kenkyusho) was weighed out to the above concentrations, and a TBE buffer solution [containing 0.089 M tris-borate and 0.002 MEDTA] was added and dissolved by heating to prepare a gel. For electrophoresis, TBE
A buffer solution was used as a running buffer solution. After electrophoresis,
The gel was immersed in a 0.5 μg/IQ ethidium bromide solution, and DNA fragments that emitted fluorescence by ultraviolet irradiation were confirmed. To elute DNA from the gel, cut out the desired band part of the gel with a knife, etc., and insert it into a dialysis tube (3500M
W cut), filled with TE buffer, and electrophoresed for 30 minutes in the same mini gel electrophoresis device.
上記で溶出されたＤＮＡを濃縮乾固後、これに少量の蒸
留水を加え、前記フェノール抽出法に従って回収した。The DNA eluted above was concentrated to dryness, a small amount of distilled water was added thereto, and the DNA was recovered according to the phenol extraction method described above.
参考例’ｌ ｐＡＰＯＡ−ＩＩの構築■ オリゴヌ
クレオチドの合成
ヒトアポＡ−Ｈの構造遺伝子を含む前記式（２）に示す
全塩基配列の構築に際し、まず同塩基配列を１４〜１９
鎖長のオリゴヌクレオチド断片３０個（Ａ−１〜Ａ−１
５及びＢ−１〜Ｂ−１５）に分け、各々の断片を同相リ
ン酸トリエステル法にて合成した。Reference Example 'l Construction of pAPOA-II ■ Synthesis of oligonucleotides When constructing the entire base sequence shown in formula (2) above, which includes the structural gene of human apoA-H, first the same base sequence was converted to 14 to 19
30 chain-length oligonucleotide fragments (A-1 to A-1
5 and B-1 to B-15), and each fragment was synthesized by the in-phase phosphotriester method.
各断片、その塩基鎖長、塩基配列は、前記第１表に示す
通りである。Each fragment, its base chain length, and base sequence are as shown in Table 1 above.
合成された各断片は、二次元ホモクロマトグラフィー・
フィンガープリント法及びマキサム−ギルバート法にて
、その塩基配列の解析を行なって確認した。Each synthesized fragment was analyzed by two-dimensional homochromatography.
The base sequence was analyzed and confirmed using the fingerprint method and the Maxam-Gilbert method.
■ 合成オリゴヌクレオチドの連結
式（２）に示す全塩基配列の構築を、サブユニットＡ（
オリゴヌクレオチドＡ−１〜Ａ−１５）とサブユニット
Ｂ（オリゴヌクレオチドＢ−１〜Ｂ−１５＞に分けて、
別々に実施した。■ Construction of the entire base sequence shown in linking formula (2) of synthetic oligonucleotides was performed using subunit A (
Oligonucleotides A-1 to A-15) and subunit B (oligonucleotides B-1 to B-15>),
carried out separately.
サブユニットＡは、上記全塩基配列の前半部、ＥＣ０Ｒ
Ｉ制限酵素認識部位よりＰＳｔＩ制限酵素認識部位まで
から構成される。また、サブユニットＢは、同全塩基配
列の後半部、ｐｓｔ工制限酵素認識部位からＭｌｕ■制
限酵素認識部位までから構成される。之等の塩基配列は
前記式（９）及び式（１０）に示す通りである。Subunit A is the first half of the above complete base sequence, EC0R
It consists of I restriction enzyme recognition site to PStI restriction enzyme recognition site. Further, subunit B is composed of the latter half of the entire base sequence, from the pst restriction enzyme recognition site to the Mlu ■ restriction enzyme recognition site. The base sequences of these are as shown in formulas (9) and (10) above.
上記各サブユニットの構築に当たっては、前述したよう
に、サブユニットＡは、これを前記式（３）〜（５）に
示したブロック１〜ブロツク３に分け、またサブユニッ
トＢは、これを前記式（６）〜（８）に示したブロック
４〜ブロツク６に分け、それぞれ別々に構築した。In constructing each of the above subunits, as mentioned above, subunit A divides it into blocks 1 to 3 shown in formulas (3) to (5) above, and subunit B divides it into blocks 1 to 3 shown in formulas (3) to (5) above, and subunit B divides it into blocks 1 to 3 shown in formulas (3) to (5) above. It was divided into blocks 4 to 6 shown in formulas (6) to (8) and constructed separately.
上記各ブロック（ブロック１〜ブロツク６）の構築は、
以下のようにして実施した。その概略は第１図に示す通
りである。図において括弧を付して示した数値は塩基鎖
長を示し、各ブロックを示す実線の末端の黒丸印（ドラ
１〜〉は、５′末端フオスフニー１〜基を示す。The construction of each of the above blocks (block 1 to block 6) is as follows:
It was carried out as follows. Its outline is as shown in FIG. In the figure, the numerical values shown in parentheses indicate the base chain length, and the black circles at the end of the solid line indicating each block (the dot 1~) indicate the 5' terminal phosphine 1~ group.
即ち、まずオリゴヌクレオチド断片の５′末喘に−Ｐを
標識し、これを３２Ｐ−ＤＮＡ溶液とした。但し、オリ
ゴヌクレオチドＡ−１、Ａ−９、Ｂ−１及びＢ−８は、
上記操作を行なわなかった。各ブロック毎（例えばブロ
ック１の場合、オリゴヌクレオチドＡ−１、Ａ−２、八
−３、Ａ−１４及びＡ−１５＞に、各々の３２Ｐ−ＤＮ
Ａ溶液１μＱをとり、１００ｍＭ ＡＴＰ１μＱと、
リガーゼ反応緩衝ｉ！！［６６０ｍＭトリス塩酸（１１
７，６＞ 、６６ｍＭ塩化マグネシウム、１１００ｔｎ
ジチオスレイトール溶液］６μＱを加え、更に水を加え
て、最終反応液量を６０μＱとし、１００’Ｃの温浴中
で２分間加熱処理後、自然冷却した。That is, first, the 5' end of the oligonucleotide fragment was labeled with -P, and this was used as a 32P-DNA solution. However, oligonucleotides A-1, A-9, B-1 and B-8 are
The above operation was not performed. For each block (for example, in the case of block 1, oligonucleotides A-1, A-2, 8-3, A-14 and A-15), each 32P-DN
Take 1μQ of solution A, add 1μQ of 100mM ATP,
Ligase reaction buffer i! ! [660mM Tris-HCl (11
7,6>, 66mM magnesium chloride, 1100tn
Dithiothreitol solution] 6 μQ was added, and water was further added to make the final reaction liquid volume 60 μQ. After heat treatment for 2 minutes in a 100'C hot bath, the mixture was naturally cooled.
次に、Ｔ４ＤＮＡリガーゼ（宝酒造社製）２．５ユニツ
トを添加して、４℃下に一晩反応させた。フェノール抽
出後、１２％ポリアクリルアミドゲル電気泳動を行ない
、オートラジオダラムをとり、目的の大きさのブロック
でおる二本鎖ＤＮＡ部分をかき取り、ＤＮＡを溶出させ
た。電気泳動条件は、２０Ｘ６０Ｃｍ、０．３５ｍＩｎ
厚さ、１２００〜１５００Ｖとし、泳動液として丁ＢＥ
緩衝液を用いた。′以上の操作により、各ブロック１〜
６を構築した。Next, 2.5 units of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was allowed to react at 4°C overnight. After phenol extraction, 12% polyacrylamide gel electrophoresis was performed, an autoradiodrum was taken, and the double-stranded DNA portion of the target size block was scraped off to elute the DNA. Electrophoresis conditions were 20X60Cm, 0.35mIn
The thickness was set to 1200 to 1500V, and the electrophoresis liquid was
A buffer solution was used. 'By the above operations, each block 1~
6 was constructed.
次に、得られた各ブロック間の組立てを以下の通り実施
した。即ち、上記で溶出させたブロック１〜６のそれぞ
れを液体シンチレーションカウンターにてカウント測定
後、５０００ｃｐｍ／μＱとなるように水で希釈し、各
２μＱ（サブユニットＡはブロック１〜３、サブユニッ
トＢはブロック４〜６）をとり、Ｔ４ＤＮＡリガーゼ及
びＡＴＰを加えて連結反応を行なった。Next, the obtained blocks were assembled as follows. That is, each of blocks 1 to 6 eluted above was counted using a liquid scintillation counter, diluted with water to 5000 cpm/μQ, and 2 μQ each (subunit A, blocks 1 to 3, subunit B took blocks 4 to 6), added T4 DNA ligase and ATP, and performed a ligation reaction.
１０％ポリアクリルアミドゲル電気泳動法にて、目的の
大きざのサブユニットを分離し、かき取り、溶出させる
ことにより、所望のザブユニッ＋−Ａ及びＢの構築を完
了した。Construction of the desired subunits A and B was completed by separating, scraping, and eluting subunits of the desired size using 10% polyacrylamide gel electrophoresis.
■ ｐＡＰＯｌ及びｐＡＰＯ２の作製
上記■で構築されたサブユニットＡをプラスミドベクタ
ー１）ＢＲ３２２に組込んだベクターをｐＡＰＯｌとし
、同サブユニットＢを同ｐＢＲ３２２に組込んだベクタ
ーをｐＡＰＯ２とした。(2) Preparation of pAPOl and pAPO2 A vector in which subunit A constructed in (1) above was integrated into plasmid vector 1) BR322 was designated as pAPOl, and a vector in which subunit B was integrated into the same pBR322 was designated as pAPO2.
まずｐＡＰＯｌの構築につき詳述する。First, the construction of pAPOl will be explained in detail.
ｐＢＲ３２２を制限酵素ｐｓｔ工及びＥＣ０ＲＩで、処
理した後、０．９％アガロースゲル電気泳動を行なって
、約３．ｅｏｈｂｐのＤＮＡ断片＜Ａ＞を得た。このＤ
ＮＡ断片＜Ａ＞と、ザブユニットＡ、Ｔ４ＤＮＡリガー
ゼ、ＡＴＰ及びリガーゼ反応用緩衝液とを混ぜ、３７°
Ｃで１時間反応させて、ＤＮＡを連結させた。この反応
組成液で大腸菌Ｈ８１０１株の形質転換を行ない、得ら
れる形質転換株を１−ｂｒｏｔｈ培地にテトラサイクリ
ン２０μｑ／ｍＱを添加した平板培地に拡げ、生育して
くるコロニーを選択した。更にこのうちの１株を４００
ｍＱ Ｌ−ｂｒｏｔｈ培地にテトラサイクリン２０μ
Ｑ／ｍＱを添加した液体培地にて振盪培養後、プラスミ
ドＤＮＡを単離、精製し、制限酵素Ｈｌ）ａＩＩ、Ｐｓ
ｔＩとＥ ｃｏＲＩ （７）二種酵素反応系で処理し、
その切断様式をポリアクリルアミドゲル電気泳動法によ
り解析した。After treating pBR322 with restriction enzymes pst and EC0RI, 0.9% agarose gel electrophoresis was performed. An eohbp DNA fragment <A> was obtained. This D
Mix NA fragment <A>, Zabuunit A, T4 DNA ligase, ATP, and ligase reaction buffer, and incubate at 37°.
The DNAs were ligated by reacting at C for 1 hour. Escherichia coli strain H8101 was transformed with this reaction composition, and the resulting transformed strain was spread on a plate medium containing 1-broth medium supplemented with 20 μq/mQ of tetracycline, and growing colonies were selected. Furthermore, one of these shares will be sold for 400
Tetracycline 20μ in mQ L-broth medium
After shaking culture in a liquid medium supplemented with Q/mQ, plasmid DNA was isolated and purified, and restriction enzymes Hl)aII, Ps
tI and EcoRI (7) treated with a two-enzyme reaction system,
The cleavage mode was analyzed by polyacrylamide gel electrophoresis.
またマキサム−ギルバート法にて直接塩基配列を解析し
た。かくして、得られた形質転換株がｐＡＰＯｌ、即ち
目的のサブユニットＡを含有し、設計した塩基配列を有
するものであることを確認した。The base sequence was also directly analyzed using the Maxam-Gilbert method. It was thus confirmed that the obtained transformed strain contained pAPOl, that is, the desired subunit A, and had the designed base sequence.
次に、ｐＡＰＯ２の構築につき詳述する。Next, the construction of pAPO2 will be explained in detail.
サブユニットＢをプラスミドベクターｌＲ３２２に導入
するに際し、構築したサブユニットＢのＣ末端側は、Ｍ
ＩＬＩ工制限酵素認識部位となっているが、ｐＢＲ３２
２は該１ｖ’ｌｌｕ工制限酵素比制限酵素認識ないため
、該サブユニットＢを直接ＤＢＲ３２２に組込むことは
できない。When subunit B was introduced into plasmid vector IR322, the C-terminal side of the constructed subunit B was
Although it is an ILI restriction enzyme recognition site, pBR32
Since subunit B does not recognize the restriction enzyme at the 1v'llu restriction enzyme ratio, subunit B cannot be directly incorporated into DBR322.
従って、まずｐＢＲ３２２にＭｌｕＩ！Ｊンカ−を挿入
したベクターｐＢＲ３２２−Ｍｌｕを以下の通り作製し
た。即ち、ｐＢＲ３２２（１０μｑＤＮＡ）にＥＣ０Ｒ
Ｉの５ユニツトを加え、３７°Ｃで２時間反応させ、フ
ェノール抽出法にてＤＮＡを回収し、更に蒸留水で溶解
させた後、Ｓ１ヌクレアーゼと共に反応させて、ｐＢＲ
３２２のＥＣ０ＲＩ制限酵素認識部位がプラントエンド
化状態になったＤＮＡ断片を得た。Therefore, first, MluI! A vector pBR322-Mlu into which a J linker was inserted was prepared as follows. That is, EC0R was added to pBR322 (10 μq DNA).
After adding 5 units of I and reacting at 37°C for 2 hours, the DNA was recovered by phenol extraction, dissolved in distilled water, and reacted with S1 nuclease to obtain pBR.
A DNA fragment in which the 322 ECORI restriction enzyme recognition site was in a plant-end state was obtained.
次に、化学合成したＭＩｕＩリンカ−［５′ＧＴＣＧＡ
ＣＧＣＧＴＣＧＡＣ３’ ］の５′末端にフォスフェー
トを導入して得たオリゴヌクレオチドを混合し、丁４Ｄ
ＮＡリガーゼを用いて、連結反応させた。Next, the chemically synthesized MIuI linker-[5'GTCGA
CGCGTCGAC3'] was mixed with an oligonucleotide obtained by introducing phosphate into the 5' end of CGCGTCGAC3'.
A ligation reaction was performed using NA ligase.
反応後、この反応組成液で大腸菌Ｈ８１０１株を形質転
換させ、得られたテトラサイクリン耐性コロニーを選択
し、そのうちの１株よりプラスミドＤＮＡＩ）ＢＲ３２
２−Ｍｌｕを単離、′（′ｙ製した。After the reaction, Escherichia coli strain H8101 was transformed with this reaction composition solution, the resulting tetracycline-resistant colonies were selected, and plasmid DNA (AI) BR32 was selected from one of the colonies.
2-Mlu was isolated and prepared.
制限酵素ＥＣ０ＲＩ、ＭｌｕＩ、５ａｌＩ、ｌ−１ｉｎ
ｆ■等で処理し、ポリアクリルアミドゲル電気泳動法に
てその切断パターンを解析し、得られたベクターがｐＢ
Ｒ３２２のＥＣ０ＲＩ制限酵素認識部位に化学合成ＭＩ
ＬＩニリンカーを挿入された目的のものであることを確
認した。Restriction enzymes EC0RI, MluI, 5alI, l-1in
The cleavage pattern was analyzed by polyacrylamide gel electrophoresis, and the resulting vector was pB.
Chemically synthesized MI at the EC0RI restriction enzyme recognition site of R322
It was confirmed that the LI linker was inserted for the purpose.
次に、このベクター１）ＢＲ３２２−Ｍｌｕを制限酵素
ＰＳｔ工及びＭｌｕＩで処理し、０．９％アガロースゲ
ル電気泳動にて、約３．６ｋｂｌ）のＤＮＡ断片＜８＞
を単離、精製した。その後、この断片とサブユニットＢ
、Ｔ４ＤＮＡリガーゼを混ぜ、３７°Ｃで１時間反応さ
せて連結した。Next, this vector 1) BR322-Mlu was treated with restriction enzymes PSt and MluI, and a DNA fragment of approximately 3.6 kbl) <8> was analyzed by 0.9% agarose gel electrophoresis.
was isolated and purified. Then this fragment and subunit B
, T4 DNA ligase was mixed, and ligation was performed by reacting at 37°C for 1 hour.
これを大腸菌Ｈ８１０１株に形質転換して、目的の１）
ＡＰＯ２を保有する菌株を得た。This was transformed into E. coli H8101 strain to achieve the objective 1).
A strain harboring APO2 was obtained.
上記で得られたｐＡＰＯ２は、ｐＡＰＯｌと同様にマキ
サム−ギルバート法にてその塩基配列を解析した結果、
サブユニットＢの存在が確認され、また目的の塩基配列
を有していることが確認された。The base sequence of pAPO2 obtained above was analyzed by the Maxam-Gilbert method in the same way as pAPOl, and as a result,
The existence of subunit B was confirmed, and it was also confirmed that it had the desired base sequence.
■ ｐＡＰＯＡ−ＩＩの構築
上記■で得たＤＡＰＯｌを、制限酵素ＰＳｔ工及びＥＣ
０ＲＩで処理し、５％ポリアクリルアミドゲル電気泳動
法にて、約０．１３ｋｂｌ）のＤＮＡ断片＜Ｃ＞を単離
、精製した。■Construction of pAPOA-II DAPOl obtained in step (■) above was treated with restriction enzyme PSt and EC.
After treatment with 0RI, DNA fragment <C> of approximately 0.13 kbl was isolated and purified by 5% polyacrylamide gel electrophoresis.
同様にしてＩ）ＡＰＯ２を、制限酵素ＰｓｔＩ及び５ａ
ｌＩで処理し、５％ポリアクリルアミドゲル電気泳動法
にて、約０．１４ｋｂｐのＤＮＡ断片＜Ｄ＞を単離、精
製した。Similarly, I) APO2 was added to the restriction enzyme PstI and 5a.
After treatment with 1I, a DNA fragment <D> of about 0.14 kbp was isolated and purified by 5% polyacrylamide gel electrophoresis.
一方、ＤＢＲ３２２を制限酵素ＥＣ０ＲＩ及びＳａＩ■
で処理し、０．９％アガロースゲル電気泳動法にて約３
．７１ｋｂｐのＤＮＡ断片＜Ｅ＞を単離、精製した。On the other hand, DBR322 was treated with restriction enzymes EC0RI and Sal■
3% by 0.9% agarose gel electrophoresis.
．． A 71 kbp DNA fragment <E> was isolated and purified.
上記で得た３種のＤＮＡ断片＜Ｃ＞、＜ ’Ｄ ＞及び
＜Ｅ＞をＴ４ＤＮＡリカーゼを用いて連結反応させた。The three DNA fragments <C>, <'D>, and <E> obtained above were ligated using T4 DNA licase.
反応物で大腸菌１１８１０１株を形質転換させ、アンピ
シリン耐性コロニーを’＞Ｂ 択し、そのうちの１株よ
りプラスミドＤＮＡを単離、精製した。E. coli strain 118101 was transformed with the reaction mixture, ampicillin-resistant colonies were selected, and plasmid DNA was isolated and purified from one of the strains.
か＜ｂ”Ｃ、ヒトアポＡ−ＩＩＩ伝子をコードする塩基
配列を有する化学合成遺伝子が、ｐＢＲ３２２のＥＣＯ
Ｒ工及びＳａｌ工制限酵素認識部位間に挿入されたプラ
スミドベクターｐＡＰｏ△−■を得た。A chemically synthesized gene having a nucleotide sequence encoding the human apoA-III gene is
A plasmid vector pAPoΔ-■ inserted between the R and Sal restriction enzyme recognition sites was obtained.
得られたベクターｐＡＰＯＡ−■につき、種々の制限酵
素（例えばＨｌ）ａＩＩ、△ａｔｎ、ＨａｅＩＩ等）に
よる切断パターン、切断部位等を、５％ポリアクリルア
ミドゲル電気泳動法により解析した結果、これが全塩基
数的３．９７ｋｂｐのヒトアポＡ−ＩＩ構造遺伝子を有
する目的のプラスミドｐＡＰＯＡ−ＩＩであることを確
認した。The obtained vector pAPOA-■ was analyzed by 5% polyacrylamide gel electrophoresis to analyze the cleavage pattern and cleavage site by various restriction enzymes (e.g., Hl, aII, Δatn, HaeII, etc.), and it was found that the entire base It was confirmed that the target plasmid pAPOA-II had a numerically 3.97 kbp human ApoA-II structural gene.
上記■及び■に示したりＡ ＰＯ１，１）ＡＰＯ２及び
ｐＡＰＯＡ−■の構築の操作の戦略を、第２図に示す。The operational strategy for the construction of APO1,1)APO2 and pAPOA-■ shown in (1) and (2) above is shown in FIG.
図においてＡｐｒはアンピシリン耐性を、ＴＣｒはテト
ラサイクリン耐性を、それぞれ示し、以降の各図におい
ても同様とする。In the figure, Apr indicates ampicillin resistance and TCr indicates tetracycline resistance, and the same applies to subsequent figures.
上記プラスミドベクター１）ＡＰＯＡ４を保有する大腸
菌Ｈ８１０１株は、通商産業省工業技術院微生物工業技
術研究所（微工研）に「Ｅｓｃｈｅｒｉｃｈｉａ ｃｏ
ｌｉ、ＨＢ −１０１、１）ＡＰＯＡ−ＩＩ−Ｎｏ、３
Ｊなる表示で寄託番号「微工研菌奇第９１６５号（ＦＥ
ＲＭ Ｐ−９１６５）Ｊとして寄託されている。The above plasmid vector 1) Escherichia coli H8101 strain carrying APOA4 was sent to the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microbial Technology Research Institute (Feikoken) as “Escherichia coli”.
li, HB-101, 1) APOA-II-No, 3
Deposit number 9165 (FE) with the designation J.
It has been deposited as RM P-9165)J.
参考例２ 大腸菌ペリプラズム分泌発現系ベクターの
構築
ｔａｃプロモーター、ｂｌａシグナルペプチドの下流に
化学合成したアポＡ−ｎ遺伝子を導入することにより、
大腸菌ペリプラズム層への分泌発現ベクターを得ること
ができるが、該ベクターの構築に当たっては、ｂｌａシ
グナルペプチドのアミノ酸配列に対するコドンの流れに
沿ってアポＡ−４構造遺伝子のコドンフレームがずれる
ことがないような結合の仕方が要求される。以下、かか
る要求を満たした分泌発現系ベクターの構築につき詳述
する。Reference Example 2 Construction of Escherichia coli periplasmic secretion expression system vector By introducing the chemically synthesized apoAn gene downstream of the tac promoter and the bla signal peptide,
An expression vector for secretion into the periplasmic layer of E. coli can be obtained, but when constructing this vector, care must be taken not to shift the codon frame of the apoA-4 structural gene along the codon flow for the amino acid sequence of the bla signal peptide. A method of connection is required. The construction of a secretory expression vector that satisfies these requirements will be described in detail below.
■ 中間プラスミドベクターｐＫＴＮの作製このベクタ
ーの構築操作の概略は、第３図に示す通りでおり、該ベ
クターは、上記要求を満たすために、ｂｌａシグナルペ
プチドの２３番目のアミン１ｍ（Ａｌａ＞の第３コドン
の後で、制限酵素Ｎａｅ工で切断できるように設計され
た。■ Preparation of intermediate plasmid vector pKTN The outline of the construction procedure for this vector is shown in Figure 3. In order to meet the above requirements, this vector was constructed using the 23rd amine 1m (Ala>) of the bla signal peptide. It was designed to be cut with the restriction enzyme Nae after the third codon.
尚、第３図において、黒矢印はｔａｃプロモーターを、
波線は旧ａシグナルペプチドをコードする塩基配列を、
白ヌキの部分はＥＧＦをコードする塩基配列をそれぞれ
示し、之等は以下の図においても同様とする。また、第
３図には上記制限酵素Ｎａｅ］−認識部位導入部分（ｂ
ｌａシグナルペプチドと合成リンカ−との結合部位）の
塩基配列を併記する。In addition, in Fig. 3, the black arrow indicates the tac promoter,
The wavy line indicates the base sequence encoding the old a signal peptide,
The white boxes indicate the base sequences encoding EGF, and the same applies to the following figures. In addition, FIG. 3 shows the above-mentioned restriction enzyme Nae]-recognition site introduction portion (b
The base sequence of the binding site between the la signal peptide and the synthetic linker is also shown.
その構築には、ｔａｃプロモーターと旧ａシグナルペプ
チドとを保有するプラスミドベクターｐＩＪＧＴＩ ５
０ （特願昭６１−１５３７８３＠、これを保有する大
腸菌ＪＭ”ｌ ０３株は微工研条奇第９７４号として寄
託されている）を利用した。For its construction, the plasmid vector pIJGTI5 carrying the tac promoter and the old a signal peptide was used.
0 (Japanese Patent Application No. 61-153783@, the Escherichia coli JM"l 03 strain possessing this has been deposited as Kaikoken Joki No. 974) was used.
ｐＵＧＴ１５０の１０／、１を、制限酵素ＮｒｕＴ及び
ＥＣ０ＲＩで処理し、０．９％アガロースゲル電気泳動
にて約０．４６ｋｂｏのＤＮＡ断片＜Ｉ＞を単離、採取
した。pUGT150 10/1 was treated with restriction enzymes NruT and EC0RI, and a DNA fragment <I> of about 0.46 kbo was isolated and collected by 0.9% agarose gel electrophoresis.
また、ｐＢＲ３２２の１０μｑを制限酵素［）ｒａ工及
びＥｃｏＲ工ｒｆｆｉ理し、１．６％アガロースゲル電
気泳動にて、約３．２３ｋｂｐのＤＮＡ断片＜Ｊ＞を単
離、採取した。Furthermore, 10 μq of pBR322 was treated with restriction enzymes [)ra and EcoR, and a DNA fragment of about 3.23 kbp <J> was isolated and collected by 1.6% agarose gel electrophoresis.
上記２種のＤＮＡ断片と、化学合成したリンカ−［５’
ＣＣＧＧＣＣＧＧ ３’ ］１μｑとを、Ｔ４ＤＮ
Ａリガーゼを用いて連結させ、連結物で大腸菌ＪＭＩ
０３株を形質転換させ、テトラサイクリン耐性を示すコ
ロニーを選択し、そのうちの１株からプラスミドＤＮＡ
を単離、精製して、目的の中間プラスミドベクターｐＫ
ＴＮを得た。The above two types of DNA fragments and a chemically synthesized linker [5'
CCGGCCGG 3']1 μq, T4DN
Ligate using A ligase and incubate E. coli JMI with the ligated product.
03 strain was transformed, colonies showing tetracycline resistance were selected, and plasmid DNA was extracted from one of the strains.
is isolated and purified to obtain the desired intermediate plasmid vector pK.
Got TN.
このベクターは、ｂｌａシグナルペプチド２３番目のア
ミノ酸の直後に、制限酵素ＮａｅＩ認識部位を有するも
のであった。その切断パターン及び切断部位の存在を確
認した。This vector had a restriction enzyme NaeI recognition site immediately after the 23rd amino acid of the bla signal peptide. The cutting pattern and the presence of the cutting site were confirmed.
上記プラスミドベクターｐＫＴＮを保有する大腸菌ＪＭ
１０３株は、通商産業省工業技術院微生物工業技術研究
所（微工研）に
［Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ、ＪＭ −１０３
，１）ＫＴＮ−２−２Ｊなる表示で寄託番号［微工研菌
奇第９１４６号（ＦＥＲＭ Ｐ−９１４６）Ｊとして
寄託されている。E. coli JM carrying the above plasmid vector pKTN
Escherichia coli, JM-103, was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
, 1) It has been deposited under the designation KTN-2-2J and the deposit number [FERM P-9146) J.
■ １）ＳＡＰ−０の作製
このベクターの構築の操作の概略は、第４図に示す通り
でおる。図において、斜線を付して示した塩基配列部分
はアポＡ−ＩＩ構造遺伝子を示す。また第４図には、ｂ
ｌａシグナルペプチドとアポＡ−Ｉ構造遺伝子との結合
部位の塩基配列をも併記する。1) Construction of SAP-0 The outline of the procedure for constructing this vector is as shown in FIG. In the figure, the hatched base sequence portion indicates the apoA-II structural gene. Also, in Figure 4, b
The base sequence of the binding site between the la signal peptide and the apoA-I structural gene is also shown.
まず、アポＡ−Ｉ遺伝子を有するプラスミドｐＡＰＯＡ
−Ｉ［の１０μＣ１を、制限酵素ＡａｔｌＩで処理し、
次に８１ヌクレアーゼ（ＤＮＡ１μΩに対して３ユニツ
ト）と反応させた後、更に制限酵素３ａｌＩで処理し、
１．６％アガロースゲル電気泳動にて約０，２４ｋｂｐ
のＤＮＡ断片＜Ｋ＞を単離、採取した。これは、アポＡ
−■の構造遺伝子をコードする塩基配列を持つＤＮＡ断
片である。First, plasmid pAPOA containing the apoA-I gene
-I [10 μC1 was treated with the restriction enzyme AatlI,
Next, after reacting with 81 nuclease (3 units per 1 μΩ of DNA), further treatment with restriction enzyme 3alI,
Approximately 0.24 kbp by 1.6% agarose gel electrophoresis
DNA fragment <K> was isolated and collected. This is appointment A
-It is a DNA fragment having a base sequence encoding the structural gene of ■.
次に、上記■で得たｐＫ丁Ｎの５μｑを制限酵素ＥＣ０
ＲＩ及びＮａｅ工で処理後、０．９％アガロースゲル電
気泳動にて、約０．４６ｋｂＤのＤＮＡ断片＜Ｌ＞を採
取した。Next, 5μq of pKdN obtained in above ① was treated with restriction enzyme EC0.
After treatment with RI and Nae, a DNA fragment <L> of approximately 0.46 kbD was collected by 0.9% agarose gel electrophoresis.
更にプラスミドベクター１）ＵＧＴｌ ５０３（特願昭
６１−３１４１５号、微工研条奇第９７５＠として寄託
された大腸菌ＪＭ”１０３株に保有される〕の５μｑを
、制限酵素ＥＣ０ＲＩ及びＸｈＯ工で処理した後、１．
６％アガロースゲル電気泳動にて、約３．２５ｋｂｐの
ＤＮＡ断片を単離、採取した。Furthermore, 5 μq of plasmid vector 1) UGTl 503 (carried in Escherichia coli JM”103 strain deposited as Japanese Patent Application No. 61-31415, FAIKEN JOKI No. 975@) was treated with restriction enzymes EC0RI and XhO. After doing that, 1.
A DNA fragment of about 3.25 kbp was isolated and collected by 6% agarose gel electrophoresis.
上記３種のＤＮＡ断片を混ぜ、丁４． Ｄ ＮＡリガー
ゼを用いて連結反応させ、反応物で大腸菌ＪＭＩ０３株
を形質転換し、テｉ・ラサイクリン耐性コロニーを選択
し、そのうちの１株からプラスミドＤＮＡを単離、精製
した。Mix the above three types of DNA fragments and mix them together.4. A ligation reaction was carried out using DNA ligase, the reaction product was used to transform Escherichia coli JMI03 strain, te-racycline-resistant colonies were selected, and plasmid DNA was isolated and purified from one of the strains.
得られたプラスミドＤＮＡにつき、種々の制限酵素（例
えば）−１ｉｎｃｌＪ等）で処理して、その切断パター
ン、切断部位の存在を検討した結果、該プラスミドはｔ
ａｃプロモーター、ｂｌａシグナルペプチドの塩基配列
及びアポＡ−ＩＩ遺伝子を同一方向に連結された塩基配
列を有する目的のベクターｐｓＡＰ−０でおることが確
認された。The obtained plasmid DNA was treated with various restriction enzymes (for example, -1inclJ, etc.) and the cleavage pattern and presence of the cleavage site were examined.
It was confirmed that the desired vector psAP-0 had a base sequence in which the ac promoter, the base sequence of the bla signal peptide, and the apoA-II gene were linked in the same direction.
上記プラスミドベクターＤＳＡＰ−０を保有する大腸菌
ＪＭ１０３株は、通商産業省工業技術院微生物工業技術
研究所（微工研）に’ｉ Ｅｓｃｈｅｒｉｃｈｉａ ｃ
ｏｌｉ、ＪＭ −１０３，ｐｓＡｐ−Ｏ−１１Ｊなる表
示で寄託番号「微工研菌奇第９１４７号（ＦＥＲＭ
Ｐ−９１４７＞Ｊとして寄託されて０る。The Escherichia coli JM103 strain carrying the above plasmid vector DSAP-0 was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
oli, JM-103, psAp-O-11J with the deposit number "FERM
Deposited as P-9147>J and 0.
実施例１ アポＡ−Ｉ［の発現及び確認■ 分泌発現
系ベクターｐＳＡＰ−０を保有する大腸菌ＪＭ１０３株
の培養
下記第２表に示す組成の液体培地〔Ｍ−９カザミノ酸培
地〕を用いた。Example 1 Expression and Confirmation of ApoA-I [2] Cultivation of Escherichia coli strain JM103 carrying the secretion expression vector pSAP-0 A liquid medium [M-9 casamino acid medium] having the composition shown in Table 2 below was used.
第２表
Ｊ 配合量ＮａＨ２Ｐ
Ｏ４５，８ＣＩ
ＫＨ２ＰＯ４３，Ｏｇ
ＮａＣＱ ５．０ｇＮ＋−１
４ＣＱ １．ＱＣ）カザミノ酸
（ディフコ社Ｗ） ５．０ｇビタミンＢ＋
１．０ｍｇプロリン
５０ｍｇグルコースＸ
５．００１Ｍ ＣａＣＱ２Ｘ ０．
１ｍＱ１Ｍ ＭｇＣ９２Ｘ １．Ｏｍ
Ｑテトラサイクリン ２０μΩ、／哩Ｈ２
０を加えて全量を１０１００Ｏとする但し表中Ｘ）印は
、別途オートクレーブ滅菌処理（１２１°Ｃで１５分間
）した試薬を使用したことを示す。Table 2 J Blend amount NaH2P
O45,8CI KH2PO43,Og NaCQ 5.0gN+-1
4CQ 1. QC) Casamino acids (Difco W) 5.0g Vitamin B+
1.0mg proline
50mg glucose
5.001M CaCQ2X 0.
1mQ1M MgC92X 1. Om
Qtetracycline 20μΩ, /哩H2
0 was added to make the total volume 10,100O. However, the mark (X) in the table indicates that a reagent that was separately sterilized in an autoclave (121°C for 15 minutes) was used.
プラスミドベクターを保有する大腸菌ＪＭ１０３株の前
培養液１ ｍＱを、上記組成のＭ−９力ザミノ酸培地１
００ｍＱを含むフラスコにｈ口え、３．７℃で往復１辰
盪培養した。培養開始後、約４時間（ＯＤ６１０＝約０
．４）にて、Ｉ ＰＴＧを０．１ｍＭｍＱとなるように
添加し、更に同様にして培養を続けた。1 mQ of the preculture of E. coli JM103 strain carrying the plasmid vector was mixed with 1 mQ of M-9 zamino acid medium having the above composition.
The cells were placed in a flask containing 00mQ and cultured at 3.7°C with one round trip. Approximately 4 hours after the start of culture (OD610 = approximately 0
．． In step 4), IPTG was added at a concentration of 0.1mMmQ, and the culture was continued in the same manner.
■ 菌体からの目的物の抽出
■で示した培養条件で培養し、Ｉ ＰＴＧ添加添加後間
時間養を停止させ、集菌（１００００回転／分回転弁間
）した。(2) Extraction of target product from bacterial cells Culture was carried out under the culture conditions shown in (2), and after addition of IPTG, incubation was stopped for a period of time, and bacteria were collected (10,000 revolutions/minute between rotating valves).
得られた菌体より浸透圧ショック法にて、ペリプラズム
画分を得た。即ち、培養液と同量の３０ｍＭ トリス塩
！ （ｐＨ８，０）−２０％ショ糖緩衝液を加えて懸濁
させた。更に最終濃度０、ＯＩＭとなるようにＥＤＴＡ
溶液を加え、ロータリーシェーカーで２４℃にて１８０
回転／分で１０分間振盪撹拌培養した。遠心分離（１０
０００回転／分回転弁間）して、菌体を集め、次いで氷
冷した水を培養液と同量加えて再懸濁させ、水中に１５
分間放置し、時々撹拌した後、遠心分離（１００００回
転／分回転弁間）により、上清液と沈渣とを分離した。A periplasmic fraction was obtained from the obtained bacterial cells by the osmotic shock method. That is, the same amount of 30mM Tris salt as the culture solution! (pH 8,0)-20% sucrose buffer was added and suspended. Furthermore, add EDTA so that the final concentration is 0, OIM.
Add the solution and shake at 180°C at 24°C on a rotary shaker.
The culture was carried out with shaking at rotation/min for 10 minutes. Centrifugation (10
000 rpm) to collect the bacterial cells, then add the same amount of ice-cold water as the culture solution to resuspend, and incubate in water for 15 minutes.
After standing for a minute and stirring occasionally, the supernatant liquid and the precipitate were separated by centrifugation (10,000 rpm between rotating valves).
得られた上滑がペリプラズム画分である。また、沈渣は
同量の氷冷した水を加えて懸濁させ、超音波処理（１０
０Ｗ、５０秒、２回）を行ない、遠心分離（１５０００
回転／分、１０分間）して、その上清両分を得た。これ
を細胞内画分とする。The obtained supernatant is the periplasmic fraction. In addition, the sediment was suspended by adding the same amount of ice-cold water, and subjected to ultrasonic treatment (10
0W, 50 seconds, twice) and centrifugation (15,000
rotation/min for 10 minutes) to obtain both supernatants. This is referred to as the intracellular fraction.
■ エンザイムイムノアツセイによるアポＡ−Ｉｔの測
定
上記■で得られた各画分のアポＡ−ＩＩの測定は、精製
ヒトアポＡ−ＩＩを標準物質として用いたアポＡ−ＩＩ
特異エンザイムイムノアッセイにより行なった。以下に
その測定法の詳細を示す。■Measurement of apoA-It by enzyme immunoassay Measurement of apoA-II in each fraction obtained in the above (■) was performed using purified human apoA-II as a standard substance.
It was performed by specific enzyme immunoassay. The details of the measurement method are shown below.
まずヒト血清より精製したアポＡ−ＩＩを抗原として、
家兎に免疫して抗血清を得た。これは、凍結乾燥ヒトア
ポＡ−ｎ１ｍＧを蒸留水１１ＴＩＩ２に溶解後、フロイ
ント完全アジュバント１ｍＧを加えて乳化させ、これを
家兎３羽の足指皮内に注射し、次に２週間毎に同量の上
記乳化アポＡ−ＩＩを家兎の背部に皮下注射して、合計
３回免疫し、最終免疫の７日後に全採血して、血清を分
離することにより実施した。かくして得られる抗血清を
以下のアッセイに用いた。First, apoA-II purified from human serum was used as an antigen.
Antiserum was obtained by immunizing a rabbit. This was done by dissolving lyophilized human apoA-n1mG in 11TII2 of distilled water, adding 1mG of Freund's complete adjuvant to emulsify it, injecting this into the toes of three domestic rabbits, and then administering the same amount every two weeks. The above emulsified ApoA-II was subcutaneously injected into the back of rabbits to immunize them three times in total. Seven days after the final immunization, whole blood was collected and serum was separated. The antiserum thus obtained was used in the following assay.
また、ペルオキシダーゼ標識アポＡ−ｍを、古式らの報
告しているマレイミド法（Ｓ。In addition, peroxidase-labeled apoA-m can be prepared using the maleimide method (S) reported by Koshiki et al.
Ｙｏｓｈｉｔａｋｅ ｅｔ ａｌ、、 Ｊ 、 Ｂｉｏ
ｃｈｅｍ、 ９２ 。Yoshitake et al., J. Bio.
chem, 92.
１４１３−１４２４ （１９８２））に従い、以下の通
り作製した。即ち、アポＡ−ＩＩを、６Ｍ尿素の存在下
で、還元剤であるジチオスレイトールで処理してＳ−８
結合を切断し、ＳＨＩを１ケ所持つ単量体分子とした。1413-1424 (1982)) as follows. That is, apoA-II was treated with dithiothreitol, a reducing agent, in the presence of 6M urea to form S-8.
The bond was cleaved to form a monomer molecule with one SHI.
一方、ペルオキシダーゼへのマレイミド基の導入は、モ
ル比で１００倍量のＮ−サクシニミジル−４−（Ｎ−マ
レイミドメチル〉シクロヘキサン−１−カルボキシレー
トとペルオキシダーゼとを反応させることにより行なっ
た。このマレイミド基導入ペルオキシダーゼを、上記で
作製したアポＡ−■単量体にモル比で１＝１になるよう
に加え、４℃で２０時間インキュベートした。その後、
バイオゲルＰ−１００（バイオラッド社製）にてゲル濾
過を行ない、ペルオキシダーゼ−アポＡ４複合体を含む
画分を集めた。On the other hand, a maleimide group was introduced into peroxidase by reacting a 100-fold molar amount of N-succinimidyl-4-(N-maleimidomethyl>cyclohexane-1-carboxylate) with peroxidase. The introduced peroxidase was added to the apoA-■ monomer prepared above at a molar ratio of 1=1, and incubated at 4° C. for 20 hours.
Gel filtration was performed using Biogel P-100 (manufactured by Bio-Rad), and fractions containing the peroxidase-apoA4 complex were collected.
アッセイに用いる抗血清及びペルオキシダーゼ標識アポ
Ａ−ｍの希釈倍率、アッセイ条件を最適化するための反
応時間、温度、抗体結合標識抗原（バウンド）と遊離標
識抗原（フリー）の分離方法等の検討を行なって、以下
の測定条件を設定した。即ち、０．５％ウシ血清アルブ
ミン（ＢＳＡ＞ 、０．１４Ｍ塩化ナトリウム及び２０
ｍＭリン酸緩衝液（ｐＨ７，２＞を希釈液として用いて
、抗ヒトアポＡ−ｎ血清（１：１２０＞５０μＱ１測定
試料又は標準ヒトアポＡ−Ｉ１１００μＱを試験管に加
え、４°Ｃで２０時間インキュベートした１多、５％（
Ｖ／Ｖ）アフィゲルプロティンＡ（バイオラッド社製）
１００μＱを加え、撹拌後、室温で１時間静置した。０
．０５％ツイーン２０及び０．１４Ｍ塩化ナトリウムを
含む２０ｍＭリン酸緩衝液（ｐＨ７，２＞（ｒＰＢｓ−
Ｔｗ２０Ｊという）１ ｍＱを加え、３０００回転／分
で１分間遠心して上清を除去した。この操作を２回繰返
すことにより試料中のベルオギシダーゼ活性に影響を及
ぼす物質を除くことができた。次にペルオキシダーゼ標
識アポＡ−ＩＩ １００ｔｌＱ （２５ｎｇ＞を加え、
撹拌後、室温で１時間静置した。その後、ＰＢＳ−丁Ｗ
２０の２ｍＱを加え、３０００回転／分で１分間遠心し
て上清を除いた。この操作を２回繰返して、遊離のペル
オキシダーゼ標識アポＡ−ＩＩを除去した。最後に０．
０３％過酸化水素及び１ｍ（１／Ｉｎ１２４−クロロ−
〇−フェニレンジアミンを含む０．０２Ｍクエン酸緩衝
液（１）Ｈ６，５＞２００μＱを加え、撹拌した。Consider the dilution ratio of the antiserum and peroxidase-labeled apoA-m used in the assay, reaction time, temperature, and separation method of antibody-bound labeled antigen (bound) and free labeled antigen (free) to optimize assay conditions. The following measurement conditions were set. i.e. 0.5% bovine serum albumin (BSA), 0.14M sodium chloride and 20%
Add anti-human apoA-n serum (1:120>50μQ1 measurement sample or standard human apoA-I1 100μQ to the test tube using mM phosphate buffer (pH 7,2>) as a diluent and incubate at 4°C for 20 hours. 1 more, 5% (
V/V) Affigel Protein A (manufactured by Bio-Rad)
After adding 100 μQ and stirring, the mixture was allowed to stand at room temperature for 1 hour. 0
．． 20mM phosphate buffer (pH 7.2>(rPBs-
1 mQ (referred to as Tw20J) was added and centrifuged at 3000 rpm for 1 minute to remove the supernatant. By repeating this operation twice, it was possible to remove substances that affected the beluogysidase activity in the sample. Next, peroxidase-labeled apoA-II 100tlQ (25ng>) was added,
After stirring, the mixture was allowed to stand at room temperature for 1 hour. After that, PBS-Ding W
20 mQ was added and centrifuged at 3000 rpm for 1 minute to remove the supernatant. This operation was repeated twice to remove free peroxidase-labeled apoA-II. Finally 0.
03% hydrogen peroxide and 1 m(1/In124-chloro-
0.02M citric acid buffer (1) H6,5>200 μQ containing 0-phenylenediamine was added and stirred.
室温で３０分間静置した後、１１１１戒を加え、反応を
停止させ、４９２ｎｌＩｌでの吸光度を測定した。標準
ヒトアポＡ−ＩＩより得られた標準曲線より、試料中の
アポＡ−ｎ免疫活性物の含量を求めた。After standing at room temperature for 30 minutes, 1111 was added to stop the reaction, and the absorbance at 492nlIl was measured. The content of apoA-n immunoreactive substance in the sample was determined from a standard curve obtained from standard human apoA-II.
■ ヒトアポＡ−ＩＩの発現量
分泌発現系ベクターを保有する大腸菌形質転換株を、上
記■及び■に従い＠善後、各画分につき、エンザイムイ
ムノアツセイを行なって、アポＡ−ＩＩの免疫活性物含
量を求めた。■ Expression level of human apoA-II After the transformed E. coli strain carrying the secretory expression vector was purified according to the above ■ and ■, enzyme immunoassay was performed on each fraction to determine the immunoactive substance of apoA-II. The content was determined.
その結果を下記第３表に示す。尚、集菌時、遠心分離し
て得られる上清画分についても、同様の試験を行なった
が、この両分には、アポ△−■は、検出されなかった。The results are shown in Table 3 below. A similar test was also conducted on the supernatant fraction obtained by centrifugation during bacterial collection, but ApoΔ-■ was not detected in both fractions.
第 ３ 表
分泌発現系ベクターを保有する大腸菌ＪＭ１０３株（単
位：μｇ、／Ｑ）
分泌発現系ベクターを保有する大腸菌ＪＭ１０３株にお
いては、ペリプラズム画分に約２０μｇ／ＱのアポＡ−
Ｉ免疫活性物の発現を認めた。また、細胞内画分にも約
２７μΩ／ＱのアポＡ−ｎ免疫活性物の発現が認められ
、細胞内にも蓄積されていることが分った。このアポＡ
−ＩＩ免疫活性物は、シグナルペプチドと連結した状態
でおるものと推測される。以上のことより、本ベクター
系においては、ペリプラズム画分へのアポＡ−Ｉ［の分
泌が認められたが、細胞内にも蓄積されていた。尚、ペ
リプラズム画分への分泌はＩ ＰＴＧ添加添加１俊すで
に同口程度の発現を示すことが確詔されている。Table 3 Escherichia coli JM103 strain harboring a secretory expression vector (unit: μg/Q) In the E. coli strain JM103 harboring a secretory expression vector, approximately 20 μg/Q of apoA-
The expression of immunoactive substances was observed. Furthermore, expression of about 27 μΩ/Q of apoA-n immunoreactive substance was observed in the intracellular fraction, indicating that it was also accumulated within the cells. This appointment A
-II immunoactive substance is presumed to be in a state linked to a signal peptide. From the above, in this vector system, secretion of apoA-I into the periplasmic fraction was observed, but it was also accumulated within the cells. It has been confirmed that the secretion into the periplasmic fraction is about the same level when IPTG is added.
実施例２ 発現したアポＡ−ＩＩ免疫活性物質の精製
■ 分泌発現系ベクターｐＳＡＰ−０を保有する大腸菌
ＪＭ１０３株の大量培養
実施例１の■に記載のＭ−９カザミノ酸培地２０Ｑを含
む３０Ｑ容ジャーファーメンタ−にて培養を行なった。Example 2 Purification of the expressed apoA-II immunoactive substance ■ Large-scale culture of Escherichia coli JM103 strain carrying the secretory expression vector pSAP-0 30Q volume containing 20Q of M-9 casamino acid medium described in Example 1 (■) Culture was carried out in a jar fermentor.
ＤＳＡＰ−０を保有する大腸菌ＪＭ１０３株を、テトラ
サイクリン２０μＱ／−を含むＬ−ｂｒｏｔｈ培地４０
培地４屹０
養し、Ｍ−９カザミノ酸培地２０Ｑに加えた。E. coli JM103 strain harboring DSAP-0 was grown in L-broth medium containing 20 μQ/- of tetracycline.
The culture medium was cultured for 4 hours and added to M-9 Casamino Acid Medium 20Q.
培養は、２００回転／分の撹拌条件下に、２０Ｑ／分の
通気を行ないながら、３７°ＣでｐＨ６、４に制御して
行なった。培養開始から４、５時間後に、Ｉ ＰＴＧを
０． ３ｍ！７／ｍＱになるように添加し、更に２時間
開条件下に培養を続けた。The culture was carried out at 37° C. under agitation conditions of 200 revolutions/minute, with aeration of 20 Q/minute, and pH controlled at 6.4. 4 to 5 hours after the start of the culture, 0.5 hours of IPTG was added. 3m! 7/mQ, and culture was continued under open conditions for an additional 2 hours.
■ 菌体からのペリプラズム画分の抽出上記で得られた
培養液２０Ｑを、３００ｍ２／分の流速で連続的に遠心
分離（８０００回転／分）して菌体を集めた。その菌体
を実施例１の■のに記載した浸透圧ショック法に従い道
理してペリプラズム画分を抽出した。(2) Extraction of periplasmic fraction from bacterial cells Culture solution 20Q obtained above was continuously centrifuged (8000 revolutions/min) at a flow rate of 300 m2/min to collect bacterial cells. The cells were processed according to the osmotic shock method described in Example 1, Section 2, and the periplasmic fraction was extracted.
■ ペリプラズム画分からの精製
上記ペリプラズム画分を、アポＡ−ＩＩエンザイムイム
７ノアッセイで測定した結果、総量２．６ｍｇ相当のア
ポＡ−ＩＩ免疫活性物が検出された。本ペリプラズム画
分抽出液からのアポＡ−ｍ免疫活性物質の回収は、疎水
性クロマトグラフィー、免疫吸着クロマトグラフィー、
高速液体クロマトグラフィー等により行なった。その概
略を以下に示す。(2) Purification from the periplasmic fraction The periplasmic fraction described above was measured by an apoA-II enzyme immunoassay, and as a result, a total amount of 2.6 mg of apoA-II immunoreactive substance was detected. The apoA-m immunoactive substance can be recovered from the periplasmic fraction extract by hydrophobic chromatography, immunoadsorption chromatography,
This was carried out by high performance liquid chromatography or the like. The outline is shown below.
即ち、まずペリプラズム画分１０Ｑに０．６Ｍ濃度とな
るように硫酸アンモニウムを加え、０．６．Ｍ硫酸アン
モニウムにて平衡化したブチルトヨパールカラム（東洋
曹達社製、４×１０ｃｍ）に流した。０．３Ｍｔ［アン
モニウム４００戒で洗浄後、蒸沼水４００鴫にてアポＡ
−Ｉｆ免疫活性物質を溶出させた。That is, first, ammonium sulfate was added to the periplasmic fraction 10Q to give a concentration of 0.6M. The mixture was passed through a butyl Toyopearl column (manufactured by Toyo Soda Co., Ltd., 4 x 10 cm) equilibrated with M ammonium sulfate. 0.3Mt [After washing with 400 ammonium, apoA with 400 swamp water
-If immunoactive substances were eluted.
次に、抗体結合カラムを用いて免疫吸着クロマトグラフ
ィーを実施した。これには抗体結合カラムとして、アフ
ィゲル１０（バイオラッド社製＞２５ｍＱと、実施例１
の■に記載した方法で得た抗血清のγ−グロブリン画分
５００ｍｇとを室温で２時間反応させて得られた抗ヒト
アポＡ−ｎ抗体結合ゲルをカラム（２，５Ｘ２５Ｃｍ）
に充填して利用した。この抗体結合カラムに、上記ブチ
ルトヨパール溶出液を流し、アポＡ−■免疫活性物質を
吸着させた。０．１４Ｍ塩化すトリウムを含む２０
ｍＭリンＭ緩衝液（ｐ）−１７，２＞にて洗）ｐ後、Ｏ
，１Ｍグリシン−塩酸緩衝液（ｐＨ２，５）にて溶出さ
せた。Next, immunoadsorption chromatography was performed using an antibody-conjugated column. This included Affigel 10 (manufactured by Bio-Rad >25mQ) and Example 1 as an antibody-binding column.
The anti-human apoA-n antibody-binding gel obtained by reacting 500 mg of the γ-globulin fraction of the antiserum obtained by the method described in ① at room temperature for 2 hours was applied to a column (2.5 x 25 cm).
It was filled and used. The butyltoyopearl eluate was passed through this antibody-binding column to adsorb the apoA-■ immunoactive substance. 20 containing 0.14M thorium chloride
After washing with mM phosphorus M buffer (p)-17,2>, O
, 1M glycine-hydrochloric acid buffer (pH 2,5).
その溶出液を１Ｍトリスにて１）Ｈ８，０に調整した後
、２０ｍＭ重炭酸アンモニウム水溶液に対して透析し、
凍結乾燥した。The eluate was adjusted to 1) H8.0 with 1M Tris, and then dialyzed against 20mM ammonium bicarbonate aqueous solution.
Lyophilized.
得られた凍結乾燥試料を、０．２Ｍ塩化ナトリウムを含
む０．１Ｍリン酸緩衝液（ｐＨ７，２）に溶解し、同援
衝液で平衡化したＧ３０ＯｏＳＷカラム（東洋曹達社製
、７．５Ｘ６００ｍｍ））を用いてゲル濾過して分画し
た。The obtained freeze-dried sample was dissolved in 0.1M phosphate buffer (pH 7.2) containing 0.2M sodium chloride, and a G30OoSW column (manufactured by Toyo Soda Co., Ltd., 7.5X600 mm) was equilibrated with the same buffer solution. ) was used for gel filtration and fractionation.
アポＡ−ｎ免疫活性を示す分画を集め、２０ｍＭ重炭酸
アンモニウム水溶液に対して透析し、凍結屹燥後、コス
モシール５０１８−３００カラム（牛丼化学社製、４．
６ｘ １５０１ｎｍ）を用いた逆相りロマトグラフィー
により最終精製を行なった。アセトニトリル４０％から
６０％の直線濃度勾配法により分画し、アポＡ−ｎ免疫
活性のある単一ピーク部分を分取し、凍結乾燥した。Fractions showing apoA-n immunoreactivity were collected, dialyzed against 20mM ammonium bicarbonate aqueous solution, freeze-dried, and coated with Cosmosil 5018-300 column (manufactured by Gyudon Kagaku Co., Ltd., 4.
Final purification was performed by reverse phase chromatography using 6x 1501 nm). Fractionation was performed using a linear concentration gradient method of 40% to 60% acetonitrile, and a single peak portion with apoAn immunoreactivity was collected and lyophilized.
■ 最終精製標品の同定
上記で得られた精製標品を、０．１％ドデシル硫酸ナト
リウムを含む１７．５％ポリアクリルアミドゲル電気泳
動法にて分析した。その結果、血清より得られたヒｉ〜
アポＡ４と同じ位置に単一バンドを示した。また、同精
製標品に、４Ｎ−メタンスルホン酸を加え、１３０’Ｃ
で４時間、加水分解を行ない、中和後、アミノ酸自動分
析計を用いて０−フタルアルデヒド法（Ｊ、 Ｒ，Ｂｅ
ｎ５ｏｎ 、 Ｐ、 Ｅ、 Ｈａｒｅ、、 Ｐｒｏｅ。(2) Identification of the final purified sample The purified sample obtained above was analyzed by electrophoresis on a 17.5% polyacrylamide gel containing 0.1% sodium dodecyl sulfate. As a result, the human blood obtained from the serum
A single band was shown at the same position as apoA4. In addition, 4N-methanesulfonic acid was added to the same purified sample, and 130'C
Hydrolysis was carried out for 4 hours, and after neutralization, the 0-phthalaldehyde method (J, R, Be
n5on, P, E, Hare,, Proe.
Ｎａｔｌ、Ａｃａｄ、Ｓｃｉ、、ＵＳＡ、７２，６１９
（１９７５＞）により、そのアミノ酸組成の分析を行な
った。Natl, Acad, Sci,, USA, 72,619
(1975>), its amino acid composition was analyzed.
結果を下記第４表に示す。尚、上記方法では、プロリン
（Ｐｒｏ）及びシスチン（ＣＶＳ）は検出できない。The results are shown in Table 4 below. Note that proline (Pro) and cystine (CVS) cannot be detected by the above method.
第４表
アミノ酸 １分子当たりの残基数 理論値精製標品
ヒトアポへ一■
Ａｓｐ＋Ａｓｎ ７． ３ ７． ４
６Ｔｈｒ １１．２ １０．５ １２Ｓ
ｅｒ １１．７ １１．６ １２ＧＩｕ＋
ＧＩｎ ３１．４ ３３．１ ３２Ｐｒｏ
検出できず 検出できず ８ＧＩＶ
７．９ ９．４ ６Ａｌａ １１．３
１２．３ １０１／２ ＣＶＳ 検出できず
検出できず ２Ｖａｌ １１．８ １１
．７ １２Ｍｅｔ １．８ ０．６
２１１ｅ ２．０ ２．１ ２
Ｌｅｕ １６．６ １６．６ １６Ｔｙｒ
７．４ ７．４ ８Ｐｈｅ
８．０ ８．０ ８Ｌｙｓ １
７．０ １８．２ １８Ｈｉｓ Ｏ，１
０，００
Ｔｒｐ Ｏ，００，００
Ａｒ Ｏ，３０，５０
また、アミノ末端アミノ酸の分−折を、ブリュワーらの
方法（Ｈ，Ｂ、 Ｂｒｅｖｅｒ、ＪＲ，ｅｔ ａｔ、。Table 4 Amino acids Number of residues per molecule Theoretical value Purified sample Human ApoE1■ Asp+Asn 7. 3 7. 4
6Thr 11.2 10.5 12S
er 11.7 11.6 12GIu+
GIn 31.4 33.1 32Pro
Unable to detect Unable to detect 8GIV
7.9 9.4 6Ala 11.3
12.3 101/2 CVS Unable to detect Unable to detect 2Val 11.8 11
．． 7 12Met 1.8 0.6
211e 2.0 2.1 2
Leu 16.6 16.6 16Tyr
7.4 7.4 8Phe
8.0 8.0 8Lys 1
7.0 18.2 18His O,1
0,00 Trp O,00,00 Ar O,30,50 In addition, the amino-terminal amino acid was separated using the method of Brewer et al.
ｐ ｒＯｃ、 Ｎ ａ口、Ａｃａｄ、Ｓｃｉ、、ＵＳＡ
、６９゜１３０４−１３０８ （１９７２））に従い決
定した。その結果、アミノ末端アミノ酸は、ヒトアポＡ
−ＩＩと同一のピログルタミン酸であると同定された。prOc, Naguchi, Acad, Sci, USA
, 69° 1304-1308 (1972)). As a result, the amino terminal amino acid is human apoA
It was identified as pyroglutamic acid, which is the same as -II.
更に、上記方法における回収量は、最終精製標品の２８
０ｎｍにおける吸光度（分子吸光係数以上の結果より、
本発明方法により検出されたアポＡ−ｎは、ヒト血清よ
り得られるアポＡ−ｎと同一物質であることが確認され
た。Furthermore, the amount recovered in the above method is 28% of the final purified sample.
Absorbance at 0 nm (from the results above the molecular extinction coefficient,
It was confirmed that apoA-n detected by the method of the present invention is the same substance as apoA-n obtained from human serum.
第１図は、本発明遺伝子を構成するサブユニットＡ及び
サブユニットＢの構築の概略を示す図でおる。
第２図は、ｐＢＲ３２２からベクター１）ＡＰＯｌ及び
１）ＡＰＯ２をそれぞれ構築し、また之等各ベクターか
らプラスミドベクターｐＡＰＯＡ−ＩＩを構築する概略
図を示す。
第３図はベクターｐＵＧＴ−１５０、合成リンカ−及び
ｐＢＲ３２２を利用してプラスミドベクターｐＫＴＮを
構築する概略図と共に、得られるＤＫＴＮのｂｌａシグ
ナルペプチド−合成リンカ−の結合部位を示す。
第４図はプラスミドベクターｐＡＰＯＡ−ＩＩ、同ｐＫ
ＴＮ及びｐＵＧＴＩ ５０を用いて分泌発現系ベクター
ｐＳＡＰ−０を構築する概略図と共に、得られるベクタ
ーのｂｌａシグナルペプチド−アポＡ−ＩＦ構造遺伝子
の結合部位を示す。
（以 上）
第３図
ｂａａシク゛ナルペプチド−／＆人リすカー米台合壱予
イ立第４図
ｂＥａジグチルペプチド゛−アポＡ−，Ｉ［不菖玲４１
イム）律吾合皆陶立手続補正書（自発）
昭和６２年５月１１日
特許庁長官 黒 １）明 雄 殿
２ 発明の名称
アポリポタンパク貿の製造法
３ 補正をする者
事件との関係 特許出願人
株式会社 大塚製薬工場
４代理人
大阪市東区平野町２の１０ 沢の鶴ビル〕
自 発
６ 補正の対象
明細書中「発明の詳細な説明」の項
７ 補正の内容
別紙添付の通り ／＃４夕りｍ−
補正の内容
１ 明細書第４４頁第１７行に「（ｐＨ７，５）ｊとあ
るを［（ｐｌ−１７，２）ＪとΔＪ正する。
２ 明細書第５９頁第１３行に「フォスフニー１へ」と
必るを［フォスフェート基−１と訂正する。
（以 上）FIG. 1 is a diagram schematically showing the construction of subunit A and subunit B constituting the gene of the present invention. FIG. 2 shows a schematic diagram of constructing the vectors 1) APO1 and 1) APO2 from pBR322, respectively, and the plasmid vector pAPOA-II from each of these vectors. FIG. 3 shows a schematic diagram of the construction of plasmid vector pKTN using vector pUGT-150, a synthetic linker, and pBR322, as well as the binding site of the bla signal peptide to the synthetic linker in the resulting DKTN. Figure 4 shows plasmid vector pAPOA-II, pK
A schematic diagram of constructing the secretion expression system vector pSAP-0 using TN and pUGTI 50 is shown, as well as the binding site of the bla signal peptide-apoA-IF structural gene in the resulting vector. (The above) Fig. 3 baa Sequential Peptide -/& Human Liquor U.S. Taiwan Association Figure 4 bEa Digtylated Peptide - Apo A-, I
Im) Written amendment to the proceedings of Rigogoe (voluntary) May 11, 1988 Commissioner of the Patent Office Kuro 1) Akio Tono 2 Name of the invention Method of manufacturing apolipoprotein trade 3 Relationship with the case of the person making the amendment Patent Applicant Co., Ltd. Otsuka Pharmaceutical Factory 4 Agent Sawanotsuru Building, 2-10 Hirano-cho, Higashi-ku, Osaka City] Spontaneous 6 Item 7 of “Detailed Description of the Invention” in the specification subject to amendment Contents of the amendment As attached in the attached sheet / #4 Evening m- Contents of correction 1 In the 17th line of page 44 of the specification, ``(pH7,5)j is corrected to [(pl-17,2)J and ΔJ.2 In the specification, page 59 of In line 13, "to phosphonye 1" should be corrected to "phosphate group-1."(that's all)
Claims (1)
子をプラスミドベクターに挿入してプロアポリポタンパ
ク質遺伝子発現プラスミド組換体を作成し、該組換体を
宿主細胞に形質転換させて形質転換体を構築し、これを
培養して発現されるアポリポタンパク質を採取すること
を特徴とするアポリポタンパク質の製造法。 【遺伝子配列があります】(1) Create a proapolipoprotein gene expression plasmid recombinant by inserting the apolipoprotein gene containing the following base sequence into a plasmid vector, transform the recombinant into a host cell to construct a transformant, and A method for producing apolipoprotein, which comprises culturing and collecting apolipoprotein expressed. [There is a gene sequence]
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7114687A JPS63237795A (en) | 1987-03-24 | 1987-03-24 | Production of apo lipoprotein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7114687A JPS63237795A (en) | 1987-03-24 | 1987-03-24 | Production of apo lipoprotein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63237795A true JPS63237795A (en) | 1988-10-04 |
Family
ID=13452166
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7114687A Pending JPS63237795A (en) | 1987-03-24 | 1987-03-24 | Production of apo lipoprotein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63237795A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2704556A1 (en) * | 1993-04-30 | 1994-11-04 | Rhone Poulenc Rorer Sa | Recombinant viruses and their use in gene therapy |
| WO2004058966A1 (en) * | 2002-12-24 | 2004-07-15 | Nitto Boseki Co., Ltd. | Marker proteins for diagnosing liver disease and method of diagnosing liver disease using the same |
-
1987
- 1987-03-24 JP JP7114687A patent/JPS63237795A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| BIOCHEM.BIOPHYS.RES.COMMUN=1984 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2704556A1 (en) * | 1993-04-30 | 1994-11-04 | Rhone Poulenc Rorer Sa | Recombinant viruses and their use in gene therapy |
| WO1994025073A1 (en) * | 1993-04-30 | 1994-11-10 | Rhone-Poulenc Rorer S.A. | Recombinant viruses and their use in gene therapy |
| WO2004058966A1 (en) * | 2002-12-24 | 2004-07-15 | Nitto Boseki Co., Ltd. | Marker proteins for diagnosing liver disease and method of diagnosing liver disease using the same |
| US7517951B2 (en) | 2002-12-24 | 2009-04-14 | Nitto Boseki Co., Ltd. | Marker proteins for diagnosing liver disease and method of diagnosing liver disease using the same |
| US7781180B2 (en) * | 2002-12-24 | 2010-08-24 | Nitto Boseki Co., Ltd. | Marker proteins for diagnosing liver disease and method of diagnosing liver disease using the same |
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