JPH022354A - Production of human b cell differentiation factor - Google Patents
Production of human b cell differentiation factorInfo
- Publication number
- JPH022354A JPH022354A JP63179119A JP17911988A JPH022354A JP H022354 A JPH022354 A JP H022354A JP 63179119 A JP63179119 A JP 63179119A JP 17911988 A JP17911988 A JP 17911988A JP H022354 A JPH022354 A JP H022354A
- Authority
- JP
- Japan
- Prior art keywords
- sequence
- human
- differentiation factor
- cell differentiation
- formula
- 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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- 238000003018 immunoassay Methods 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
Ｂ細胞分化因子（以下、ＢＳＦ−２と略称する）は、Ｔ
リンパ球代替因子、ＴＲＦとしてマウスのリンパ球混合
物培養後の培養上清又は、抗原やマイトゲンにより刺激
を受けたマウスのリンパ球上清中に、マウスのＴ細胞を
除去されたリンパ球細胞集団やヌードマウス由来のリン
パ球のヒツジ赤血球に対する一次免疫応答を増幅させる
物質として見出された。それ以来、ＴＲＦ’は抗原非特
異的に主要組織適合遺伝子複合体 （以下、ＭＨＣと略
称する。）の一致を必要としない様式で、Ｂ細胞に作用
し、Ｂ細胞の***増殖を誘導せず、Ｂ細胞の抗体産生細
胞への分化を誘導する液性因子であると定義されている
。現在では、上述のように定義されたＢ細胞を抗体産生
細胞へ分化させる因子をＢｓＦ−２と称するようになっ
た。この上うなり５Ｆ−２は、人の体内でＢ細胞の抗体
産生機能に重要な働きをしている。[Detailed Description of the Invention] (Industrial Application Field) B cell differentiation factor (hereinafter abbreviated as BSF-2) is T
Lymphocyte replacement factor, TRF, is used in the culture supernatant after culture of a mouse lymphocyte mixture, or in the lymphocyte supernatant of mice stimulated with antigens or mitogens, or a lymphocyte cell population from which mouse T cells have been removed. It was discovered as a substance that amplifies the primary immune response of lymphocytes derived from nude mice to sheep red blood cells. Since then, TRF' has been shown to act on B cells in a non-antigen-specific manner that does not require major histocompatibility complex (MHC) matching, and does not induce B cell division and proliferation. , is defined as a humoral factor that induces the differentiation of B cells into antibody-producing cells. Currently, the factor that differentiates B cells into antibody-producing cells as defined above is called BsF-2. Furthermore, Unauri 5F-2 plays an important role in the antibody production function of B cells in the human body.
ＢＳＦ−２の臨床への応用としては、１）ＢＳＦ２によ
りＢＳＦ−２抗体を作り、Ｂ５１２と抗ＢＳＦ−２抗体
によるＢＳＦ−２のイムノアッセイ系を用いて免疫学的
な病態の解析に用いる、２）各種疾患の治療への応用で
、例えば、Ｔ細胞のヘルパー機能低下にとらなうＢ細胞
抗体産生能低下による免疫不全症患者にＢＳＦ−２単独
または、他のリンホカインと共に投与することにより、
抗体産生機能を正常にもどす、３）Ｂ細胞増殖因子、そ
の他のリンホカインを含むＴ細胞因子を培地に加えるこ
とにより正常Ｂ細胞あるいは、ＥＢウィルスで形質転換
したＢ細胞に対し適当な時期にＢＳＦ−２を作用させる
ことにより、生体外で抗体を産生させる等が考えられろ
。さらに、特定の抗体、例えば、病原細菌、病原ウィル
ス、病原原虫、癌細胞などの表面にある特定抗原を認識
する抗体を産生ずるＢ細胞をモノクロナール化し、クロ
ーン化正常Ｂ細胞または、ＥＢウィルスで形質転換した
細胞をＢＳＦ’−２とその他のリンホカインを組み合わ
せて培養し、有用なモノクロナール抗体を産生させるこ
とが出来る。これらの抗体は感染症や癌の治療および、
診断に利用できる。Clinical applications of BSF-2 include 1) producing BSF-2 antibodies using BSF2 and using a BSF-2 immunoassay system using B512 and anti-BSF-2 antibodies to analyze immunological pathological conditions; 2) ) Application to the treatment of various diseases, for example, by administering BSF-2 alone or together with other lymphokines to patients with immunodeficiency due to a decrease in the ability of B cells to produce antibodies due to a decrease in the helper function of T cells.
3) By adding T cell factors including B cell growth factors and other lymphokines to the culture medium, BSF- It is conceivable that antibodies could be produced in vitro by causing 2 to act. Furthermore, B cells that produce specific antibodies, such as antibodies that recognize specific antigens on the surface of pathogenic bacteria, pathogenic viruses, pathogenic protozoa, cancer cells, etc., are monoclonalized and cloned with normal B cells or EB viruses. Transformed cells can be cultured with BSF'-2 in combination with other lymphokines to produce useful monoclonal antibodies. These antibodies can be used to treat infectious diseases and cancer, and
Can be used for diagnosis.
本発明は、このＢＳＦ−２の製造方法に関するものであ
る。The present invention relates to a method for producing BSF-2.
（従来の技術）
ＢＳＦ−２を得る方法としては、人末梢血などより分離
した正常人Ｔ細胞をマイトゲン刺激することによりＢＳ
Ｆ−２を産生させる方法、人Ｔ細胞を人癌と細胞融合し
て人Ｔ融合細胞を得、これによりＢＳＦ−２を産生させ
る方法、さらにＢＳＦ −２をコードするＤＮＡを大腸
菌で発現せしめる方法として、トリプトファンプロモー
ター支配下にＢＳＦ−２を直接発現する方法と、ヒトＩ
Ｌ２の部分Ｎ末端ペプチドとの融合蛋白として発現する
方法が知られている。(Prior art) As a method for obtaining BSF-2, BSF-2 is obtained by stimulating normal human T cells isolated from human peripheral blood with mitogens.
A method for producing F-2, a method for fusing human T cells with human cancer to obtain human T fusion cells, and producing BSF-2 thereby, and a method for expressing DNA encoding BSF-2 in Escherichia coli. As a method for directly expressing BSF-2 under the control of a tryptophan promoter, and for human I
A method of expressing it as a fusion protein with a partial N-terminal peptide of L2 is known.
本発明者は、以上のような状況に鑑み、Ｂ５Ｆ２をヒト
成長ホルモンと融合した形で製造する方法を完成させた
。すなわち、本発明は構造遺伝子の発現を調節する制御
遺伝子の下流にリボゾーム結合部位、およびヒト成長ホ
ルモンＡドメインおよび、ヒト成長ホルモンＢドメイン
をコードする遺伝子、ＢＳＦ−２をコードする遺伝子、
終止コドンを含んだプラスミドで形質転換させた大腸菌
を培養し、発現したヒト成長ホルモンＡドメイン、ヒト
成長ホルモンＢドメイン、ＢＳＦ−２の融合蛋白を酵素
で処理してＢＳＦ−２を得ることを特徴とするＢＳＦ−
２の製造法に関するものである。以下、さらに本発明の
詳細な説明する。In view of the above circumstances, the present inventors have completed a method for producing B5F2 in a form fused with human growth hormone. That is, the present invention provides a ribosome binding site downstream of a regulatory gene that regulates the expression of structural genes, a gene encoding human growth hormone A domain and human growth hormone B domain, a gene encoding BSF-2,
BSF-2 is obtained by culturing Escherichia coli transformed with a plasmid containing a stop codon, and treating the expressed fusion protein of human growth hormone A domain, human growth hormone B domain, and BSF-2 with an enzyme. BSF-
This relates to the manufacturing method of No. 2. The present invention will be further explained in detail below.
（発明の構成）
「ＢＳＦ−２遺伝子セグメントとその調製」即ち、
本発明は第一に、
式
％式％
て表される塩基配列からなるＢＳＦ−２遺伝子セグメン
ト［以下式（１）という、式中Ａはデオキシアデニル酸
の、Ｃはデオキシシチジル酸の、Ｇはデオキシグアニル
酸の、Ｔはデオキシチミジル酸の各残基を表しく以下同
じ）、塩基配列上のアミノ酸略号はこの塩基配列により
コードされているアミノ酸配列を示す］を提供するもの
である。(Structure of the Invention) "BSF-2 Gene Segment and Preparation thereof" That is, the present invention first relates to a BSF-2 gene segment [hereinafter referred to as formula (1)] consisting of a base sequence represented by the formula % formula % In the middle, A is deoxyadenylic acid, C is deoxycytidylic acid, G is deoxyguanylic acid, and T is deoxythymidylic acid. The encoded amino acid sequence is shown below.
このＢＳＦ−２遺伝子セグメントは好ましくは、その上
流末端に、式
％式％
（式中ＸはＮ末端よりｌｌｅ Ｇｌｕ Ｇｌｙ Ａｒｇ
で表されるアミノ酸配列をコードするＤＮＡ配列を、ま
たその下流に停止コドンを含み、さらに、その下流に制
限酵素開裂末端で終わる、式
て表されるＤＮＡ配列をもつものを提供するものである
。This BSF-2 gene segment preferably has at its upstream end a sequence of the formula % (where X is from the N-terminus to lle Glu Gly Arg
The present invention provides a DNA sequence that encodes the amino acid sequence represented by the following formula, contains a stop codon downstream thereof, and further terminates with a restriction enzyme cleavage end downstream of the stop codon. .
上流側に付加されるＤＮＡ配列のＸ及びＸ′とじては、
式
％式％
て表される配列を例示することかできる。As for the DNA sequence added to the upstream side, X and X',
An example of an array represented by the expression % expression % can be given.
下流側に付加される、停止コドンを含み、さらにその下
流に制限酵素開裂末端で終わるＤＮＡ配列のＹ及びＹ゛
とじては制限酵素ＢａｍＨＩ の開裂末端の配列、式
％式％
で表される配列を含むものを例示することができる。Y and Y of the DNA sequence that is added to the downstream side and includes a stop codon and ends further downstream with a restriction enzyme cleavage end is the sequence of the cleavage end of the restriction enzyme BamHI, a sequence represented by the formula % formula % Examples include:
上記（２）式では上流末端がＢａｍＨＩ の末端で終わ
っている。（１）の上流側に付加されろ、式％式％
で表されるＤＮＡ配列をもつオリゴヌクレオチドは、以
下に述べる様な方法で調製することができる。In formula (2) above, the upstream end ends at the BamHI end. An oligonucleotide having a DNA sequence represented by the formula %, which is added to the upstream side of (1), can be prepared by the method described below.
まずこれらのオリゴヌクレオチドの各−本鎖部分を合成
し、水素結合させればよい。この場合、リガーゼを用い
てオリゴヌクレオチド、または二本鎖ＤＮＡ断片を結合
させる際には、あらかじめこれらの５゛側の反応ににあ
ずかる成分の５−末端はリン酸化させておく。リン酸化
は慣用の手法および条件で行うことができる。First, each of the main chain portions of these oligonucleotides may be synthesized and hydrogen bonded. In this case, when oligonucleotides or double-stranded DNA fragments are linked using ligase, the 5-ends of the components that participate in the 5'-end reaction are phosphorylated in advance. Phosphorylation can be performed using conventional techniques and conditions.
得られたＢＳＦ−２遺伝子セグメントは適当なプラスミ
ド中へ挿入して大腸菌等に形質転換し、薬剤感受性など
の指標と、ラピッドアイソレーション等の手法を用いて
クロン化し増幅することができる。The obtained BSF-2 gene segment can be inserted into an appropriate plasmid, transformed into E. coli, etc., and cloned and amplified using indicators such as drug sensitivity and techniques such as rapid isolation.
「プラスミド及びその調製」
本発明の第二は、ＢＳＦ’−２遺伝子セグメントを含む
組み換えプラスミドを提供するものである。"Plasmid and its preparation" The second aspect of the present invention provides a recombinant plasmid containing the BSF'-2 gene segment.
ヒ１】ち、式（１）で表される塩基配列のＢＳＦ−２遺
伝子セグメントを含み、微生物中で自己増殖可能な組み
換えプラスミドを提供するものである。そして好ましく
は、ＢＳＦ−２遺伝子の上流にこの遺伝子を発現可能に
支配するプロモーター・オペレーター系を含み、そのプ
ロモーター・オペレーターの上流とＢＳＦ−２遺伝子の
下流との間に、プラスミドを自己増殖可能とする部分を
含むものである。プロモーター・オペレーター系として
は、従来、トリプトファンプロモーター／オペレーター
系、ラクトースプロモーター／オペレーター系、あるい
は、トリプトファンプロモーター／ラクトースオペレー
ター系を用いればよい。本発明の組み換えプラスミドは
、ＢＳＦ−２遺伝子セグメントを慣用の方法で適当なベ
クタープラスミドに組み込むことにより調製することが
できる。特に、その上流及び、下流に制限酵素開裂末端
を持っＢＳＦ−２遺伝子セグメントは、その様なプラス
ミドの相当する開裂部位、例えば、ＢｇｌＩＩ等の部位
へ容易に組み込むことができる。[1] The present invention provides a recombinant plasmid that contains a BSF-2 gene segment having the base sequence represented by formula (1) and is capable of self-replication in microorganisms. Preferably, a promoter/operator system that controls the expression of this gene is included upstream of the BSF-2 gene, and between the upstream of the promoter/operator and the downstream of the BSF-2 gene, the plasmid is capable of self-replication. It includes a part that does. As the promoter/operator system, a conventional tryptophan promoter/operator system, lactose promoter/operator system, or tryptophan promoter/lactose operator system may be used. Recombinant plasmids of the invention can be prepared by incorporating the BSF-2 gene segment into a suitable vector plasmid in a conventional manner. In particular, a BSF-2 gene segment having restriction enzyme cleavage ends upstream and downstream thereof can be easily integrated into the corresponding cleavage sites of such plasmids, such as BglII sites.
本発明の組み換えプラスミドは大腸菌などの微生物に形
質転換して、本発明のヒトＢＳＦ−２遺伝子の産物を融
合蛋白質の形で産生させることができろ。例えば、ＢＳ
Ｆ−２遺伝子セグメントで、上流に式（２）で表される
末端を持つものは、プラスミドの発現され得る蛋白質を
コードするＤＮＡ配列中に制限酵素Ｂｇｌｌｌ の部位
を持つもの、ＢｇｌＩＩの部位へに読取位相の一致する
様に組み入れることによって、その蛋白質のＮ末端側の
一部とアミノ酸配列１１ｅ Ｇｌｕ Ｇｌｙ Ａｒｇを
介して連結された、ＢＳＦ−２蛋白質との融合蛋白を産
生させることのできるプラスミドを構築することができ
る。この様な蛋白質としてはヒト成長ホルモンを例示す
ることができ、Ｂｇ１１１部位を含むＤＮＡ配列として
は、特開昭６０−２３４５８４号明細書中で開示されて
いるｐＧＩＩ−Ｌシリーズのプラスミドを例示すること
かできる。The recombinant plasmid of the invention can be transformed into a microorganism such as E. coli to produce the product of the human BSF-2 gene of the invention in the form of a fusion protein. For example, B.S.
F-2 gene segments that have an upstream end represented by formula (2) have a restriction enzyme Bglll site in the DNA sequence encoding a protein that can be expressed on the plasmid, or a BglII site. A plasmid that can produce a fusion protein between a part of the N-terminal side of the protein and the BSF-2 protein, which is linked via the amino acid sequence 11e Glu Gly Arg, by integrating the protein so that the reading phases match. Can be built. An example of such a protein is human growth hormone, and an example of a DNA sequence containing a Bg111 site is the pGII-L series plasmid disclosed in JP-A-60-234584. I can do it.
こうして構築される組み換えプラスミドのプロモーター
・オペレーター系のメツセンジャーＲＮＡ転写開始点よ
りＦ３９Ｐ−２遺伝子までのＤＮＡ配列が、式
％式％
（Ｘ及びＸ′は前記同様の意味を表す）であるものが特
に好ましい。この様な配列を持つプラスミドは、例えば
、ｐＧＩ（−１，９プラスミドをＢｇｌＩＩ消化したも
のにＢＳＦ−２遺伝子セグメントで上流側に式（２）で
表される末端配列をもち、下流側に式（３）で表される
末端配列をもつものを接着閉環させることによって調製
できる。第（１）図にこの調製を示した。この様にして
プラスミドｐＧＢＳａ ＩＩ製することができる。なお
、プラスミドｐＧＨ−Ｌ９を持つＥ、コリ菌株、Ｅ、ｃ
ｏｌｉ ｐＧＨ−Ｌ９は通商産業省工業技術院微生物工
業技術研究所に、微工研菌寄第７６０６号として寄託さ
れている。The recombinant plasmid thus constructed has a DNA sequence from the Messenger RNA transcription start point of the promoter-operator system to the F39P-2 gene of the formula % (X and X' represent the same meanings as above). Particularly preferred. A plasmid having such a sequence is, for example, a BSF-2 gene segment obtained by digesting pGI(-1,9 plasmid with BglII), which has a terminal sequence represented by formula (2) on the upstream side, and a terminal sequence represented by formula (2) on the downstream side. It can be prepared by adhesive ring-closing a terminal sequence represented by (3). This preparation is shown in Figure (1). Plasmid pGBSa II can be prepared in this way. Plasmid pGH E.coli strain with -L9, E.c.
oli pGH-L9 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, as Microtechnology Research Institute No. 7606.
「形質転換体」
本発明の組み換えプラスミドｐＧＢＳ３でＥ、 ｃｏｌ
ｉ株、例えば、Ｒ８７９１株等を形質転換し、これを栄
養培地中で培養することによってＢＳＦ−２遺伝子を効
率よく増幅することができる。また、本発明のＢＳＦ−
２遺伝子の上流にその発現を支配するプロモーター・オ
ペレーター系、特にトリプトファン・プロモーター・オ
ペレーター系を持つものは、ＢＳＦ−２をヒト成長ホル
モンのＮ末端側の一部との融合蛋白質の形で発現し、菌
体内に蓄積する。"Transformant" E, col with the recombinant plasmid pGBS3 of the present invention
The BSF-2 gene can be efficiently amplified by transforming an i strain, such as the R8791 strain, and culturing it in a nutrient medium. Moreover, the BSF-
Those with a promoter-operator system, especially a tryptophan promoter-operator system, that controls the expression of BSF-2 upstream of the gene express BSF-2 in the form of a fusion protein with a part of the N-terminal side of human growth hormone. , accumulates within the bacterial body.
この場合（トリプトファン・プロモーター・オベレーク
−系のとき）ＢＳＦ−２の産生（ｍＲＮＡ合成）はイン
ドールアクリル酸の添加によって強く誘導される。In this case (in the case of tryptophan promoter overlay system), BSF-2 production (mRNA synthesis) is strongly induced by the addition of indoleacrylic acid.
菌体内に蓄積されたＢＳＦ−２及びヒト成長ホルモンと
の融合蛋白質は菌体を溶菌させたのち、通常の生理活性
蛋白質回収法によって分離回収することができる。例え
ば培養終了後、菌体を遠心分離により集菌し、トリス−
塩酸緩衝液に懸濁し、破砕後、尿素等の処理をして得ら
れる上清液をゲルろ過等により目的の蛋白質分画をうる
。The fusion protein of BSF-2 and human growth hormone accumulated in the bacterial cells can be separated and recovered by a normal bioactive protein recovery method after the bacterial cells are lysed. For example, after culturing, the bacterial cells are collected by centrifugation, and Tris-
The protein is suspended in a hydrochloric acid buffer, crushed, and treated with urea, etc., and the resulting supernatant is subjected to gel filtration or the like to obtain the desired protein fraction.
本発明の微生物によるヒト成長ホルモンとの融合蛋白質
はその融合部位に血液凝固因子Ｘａの認識部位（Ｉ ｌ
ｅ−Ｇｌｕ−Ｇｌｙ−Ａｒｇ）を有し、この酵素で処理
することによってＮ末端側から一個のアミノ酸残Ｗ Ａ
ｌ ａの付加したＢＳＦ−２とすることかできろ。The microorganism-mediated fusion protein of the present invention with human growth hormone has a blood coagulation factor Xa recognition site (I l
e-Glu-Gly-Arg), and by treatment with this enzyme, one amino acid residue W A from the N-terminal side
Could it be BSF-2 with added la?
実施例１
（オリゴヌクレオチドのｆＪＥＩ製）
（１） ” ＧＡＴＣＣＴＣＡＴＣＧＡＡＧＧＴ
ＣＧＴＧＣＴＣＣＧＧＴＴＣＣＧ ’ −（２）
’ −ＣＣＴＧＧＣＧＧＡΔＣＣＧＧＡＧＣＡＣＧＡ
ＣＣＴＴＣＧＡＴＧＡＧ３−の調製
上記のオリゴヌクレオチド（１）、（２）をＤ Ｎ Ａ
シンセサイザーｍ ｏ ｄ ｅ ｌ −３８１Ａ（Ａｐ
ｐｌｉｅｄＢｉｏｓｙｓｔｅｍ社製）を用いて合成した
。合成終了後、カラム中の担体に吸着したオリゴヌクレ
オチドをアンモニア水１ｍｌで一時間処理し溶出した。Example 1 (Oligonucleotide manufactured by fJEI) (1) ”GATCCTCATCGAAGGT
CGTGCTCCGGTTCCG' - (2)
' -CCTGGCGGAΔCCGGAGCACGA
Preparation of CCTTCGATGAG3- The above oligonucleotides (1) and (2) were converted into DNA
Synthesizer model -381A (Ap
(manufactured by pliedBiosystem). After the synthesis was completed, the oligonucleotide adsorbed on the carrier in the column was treated with 1 ml of aqueous ammonia for one hour and eluted.
スクリューバイアル付きの容器にこのアンモニア溶液を
入れ５５℃で一夜反応させた。減圧下で溶媒を留去し、
残さを蒸留水５００μｌに溶解し、三度ジエチルエーテ
ル５００μｌで不要物を抽出後、再び溶媒を減圧留去し
た。残さを１０ｍＭ）リス−塩酸緩衝溶液（ｐＨ７，５
）／１ｍエチレンジアミンテトラアセテート２００μｌ
に溶解し、アニ−リング２６０ｎｍで吸光度を測定し濃
度検定を行い、各々］Ｏｐｍｏｌ／μｌの溶液を調製し
た。合成により得られた上述のオリゴヌクレオチド（１
）、（２）を用いて次の条件で反応を行い、合成フラグ
メントを得た。This ammonia solution was placed in a container equipped with a screw vial and reacted overnight at 55°C. The solvent was distilled off under reduced pressure,
The residue was dissolved in 500 μl of distilled water, unnecessary substances were extracted three times with 500 μl of diethyl ether, and the solvent was again distilled off under reduced pressure. The residue was dissolved in 10mM) Lis-HCl buffer solution (pH 7.5
)/1m ethylenediaminetetraacetate 200μl
The absorbance was measured at 260 nm during annealing to determine the concentration, and a solution of Opmol/μl was prepared for each. The above-mentioned oligonucleotide (1
) and (2) under the following conditions to obtain a synthetic fragment.
合成オリゴヌクレオチド（ｌσｐｍｏｌ） ５μＱ５
×カイネーシヨン緩衝液 １０μＱＯ−０１Ｍ
ＡＴＰ
５ｐＱＴ４ポリヌクレオチドカイネース ２μ
ｅ（全酒造社製、ｆｏｕｌ／μｍ）
カイネーション緩衝液の組成は、５０ｍＭ）リス塩酸緩
衝溶液（ｐＨ９，６）、１０ｍＭ塩化マグネシウム、２
ｍＭスペルミジン、１０ｍＭジチオスレトール、１００
ｍＭ塩化カリウムで、３７”Ｃ２０分間インキュベート
した。インキュベート後、合成オリゴヌクレオチド（１
）、（２）溶液を混合し６５℃ｌＯ分間反応させ、それ
からゆっくり室温まで冷却しアニーリングを行った。こ
の操作により、５ｐｍｏｌ／μｌの濃度の合成フラグメ
ントを得た。Synthetic oligonucleotide (lσpmol) 5μQ5
×Kination buffer 10μQO-01M
ATP
5pQT4 polynucleotide kinase 2μ
e (manufactured by Zenshuzo Co., Ltd., foul/μm) The composition of the caination buffer is: 50mM) Liss-HCl buffer solution (pH 9,6), 10mM magnesium chloride, 2
mM spermidine, 10mM dithiothretol, 100
Incubate with mM potassium chloride for 20 minutes at 37"C. After incubation, synthetic oligonucleotide (1
), (2) The solutions were mixed and reacted for 65° C. 10 minutes, and then slowly cooled to room temperature for annealing. This operation yielded a synthetic fragment at a concentration of 5 pmol/μl.
実施例２
（組換プラスミド及び形質転換株の調製）本発明の組換
プラスミドは融合蛋白質発現型であり、以下実施例で詳
細に述べる。Example 2 (Preparation of recombinant plasmid and transformed strain) The recombinant plasmid of the present invention is a fusion protein expression type, and will be described in detail in the following example.
既知のプラスミドｐＢＳＦ２，３８を制限酵素ＡｌｕＩ
及びＢａｍＨＩで消化して約１０００塩基対のＡｌｕｌ
ＢａｍＨＩ断片を取り出した。The known plasmid pBSF2,38 was digested with the restriction enzyme AluI.
and digested with BamHI to create approximately 1000 base pairs of Alul
The BamHI fragment was extracted.
一方、公知のプラスミドｐＧＥＭ４をＳｍａＩ及びＢａ
ｍＨＩで消化して開裂させた。切り出したＡｌｕｌ−Ｂ
ａｍＨ１断片をＴ、ＤＮＡリガーゼの存在下でｐＧＥＭ
４のＳｍａ １％ ＢａｍＨ１切断部に挿入し、プラス
ミドｐＢＳＦ２．３８−１を調製した。続いて該プラス
ミドをＥ ｃ ｏ ＲＩＩ及びＢ ａ ｍＨｌで消化し
て、約１０００塩基対の翻訳開始コドンを取り除いたＥ
ｃｏＲＩＩ−ＢａｍＨＩ断片を得た。この断片をＡとす
る。On the other hand, the known plasmid pGEM4 was used with SmaI and Ba
Digested and cleaved with mHI. Cut out Alul-B
amH1 fragment into T, pGEM in the presence of DNA ligase
4 into the Sma 1% BamH1 cleavage site to prepare plasmid pBSF2.38-1. The plasmid was then digested with E co RII and B a mHl to remove approximately 1000 base pairs of the translation initiation codon.
A coRII-BamHI fragment was obtained. Let this fragment be A.
次に翻訳開始コドンを含んだ式、
’ −ＧＡＴＣＣＴＣＡＴＣＧＡＡＧＧＴＣＧＴＧＣＴ
ＣＣＧＧＴＴＣＣＧ ３−ＧＡＧＴＡＧＣＴＴＣＣＡＧ
ＣＡＣＧＡＧＧＣＣＡＡＧＧＣＧＧＴＣＣで表されるオ
リゴヌクレオチドを合成した。このオリゴヌクレオチド
をＢとする。上記オリゴヌクレオチドは５″末端がＢａ
ｍＨＩ構造、３゛末端がＥ ｃ ｏ Ｒｎ構造を有し、
Ｉｌｅ Ｇｌｕ Ｇｌｙ Ａｒｇで表される血液
凝固因子Ｘａの消化認識アミノ酸配列および、Ａｌａ
Ｐｒｏ ＶａｌＰｒｏ Ｐｒｏで表されるアミ入
酸配列をコードする。Next, the expression containing the translation start codon is '-GATCCTCATCGAAGGTCGTGCT
CCGGTTCCG 3-GAGTAGCTTCCAG
An oligonucleotide represented by CACGAGGCCAAGGCGGTCC was synthesized. This oligonucleotide is designated as B. The 5″ end of the above oligonucleotide is Ba.
mHI structure, the 3′ end has an E co Rn structure,
Digestion recognition amino acid sequence of blood coagulation factor Xa represented by Ile Glu Gly Arg and Ala
Encodes an amidoic acid sequence represented by Pro ValPro Pro.
（融合発現型 ｐＧＢｓ−３）
プラスミドｐＧＨ−Ｌ９ （Ｅ、コリ ｐＧＨ−Ｌ９
、微工研菌寄第７６０６号）３０μｇｆ！−１００ユニ
ットのＢｇｌＩＩ（全酒造社製）と３７℃、３時間反応
させた。反応液量１００μｌで組成は、１０ｍＭ）リス
−塩酸緩衝液（ｐＨ７，５）、７ｍＭ塩化マグネシウム
、１００ｍＭ塩化ナトリウム、７ｍＭ ２−メルカプ
トエタノール、１００μｚ／ｍｌ牛血清アルブミンであ
った。エタノールにて沈澱させＤＮＡを回収後、１００
μｌのトリス−塩酸緩衝溶液に溶解し、０，９ユニツト
のアルカリフォスファターゼ（全酒造社製）で６５℃、
３０分反応させ、５゛末端のリン酸基を除去した。さら
に、フェノール処理、クロロフォルム処理を行い、エタ
ノールにて沈澱させＤＮＡを回収し、ベクターＤＮＡｐ
ＧＨ−Ｌ９／Ｂｇ ｌ ＩＩを得た。得られたベクター
Ｄ Ｎ Ａ ｐ Ｇ Ｈ−Ｌ ９ ／ Ｂｇ Ｉ ＩＩ
（０，５μｇ／ｍｌ ） １ ｔｔ １と断片Ａ（０
゜５μｇ／ｍ ｌ）１ ｕ ｌとオリゴヌクレオチドＢ
（ＯＩμｇ／ｍ１）ｌμｌを混合しライゲーションキッ
ト（全酒造社製）を用いて１６℃３０分反応させた。こ
の反応溶液ｌＯμｍを大腸菌に形質転換し、形質転換株
Ｅ、コリ／ｐＧＢＳ−３を得た（図２）。(Fusion expression type pGBs-3) Plasmid pGH-L9 (E. coli pGH-L9
, Microtechnical Research Institute No. 7606) 30μgf! The mixture was reacted with -100 units of BglII (manufactured by Zenshuzo Co., Ltd.) at 37°C for 3 hours. The reaction solution volume was 100 μl, and the composition was 10 mM) Lis-HCl buffer (pH 7.5), 7 mM magnesium chloride, 100 mM sodium chloride, 7 mM 2-mercaptoethanol, and 100 μz/ml bovine serum albumin. After collecting the DNA by precipitation with ethanol,
Dissolve in μl of Tris-HCl buffer solution, add 0.9 units of alkaline phosphatase (manufactured by Zenshuzo Co., Ltd.) at 65°C.
The reaction was allowed to proceed for 30 minutes, and the phosphoric acid group at the 5' end was removed. Furthermore, the DNA was collected by phenol treatment and chloroform treatment, and precipitated with ethanol, and the vector DNAp
GH-L9/Bg l II was obtained. The obtained vector DNApGHL9/BgIII
(0.5 μg/ml) 1 tt 1 and fragment A (0
゜5μg/ml) 1ul and oligonucleotide B
(OIμg/ml) was mixed and reacted for 30 minutes at 16°C using a ligation kit (manufactured by Zenshuzo Co., Ltd.). 10 μm of this reaction solution was transformed into Escherichia coli to obtain transformed strain E, coli/pGBS-3 (FIG. 2).
実施例３
（発現）
実施例Ｉで得られたＥ、コリ／ｐＧＢＳ−３をＬ培地５
ｍｔにて３７°Ｃ−晩培養した。得られた培養液５０μ
！をＬ培地５ｍｌに加え培養し、アブソーバンス６００
ｎｍで吸光度０．４〜０．５となった時点で、インドー
ルアクリル酸（１０ｍｇ／ｍ１）５０μｌを加えさらに
４時間培養した。Example 3 (Expression) E. coli/pGBS-3 obtained in Example I was added to L medium 5
The cells were cultured overnight at 37°C. Obtained culture solution 50μ
! was added to 5 ml of L medium, cultured, and Absorbance 600
When the absorbance at nm reached 0.4 to 0.5, 50 μl of indoleacrylic acid (10 mg/ml) was added and cultured for an additional 4 hours.
培養終了後、遠心分離機で１０．００Ｏｒｐｍ、１０分
間遠心し面体を集めた。その一部をサンプルバッファー
（６２，５ｍｌ トリス−塩酸緩衝液（ｐＨ６，８）
、２％ＳＤＳ、５ｍＭエチレンデアミンチトラアセテー
ト、１０％グリセロール、３５ｍＭ２−メルカプトエタ
ノール、０．００１％ブロモフェノールブルー）に懸濁
、沸騰水中で５分間加熱した後、０．１％５ＤＳ−１２
，５％ＰＡＧＥで電気泳動を行い、分子量３．６Ｘ１０
４のものが得られた（図３）。After the culture was completed, the face pieces were collected by centrifugation at 10.00 rpm for 10 minutes using a centrifuge. A portion of it was added to sample buffer (62.5 ml Tris-HCl buffer (pH 6.8)).
, 2% SDS, 5mM ethylenedeamine chitraacetate, 10% glycerol, 35mM 2-mercaptoethanol, 0.001% bromophenol blue), heated for 5 min in boiling water, then 0.1% 5DS-12.
, electrophoresis was performed on 5% PAGE, and the molecular weight was 3.6X10.
4 were obtained (Figure 3).
実施例４
（血液凝固因子Ｘａによる融合蛋白質からのＢＳＦ−２
の切断）
５Ｑのし培地中、Ｅ、コリ／ｐＧＢＳ−３をインドール
アクリル酸で誘導し４時間培養後、集菌し、約ｌｏｇ（
湿重量）を得た。得られた図体を５０ｍＭトリス−塩酸
緩衝液（ｐＨ８，０）に懸渇し、破砕後、遠心し沈澱を
得た。得られた沈澱を５０ｍＭグリシン−水酸化ナトリ
ウム緩衝液（ｐＨ９，０）に懸濁し、さらに尿素の最終
濃度が８Ｍとなるように緩衝溶液を調製した。６０℃で
１時間加温し、ＩＭの尿素を含んだ透析液（１Ｍ尿素、
５０ｍＭグリシン−水酸化ナトリウム緩衝液（ｐＨ９，
０）、２ｍＭ還元型グルタチオン、０゜２ｍＭ酸化型グ
ルクチオン、１ｒｎＭエチレンジアミンテトラアセテー
ト）で１２時間透析後、５０ｍＭ）リス−塩酸緩衝液（
ｐＨ８，０）で６時間透析した。透析内液を遠心し、上
清を最終濃度１００ｍＭ塩化ナトリウム、１ｍＭ塩化カ
ルシウム、５０ ｍＭ ）リス−塩酸緩衝液（ｐＨ８，
０）になるように調製した。この溶液に、血液凝固因子
Ｘａ（ベーリンガーマンハイム山）自社製）を蛋白質５
０ｍｇあたりｌＯユニット加え、３７℃１０時間反応さ
せた。反応液を遠心後、その上清をＤｉａｆｌｏ Ｙ
Ｍ−１０（Ａｍｉｃｏｎ社製）を用いて約１０倍濃縮し
、最終的に５０ｍＭ）リス−塩酸緩衝液（ｐＨ７，６）
、２ｍＭジチオスレトール、６Ｍグアニジン塩酸塩、Ｏ
，ｉｌ塩化ナトリウム溶液を調製した。この溶液をＴＳ
Ｋ Ｇｅｌ Ｇ３０００ＳＷ カラム（２５ｍｍ
Ｘ６００ｍｍ）にかけ、Ｂ細胞分化因子溶出画分を１Ｍ
グアニジン塩酸、５０ｍＭ）リス−塩酸緩衝溶液（ｐＨ
８，０）、１ｍＭエチレンジアミンテトラアセテート、
続いて１０ｍＭ）リス−塩酸緩衝液（ｐＨ８，５）で透
析した。この透析液をＴＳＫＧｅｌ ＤＥＡＥ−５Ｐ
Ｗ カラム（２５ｍｍＸ２００ｍｍ）にかけ、０から
０．８Ｍの塩化ナトリウム溶液による直線濃度勾配法に
より溶出した。溶出フラクションをアクリルアミド電気
泳動に付し、Ｂ細胞分化因子と推定される分子量２１゜
０００の画分を得た（図３）。Example 4 (BSF-2 from fusion protein with blood coagulation factor Xa)
E. coli/pGBS-3 was induced with indole acrylic acid in 5Q nori medium, and after culturing for 4 hours, the bacteria were harvested and approximately log(
Wet weight) was obtained. The obtained bodies were suspended in 50 mM Tris-HCl buffer (pH 8,0), crushed, and centrifuged to obtain a precipitate. The obtained precipitate was suspended in 50 mM glycine-sodium hydroxide buffer (pH 9,0), and a buffer solution was prepared so that the final concentration of urea was 8M. Dialysate containing IM urea (1M urea,
50mM glycine-sodium hydroxide buffer (pH 9,
After dialysis for 12 hours with 0), 2mM reduced glutathione, 0゜2mM oxidized glucthione, 1rnM ethylenediaminetetraacetate), 50mM) Lis-HCl buffer (
Dialysis was performed for 6 hours at pH 8.0). The dialyzed fluid was centrifuged, and the supernatant was diluted with Lis-HCl buffer (pH 8,
0). To this solution, add blood coagulation factor Xa (manufactured by Boehringer Mannheim) to protein 5
10 units per 0 mg were added and the reaction was carried out at 37°C for 10 hours. After centrifuging the reaction solution, the supernatant was transferred to Diaflo Y.
Concentrate approximately 10 times using M-10 (manufactured by Amicon) to a final concentration of 50 mM) Lis-HCl buffer (pH 7.6)
, 2mM dithiothretol, 6M guanidine hydrochloride, O
, il sodium chloride solution was prepared. This solution is TS
K Gel G3000SW column (25mm
x600mm) and the B cell differentiation factor elution fraction was diluted with 1M
Guanidine hydrochloride, 50mM) Lis-HCl buffer solution (pH
8,0), 1mM ethylenediaminetetraacetate,
Subsequently, the mixture was dialyzed against 10 mM) Lis-HCl buffer (pH 8.5). This dialysate was added to TSKGel DEAE-5P.
It was applied to a W column (25 mm x 200 mm) and eluted using a linear concentration gradient method using a 0 to 0.8 M sodium chloride solution. The eluted fraction was subjected to acrylamide gel electrophoresis to obtain a fraction with a molecular weight of 21.000, which is estimated to be a B cell differentiation factor (FIG. 3).
（発明の効果）
本発明の組換プラスミド（発現型）はＢＳＦ−２遺伝子
セグメントを効率よく増幅し、これによって形質転換さ
れた微生物に、ＢＳＦ−２蛋白質をヒト成長ホルモンと
の融合蛋白質の形での産生能を与えることができる。
また、本発明のＢＳＦ−２融合蛋白質は血液凝固因子Ｘ
λで処理することにより、Ｎ末端側から一個のアミノ酸
残基Ａｌａの付加したＢＳＦ−２とすることができる。(Effects of the Invention) The recombinant plasmid (expression type) of the present invention efficiently amplifies the BSF-2 gene segment, and transforms the BSF-2 protein into a fusion protein with human growth hormone into a microorganism transformed thereby. It can give the production ability in
In addition, the BSF-2 fusion protein of the present invention has blood coagulation factor
By treatment with λ, BSF-2 with one amino acid residue Ala added from the N-terminus can be obtained.
第１図は本発明のヒトＢＳＦ−２融合発現型プラスミド
の調製を説明する図であり、第２図は本発明で得られる
ＢＳＦ−２融合蛋白質のアクリルアミド電気泳動パター
ンを説明する図であり、第３図はＢＳＦ−２融合蛋白質
を血液凝固因子Ｘａで処理後、精製して得られたＢＳＦ
−２蛋白質のアクリルアミド電気泳動パターンを説明す
る図である。FIG. 1 is a diagram illustrating the preparation of the human BSF-2 fusion expression plasmid of the present invention, and FIG. 2 is a diagram illustrating the acrylamide electrophoresis pattern of the BSF-2 fusion protein obtained by the present invention. Figure 3 shows BSF obtained by treating BSF-2 fusion protein with blood coagulation factor Xa and then purifying it.
It is a figure explaining the acrylamide electrophoresis pattern of -2 protein.
Claims (1)
伝子セグメント。 （２）ヒトＢ細胞分化因子遺伝子の上流側末端に、式 【遺伝子配列があります】 （式中ＸはＮ末端よりＩｌｅＧｌｕＧｌｙＡｒｇで表さ
れるアミノ酸配列をコードするＤＮＡ配列を、Ｘ′はそ
の相補配列を示す）で表されるＤＮＡ配列を、また、そ
の下流側末端に停止コドンを含み、さらにその下流に制
限酵素開裂末端で終わる、式、【遺伝子配列があります
】 （式中Ｙは最初の停止コドンの第二の塩基から制限酵素
開裂末端の３′末端までを表し、Ｙ′はＹの相補配列で
、制限酵素開裂末端の５′末端までを表す）で表される
ＤＮＡ配列を有する特許請求の範囲第１項記載のヒトＢ
細胞分化因子遺伝子セグメント。 （３）上流側に付加されるＤＮＡ配列のＸ及びＸ′が、
式 【遺伝子配列があります】 で表される配列である特許請求の範囲第２項記載のヒト
Ｂ細胞分化因子遺伝子セグメント。 （４）下流側に付加されるＤＮＡ配列のＹ及びＹ′が停
止コドンの第二、第三の塩基配列を含んだ配列である特
許請求の範囲第２項記載のヒトＢ細胞分化因子遺伝子セ
グメント。（５）下流側に付加されるＤＮＡ配列のＹ及
びＹ′の制限酵素開裂末端が、式、 【遺伝子配列があります】 で表される配列を含む特許請求の範囲第２項記載のヒト
Ｂ細胞分化因子遺伝子セグメント。 （６） 【遺伝子配列があります】 で表されるＤＮＡ配列からなる、ヒトＢ細胞分化因子遺
伝子セグメントの製造法。 （７）ヒトＢ細胞分化因子遺伝子の上流側末端に、式 【遺伝子配列があります】 （式中ＸはＮ末端よりＩｌｅＧｌｕＧｌｙＡｒｇで表さ
れるアミノ酸配列をコードするＤＮＡ配列を、Ｘ′はそ
の相補配列を示す）で表されるＤＮＡ配列を、また、そ
の下流側末端に停止コドンを含み、さらにその下流に制
限酵素開裂末端で終わる、式、【遺伝子配列があります
】 （式中Ｙは最初の停止コドンの第二の塩基から制限酵素
開裂末端の３′末端までを表し、Ｙ′はＹの相補配列で
、制限酵素開裂末端の５′末端までを表す）で表される
ＤＮＡ配列を有する特許請求の範囲第６項記載のヒトＢ
細胞分化因子遺伝子セグメントの製造法。 （８）上流側に付加されるＤＮＡ配列のＸ及びＸ′が、
式 【遺伝子配列があります】 で表される配列である特許請求の範囲第７項記載のヒト
Ｂ細胞分化因子遺伝子セグメントの製造法。 （９）下流側に付加されるＤＮＡ配列のＹ及びＹ′が停
止コドンの第二、第三の塩基配列を含んだ配列である特
許請求の範囲第７項記載のヒトＢ細胞分化因子遺伝子セ
グメントの製造法。 （１０）下流側に付加されるＤＮＡ配列のＹ及びＹ′の
制限酵素開裂末端が、式、 【遺伝子配列があります】 で表される配列を含む特許請求の範囲第７項記載のヒト
Ｂ細胞分化因子遺伝子セグメントの製造法。 （１１） 【遺伝子配列があります】 で表されるＤＮＡ配列からなる、ヒトＢ細胞分化因子遺
伝子セグメントを含み、微生物中で自己増殖可能な組換
プラスミド。 （１２）ヒトＢ細胞分化因子遺伝子の上流側末端に、式 【遺伝子配列があります】 （式中ＸはＮ末端よりＩｌｅＧｌｕＧｌｙＡｒｇで表さ
れるアミノ酸配列をコードするＤＮＡ配列を、Ｘ′はそ
の相補配列を示す）で表されるＤＮＡ配列を、また、そ
の下流側末端に停止コドンを含み、さらにその下流に制
限酵素開裂末端で終わる、式、【遺伝子配列があります
】 （式中Ｙは最初の停止コドンの第二の塩基から制限酵素
開裂末端の３′末端までを表し、Ｙ′はＹの相補配列で
、制限酵素開裂末端の５′末端までを表す）で表される
ＤＮＡ配列を有するヒトＢ細胞分化因子遺伝子セグメン
ト含むものである特許請求の範囲第１１項記載の組換プ
ラスミド。 （１３）上流側に付加されるＤＮＡ配列のＸ及びＸ′が
、式 【遺伝子配列があります】 で表される配列であるヒトＢ細胞分化因子遺伝子セグメ
ントを含むものである特許請求の範囲第１２項記載の組
換プラスミド。 （１４）下流側に付加されるＤＮＡ配列のＹ及びＹ′が
停止コドンの第二、第三の塩基配列を含んだ配列である
Ｂ細胞分化因子遺伝子セグメントを含むものである特許
請求の範囲第１２項記載の組換プラスミド。 （１５）下流側に付加されるＤＮＡ配列のＹ及びＹ′の
制限酵素開裂末端が、式、 【遺伝子配列があります】 で表される配列を含むヒトＢ細胞分化因子遺伝子セグメ
ントを含むものである特許請求の範囲第１２項記載の組
換プラスミド。 （１６）プロモーター／オペレーター系のメッセンジャ
ーＲＮＡ転写開始点よりヒトＢ細胞分化因子遺伝子まで
のＤＮＡ配列が、式 【遺伝子配列があります】 （式中ＸはＮ末端よりＩｌｅ Ｇｌｕ Ｇｌｙ Ａｒｇ
で表されるアミノ酸配列をコードするＤＮＡ配列を、Ｘ
′はその相補配列を示す）で表される配列である組換プ
ラスミド。（１７）式 【遺伝子配列があります】 で表される配列のヒトＢ細胞分化因子遺伝子を含み、微
生物中で自己増殖可能な組換プラスミドを微生物中に保
有するエシャリシャ属に属する微生物。 （１８）組換プラスミドのヒトＢ細胞分化因子遺伝子の
上流側末端に、式 【遺伝子配列があります】 （式中ＸはＮ末端よりＩｌｅ Ｇｌｕ Ｇｌｙ Ａｒｇ
で表されるアミノ酸配列をコードするＤＮＡ配列を、Ｘ
′はその相補配列を示す）で表されるＤＮＡ配列を、ま
た、その下流側末端に停止コドンを含み、さらにその下
流に制限酵素開裂末端で終わる、式、＾５′ＴＹ＾３′ ＡＹ′ （式中Ｙは最初の停止コドンの第二の塩基から制限酵素
開裂末端の３′末端までを表し、Ｙ′はＹの相補配列で
、制限酵素開裂末端の５′末端までを表す）で表される
ＤＮＡ配列を有する特許請求の範囲第１７項記載の微生
物。 （１９）上流側に付加されるＤＮＡ配列のＸ及びＸ′が
、式 【遺伝子配列があります】 で表される配列であるヒト細胞分化因子遺伝子を含む組
換プラスミドを保有する特許請求の範囲第１８項記載の
微生物。 （２０）下流側に付加されるＤＮＡ配列のＹ及びＹ′が
停止コドンの第二、第三の塩基配列を含む組換プラスミ
ドを保有するものである特許請求の範囲第１８項記載の
微生物。 （２１）下流側に付加されるＤＮＡ配列のＹ及びＹ′の
制限酵素開裂末端が、式、 【遺伝子配列があります】 で表される配列であるヒトＢ細胞分化因子遺伝子セグメ
ントを含む組換プラスミドを保有する特許請求の範囲第
１８項記載の微生物。 （２２）プロモーター／オペレーター系のメッセンジャ
ーＲＮＡ転写開始点よりヒトＢ細胞分化因子遺伝子まで
のＤＮＡ配列が、式 【遺伝子配列があります】 （式中ＸはＮ末端よりＩｌｅＧｌｕＧｌｙＡｒｇで表さ
れるアミノ酸配列をコードするＤＮＡ配列を、Ｘ′はそ
の相補配列を示す）で表される配列である組換プラスミ
ドを保有する微生物。 （２３）式 【遺伝子配列があります】 で表されるＤＮＡ配列のヒトＢ細胞分化因子遺伝子を含
み、微生物中で自己増殖可能でかつヒトＢ細胞分化因子
を発現することのできる組換プラスミドを細胞中に保有
する、エシャリシャ属に属する微生物を栄養培地に培養
して、ヒトＢ細胞分化因子蛋白を蓄積させてこれを採取
し、必要に応じて血液凝固因子Ｘａで消化することを特
徴とするヒトＢ細胞分化因子の製造法。 （２４）ヒトＢ細胞分化因子遺伝子の上流側末端に、式 【遺伝子配列があります】 （式中ＸはＮ末端よりＩｌｅＧｌｕＧｌｙＡｒｇで表さ
れるアミノ酸配列をコードするＤＮＡ配列を、Ｘ′はそ
の相補配列を示す）で表されるＤＮＡ配列を、また、そ
の下流側末端に停止コドンを含み、さらにその下流に制
限酵素開裂末端で終わる、式、【遺伝子配列があります
】 （式中Ｙは最初の停止コドンの第二の塩基から制限酵素
開裂末端の３′末端までを表し、Ｙ′はＹの相補配列で
、制限酵素開裂末端の５′末端までを表す）で表される
ＤＮＡ配列からなるプラスミドを保有する微生物を用い
る特許請求の範囲第２３項記載の製造法。 （２５）上流側に付加されるＤＮＡ配列のＸ及びＸ′が
、式 【遺伝子配列があります】 で表される配列であるヒトＢ細胞分化因子遺伝子セグメ
ントを含む組換プラスミドを保有する微生物を用いる特
許請求の範囲第２４項記載の製造法。 （２６）下流側に付加されるＤＮＡ配列のＹ及びＹ′が
停止コドンの第二、第三の塩基配列を含んだ配列である
Ｂ細胞分化因子遺伝子セグメントを含む組換プラスミド
を保有する微生物を用いる特許請求の範囲第２４項記載
の製造法。 （２７）下流側に付加されるＤＮＡ配列のＹ及びＹ′の
制限酵素開裂末端が、式、 【遺伝子配列があります】 で表される配列を含むヒトＢ細胞分化因子遺伝子セグメ
ントを含む組換プラスミドを保有する微生物を用いる特
許請求の範囲第２４項記載の製造法。 （２８）ヒト成長ホルモンのＮ末端側の一部とＮ末端よ
りＩｌｅＧｌｕＧｌｙＡｒｇで表されるＤＮＡ[Claims] (1) A human B cell differentiation factor gene segment consisting of a DNA sequence represented by the formula [There is a gene sequence]. (2) At the upstream end of the human B cell differentiation factor gene, there is a gene sequence with the formula (where X is the DNA sequence encoding the amino acid sequence expressed by IleGluGlyArg from the N terminus, and There is also a DNA sequence represented by the formula [gene sequence], which contains a stop codon at its downstream end and ends with a restriction enzyme cleaved end downstream of it (in the formula, Y represents the first stop). A patent claim having a DNA sequence from the second base of the codon to the 3' end of the restriction enzyme cleaved end, where Y' is a complementary sequence of Y and represents the 5' end of the restriction enzyme cleaved end. Human B according to item 1 within the scope of
Cell differentiation factor gene segment. (3) The DNA sequences X and X' added to the upstream side are
The human B cell differentiation factor gene segment according to claim 2, which has a sequence represented by the formula [There is a gene sequence]. (4) The human B cell differentiation factor gene segment according to claim 2, wherein Y and Y' of the DNA sequence added to the downstream side are sequences containing the second and third base sequences of a stop codon. . (5) A human B cell according to claim 2, in which the restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side include a sequence represented by the formula: [There is a gene sequence] Differentiation factor gene segment. (6) [There is a gene sequence] A method for producing a human B cell differentiation factor gene segment consisting of the DNA sequence represented by. (7) At the upstream end of the human B cell differentiation factor gene, there is a gene sequence with the formula (where X is the DNA sequence encoding the amino acid sequence expressed by IleGluGlyArg from the N-terminus, and X' is its complementary sequence. There is also a DNA sequence represented by the formula [gene sequence], which contains a stop codon at its downstream end and ends with a restriction enzyme cleaved end downstream of it (in the formula, Y represents the first stop). A patent claim having a DNA sequence from the second base of the codon to the 3' end of the restriction enzyme cleaved end, where Y' is a complementary sequence of Y and represents the 5' end of the restriction enzyme cleaved end. Human B according to item 6 within the scope of
Method for producing cell differentiation factor gene segments. (8) The DNA sequences X and X' added to the upstream side are
A method for producing a human B cell differentiation factor gene segment according to claim 7, which has a sequence represented by the formula [there is a gene sequence]. (9) The human B cell differentiation factor gene segment according to claim 7, wherein Y and Y' of the DNA sequence added to the downstream side are sequences containing the second and third base sequences of a stop codon. manufacturing method. (10) A human B cell according to claim 7, in which the restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side include a sequence represented by the formula: [There is a gene sequence] Method for producing differentiation factor gene segments. (11) [There is a gene sequence] A recombinant plasmid that contains the human B cell differentiation factor gene segment and is capable of self-replication in microorganisms, consisting of the DNA sequence represented by the following. (12) At the upstream end of the human B cell differentiation factor gene, there is a gene sequence with the formula (where X is the DNA sequence encoding the amino acid sequence expressed by IleGluGlyArg from the N-terminus, and X' is its complementary sequence). There is also a DNA sequence represented by the formula [gene sequence], which contains a stop codon at its downstream end and ends with a restriction enzyme cleaved end downstream of it (in the formula, Y represents the first stop). A human B with a DNA sequence represented by the following: from the second base of the codon to the 3' end of the restriction enzyme cleaved end, where Y' is the complementary sequence of Y and represents the 5' end of the restriction enzyme cleaved end. The recombinant plasmid according to claim 11, which contains a cell differentiation factor gene segment. (13) Claim 12, wherein the DNA sequences X and X' added to the upstream side include a human B cell differentiation factor gene segment that is a sequence represented by the formula: [There is a gene sequence] recombinant plasmid. (14) Claim 12, which includes a B cell differentiation factor gene segment in which Y and Y' of the DNA sequence added to the downstream side is a sequence containing the second and third base sequences of a stop codon. Recombinant plasmids as described. (15) A patent claim in which the restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side include a human B cell differentiation factor gene segment containing the sequence represented by the formula: [There is a gene sequence] The recombinant plasmid according to item 12. (16) The DNA sequence from the messenger RNA transcription start point of the promoter/operator system to the human B cell differentiation factor gene is expressed as follows:
A DNA sequence encoding the amino acid sequence represented by
' indicates its complementary sequence). (17) A microorganism belonging to the genus Esharicha that contains a human B cell differentiation factor gene with the sequence represented by the formula [gene sequence is available] and has a recombinant plasmid capable of self-replication in the microorganism. (18) At the upstream end of the human B cell differentiation factor gene of the recombinant plasmid, there is the formula [gene sequence] (where X is from the N terminus to Ile Glu Gly Arg
A DNA sequence encoding the amino acid sequence represented by
' indicates its complementary sequence), which also contains a stop codon at its downstream end and ends with a restriction enzyme cleavage end downstream of it, with the formula ^5'TY^3'AY' (In the formula, Y represents the sequence from the second base of the first stop codon to the 3' end of the restriction enzyme cleaved end, and Y' is the complementary sequence of Y and represents the region up to the 5' end of the restriction enzyme cleaved end.) 18. The microorganism according to claim 17, having a DNA sequence. (19) Claims No. 1 possessing a recombinant plasmid containing a human cell differentiation factor gene in which the DNA sequences X and X' added to the upstream side are the sequences represented by the formula [There is a gene sequence] The microorganism according to item 18. (20) The microorganism according to claim 18, which has a recombinant plasmid in which the DNA sequences Y and Y' added to the downstream side include the second and third base sequences of a stop codon. (21) A recombinant plasmid containing a human B cell differentiation factor gene segment in which the restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side are the sequence represented by the formula: [There is a gene sequence] The microorganism according to claim 18, which has the following. (22) The DNA sequence from the messenger RNA transcription start point of the promoter/operator system to the human B cell differentiation factor gene is expressed as follows: A microorganism that possesses a recombinant plasmid having a DNA sequence represented by the following sequence (X' indicates its complementary sequence). (23) A recombinant plasmid containing the human B cell differentiation factor gene with the DNA sequence represented by the formula [gene sequence is available], capable of self-replication in microorganisms, and capable of expressing the human B cell differentiation factor is injected into cells. A microorganism belonging to the genus Esharicia, which is present in the human body, is cultured in a nutrient medium to accumulate human B cell differentiation factor protein, which is collected and digested with blood coagulation factor Xa as necessary. Method for producing B cell differentiation factor. (24) At the upstream end of the human B cell differentiation factor gene, there is a gene sequence with the formula There is also a DNA sequence represented by the formula [gene sequence], which contains a stop codon at its downstream end and ends with a restriction enzyme cleaved end downstream of it (in the formula, Y represents the first stop). A plasmid consisting of a DNA sequence from the second base of the codon to the 3' end of the restriction enzyme cleaved end, where Y' is the complementary sequence of Y and represents the 5' end of the restriction enzyme cleaved end. 24. The production method according to claim 23, using the microorganisms possessed. (25) Using a microorganism carrying a recombinant plasmid containing a human B cell differentiation factor gene segment in which the DNA sequences X and X' added to the upstream side are expressed by the formula [There is a gene sequence] The manufacturing method according to claim 24. (26) A microorganism carrying a recombinant plasmid containing a B cell differentiation factor gene segment in which Y and Y' of the DNA sequence added to the downstream side is a sequence containing the second and third base sequences of the stop codon. The manufacturing method according to claim 24 used. (27) A recombinant plasmid containing a human B cell differentiation factor gene segment whose restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side contain a sequence represented by the formula: [There is a gene sequence] 25. The production method according to claim 24, which uses a microorganism having the following. (28) Part of the N-terminal side of human growth hormone and DNA expressed by IleGluGlyArg from the N-terminus
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63179119A JPH022354A (en) | 1988-03-31 | 1988-07-20 | Production of human b cell differentiation factor |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63-75991 | 1988-03-31 | ||
| JP7599188 | 1988-03-31 | ||
| JP63179119A JPH022354A (en) | 1988-03-31 | 1988-07-20 | Production of human b cell differentiation factor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH022354A true JPH022354A (en) | 1990-01-08 |
Family
ID=26417135
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63179119A Pending JPH022354A (en) | 1988-03-31 | 1988-07-20 | Production of human b cell differentiation factor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH022354A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993006840A1 (en) * | 1991-10-09 | 1993-04-15 | Toray Industries, Inc. | Medicine for preventing and treating bleeding tendency |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1988000206A1 (en) * | 1986-07-08 | 1988-01-14 | Genetics Institute, Inc. | Production and use of il-6 |
-
1988
- 1988-07-20 JP JP63179119A patent/JPH022354A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1988000206A1 (en) * | 1986-07-08 | 1988-01-14 | Genetics Institute, Inc. | Production and use of il-6 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993006840A1 (en) * | 1991-10-09 | 1993-04-15 | Toray Industries, Inc. | Medicine for preventing and treating bleeding tendency |
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