【０００１】[0001]
【産業上の利用分野】本発明は新規ＤＮＡおよびポリペ
プチドに関する。FIELD OF THE INVENTION The present invention relates to novel DNAs and polypeptides.
【０００２】[0002]
【従来の技術】ヒトＢ型肝炎は、ＤＮＡウイルスの１種
であるＢ型肝炎ウイルス（Ｈｅｐａｔｉｔｉｓ Ｂ Ｖ
ｉｒｕｓ；以下、ＨＢＶと称することもある）の感染に
よって発症する。ＨＢＶは、デン粒子としてその性状が
知られており、このウイルス粒子の表面にはＨＢＶ表面
抗原（以下、ＨＢｓＡｇと略称する）、粒子内部に一部
分単鎖部分をもつ二重鎖環状ＤＮＡ、ＨＢＶコア抗原
（以下、ＨＢｃＡｇと略称する）、ＨＢＶｅ抗原（ＨＢ
ｅＡｇ）が存在し、また二重鎖ＤＮＡの単鎖部分を修復
する酵素として内存性ＤＮＡ合成酵素の活性が検出され
ている。ＨＢＶ ＤＮＡは分子量２．１×１０6ダルトン
で約３,２００の塩基対を有する。ＨＢｓＡｇの抗原性
は共通抗原のａを中心にａｄｒ，ａｄｗ，ａｙｒ，ａｙ
ｗの４種類が知られている。 ＨＢｃＡｇはＨＢＶ感染
の早期診断のための診断用試薬として使用できる。すな
わち、ＨＢＶに感染すると抗ＨＢｓ抗体が血中に現われ
る前に、抗ＨＢｃ抗体が出現するので、これを検出する
ことによりＨＢＶ感染の有無を早期に判定できる。ま
た、ＨＢｃＡｇは最近、ＨＢＶの感染防御に有効である
との報告もなされている。一方、ＨＢｅＡｇを含有する
血液はデン粒子の含有量も多く、ＨＢＶの感染性のある
血液として危険性が高い。それ故、ＨＢｅＡｇの存在の
有無を知ることはＨＢＶ感染患者の治療においても重要
であり、ＨＢＶ感染の診断に必須である。BACKGROUND OF THE INVENTION Human hepatitis B is one of the DNA viruses, Hepatitis B virus.
irus; hereinafter sometimes referred to as HBV). The properties of HBV are known as den particles, and the surface of this virus particle is HBV surface antigen (hereinafter abbreviated as HBsAg), double-stranded circular DNA partially having a single-stranded portion, and HBV core. Antigen (hereinafter abbreviated as HBcAg), HBVe antigen (HB
eAg) is present, and the activity of endogenous DNA synthase has been detected as an enzyme that repairs the single-stranded portion of double-stranded DNA. HBV DNA has a molecular weight of 2.1 × 106 Daltons and has about 3,200 base pairs. The antigenicity of HBsAg is adr, adw, ayr, ay centered on the common antigen a.
Four types of w are known. HBcAg can be used as a diagnostic reagent for early diagnosis of HBV infection. That is, when infected with HBV, the anti-HBc antibody appears before the anti-HBs antibody appears in the blood. Therefore, the presence or absence of HBV infection can be determined early by detecting this. In addition, it has recently been reported that HBcAg is effective in preventing HBV infection. On the other hand, blood containing HBeAg also has a high content of den particles, and is highly dangerous as blood having an HBV infectivity. Therefore, knowing the presence or absence of HBeAg is important in the treatment of HBV-infected patients and is essential for the diagnosis of HBV infection.
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、ＨＢｃ
ＡｇおよびＨＢｅＡｇを得るには一度デン粒子を集め、
この中のＨＢｃＡｇ，ＨＢｅＡｇを分離しなければなら
ないが、デン粒子中に含まれるＨＢｃＡｇおよびＨＢｅ
Ａｇは微量であるため、これらを安全に、しかも大量に
得る方法の確立が望まれていた。[Problems to be Solved by the Invention] However, HBc
To obtain Ag and HBeAg, once collect the den particles,
HBcAg and HBeAg in this must be separated, but HBcAg and HBe contained in the den particles are separated.
Since the amount of Ag is very small, it has been desired to establish a method for safely obtaining a large amount of Ag.
【０００４】[0004]
【課題を解決するための手段】本発明者等は上記課題を
解決すべく研究を重ねた結果、ＨＢｃＡｇおよびＨＢｅ
Ａｇを遺伝子工学的方法により、これらを大量に得る方
法を提供することに成功したものである。即ち、本発明
はＢ型肝炎ウイルスｅ抗原性を示す新規なポリペプチ
ド、該ポリペプチドをコードするＤＮＡを含有する組換
えＤＮＡ、該組換えＤＮＡを含有する形質転換体、該形
質転換体を培養するＢ型肝炎ウイルスｅ抗原性を示すポ
リペプチドの製造法、Ｂ型肝炎ウイルスｃ抗原性を示す
ポリペプチドをコードするＤＮＡを含有する組換えＤＮ
Ａ、該組換えＤＮＡを含有する形質転換体、該形質転換
体を培養するＢ型肝炎ウイルスｃ抗原性を示すポリペプ
チドの製造法に関し、更に詳しくは （１）次のアミノ酸配列： Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Ile Thr Asn、 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Arg Ile Leu Glu Asp Glu Arg Ala Ser、 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Arg Ile Leu Glu Asp Glu Arg Ala Ser、または Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser 、 を有するＢ型肝炎ウイルスｅ抗原性を示すポリペプチ
ド、（２）プロモーター、該プロモーターの下流に、翻
訳開始コドン、Ｂ型肝炎ウイルスｅ抗原性を示すポリペ
プチドをコードするＤＮＡおよびその直後に停止コドン
を連結してなる組換えＤＮＡを含有する形質転換体を培
養し、該培養物からＢ型肝炎ウイルスｅ抗原性を示すポ
リペプチドを回収することを特徴とする、Ｂ型肝炎ウイ
ルスｅ抗原性を示すポリペプチドの製造法、（３）プロ
モーターのＳＤ配列と翻訳開始コドンとの間の配列がＡ
ＴＣＧＧＧＣである、（２）記載のＢ型肝炎ウイルスｅ
抗原性を示すポリペプチドの製造法、（４）プロモータ
ー、該プロモーターの下流に、翻訳開始コドン、Ｂ型肝
炎ウイルスｃ抗原性を示すポリペプチドをコードするＤ
ＮＡおよびその直後に停止コドンを連結してなる組換え
ＤＮＡにおいて、該プロモーターのＳＤ配列と翻訳開始
コドンとの間の配列がＡＴＣＧＧＧＣであることを特徴
とする組換えＤＮＡ、（５）次のプラスミド： ｐＴＢ(４)３６９（ＦＥＲＭ ＢＰ−１１５３） ｐＴＢ(４)３６９−４ ｐＴＢ(４)３６９−３２およびｐＴＢ(４)３６９−４１ から選ばれるものである、（４）記載の組換えＤＮＡ、
（６）プロモーター、該プロモーターの下流に、翻訳開
始コドン、Ｂ型肝炎ウイルスｃ抗原性を示すポリペプチ
ドをコードするＤＮＡおよびその直後に停止コドンを連
結してなる組換えＤＮＡであって、該プロモーターのＳ
Ｄ配列と翻訳開始コドンとの間の配列がＡＴＣＧＧＧＣ
であることを特徴とする組換えＤＮＡを含有する形質転
換体、（７）プロモーター、該プロモーターの下流に、
翻訳開始コドン、Ｂ型肝炎ウイルスｃ抗原性を示すポリ
ペプチドをコードするＤＮＡおよびその直後に停止コド
ンを連結してなる組換えＤＮＡにおいて、該プロモータ
ーのＳＤ配列と翻訳開始コドンとの間の配列がＡＴＣＧ
ＧＧＣであることを特徴とする組換えＤＮＡを含有する
形質転換体を培養し、該培養物からＢ型肝炎ウイルスｃ
抗原性を示すポリペプチドを回収することを特徴とす
る、Ｂ型肝炎ウイルスｃ抗原性を示すポリペプチドの製
造法、を提供するものである。Means for Solving the Problems As a result of repeated studies to solve the above problems, the present inventors have found that HBcAg and HBe
The inventors have succeeded in providing a method for obtaining Ag in a large amount by a genetic engineering method. That is, the present invention relates to a novel polypeptide exhibiting hepatitis B virus e antigenicity, recombinant DNA containing DNA encoding the polypeptide, transformant containing the recombinant DNA, and culture of the transformant. Method for producing polypeptide showing hepatitis B virus e antigenicity, recombinant DN containing DNA encoding polypeptide showing hepatitis B virus c antigenicity
A, a transformant containing the recombinant DNA, and a method for producing a polypeptide showing hepatitis B virus c antigenicity, which comprises culturing the transformant, more specifically, (1) the following amino acid sequence: Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Ile Thr Asn, Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu S er Pro Glu His Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Arg Ile Leu Glu Asp Glu Arg Ala Ser, Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Arg Ile Leu Glu Asp Glu Arg Ala Ser, or Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pr o His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser, a polypeptide exhibiting hepatitis B virus e antigenicity, (2) promoter, translation initiation downstream of the promoter A transformant containing a codon, a DNA encoding a polypeptide having hepatitis B virus e antigenicity, and a recombinant DNA having a stop codon immediately linked thereto is cultured, and the hepatitis B virus e is cultured from the culture. A method for producing a polypeptide exhibiting hepatitis B virus e antigenicity, which comprises recovering a polypeptide exhibiting antigenicity, (3) a sequence between the SD sequence of a promoter and a translation initiation codon is A
Hepatitis B virus e according to (2), which is TCGGGC
A method for producing a polypeptide having antigenicity, (4) a promoter, a translation initiation codon downstream of the promoter, and D encoding a polypeptide having hepatitis B virus c antigenicity
Recombinant DNA comprising NA and a stop codon immediately thereafter, wherein the sequence between the SD sequence of the promoter and the translation initiation codon is ATCGGGC, (5) The following plasmid A recombinant DNA according to (4), which is selected from pTB (4) 369 (FERM BP-1153) pTB (4) 369-4 pTB (4) 369-32 and pTB (4) 369-41.
(6) A promoter, a recombinant DNA comprising a translation initiation codon, a DNA encoding a polypeptide exhibiting hepatitis B virus c antigenicity, and a stop codon immediately after the promoter, which is downstream of the promoter. Of S
The sequence between the D sequence and the translation initiation codon is ATCGGGGC.
A transformant containing the recombinant DNA, wherein (7) a promoter and downstream of the promoter,
In a recombinant initiation codon, a DNA encoding a polypeptide exhibiting hepatitis B virus c antigenicity, and a recombinant DNA obtained by ligating a stop codon immediately after that, the sequence between the SD sequence of the promoter and the translation initiation codon is ATCG
A transformant containing a recombinant DNA characterized by being GGC is cultured, and the hepatitis B virus c is obtained from the culture.
The present invention provides a method for producing a polypeptide having hepatitis B virus c antigenicity, which comprises recovering a polypeptide having antigenicity.
【０００５】本発明のＢ型肝炎ウイルスｅ抗原性を示す
ポリペプチドを生産する形質転換体の宿主たる微生物と
してはＢ型肝炎ウイルスｅ抗原性を示すポリペプチドを
生産する能力を有するものであればいかなるものであっ
てもよいが、プロモーターの下流に、翻訳開始コドン、
Ｂ型肝炎ウイルスｅ抗原性を示すポリペプチドをコード
するＤＮＡおよびその直後に停止コドンを連結してなる
組換えＤＮＡを保持する大腸菌、酵母などがあげられる
が、なかでも上記組換えＤＮＡを保持する大腸菌が好ま
しい。プロモーターとしては、ＲＮＡポリメラーゼが結
合することによってｍＲＮＡ合成を開始させるのに必要
な部位を含むものであれば、いかなるものであってもよ
い。たとえば大腸菌を宿主として用いる場合に使用する
プロモーターとしてはｔｒｐプロモーター，ｒｅｃＡプ
ロモーター，ｌａｃプロモーター，λＰLプロモータ
ー，ｔｕｆＢプロモーターなどがあげられ、とりわけｔ
ｒｐプロモーターが好適である。さらに好ましくは、プ
ロモーター中のＳＤ〔シャイン−ダルガーノ（Shine-Da
lgarno)〕配列と、後述の翻訳開始コドンとの間の配列
がＡＴＣＧＧＧＣであるプロモーターがあげられる。た
とえば酵母を宿主として使用する場合においては酵母で
機能しうるプロモーターであればいかなるものであって
もよい。翻訳開始コドンとしては好ましくはＡＴＧがあ
げられる。Ｂ型肝炎ウイルスｃ抗原性を示すポリペプチ
ドをコードするＤＮＡとしてはいかなるサブタイプのＢ
型肝炎ウイルスｃ抗原性を示すポリペプチドをコードす
るＤＮＡであってもよいが、図２に示されるポリペプチ
ドをコードするＤＮＡ（ａｄｗ型）が好ましい。さらに
好ましくは、図１（ａｄｗ型ＨＢＶ ＤＮＡの全塩基配
列を示す）に示される塩基配列順序１９０１〜２４５５
のＤＮＡ（ａｄｗ型）がさらに好ましい。Ｂ型肝炎ウイ
ルスｅ抗原性を示すポリペプチドをコードするＤＮＡと
してはいかなるサブタイプのＢ型肝炎ウイルスｅ抗原性
を示すポリペプチドをコードするＤＮＡであってもよい
が、図２に示されるアミノ酸配列順序１より始まり９５
ないし１８４のポリペプチド（ａｄｗ型）をコードする
ＤＮＡが好ましい。さらに好ましくは、図１に示される
塩基配列順序１９０１より始まり２２６３（２２６３番
目の塩基はＣでもよい）ないし２３５６のＤＮＡ（ａｄ
ｗ型）がさらに好ましい。 Ｂ型肝炎ウイルスｃ抗原性
またはｅ抗原性を示すポリペプチドをコードするＤＮＡ
は、生産されるポリペプチドの抗原性が失なわれない限
り、一部のアミノ酸，ペプチドを欠除、他のアミノ酸，
ペプチドへの置換、あるいは他のアミノ酸，ペプチドの
付加がなされているポリペプチドをコードするＤＮＡで
あってもよい。停止コドンとしてはＴＡＡ，ＴＡＧ，Ｔ
ＧＡがあげられる。The microorganism that is a host of the transformant that produces the polypeptide showing hepatitis B virus e antigenicity of the present invention is any microorganism that has the ability to produce a polypeptide showing hepatitis B virus e antigenicity. It may be any, but a translation initiation codon,
Escherichia coli, yeast, etc., which retains a DNA encoding a polypeptide exhibiting hepatitis B virus e antigenicity and a recombinant DNA formed by ligating a stop codon immediately after that, include, among others, the above recombinant DNAs. E. coli is preferred. The promoter may be any promoter as long as it contains a site necessary for initiating mRNA synthesis by binding with RNA polymerase. For example, when Escherichia coli is used as a host, examples of the promoter used include trp promoter, recA promoter, lac promoter, λPL promoter, tufB promoter and the like.
The rp promoter is preferred. More preferably, SD [Shine-Darugano (Shine-Da
lgarno)] sequence and the sequence between the translation initiation codon described below is ATCGGGC. For example, when yeast is used as a host, any promoter may be used so long as it can function in yeast. The translation initiation codon is preferably ATG. Any subtype of B can be used as a DNA encoding a polypeptide showing hepatitis B virus c antigenicity.
Although it may be a DNA encoding a polypeptide exhibiting hepatitis C virus c antigenicity, a DNA encoding the polypeptide shown in FIG. 2 (adw type) is preferable. More preferably, the nucleotide sequence order 1901 to 2455 shown in FIG. 1 (which shows the entire nucleotide sequence of adw type HBV DNA).
Is more preferable (adw type). The DNA encoding a polypeptide exhibiting hepatitis B virus e antigenicity may be a DNA encoding a polypeptide exhibiting hepatitis B virus e antigenicity of any subtype, and the amino acid sequence shown in FIG. Start from sequence 1 95
DNAs encoding polypeptides 184 to 184 (adw type) are preferred. More preferably, it starts from the base sequence order 1901 shown in FIG. 1 and starts from 2263 (the 2263rd base may be C) to 2356 DNA (ad
w type) is more preferable. DNA encoding a polypeptide showing hepatitis B virus c antigenicity or e antigenicity
Is a deficiency of some amino acids and peptides, other amino acids, unless the antigenicity of the produced polypeptide is lost.
It may be a DNA encoding a polypeptide having a substitution with a peptide, or addition of another amino acid or peptide. TAA, TAG, T as stop codon
GA can be given.
【０００６】たとえばａｄｗ型ＨＢｃ抗原性を示すポリ
ペプチドをコードするＤＮＡは、ヌクレイック・アシッ
ズ・リサーチ（Nucleic Acids Res.)，１１巻，１７４
７頁（１９８３年）に記載されているｐＢＲ３２２−Ｅ
ｃｏＲＩ／ＨＢＶ９３３（ｐＨＢＶ９３３と略称）を制
限酵素ＨｈａＩ消化により断片化し、得られるＤＮＡ断
片のうちＨＢｃ抗原性を示すポリペプチドをコードする
ＤＮＡを含有する約１キロ塩基対のＤＮＡを分離し、Ｅ
ｃｏＲＩリンカーを結合した後、トリプトファンプロモ
ーターを含むｐｔｒｐ７８１〔ｐｔｒｐ７０１（ヨーロ
ッパ特許公開第００６８７１９号公報参照）を制限酵素
ＥｃｏＲＩ消化後、ＣｌａＩで部分分解し、生じたのり
しろ部分をＤＮＡポリメラーゼＩラージフラグメントで
修復し、Ｔ４ＤＮＡリガーゼを用いて環状化されたプラ
スミドであって、ＥｃｏＲＩ部位に近い方のＣｌａＩ部
位がこわれたもの〕のＥｃｏＲＩ部位に結合されたプラ
スミドｐＨＥ１を得る。さらに該プラスミドｐＨＥ１を
ＥｃｏＲＩ消化し、さらにヌクレアーゼＢａｌ ３１で
消化した後、ＣｌａＩリンカーを結合させ、ｔｒｐプロ
モーターを含むｐｔｒｐ７７１（ヨーロッパ特許公開第
００６８７１９号公報参照）のＣｌａΙ部位に挿入すれ
ば目的のＨＢｃ抗原性を示すポリペプチドをコードする
ＤＮＡを含有するプラスミドｐＴＢ３６８を得ることが
できる（図４参照）。該プラスミドで大腸菌（ＤＨ１，
χ１７７６，Ｃ６００など）を形質転換することにより
ＨＢｃ抗原性を示すポリペプチドの生産能を有する大腸
菌株が得られる。ａｄｗ型ＨＢｅ抗原性を示すポリペプ
チドをコードするＤＮＡは、たとえば適当な制限酵素
（例、ＡｖａＩ，ＡｖａII）でプラスミドｐＴＢ３６８
を切断し、必要によりさらにヌクレアーゼＢａｌ ３１
で消化した後、停止コドンを含む適当なリンカーを結合
させ、ｐｔｒｐ ７７１に組み込むことにより、目的の
ＨＢｅ抗原性を示すポリペプチドをコードするＤＮＡ含
有するプラスミドを得ることができる。得られたプラス
ミドで大腸菌（ＤＨ１，χ１７７６，Ｃ６００など）を
形質転換することによりＨＢｅ抗原性を示すポリペプチ
ドの生産能を有する大腸菌株が得られる（図５参照）。
ａｄｗ型以外のサブタイプのＨＢｃ抗原性あるいはＨＢ
ｅ抗原性を示すポリペプチドをコードするＤＮＡも上記
と同様にして得ることができ、また、大腸菌以外の微生
物を宿主として用いる場合においても、該宿主において
機能しうるプロモーターを含有するプラスミドに上記Ｄ
ＮＡを挿入することにより目的とするポリペプチドの生
産能を有する微生物が得られる。プロモーターのＳＤ配
列と翻訳開始コドンとの間の配列がＡＴＣＧＧＧＣであ
る組換えＤＮＡはたとえば、適当な制限酵素（例、Ｅｃ
ｏＲＩ）で処理した後、Ｓ１ヌクレアーゼを作用させ、
ライゲーション反応を行うことにより得ることもできる
が、オリゴヌクレチオド・ミュータジェネシス（oligon
ucleotide mutagenesis）などの公知の方法を用いて作
製することもできる。[0006] For example, DNA encoding a polypeptide exhibiting adw-type HBc antigenicity is described in Nucleic Acids Res., Vol. 11, 174.
PBR322-E described on page 7 (1983).
coRI / HBV933 (abbreviated as pHBV933) was fragmented by digestion with a restriction enzyme HhaI, and about 1 kilobase pair DNA containing a DNA encoding a polypeptide showing HBc antigenicity was isolated from the resulting DNA fragment.
After the coRI linker was ligated, ptrp781 containing a tryptophan promoter [ptrp701 (see European Patent Publication No. 0068719) was digested with the restriction enzyme EcoRI and partially digested with ClaI, and the resulting margin was repaired with DNA polymerase I large fragment. , A plasmid circularized with T4 DNA ligase, in which the ClaI site closer to the EcoRI site was broken], resulting in the plasmid pHE1 ligated to the EcoRI site. Further, the plasmid pHE1 is digested with EcoRI, further digested with nuclease Bal31, and then a ClaI linker is ligated thereto, and the desired HBc antigen can be obtained by inserting it into the ClaI site of ptrp771 containing the trp promoter (see European Patent Publication No. 0068719). A plasmid pTB368 containing a DNA encoding a sex-providing polypeptide can be obtained (see FIG. 4). E. coli (DH1,
χ1776, C600, etc.) to obtain an E. coli strain having the ability to produce a polypeptide showing HBc antigenicity. The DNA encoding the polypeptide exhibiting adw-type HBe antigenicity can be obtained by, for example, using an appropriate restriction enzyme (eg, AvaI, AvaII) to prepare plasmid pTB368.
And nuclease Bal 31 if necessary.
After digestion with, a suitable linker containing a stop codon is ligated and incorporated into ptrp 771 to obtain a plasmid containing a DNA encoding the desired polypeptide having HBe antigenicity. By transforming Escherichia coli (DH1, χ1776, C600, etc.) with the obtained plasmid, an Escherichia coli strain capable of producing a polypeptide showing HBe antigenicity can be obtained (see FIG. 5).
HBc antigenicity or HB of subtypes other than adw type
DNA encoding a polypeptide exhibiting e-antigenicity can be obtained in the same manner as described above, and when a microorganism other than Escherichia coli is used as a host, the above-mentioned D
By inserting the NA, a microorganism having the ability to produce the target polypeptide can be obtained. Recombinant DNA in which the sequence between the SD sequence of the promoter and the translation initiation codon is ATCGGGC is, for example, a suitable restriction enzyme (eg, Ec
and then treated with S1 nuclease,
Although it can also be obtained by carrying out a ligation reaction, it is possible to use oligonucleotide mutagenesis (oligon
ucleotide mutagenesis).
【０００７】得られた大腸菌の形質転換体は自体公知の
培地で培養することができる。培地としてはＬブロス，
ペナセイ（Penassay）ブロス，グルコース，カザミノ酸
を含むＭ−９培地などの公知の培地があげられる。必要
によりプロモーターを効率よく働かせるために、たとえ
ば、３β−インドリルアクリル酸のような薬剤を加える
ことができる。培養は通常１５〜４３℃、好ましくは２
８〜４０℃で２〜２４時間、好ましくは３〜８時間行
い、必要により通気や攪拌を加えることもできる。培養
後、公知の方法で菌体を集め、緩衝液に懸濁し、超音波
処理、リゾチームおよび（または）凍結融解によって菌
体を破壊したのち、遠心分離により目的とするポリペプ
チドを含む抽出液を得る方法などの公知の方法を用いる
ことができる。また抽出液からの目的とするポリペプチ
ドの分離、精製も自体公知の方法により行なわれる。大
腸菌以外の微生物の場合も同様にして目的とするポリペ
プチドを得ることができる。The obtained E. coli transformant can be cultured in a medium known per se. L broth as the medium,
Known media such as M-9 medium containing Penassay broth, glucose and casamino acid can be mentioned. If necessary, a drug such as 3β-indolylacrylic acid can be added to allow the promoter to work efficiently. Culturing is usually 15 to 43 ° C., preferably 2
It is carried out at 8 to 40 ° C. for 2 to 24 hours, preferably 3 to 8 hours, and aeration and stirring can be added if necessary. After culturing, cells are collected by a known method, suspended in a buffer solution, disrupted by sonication, lysozyme and / or freeze-thawing, and then centrifuged to obtain an extract containing the polypeptide of interest. A known method such as a method of obtaining the same can be used. Further, separation and purification of the desired polypeptide from the extract are also carried out by a method known per se. In the case of microorganisms other than E. coli, the target polypeptide can be obtained in the same manner.
【０００８】[0008]
【作用】本発明のＨＢｃ抗原性あるいはＨＢｅ抗原性を
示すポリペプチドは天然由来のＨＢｃ，ＨＢｅ抗原と同
様にＢ型肝炎の診断剤，Ｂ型肝炎ウイルスの感染の防御
（予防）剤として使用することができる。プロモーター
のＳＤ配列と翻訳開始コドンとの間の配列がＡＴＣＧＧ
ＧＣである組換えＤＮＡにおいては、ＨＢｃ抗原性ある
いはＨＢｅ抗原性を示すポリペプチドの生産量が増大
し、ＨＢｃ抗原あるいはＨＢｅ抗原の製造に有利であ
る。The polypeptide of the present invention showing HBc antigenicity or HBe antigenicity is used as a hepatitis B diagnostic agent and a hepatitis B virus infection preventive (preventive) agent in the same manner as naturally occurring HBc and HBe antigens. be able to. The sequence between the SD sequence of the promoter and the translation initiation codon is ATCGG.
In the recombinant DNA which is GC, the production amount of the polypeptide showing HBc antigenicity or HBe antigenicity is increased, which is advantageous for the production of HBc antigen or HBe antigen.
【０００９】[0009]
【実施例】以下に実施例を示して本発明をさらに具体的
に説明するが、本発明はこれらに限定されるべきもので
はない。 実施例１ プラスミドｐＨＢＶ９３３を制限酵素ＥｃｏＲＩおよび
ＨｈａＩで切断し、１％アガロースゲル電気泳動によ
り、ＨＢｃ抗原遺伝子を含むＤＮＡ断片（１００５ｂ
ｐ）を分離した。緩衝液中（１０ｍＭ酢酸ナトリウム，
１５０ｍＭ ＮａＣｌ，０．０５ｍＭ ＺｎＳＯ4，ｐＨ
４．０）でヌクレアーゼＳ１処理した後、ＥｃｏＲＩリ
ンカー ｄ（ＧＧＡＡＴＴＣＣ）を結合し、さらにＥｃ
ｏＲＩ処理した。このＤＮＡ断片をプラスミドｐｔｒｐ
７８１のＥｃｏＲＩ部位に挿入した後、これを用いて大
腸菌ＤＨ１を形質転換した。ＨＢｃＡｇ遺伝子が組み込
まれたプラスミド（ｐＨＢｃＨＥ１）を保持するクロー
ンよりプラスミドを抽出し、該プラスミドよりＥｃｏＲ
Ｉ ＤＮＡ断片を切り出した。５μｇの該ＤＮＡ断片を
緩衝液中（６００ｍＭ ＮａＣ１，２０ｍＭトリス・Ｈ
Ｃ１，ｐＨ８．０，１２ｍＭ ＣａＣ１2，１２ｍＭ Ｍ
ｇＣ１2，１．０ｍＭ ＥＤＴＡ）で２ユニットのＢａｌ
３１で２４℃，１分間処理した。Ｃ１ａＩリンカー ｄ
（ＣＡＴＣＧＡＴＧ）を結合したのち、Ｃ１ａＩで消化
した。該ＤＮＡ断片をｐｔｒｐ ７７１のＣ１ａＩ部位
に挿入し、これを用いて大腸菌ＤＨ１を形質転換させ、
テトラサイクリン耐性の形質転換体（Escherichia coli
ＤＨ１／ｐＴＢ３６８）を得た。The present invention will be described in more detail with reference to the following examples, but the present invention should not be limited thereto. Example 1 Plasmid pHBV933 was cleaved with restriction enzymes EcoRI and HhaI and subjected to 1% agarose gel electrophoresis to obtain a DNA fragment containing the HBc antigen gene (1005b
p) was separated. In buffer (10 mM sodium acetate,
150 mM NaCl, 0.05 mM ZnSO4 , pH
After treatment with nuclease S1 with 4.0), EcoRI linker d (GGAATTCC) was ligated, and Ec was further added.
It was subjected to oRI treatment. This DNA fragment is used as the plasmid ptrp.
After insertion into the EcoRI site of 781, this was used to transform E. coli DH1. A plasmid was extracted from a clone having a plasmid (pHBcHE1) in which the HBcAg gene was integrated, and EcoR was extracted from the plasmid.
The I DNA fragment was excised. 5 μg of the DNA fragment in buffer (600 mM NaCl, 20 mM Tris.H
C1, pH 8.0, 12 mM CaC12 , 12 mM M
gC12 , 1.0 mM EDTA) with 2 units of Bal
It was treated with 31 at 24 ° C. for 1 minute. C1aI linker d
After ligating (CATCGATG), it was digested with C1aI. The DNA fragment was inserted into the C1aI site of ptrp 771 and used to transform Escherichia coli DH1,
Transformants resistant to tetracycline (Escherichia coli
DH1 / pTB368) was obtained.
【００１０】実施例２ 実施例１で得られた形質転換体を８μｇ／ｍｌのテトラ
サイクリンを含むＭ−９培地（１０ｍｌ）中、３７℃で
培養し、クレット・ユニット(Klett unit）［５８０ｎ
ｍの吸光度］が１８０〜２００の時、３β−インドリル
アクリル酸を２５μｇ／ｍｌの濃度に加えて、培養をさ
らに２〜３時間続けた。培養液を５０００ｒｐｍ，１０
分間遠心分離して菌体を集め、１ｍｌの緩衝液（２０ｍ
Ｍ トリス・ＨＣｌ，ｐＨ７．６，２０％ショ糖）に懸
濁し、リゾチーム（３ｍｇ／ｍｌ），ＥＤＴＡ（最終濃
度５ｍＭ）およびＰＭＳＦ（最終濃度１ｍＭ）を添加し
て、よく攪拌したのち、４℃で１時間静置した。超音波
破砕により菌体をさらに破壊し、１５０００ｒｐｍで２
０分間遠心を行い（２回）、得られた上清（抽出液）を
アボット社のＨＢｅリアキットを使用してＨＢｃ抗原価
の測定に使用した。その結果、該形質転換体はＨＢｃ抗
原発現クローンであることがわかった。該形質転換体の
菌体抽出液を生理食塩水で希釈し、その抗原価を測定し
た結果、少なくとも２５００倍希釈の濃度でもＨＢｃ抗
原の検出は可能であった。Example 2 The transformant obtained in Example 1 was cultivated at 37 ° C. in M-9 medium (10 ml) containing 8 μg / ml tetracycline to obtain a Klett unit [580n].
m absorbance] of 180-200, 3β-indolylacrylic acid was added to a concentration of 25 μg / ml, and the culture was continued for another 2-3 hours. Culture medium at 5000 rpm, 10
Collect the cells by centrifugation for 1 minute and collect 1 ml of buffer solution (20 m
Suspend in M Tris.HCl, pH 7.6, 20% sucrose), add lysozyme (3 mg / ml), EDTA (final concentration 5 mM) and PMSF (final concentration 1 mM), stir well, and then at 4 ° C. It was left still for 1 hour. The cells are further destroyed by ultrasonic disruption, and 2 at 15000 rpm
Centrifugation was performed for 0 minutes (twice), and the obtained supernatant (extract) was used for measurement of HBc antigen titer using HBe rear kit of Abbott. As a result, it was found that the transformant was an HBc antigen-expressing clone. The cell extract of the transformant was diluted with physiological saline and the antigen titer was measured. As a result, it was possible to detect the HBc antigen even at a concentration of at least 2500 times.
【００１１】実施例３ 上記形質転換体（Escherichia coli ＤＨ１／ｐＴＢ３
６８）よりプラスミド（ｐＴＢ３６８）を抽出し、該プ
ラスミドを制限酵素ＡｖａΙで切断し、ヌクレアーゼＢ
ａｌ ３１で３０〜９０秒間処理した。停止コドンを有
するＥｃｏＲΙリンカーを結合し、ｐｔｒｐ７７１のＣ
ｌａΙ・ＥｃｏＲΙ部位に挿入したのち、大腸菌ＤＨ１
を形質転換した。得られた形質転換体をＭ−９培地で培
養し、実施例２に記載の方法でＨＢｅ抗原価を測定し
た。ＨＢｅ抗原を産生する形質転換体（Escherichia co
li ＤＨ１／ｐＴＢ４４１，Escherichia coli ＤＨ１／
ｐＴＢ４４９，Escherichia coli ＤＨ１／ｐＴＢ５５
２）を得た。 実施例４ 形質転換体の菌体抽出液を生理食塩水で５倍に希釈し、
アボット社のＨＢｅリアキットで抗原価を測定した。結
果を以下に示す。Ｐ／Ｎ＝（ポジティブ ｃｐｍ）／（ネガティブ・コン
トロール ｃｐｍ）。Example 3 The above transformant (Escherichia coli DH1 / pTB3
68), a plasmid (pTB368) is extracted, and the plasmid is cleaved with a restriction enzyme AvaI to obtain nuclease B.
Al 31 for 30-90 seconds. An EcoRl linker with a stop codon was attached to the C of ptrp771.
E. coli DH1 after insertion at the laI / EcoRΙ site
Was transformed. The obtained transformant was cultured in M-9 medium, and the HBe antigen titer was measured by the method described in Example 2. Transformants that produce HBe antigen (Escherichia co
li DH1 / pTB441, Escherichia coli DH1 /
pTB449, Escherichia coli DH1 / pTB55
2) was obtained. Example 4 A bacterial cell extract of the transformant was diluted 5 times with physiological saline,
The antigen titer was measured with the HBe rear kit from Abbott. The results are shown below. P / N = (positive cpm) / (negative control cpm).
【００１２】実施例５ ｉ）大腸菌ＤＨ１／ｐＴＢ３６８の菌体抽出液をベック
マン超遠心機（ＳＷ５５ローター）で４０，０００ｒｐ
ｍ，４℃で６時間遠心し、沈殿物を緩衝液（２０ｍＭト
リス・ＨＣ１，１ｍＭ ＥＤＴＡ，０．１５Ｍ ＮａＣ
１，ｐＨ７．６）に溶解した。試料５０μｌに２倍濃度
のＳＤＳポリアクリルアミド用緩衝液（０．１５Ｍトリ
ス・ＨＣ１，ｐＨ６．８，４％ＳＤＳ，２０％グリセロ
ール，１０％ ２−メルカプトエタノール，０．００２
％ブロモフェノールブルー）５０μｌを加え、１００
℃，５〜１０分間加熱した後、ポリアクリルアミドゲル
電気泳動を行った。ニトロセルロースフィルターに上記
の分離された蛋白を電気的に吸着させ、125Ι−抗−Ｈ
Ｂｅ抗血清を反応させ、洗浄後オートラジオグラフを取
った。その結果、該形質転換体が産生する蛋白は分子量
が２１,５００〜２２,０００ダルトンと推定された（図
６参照）。 ii）大腸菌ＤＨ１／ｐＴＢ３６８の菌体抽出液に固形の
セシウムクロリドを加え、平均ρ＝１．２０となるよう
にした後、３８,０００ｒｐｍで７２時間遠心し、分画
した後、ＨＢｃ抗原の抗原性の存在する分画を検討した
結果、ＨＢｃ抗原はρ＝１．３４付近にピークとして分
画された（図７参照）。以上、ｉ，iiの結果から、該形
質転換体の産物はＨＢｃＡｇの粒子を形成していると推
定される。Example 5 i) A bacterial cell extract of Escherichia coli DH1 / pTB368 was subjected to 40,000 rp with a Beckman ultracentrifuge (SW55 rotor).
Centrifuge at 4 ° C. for 6 hours and the precipitate is buffered (20 mM Tris-HC1, 1 mM EDTA, 0.15 M NaC
1, pH 7.6). A buffer solution for SDS polyacrylamide having a double concentration (0.15 M Tris-HC1, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.002) was added to 50 μl of the sample.
% Bromophenol blue) 50 μl was added to 100
After heating at 5 ° C for 5 to 10 minutes, polyacrylamide gel electrophoresis was performed. The nitrocellulose filter is electrically adsorbed with the above-mentioned separated protein, and125 I-anti-H
After reacting with Be antiserum, an autoradiograph was taken after washing. As a result, the protein produced by the transformant was estimated to have a molecular weight of 21,500 to 22,000 daltons (see FIG. 6). ii) After adding solid cesium chloride to the bacterial cell extract of E. coli DH1 / pTB368 so that the average ρ = 1.20, centrifuge at 38,000 rpm for 72 hours to fractionate the antigen of HBc antigen. As a result of examining the fraction having sex, the HBc antigen was fractionated as a peak near ρ = 1.34 (see FIG. 7). As described above, from the results of i and ii, it is estimated that the product of the transformant forms particles of HBcAg.
【００１３】iii）大腸菌ＤＨ１／ｐＴＢ３６８，大腸
菌ｐＴＢ４４１の菌体抽出液０．５ｍｌを５〜２５％の
ショ糖濃度勾配液（２０ｍＭ トリス，ｐＨ７．６，
０．１５ＭＮａＣｌ，０．００１Ｍ ＥＤＴＡ）３５ｍ
ｌ上に重層し、２４，０００ｒｐｍ，４℃で４時間遠心
した後、３０本に分画した。それぞれの分画の２００μ
ｌずつをＨＢｃＡｇのＥＩＡ（Enzyme Immunoassayキッ
ト，アボット社）法によりＨＢｃＡｇ，ＨＢｅＡｇの検
出を行った。大腸菌ＤＨ１／ｐＴＢ３６８の産物は主に
分画９，１０に、大腸菌ＤＨ１／ｐＴＢ４４１の産物は
分画２０，２１，２２，２３に分画され（図８参照）、
前者は粒子状、後者は粒子の形態をとらないペプチドと
推定された。 iv）大腸菌ＤＨ１／ｐＴＢ３６８の菌体抽出液にＨＢＶ
感染チンパンジーの肝臓から抽出したＨＢｃＡｇ粒子で
免疫して得た抗ＨＢｃ抗血清を加えた後、抗体価の減少
を抗ＨＢｃ測定用キット（アボット社）で測定した結
果、抽出物は抗ＨＢｃ抗体と反応し、抗ＨＢｃ抗体を中
和することが明らかとなった。一方、大腸菌ＤＨ１／ｐ
ＴＢ４４１， 大腸菌ＤＨ１／ｐＴＢ４４９，大腸菌Ｄ
Ｈ１／ｐＴＢ５５２ の菌体抽出物は殆ど抗ＨＢｃ抗体
と反応しなかった。各形質転換体の抽出物と125Ι−抗
−ＨＢｃ抗体とを反応させ、125Ι−抗−ＨＢｃ抗体の
残存量を抗ＨＢｃ 測定用キット（アボット社）で検討
した結果を以下に示す。Iii) 0.5 ml of the cell extract of Escherichia coli DH1 / pTB368 and E. coli pTB441 was added to a sucrose concentration gradient solution (20 mM Tris, pH 7.6) of 5 to 25%.
0.15M NaCl, 0.001M EDTA) 35m
It was layered on 1 and centrifuged at 24,000 rpm at 4 ° C. for 4 hours, and then fractionated into 30 tubes. 200μ of each fraction
HBcAg and HBeAg were detected by the EIA (Enzyme Immunoassay kit, Abbott) method for HBcAg. The product of E. coli DH1 / pTB368 was mainly fractionated into fractions 9 and 10, and the product of E. coli DH1 / pTB441 was fractionated into fractions 20, 21, 22, and 23 (see FIG. 8).
The former was presumed to be in the form of particles and the latter was presumed to be in the form of particles. iv) HBV was added to the bacterial cell extract of E. coli DH1 / pTB368.
After addition of anti-HBc antiserum obtained by immunization with HBcAg particles extracted from the liver of an infected chimpanzee, the decrease in antibody titer was measured with an anti-HBc measurement kit (Abbott), and the extract was identified as anti-HBc antibody. It was revealed that they reacted and neutralized the anti-HBc antibody. On the other hand, E. coli DH1 / p
TB441, E. coli DH1 / pTB449, E. coli D
The H1 / pTB552 cell extract hardly reacted with the anti-HBc antibody. The results obtained by reacting the extract of each transformant with125 I-anti-HBc antibody and examining the remaining amount of125 I-anti-HBc antibody with an anti-HBc assay kit (Abbott) are shown below.
【００１４】実施例７ 予め、約２００００ｃｐｍを示すよう生理食塩水で希釈
した菌体抽出物（大腸菌ＤＨ１／ｐＴＢ３６８，６４倍
希釈；大腸菌ＤＨ１／ｐＴＢ４４１，４倍希釈）および
血清ＨＢｅＡｇ（未希釈）１００μｌに、生理食塩水で
１００倍，５００倍，１０００倍に希釈したウサギ抗Ｈ
Ｂｃ抗体およびコントロールとして生理食塩水，固相Ｈ
Ｂｅ（ＨＢｃ）抗体を加え、室温一晩反応後、固相に結
合した抗原を測定した。結果はコントロールとして生理
食塩水を加えた系のカウントを１００％として％（パー
セント）で図１０に示した。なお、コントロールのカウ
ントは各々、大腸菌ＤＨ１／ｐＴＢ３６８，２２３４０
ｃｐｍ，大腸菌ＤＨ１／ｐＴＢ４４１，２３４７１ｃｐ
ｍ，血清ＨＢｅＡｇ，９４９１ｃｐｍであった。血清Ｈ
ＢｅＡｇはカウントで、他の約１／２量の抗原しか加え
ていないのにもかかわらず、図１０から明らかなように
抗ＨＢｃ抗体を添加してもカウントは最大１０％程度し
か低下しなかった。従ってこの抗ＨＢｃ抗体はＨＢｃＡ
ｇの検出にはほとんど影響を及ぼさないものと考えられ
る。大腸菌ＤＨ１／ｐＴＢ３６８抽出物では抗ＨＢｃ抗
体の添加により明らかにカウントの低下がみられ、産生
物がＨＢｃ抗原性を有していることがわかる。大腸菌Ｄ
Ｈ１／ｐＴＢ４４１抽出物では大腸菌ＤＨ１／ｐＴＢ３
６８抽出物のような明らかなカウントの低下はみられ
ず、産生物は主にＨＢｅ抗原性を示していることが示唆
される。Example 7 A bacterial cell extract (E. coli DH1 / pTB368, 64 times diluted; E. coli DH1 / pTB441, 4 times diluted) previously diluted with physiological saline to show about 20,000 cpm and 100 μl of serum HBeAg (undiluted) Rabbit anti-H diluted 100 times, 500 times and 1000 times with physiological saline
Bc antibody and saline as control, solid phase H
A Be (HBc) antibody was added, and the mixture was reacted overnight at room temperature, and then the antigen bound to the solid phase was measured. The results are shown in FIG. 10 as a percentage (percentage) with the count of the system containing physiological saline as a control taken as 100%. The control counts were E. coli DH1 / pTB368 and 22340, respectively.
cpm, E. coli DH1 / pTB441, 23471 cp
m, serum HBeAg, 9491 cpm. Serum H
As for BeAg, the count was reduced only by about 10% at maximum even when the anti-HBc antibody was added, as is clear from FIG. 10, although the other about 1/2 amount of the antigen was added. . Therefore, this anti-HBc antibody is HBcA
It is considered that it has almost no effect on the detection of g. In the E. coli DH1 / pTB368 extract, the count was clearly decreased by the addition of the anti-HBc antibody, indicating that the product has HBc antigenicity. E. coli D
E. coli DH1 / pTB3 in H1 / pTB441 extract
No apparent decrease in count was seen as with 68 extract, suggesting that the product is predominantly HBe antigenic.
【００１５】実施例８ プラスミドpＴＢ４４１，ｐＴＢ４４９およびｐＴＢ５
５２に挿入されているＨＢeＡgをコードするＤＮＡの
3′末端側の塩基配列を以下に示す。 ｉ)pＴＢ４４１ ＣＣＡ・ＣＣＡ・ＡＡＴ・ＧＣＣ・ＣＣＴ・ＡＧＡ・ＡＴＴ・ＣＴＴ・ Pro Pro Asn Ala Pro Arg Ile Leu (134) ＧＡＡ・ＧＡＣ・ＧＡＡ・ＡＧＧ・ＧＣＣ・ＴＣＧ・ＴＧＡ Glu Asp Glu Arg Ala Ser − すなわち、該プラスミドにより生産されるペプチドはＨ
ＢcＡgのＣ末端側のアミノ酸47個が欠損（天然のＨＢe
ＡgのＣ末端側のアミノ酸 7個が欠損)し、9個の別のア
ミノ酸が付加したものである。 ii）pＴＢ４４９ ＡＧＡ・ＣＧＡ・ＣＧＧ・ＣＴＡ・ＧＡＡ・ＴＴＣ・ＴＴＧ・ＡＡＧ・ (2348) ＡＣＧ・ＡＡＡ・ＧＧＧ・ＣＣＴ・ＣＧＴ・ＧＡＴ・ＡＣＧ・ＣＣＴ・ ＡＴＴ・ＴＴＴ・ＡＴＡ・ＧＧＴ・ＴＡＡ すなわち、ＨＢcＡgのＣ末端側のアミノ酸33個が欠損
し、１６個の別のアミノ酸が付加している。 iii）pＴＢ５５２ ＧＴＡ・ＣＴＴ・ＧＡＡ・ＴＡＴ・ＴＴＧ・ＧＴＣ・ＴＣＣ・ＴＡＧ (2243) すなわち、ＨＢcＡgのＣ末端側のアミノ酸64個を欠損し
ている。Example 8 Plasmids pTB441, pTB449 and pTB5
Of the DNA encoding HBeAg inserted at 52
The base sequence on the 3'end side is shown below. i) pTB441 CCA, CCA, AAT, GCC, CCT, AGA, ATT, CTT, Pro Pro Asn Ala Pro Arg Ile Leu (134) GAA, GAC, GAA, AGG, GCC, TCG, TGA Glu Asp Glu Arg Ala Ser- That is, the peptide produced by the plasmid is H
47 amino acids at the C-terminal side of BcAg are deleted (natural HBe
(7 amino acids at the C-terminal side of Ag are deleted), and 9 other amino acids are added. ii) pTB449 AGA, CGA, CGG, CTA, GAA, TTC, TTG, AAG, (2348) ACG, AAA, GGG, CCT, CGT, GAT, ACG, CCT, ATT, TTT, ATA, GGT, TAA, that is, HBcAg. 33 amino acids on the C-terminal side are deleted and 16 other amino acids are added. iii) pTB552 GTA / CTT / GAA / TAT / TTG / GTC / TCC / TAG (2243) That is, 64 amino acids at the C-terminal side of HBcAg are deleted.
【００１６】実施例９ プラスミドｐＴＢ３６８を制限酵素ＥｃｏＲΙで切断し
た後、エクソヌクレアーゼＢａｌ３１で処理した。次
に、ＰｓｔＩリンカーｄ（ＧＣＴＧＣＡＧＣ）を結合
し、ＣｌａＩおよびＰｓｔＩで処理してＨＢｃＡｇ遺伝
子を含むＣｌａＩ・ＰｓｔＩＤＮＡ断片を得た。該ＤＮ
Ａ断片をｐｔｒｐ７７１のＣｌａＩ・ＰｓｔＩ部位に組
込んだ後、ＣｌａＩで切断し、ＥｃｏＲＩリンカーｄ
（ＧＧＡＡＴＴＣＣ）を結合させ、ＥｃｏＲＩおよびＰ
ｓｔＩ処理で得られたＤＮＡ断片をｐｔｒｐ７８１のＥ
ｃｏＲＩ部位に組込み、これを用いて大腸菌ＤＨ１を形
質転換させ、プラスミドｐＴＢ（４）３６８−１を保持
する形質転換体（Escherichia coli ＤＨ１／ｐＴＢ
（４）３６８−１）を得た。 該形質転換体をＭ−９培
地で培養し、菌体抽出物のＨＢｃＡｇ産生量を測定した
結果、該形質転換体は大腸菌ＤＨ１／ｐＴＢ３６８の約
１０倍のＨＢｃＡｇ生産量が確認された。プラスミドｐ
ＴＢ（４）３６８−１のプロモーター部位のＳＤ配列か
らＨＢｃＡｇ遺伝子の翻訳開始コドンまでの塩基配列を
調べた結果、以下のとおり１４ｂｐであった。 ５’ ＡＡＡＡＧＧＧＴＡＴＣＧＡＡＧＧＣＣＧＧＧＣＡＴＧ ＳＤ ＳＤ配列とＡＴＧとの間の塩基数を減らすためにｐＴＢ
（４）３６８−１をＥｃｏＲＩで切断し、Ｓ１ヌクレア
ーゼ処理した後、ライゲーション（ligation)反応を行
い、該反応液を用いて、大腸菌ＤＨ１を形質転換し、プ
ラスミドｐＴＢ（４）３６９を保持する大腸菌／ｐＴＢ
（４）３６９を得た。該形質転換体のＨＢｃＡｇ生産量
を測定した結果、大腸菌ＤＨ１／ｐＴＢ３６８の１０５
倍量のＨＢｃＡｇを生産することがわかった。プラスミ
ドｐＴＢ（４）３６９のＳＤ配列とＡＴＧとの間の塩基
配列は以下のとおり７ｂｐであった。Example 9 The plasmid pTB368 was digested with the restriction enzyme EcoRΙ and then treated with the exonuclease Bal31. Next, PstI linker d (GCTGCAGC) was ligated and treated with ClaI and PstI to obtain a ClaI.PstI DNA fragment containing the HBcAg gene. The DN
The A fragment was integrated into the ClaI and PstI sites of ptrp771 and then cut with ClaI to obtain EcoRI linker d.
(GGAATTCC), EcoRI and P
The DNA fragment obtained by the stI treatment was treated with E of ptrp781.
A transformant (Escherichia coli DH1 / pTB) carrying plasmid pTB (4) 368-1 which was integrated into the coRI site and used to transform Escherichia coli DH1.
(4) 368-1) was obtained. The transformant was cultured in M-9 medium, and the HBcAg production amount of the bacterial cell extract was measured. As a result, the transformant was confirmed to have an HBcAg production amount about 10 times higher than that of Escherichia coli DH1 / pTB368. Plasmid p
As a result of examining the nucleotide sequence from the SD sequence of the promoter site of TB (4) 368-1 to the translation initiation codon of the HBcAg gene, it was 14 bp as follows.5'AA AAGGGT ATCGAAGGCCGGGCATG SD pTB to reduce the number of bases between the SD sequence and ATG
(4) Escherichia coli harboring plasmid pTB (4) 369 by cutting 368-1 with EcoRI and treating with S1 nuclease, and then carrying out a ligation reaction, and transforming Escherichia coli DH1 with the reaction solution. / PTB
(4) 369 was obtained. As a result of measuring the amount of HBcAg produced by the transformant, the yield of E. coli DH1 / pTB368 was 105.
It was found to produce double the amount of HBcAg. The base sequence between the SD sequence of plasmid pTB (4) 369 and ATG was 7 bp as shown below.
【００１７】実施例１０ （ｉ）プラスミドｐＴＢ（４）３６９をＡｖａＩで切断
し、Klenow フラグメントで単鎖部分を二重鎖とした
後、ＰｓｔＩで処理し、大断片を分離し、ＰｓｔＩ認識
部位を有するＰｓｔＩストップリンカー5 'ＡＴＡＡＣＴＡＡＣＴＡＡＣＴＧＣＡ3'3 'ＴＡＴＴＧＡＴＴＧＡＴＴＧ5' を結合した。ＰｓｔＩで処理し、大断片を分離し、ライ
ゲーション反応を行い、プラスミドｐＴＢ（４）３６９
−４を得た（図１１参照）。 （ii）また、ｐＴＢ（４）３６９をＳｔｙＩで処理し、
小断片を得、該断片をＡｖａIIで処理した後、Klenow
フラグメントで修復した。前記のＰｓｔＩストップリン
カーを結合した後、ＨｐａＩおよびＰｓｔＩで処理し、
５０２ｂｐ断片を分離し、該断片をｐｔｒｐ７８１のＨ
ｐａＩ・ＰｓｔＩ部位に組込み、ｐＴＢ（４）３６９−
３２を得た（図１２参照）。 （iii）また、ｐＴＢ（４）３６９のＳｔｙＩ処理で得
られた小断片をＨｐａIIで処理した後、Klenow フラグ
メントで修復した。前記のＰｓｔＩストップリンカーを
結合した後、ＨｐａＩおよびＰｓｔＩで処理し、４７２
ｂｐ断片を分離し、該断片をｐｔｒｐ７７１のＨｐａＩ
・ＰｓｔＩ部位に組込み、ｐＴＢ（４）３６９−４１を
得た（図１２参照）。 （iv）ｐＴＢ４４１のＨＢｅＡｇ遺伝子中に２つ存在す
るＢｇｌII部位の５’側のＢｇｌII部位とベクター中の
ＰｓｔＩ部位間のＤＮＡ断片をｐＴＢ（４）３６９のＢ
ｇｌII（５’側）・ＰｓｔＩ部位に組込み、ｐＴＢ
（４）４４１−１を得た（図１３参照）。 （ｖ）ｐＴＢ４４１のＨＢｅＡｇ遺伝子中に２つ存在す
るＢｇｌII部位の３’側のＢｇｌII部位とベクター中の
ＰｓｔＩ部位間のＤＮＡ断片をｐＴＢ（４）３６９のＢ
ｇｌII（５’側）・ＰｓｔＩ部位に組込み、ｐＴＢ
（４）４４１−８を得た（図１４参照）。 （vi）ｐＴＢ５５２のＨＢｅＡｇ遺伝子中に２つ存在す
るＢｇｌII部位の５’側のＢｇｌII部位とベクター中の
ＰｓｔＩ部位間のＤＮＡ断片をｐＴＢ（４）３６９のＢ
ｇｌII（５’側）・ＰｓｔＩ部位に組込み、ｐＴＢ
（４）５５２−８を得た（図１５参照）。Example 10 (i) The plasmid pTB (4) 369 was cleaved with AvaI, the single-stranded portion was made into a double strand with Klenow fragment, and then treated with PstI to separate the large fragment, and the PstI recognition site was isolated. combines the PstI stop linker5 'ATAACTAACTAACTGCA 3' 3 'TATTGATTGATTG 5' with. Treatment with PstI, the large fragment was isolated, ligation reaction was performed, and the plasmid pTB (4) 369 was isolated.
-4 was obtained (see FIG. 11). (Ii) In addition, pTB (4) 369 was treated with StyI,
After obtaining a small fragment and treating the fragment with AvaII, Klenow
Repaired with fragment. After ligation of the PstI stop linker described above, treatment with HpaI and PstI,
The 502 bp fragment was isolated and the fragment was labeled with H of ptrp781.
Incorporated into the paI / PstI site, pTB (4) 369-
32 was obtained (see FIG. 12). (Iii) In addition, the small fragment obtained by the StyI treatment of pTB (4) 369 was treated with HpaII and then repaired with Klenow fragment. After ligation with the PstI stop linker described above, treated with HpaI and PstI, 472
The bp fragment was isolated and the fragment was isolated from HpaI of ptrp771.
-Incorporation into the PstI site gave pTB (4) 369-41 (see Figure 12). (Iv) The DNA fragment between the BglII site on the 5'side of the two BglII sites present in the HBeAg gene of pTB441 and the PstI site in the vector was designated as B of pTB (4) 369.
glII (5 'side) -Integrated at PstI site, pTB
(4) 441-1 was obtained (see FIG. 13). (V) The DNA fragment between the BglII site 3'of the two BglII sites present in the HBeAg gene of pTB441 and the PstI site in the vector was designated as B of pTB (4) 369.
glII (5 'side) -Integrated at PstI site, pTB
(4) 441-8 was obtained (see FIG. 14). (Vi) A DNA fragment between the BglII site on the 5'side of the two BglII sites present in the HBeAg gene of pTB552 and the PstI site in the vector was designated as B of pTB (4) 369.
glII (5 'side) -Integrated at PstI site, pTB
(4) 552-8 was obtained (see FIG. 15).
【００１８】実施例１１ 実施例９および１０で得られたプラスミドを保持する大
腸菌ＤＨ１を実施例２と同様に培養した後、菌体を集
め、ＥＤＴＡおよびリゾチームを加え、超音波破砕によ
り菌体抽出液を得た。アボット社のＨＢｅ・ＥＩＡおよ
びコアザイム（Corzyme）を用いてＨＢｃＡｇおよびＨ
ＢｅＡｇの測定を行った結果、ｐＴＢ(４)３６９，ｐＴ
Ｂ(４)３６９−４，ｐＴＢ(４)３６９−３２またはｐＴ
Ｂ(４)３６９−４１を保持する大腸菌ＤＨ１はＨＢｃＡ
ｇおよびＨＢｅＡｇを産生していることが、また、ｐＴ
Ｂ(４)４４１−１またはｐＴＢ(４)５５２−８を保持す
る大腸菌ＤＨ１はＨＢｅＡｇを産生していることがわか
った。ｐＴＢ（４）４４１−８を保持する大腸菌ＤＨ１
の菌体抽出液を希釈し、アボット社のＲＩＡ ＨＢｅ測
定キットで抗原価を測定した。結果を以下に示す。Example 11 Escherichia coli DH1 harboring the plasmids obtained in Examples 9 and 10 were cultured in the same manner as in Example 2, then the cells were collected, EDTA and lysozyme were added, and the cells were extracted by ultrasonication. A liquid was obtained. HBcAg and H using HBe EIA and Corezyme (Corzyme) from Abbott
As a result of measuring BeAg, pTB (4) 369, pT
B (4) 369-4, pTB (4) 369-32 or pT
E. coli DH1 carrying B (4) 369-41 is HBcA
g and HBeAg producing pT
It was found that E. coli DH1 carrying B (4) 441-1 or pTB (4) 552-8 produced HBeAg. E. coli DH1 harboring pTB (4) 441-8
The microbial cell extract was diluted and the antigen titer was measured with a RIA HBe measurement kit from Abbott. The results are shown below.
【００１９】実施例１２ プラスミドｐＴＢ（４）３６９−４，ｐＴＢ（４）３６
９−３２および ｐＴＢ３６９−４１に挿入されている
ＨＢｅＡｇをコードするＤＮＡの３’末端側の塩基配列
を以下に示す。プラスミドｐＴＢ（４）４４１−８はＨＢｃＡｇ遺伝子
中の２９〜８２番目のアミノ酸をコードする部位が欠損
し、Ｃ末端側のアミノ酸４７個をコードする部分が欠損
し、９個の別のアミノ酸をコードする塩基配列を有して
いる。Example 12 Plasmids pTB (4) 369-4, pTB (4) 36
The nucleotide sequences of the 3'-terminal side of the DNA encoding HBeAg inserted in 9-32 and pTB369-41 are shown below. The plasmid pTB (4) 441-8 lacks the site encoding the 29th to 82nd amino acids in the HBcAg gene, lacks the part encoding the 47 amino acids at the C-terminal side, and encodes 9 other amino acids. It has a base sequence that
【００２０】実施例１３ ｐＴＢ（４）４４１−１またはｐＴＢ（４）５５２−８
を保持する大腸菌ＤＨ１のＨＢｅＡｇ抗原性を示す産生
物の分子量を125Ｉ−抗ＨＢｅ 抗血清を使用したウエ
スタン・ブロッテング（Western blotting）法〔Towbi
n,H, 「プロシージング オブ ナショナル アカデミー オ
ブ サイエンス(Proc.Natl.Acad.Sci. U.S.A.)７６巻、
９号、４３５０〜４３５４頁(１９７９)〕およびヒト抗
ＨＢｅ抗血清を使用した免疫沈降法により決定した。す
なわち、ウエスタン・ブロッテング 法によりｐＴＢ
（４）４４１−１を保持する大腸菌の産生物の分子量は
１５，０００〜１５，５００ダルトンであることがわか
った（図１６参照）。また、ｐＴＢ（４）５５２−８を
保持する大腸菌１を14Ｃ−アミノ酸存在下にＭ−９培地
で培養し、菌体抽出物にヒト抗ＨＢｅ抗血清を加えて３
７℃で反応させた後、ＰｒｏｔｅｉｎＡ（１０％溶液）
を加えて抗原抗体反応物を回収し、ポリアクリルアミド
ゲル（１７％）電気泳動を行い、オートラジオグラフィ
−により、産生物の分子量を測定した結果１３，０００
〜１３，５００ダルトンであることがわかった（図１
７）。なお、形質転換体の寄託機関および受託番号は以
下のとおりである。なお、大腸菌ＤＨ１は公知の微生物である〔Selson,M.
E.ら，ネイチャー，第２１７巻，１１１０頁（１９６８
年）〕。ＩＦＯは財団法人発酵研究所の受託番号を、Ｆ
ＥＲＭは通商産業省工業技術院生命工学工業技術研究所
の受託番号を表す。Example 13 pTB (4) 441-1 or pTB (4) 552-8
The molecular weight of the Escherichia coli DH1 product exhibiting HBeAg antigenicity was determined by the Western blotting method using125 I-anti-HBe antiserum [Towbi
n, H, “Procedure of National Academy of Science (Proc.Natl.Acad.Sci. USA) 76 volumes,
No. 9, 4350-4354 (1979)] and an immunoprecipitation method using a human anti-HBe antiserum. That is, pTB was determined by the Western blotting method.
(4) The molecular weight of the Escherichia coli product retaining 441-1 was found to be 15,000 to 15,500 daltons (see FIG. 16). In addition, Escherichia coli 1 harboring pTB (4) 552-8 was cultured in M-9 medium in the presence of14 C-amino acids, and human anti-HBe antiserum was added to the bacterial cell extract to obtain 3
After reacting at 7 ° C, Protein A (10% solution)
Was added to collect the antigen-antibody reaction product, polyacrylamide gel (17%) electrophoresis was performed, and the molecular weight of the product was measured by autoradiography.
It was found to be ~ 13,500 daltons (Fig. 1
7). The depositary institutions and deposit numbers of the transformants are as follows. Escherichia coli DH1 is a known microorganism [Selson, M.
E. et al., Nature, 217, 1110 (1968).
Year)〕. IFO is the Fermentation Institute's trust number, F
ERM represents the consignment number of the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.
【００２１】[0021]
【発明の効果】ＨＢｃ抗原，ＨＢｅ抗原は天然にはＨＢ
Ｖ感染後のヒトあるいはチンパンジーのみから得ること
しかできず、量的にも制約されてきた。また、これらの
疾患動物の取扱いはＨＢＶ感染の危険を伴い、不都合で
あった。本発明はこれらの問題を解決し、本発明によれ
ば抗原性が天然のものに近く、安価で、安全で、しかも
大量にＨＢｃ抗原性あるいはＨＢｅ抗原性を示すポリペ
プチドが得られる。EFFECT OF THE INVENTION HBc antigen and HBe antigen are naturally HB
It can only be obtained from humans or chimpanzees after V infection, and has been quantitatively limited. In addition, handling of these diseased animals is inconvenient because of the risk of HBV infection. The present invention solves these problems, and according to the present invention, a polypeptide having an antigenicity close to that of natural one, inexpensive, safe, and large in amount showing HBc antigenicity or HBe antigenicity can be obtained.
【００２２】[0022]
【配列表】配列番号：１ 配列の長さ：147 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：タンパク質 配列 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Ile Thr Asn 145。[Sequence Listing] SEQ ID NO: 1 Sequence length: 147 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Ile Thr Asn 145.
【００２３】配列番号：１ 配列の長さ：147 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：タンパク質 配列 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Arg Ile Leu Glu Asp Glu 130 135 140 Arg Ala Ser 145。SEQ ID NO: 1 Sequence length: 147 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Arg Ile Leu Glu Asp Glu 130 135 140 Arg Ala Ser 145.
【００２４】配列番号：３ 配列の長さ：93 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：タンパク質 配列 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Val Val 20 25 30 Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp 35 40 45 Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr 50 55 60 Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro 65 70 75 80 Pro Asn Ala Pro Arg Ile Leu Glu Asp Glu Arg Ala Ser 85 90。SEQ ID NO: 3 Sequence length: 93 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Val Val 20 25 30 Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp 35 40 45 Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr 50 55 60 Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro 65 70 75 80 Pro Asn Ala Pro Arg Ile Leu Glu Asp Glu Arg Ala Ser 85 90.
【００２５】配列番号：４ 配列の長さ：121 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：タンパク質 配列 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser 115 120。SEQ ID NO: 4 Sequence length: 121 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gln Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser 115 120.
【図１−１】ａｄｗ型ＨＢＶ ＤＮＡの全塩基配列を示
し、図１−２に続く。FIG. 1-1 shows the entire nucleotide sequence of adw-type HBV DNA, which is continued from FIG. 1-2.
【図１−２】ａｄｗ型ＨＢＶ ＤＮＡの全塩基配列を示
し、図１−３に続く。1-2 shows the entire base sequence of adw-type HBV DNA, which is continued from FIG. 1-3.
【図１−３】ａｄｗ型ＨＢＶ ＤＮＡの全塩基配列を示
し、図１−４に続く。FIG. 1-3 shows the entire base sequence of adw-type HBV DNA, which is continued from FIG. 1-4.
【図１−４】ａｄｗ型ＨＢＶ ＤＮＡの全塩基配列を示
し、図１−５に続く。FIG. 1-4 shows the entire base sequence of adw-type HBV DNA, which is continued from FIG. 1-5.
【図１−５】ａｄｗ型ＨＢＶ ＤＮＡの全塩基配列を示
し、図１−６に続く。FIG. 1-5 shows the entire nucleotide sequence of adw-type HBV DNA, which is continued from FIG. 1-6.
【図１−６】ａｄｗ型ＨＢＶ ＤＮＡの全塩基配列を示
し、図１−７に続く。FIG. 1-6 shows the entire base sequence of adw-type HBV DNA, which is continued from FIG. 1-7.
【図１−７】ａｄｗ型ＨＢＶ ＤＮＡの全塩基配列を示
し、図１−１から図１−７にて完了する。FIG. 1-7 shows the entire nucleotide sequence of adw-type HBV DNA, which is completed in FIGS. 1-1 to 1-7.
【図２】ａｄｗ型ＨＢｃＡｇのアミノ酸配列を示す。FIG. 2 shows the amino acid sequence of adw-type HBcAg.
【図３】ａｄｗ型ＨＢｃＡｇをコードするＤＮＡの塩基
配列を示す。FIG. 3 shows the nucleotide sequence of DNA encoding adw-type HBcAg.
【図４】ｐＴＢ３６８の構築図である。FIG. 4 is a construction diagram of pTB368.
【図５】ＨＢｅＡｇ発現用プラスミドの構築図である。FIG. 5 is a construction diagram of a plasmid for expressing HBeAg.
【図６】レーン１，２とも大腸菌ＤＨ１／ｐＴＢ３６８
の産生物のウエスタン・ブロッティング（Western blot
ting）の結果を示す。FIG. 6: E. coli DH1 / pTB368 in both lanes 1 and 2.
Western blot of the product of
ting) results.
【図７】大腸菌ＤＨ１／ｐＴＢ３６８の産生物のセシウ
ムクロリド平衡密度勾配遠心による解析結果を示す。FIG. 7 shows the analysis results of the product of Escherichia coli DH1 / pTB368 by cesium chloride equilibrium density gradient centrifugation.
【図８】大腸菌ＤＨ１／ｐＴＢ３６８の産生物のショ糖
濃度勾配遠心による解析結果を示す。FIG. 8 shows the analysis results of the product of Escherichia coli DH1 / pTB368 by sucrose gradient centrifugation.
【図９】大腸菌ＤＨ１／ｐＴＢ４４１の産生物のショ糖
濃度勾配遠心による解析結果を示す。FIG. 9 shows the results of analysis of the product of Escherichia coli DH1 / pTB441 by sucrose gradient centrifugation.
【図１０】ウサギ抗ＨＢｃ抗体によるＨＢｅ（ＨＢｃ）
ＲＩＡの中和試験結果を示す。FIG. 10: HBe (HBc) by rabbit anti-HBc antibody
The result of the neutralization test of RIA is shown.
【図１１】ｐＴＢ（４）３６９−４の構築図である。FIG. 11 is a construction diagram of pTB (4) 369-4.
【図１２】ｐＴＢ（４）３６９−３２及びｐＴＢ（４）
３６９−４１の構築図である。FIG. 12: pTB (4) 369-32 and pTB (4)
369-41 is a construction drawing.
【図１３】ｐＴＢ（４）４４１−１の構築図である。FIG. 13 is a construction diagram of pTB (4) 441-1.
【図１４】ｐＴＢ（４）４４１−８の構築図である。FIG. 14 is a construction diagram of pTB (4) 441-8.
【図１５】ｐＴＢ（４）５５２−８の構築図である。FIG. 15 is a construction diagram of pTB (4) 552-8.
【図１６】本発明産生物の分子量のウェスタン・ブロッ
ティング法による解析図である。FIG. 16 is an analysis diagram of the molecular weight of the product of the present invention by Western blotting.
【図１７】本発明産生物の分子量の免疫沈降法による解
析図である。FIG. 17 is an analysis diagram of the molecular weight of the product of the present invention by an immunoprecipitation method.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｐ 21/02 Ｃ 9282−4Ｂ //(Ｃ１２Ｎ 1/21 Ｃ１２Ｒ 1:91） (Ｃ１２Ｎ 15/09 ＺＮＡ Ｃ１２Ｒ 1:92） (Ｃ１２Ｐ 21/02 Ｃ１２Ｒ 1:91） Ｃ１２Ｒ 1:92）─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl.6 Identification code Internal reference number FI Technical indication C12P 21/02 C 9282-4B // (C12N 1/21 C12R 1:91) (C12N 15/09 ZNA C12R 1:92) (C12P 21/02 C12R 1:91) C12R 1:92)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7067521AJP2648122B2 (en) | 1985-10-03 | 1995-03-27 | Novel polypeptide and method for producing the same |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60-221518 | 1985-10-03 | ||
| JP22151885 | 1985-10-03 | ||
| JP7067521AJP2648122B2 (en) | 1985-10-03 | 1995-03-27 | Novel polypeptide and method for producing the same |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61205036ADivisionJPH084507B2 (en) | 1985-10-03 | 1986-09-02 | Novel DNA and polypeptide |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9055857ADivisionJP2749010B2 (en) | 1985-10-03 | 1997-03-11 | Novel DNA and method for producing polypeptide using the same |
| Publication Number | Publication Date |
|---|---|
| JPH0859695Atrue JPH0859695A (en) | 1996-03-05 |
| JP2648122B2 JP2648122B2 (en) | 1997-08-27 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7067521AExpired - Fee RelatedJP2648122B2 (en) | 1985-10-03 | 1995-03-27 | Novel polypeptide and method for producing the same |
| Country | Link |
|---|---|
| JP (1) | JP2648122B2 (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1764369A1 (en)* | 2005-09-16 | 2007-03-21 | Rhein Biotech Gesellschaft für neue biotechnologische Prozesse und Produkte mbH | Vaccines comprising truncated HBC core protein plus saponin-based adjuvant |
| WO2008031878A1 (en)* | 2006-09-15 | 2008-03-20 | Rhein Biotech Gesellschaft für neue Biotechnologische Prozesse und Produkte mbH | A composition for therapy and/or for prophylaxis of hbv-infections and hbv-mediated diseases |
| EP1902727A1 (en)* | 2006-09-22 | 2008-03-26 | Rhein Biotech Gesellschaft für neue biotechnologische Prozesse und Produkte mbH | Vaccines comprising truncated HBC core protein plus saponin-based adjuvants |
| Title |
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| JOURNAL OF IMMUNOLOGY V130-6=1983* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1764369A1 (en)* | 2005-09-16 | 2007-03-21 | Rhein Biotech Gesellschaft für neue biotechnologische Prozesse und Produkte mbH | Vaccines comprising truncated HBC core protein plus saponin-based adjuvant |
| WO2007031334A3 (en)* | 2005-09-16 | 2007-05-18 | Rhein Biothech Ges Fuer Neue B | Vaccines comprising truncated hbc core protein plus saponin-based adjuvants |
| AU2006291430B2 (en)* | 2005-09-16 | 2011-10-20 | Rhein Biotech Gesellschaft Fur Neue Biotechnologische Prozesse Und Produkte Mbh | Vaccines comprising truncated HBC core protein plus saponin-based adjuvants |
| WO2008031878A1 (en)* | 2006-09-15 | 2008-03-20 | Rhein Biotech Gesellschaft für neue Biotechnologische Prozesse und Produkte mbH | A composition for therapy and/or for prophylaxis of hbv-infections and hbv-mediated diseases |
| EP1902727A1 (en)* | 2006-09-22 | 2008-03-26 | Rhein Biotech Gesellschaft für neue biotechnologische Prozesse und Produkte mbH | Vaccines comprising truncated HBC core protein plus saponin-based adjuvants |
| Publication number | Publication date |
|---|---|
| JP2648122B2 (en) | 1997-08-27 |
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| US4710463A (en) | Recombinant DNA molecules capable of expressing HBV core and surface antigens | |
| EP0244924B1 (en) | Production of vaccines against Hepatitis B virus | |
| US4977092A (en) | Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells | |
| Galibert et al. | Nucleotide sequence of a cloned woodchuck hepatitis virus genome: comparison with the hepatitis B virus sequence | |
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| EP0174444A2 (en) | Hepatitis surface antigen particle vaccine | |
| Murray | Application of recombinant DNA techniques in the development of viral vaccines | |
| JPH084507B2 (en) | Novel DNA and polypeptide | |
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| JP2648122B2 (en) | Novel polypeptide and method for producing the same | |
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| US6270955B1 (en) | Pharmaceutical compositions and methods for producing antibodies to hepatitis b virus and kits and methods for detecting antibodies to hepatitis b virus | |
| JP2749010B2 (en) | Novel DNA and method for producing polypeptide using the same | |
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| IE59389B1 (en) | Method for producing hepatitis b virus core antigen (HBcAg) in yeast | |
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| WO1986003411A1 (en) | Process for preparing novel protein | |
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