JPH02186990A - Cdna clone of post-transfusion non-a non-b hepatitis virus (nanb) and use thereof - Google Patents
Cdna clone of post-transfusion non-a non-b hepatitis virus (nanb) and use thereofInfo
- Publication number
- JPH02186990A JPH02186990A JP1004059A JP405989A JPH02186990A JP H02186990 A JPH02186990 A JP H02186990A JP 1004059 A JP1004059 A JP 1004059A JP 405989 A JP405989 A JP 405989A JP H02186990 A JPH02186990 A JP H02186990A
- Authority
- JP
- Japan
- Prior art keywords
- hepatitis
- nanb
- cdna
- fraction
- precipitate
- 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
- 241000724832 Non-A, non-B hepatitis virus Species 0.000 title abstract 3
- 239000002299 complementary DNA Substances 0.000 claims abstract description 22
- 239000002244 precipitate Substances 0.000 claims abstract description 16
- 239000012634 fragment Substances 0.000 claims abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 210000002966 serum Anatomy 0.000 claims abstract description 6
- 239000002245 particle Substances 0.000 claims abstract description 5
- 239000013598 vector Substances 0.000 claims abstract description 5
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 3
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 3
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims abstract 3
- 230000003381 solubilizing effect Effects 0.000 claims abstract 3
- 239000003795 chemical substances by application Substances 0.000 claims abstract 2
- 208000006454 hepatitis Diseases 0.000 claims description 18
- 231100000283 hepatitis Toxicity 0.000 claims description 18
- 241000700605 Viruses Species 0.000 claims description 15
- 102000036639 antigens Human genes 0.000 claims description 14
- 108091007433 antigens Proteins 0.000 claims description 14
- 239000000427 antigen Substances 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 238000005199 ultracentrifugation Methods 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 claims description 4
- 238000000703 high-speed centrifugation Methods 0.000 claims description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 3
- 102000039446 nucleic acids Human genes 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims description 3
- 230000009257 reactivity Effects 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 2
- 229920002527 Glycogen Polymers 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- VOAXAOULFRTTAM-UHFFFAOYSA-N chloroform phenol Chemical compound C1(=CC=CC=C1)O.C(Cl)(Cl)Cl.C1(=CC=CC=C1)O.C1(=CC=CC=C1)O VOAXAOULFRTTAM-UHFFFAOYSA-N 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 229940096919 glycogen Drugs 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 239000013612 plasmid Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 229940124597 therapeutic agent Drugs 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims 2
- 229930006000 Sucrose Natural products 0.000 claims 2
- 239000000872 buffer Substances 0.000 claims 2
- 230000029087 digestion Effects 0.000 claims 2
- 108091008146 restriction endonucleases Proteins 0.000 claims 2
- 238000006467 substitution reaction Methods 0.000 claims 2
- 239000005720 sucrose Substances 0.000 claims 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims 1
- 238000007865 diluting Methods 0.000 claims 1
- 238000003780 insertion Methods 0.000 claims 1
- 230000037431 insertion Effects 0.000 claims 1
- 238000002955 isolation Methods 0.000 claims 1
- 239000008280 blood Substances 0.000 abstract description 5
- 210000004369 blood Anatomy 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 5
- 230000002265 prevention Effects 0.000 abstract description 3
- 206010019786 Hepatitis non-A non-B Diseases 0.000 abstract 2
- 239000000047 product Substances 0.000 abstract 2
- 239000007853 buffer solution Substances 0.000 abstract 1
- 239000012459 cleaning agent Substances 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 239000012629 purifying agent Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、輸血後非Ａ非Ｂ型肝炎ウィルス（以下ＮＡＮ
Ｂと記載）のｃＤＮＡクローン及びその用途に係る。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention is directed to the treatment of non-A, non-B hepatitis virus (hereinafter NAN) after blood transfusion.
cDNA clone described as B) and its uses.
本発明によるｃＤＮＡクローンは酵母等に組込む事によ
り、あるいは、塩基配列から推定できるアミノ酸配列を
もったポリペプチドを化学合成する事により、非Ａ非Ｂ
型肝炎ウィルス抗原近似の蛋白質が得られるため、その
反応性を利用し、かつ抗体を製造する事により、非Ａ非
Ｂ型肝炎の診断、治療薬、輸血用血液の浄化剤、ワクチ
ン等として利用できるものである。The cDNA clone according to the present invention can be produced by integrating into yeast etc. or by chemically synthesizing a polypeptide having an amino acid sequence that can be deduced from the base sequence.
Since a protein similar to the hepatitis virus antigen can be obtained, by utilizing its reactivity and producing antibodies, it can be used as a diagnostic and therapeutic drug for non-A and non-B hepatitis, a purifying agent for blood for transfusion, a vaccine, etc. It is possible.
（従来技術）
ＮＡＮＢは輸血関連肝炎の９（）−９５％を示し治療法
も未だ確立されておらず、非Ａ非Ｂ型肝炎の診断、治療
薬、輸血用血液の浄化剤、ワクチン等の開発が望まれて
いる状況である。(Prior art) NANB accounts for 9-95% of transfusion-related hepatitis, and no treatment has been established yet. The situation is such that development is desired.
近年、ＮＡＮＢの抗原研究が進み一万塩基程度のＲＮＡ
−本領ウイルスの存在が報告されているものの、非Ａ非
Ｂ型肝炎ウィルスの構造遺伝子である確証は得られてい
ないし、他の原因ウィルスの存在も不明である。In recent years, research on NANB antigens has progressed, and RNA of approximately 10,000 bases has been developed.
-Although the existence of the main virus has been reported, there is no confirmation that it is a structural gene of non-A, non-B hepatitis virus, and the existence of other causative viruses is also unknown.
（特開昭６２−２４９９９９号、特開昭６３−９１３２
８号等）（発明が解決しようとする課題及び発明の目的
）本発明はＮＡＮＢｃＤＮＡクローン及びその断片を提
供し、バイオテクノロジー技術を応用して、非Ａ非Ｂ型
肝炎ウィルス抗原、あるいは抗体性物質の工業的生産方
法を確立するものである。(JP-A-62-249999, JP-A-63-9132
(No. 8, etc.) (Problems to be Solved by the Invention and Objectives of the Invention) The present invention provides NANB cDNA clones and fragments thereof, and applies biotechnology techniques to produce non-A, non-B hepatitis virus antigens or antibody substances. The aim is to establish an industrial production method for
（課題を解決し、目的を達成する手段及び作用）本発明
等は鋭意研究の結果、ＮＡＮＢ ＤＮＡのクローン化
に初めて成功し、この構造決定がなされ、これによって
基本的な問題点を解決するに至ったのである。(Means and effects for solving the problem and achieving the object) As a result of intensive research, the present invention succeeded in cloning NANB DNA for the first time, determined its structure, and thereby solved the fundamental problem. It has come to this.
即ち１本発明によれば、基本的問題点はＮＡＮＢのアミ
ノ酸配列をコードしている約８００個のヌクレオチドを
含むクローン化−本領ＤＮＡ又はこの−本領ＤＮＡと相
補ＤＮＡからなるクローン化二本鎖ＤＮＡにより解決さ
れるのである。That is, according to the present invention, the basic problem is that the cloned original DNA containing approximately 800 nucleotides encoding the amino acid sequence of NANB or the cloned double-stranded DNA consisting of this original DNA and complementary DNA. This is solved by
（発明の効果）
本発明による非Ａ非Ｂ型肝炎ウィルス−本領ＤＮＡ又は
その断片は、これをブローベとして用いる事により、遺
伝子レベルでの診断に利用する事も可能である。(Effects of the Invention) The non-A, non-B hepatitis virus-main DNA or its fragment according to the present invention can also be used for diagnosis at the genetic level by using it as a probe.
本発明による二本鎖ＤＮＡ又はその断片を組込んだプラ
スミドを微生物又は動物細胞に組込ませて培養すれば、
抗原性物質を大量生産する事ができる。又、これを抗原
として、抗体性物質を多量に生産する事もできる。If the plasmid incorporating the double-stranded DNA or its fragment according to the present invention is incorporated into microorganisms or animal cells and cultured,
Antigenic substances can be mass-produced. Furthermore, using this as an antigen, it is also possible to produce large amounts of antibody-based substances.
主な応用範囲は以下の通りである。The main application ranges are as follows.
１）ｃＤＮＡ配列から得られるアミノ酸配列に基ずく人
口合成ペプチド又はそのｃＤＮＡ配列を組込んだ発現ベ
クターによる大腸菌及び酵母の
ｉｎ ｖｉｖｏ発現系で得られたペプチド及びその修飾
誘導体を用いた免疫原物質、ＮＡＮＢウィルス吸着阻害
剤としての利用。1) Immunogens using artificially synthesized peptides based on amino acid sequences obtained from cDNA sequences or peptides and modified derivatives thereof obtained by in vivo expression systems of Escherichia coli and yeast using expression vectors incorporating the cDNA sequences; Use as a NANB virus adsorption inhibitor.
ａ）ワクチンによるＮＡＮＢ感染予防。a) Prevention of NANB infection by vaccination.
ｂ）抗体価の測定による利用：肝炎、肝癌における病状
、予後の診断、供血試料、のスクリーニング。b) Utilization by measuring antibody titer: diagnosis of pathology and prognosis of hepatitis and liver cancer, screening of donated blood samples.
ｃ）ＮＡＮＢウィルスの細胞への感染防止を目的とした
肝炎及び肝癌治療剤。c) A therapeutic agent for hepatitis and liver cancer aimed at preventing NANB virus infection of cells.
ｄ ） ｉｎ ｖｉｖｏ及びｉｎ Ｖｉｔｒ’Ｏ感染系
を用いた抗ウィルス剤の検定に於ける効果の判定試薬。d) Reagent for determining effectiveness in assaying antiviral agents using in vivo and in Vitr'O infection systems.
２）合成ペプチドまたはｉｎ ｖｉｔｒｏ発現系で得た
ペプチドを抗原として作製されたポリクローナルまたは
モノクローナル抗体を用いた利用。2) Utilization using polyclonal or monoclonal antibodies produced using synthetic peptides or peptides obtained using an in vitro expression system as antigens.
ａ）ウィルス存在の証明による肝炎及び肝患者中のキャ
リヤーの診断、供血試料の検定診断、ウィルス局在等の
免疫生化学試薬。a) Immunobiochemical reagents for diagnosis of hepatitis and liver carriers in patients by proving the presence of viruses, assay diagnosis of donated blood samples, virus localization, etc.
ｂ）感染事故後の緊急発病防止を目的とした抗体血清の
利用。b) Utilization of antibody serum for the purpose of emergency prevention of illness after an infection accident.
ｃ ） ｉｎ ｖｉｖｏ及び１ｎｖｉｔｒｏ感染系を用
いた抗ウィルス剤の検定に於ける効果の判定試薬。c) Reagent for determining effectiveness in assaying antiviral agents using in vivo and 1 in vitro infection systems.
（実施例等）
次に、製造例及び構造決定のための試験例に関連して本
発明を更に詳細に説明する。(Examples, etc.) Next, the present invention will be described in further detail with reference to manufacturing examples and test examples for determining the structure.
（１）ウィルスの調製法
ＮＡＮＢ肝炎患者由来の血漿に２０％ポリエヂレングリ
コール（４０００）溶液（ＴＥＮ緩街液：Ｔｒｉ ５−
ＨＣ１ｐＨ８，０，５０ｍＭ、２ｍＭＥＤＴＡ、１５０
ｎＭ ＮａＣ１，１ｍＭＰＭＳＦ）を加え、攪拌、約
３０分放置、高速遠心にて沈澱画分を分取する。(1) Virus preparation method Add 20% polyethylene glycol (4000) solution (TEN loose solution: Tri 5-
HC1 pH 8, 0, 50mM, 2mM EDTA, 150
Add nM NaCl, 1mM PMSF), stir, leave for about 30 minutes, and collect the precipitate fraction by high-speed centrifugation.
沈澱の５倍量のＴＥＮ緩街液により、沈澱の可溶化、高
速遠心により不溶画分を除去する。The precipitate is solubilized with TEN loose solution in an amount 5 times the amount of the precipitate, and the insoluble fraction is removed by high-speed centrifugation.
１５％、６０％５ｕｃｒｏｓｅ ｉｎ ＴＥＮのｃｕ
ｓｈｉ。15%, 60%5ucrose in TEN cu
shi.
ｎに重層し、ＳＷ２８ローターを用いて、２５０００回
転 １２時間超遠心により、沈澱画分を得てＲＮＡ画分
とする。The precipitate fraction was obtained as an RNA fraction by ultracentrifugation at 25,000 rpm for 12 hours using an SW28 rotor.
ＴＥＮにより希釈し１５％、６０％５ｕｃｒｏｓｅｎ
ＴＥＮのｃｕｓｈｉｏｎに重層し、ＳＷ２８ローター
を用いて、２５０００回転 １２時間超遠心により、沈
澱画分を得てＲＮＡ画分とする操作を繰返す。 粒子画
分を１５％５ｕｃｒｏｓｅ １ｎＴＥＮのｃｕｓｈｉｏ
ｎに重層し、ＳＷ２８ローターを用いて、２５０００回
転 １２時間超遠心により、沈澱画分を得ＴＥＮにより
沈澱を可溶化する。Diluted with TEN 15%, 60% 5ucrosen
The procedure of layering on a TEN cushion and ultracentrifuging at 25,000 rpm for 12 hours using an SW28 rotor to obtain a precipitate fraction and use it as an RNA fraction is repeated. Particle fraction 15% 5ucrose 1nTEN cushio
The precipitate fraction was obtained by ultracentrifugation using an SW28 rotor at 25,000 rpm for 12 hours, and the precipitate was solubilized using TEN.
該試料にＧＩＴ液を加え、ＣｓＣｌ（密度１゜７）ｉｎ
ＴＥＮに重層し、ＳＷ４０ローターを用いて、３５００
０回転 １２時間超遠心により、沈殿部分を得て、ＲＮ
Ａ画分とする。GIT solution was added to the sample, and CsCl (density 1°7) in
Layered on TEN and using SW40 rotor, 3500
Obtain the precipitate by ultracentrifugation at 0 rotations for 12 hours, and
It is called A fraction.
Ｃｈｌｏｒｏｆｏｒｍ−ｐｈｅｎｏｌ 、 ツいでＣｈ
ｌｏｒｏｆｏｒｍによる有機溶媒による抽出後、水溶液
層にグリコーゲン（最終濃度：２０μｇ／ｍｌをキャリ
ヤーとして加え、エタノールにより核酸を沈殿し最終製
品とする。（２）ｃＤＮＡクローン作製法上記核酸を鋳
型としてｃＤＮＡを作成しベクター＾ｇｔｌＯに組み込
む方法はＡｍｅｒｓｈａｍ社の！’ｃＤＮＡクローニン
グシステムλｇｔ１０ＪＪに準じた。Chloroform-phenol, Tsuide Ch
After extraction with an organic solvent using loroform, add glycogen (final concentration: 20 μg/ml) as a carrier to the aqueous solution layer, and precipitate the nucleic acid with ethanol to obtain the final product. (2) cDNA cloning method Create cDNA using the above nucleic acid as a template. The method for integrating it into the vector ^gtlO was based on Amersham's !' cDNA cloning system λgt10JJ.
第１図はｍＹＳＢ−１の塩基配列を示したもので゛ある
。
第２図はｍＹＳＢ−２の塩基配列を示したもので°ある
。
第３図はｍＹＳＢ−１から翻訳したアミノ酸配列を示し
たものである。
特 許１！）騒人 株式会社三和化学研究所第３図
Ｌｅｕ（ｒｐ−Ａｌａ−Ｔｈｒ−Ｍｅｔ−Ｇｌｎ−Ｖａ
ｌ−Ｌｙｓ−Ｔｙｒ−Ｇｌｙ−Ｍｅｔ−Ｌｅｕ −Ｈａ
ｔ−Ｐｒｏ−丁ｙｒ−Ａｒｇ−へ１ａ−Ａｓｐ−Ｃｙｓ
−Ｐｒｏ−Ｓｅｒ−Ｐｒ。
Ｔｈｒ−Ｐｒｏ−Ａｌａ−Ｐｒｏ−Ａｓｎ−Ｐｒｏ−Ｈ
ｌ ５−Ａｓｎ−Ｇｌｎ−Ｔｈｒ−Ｔｈｒ−５ｅｒ−八
１ａ−Ｇｉｎ−Ｔｈｒ−Ｇｌｕ−Ｖａｌ−Ｔｈｒ−Ｇｌ
ｙ−Ｌｙｓ−Ｖａｌ−ＰｈｅＬｅｕ−Ｐｈ ｅ−３ｅ
ｒ−Ｐｈ ａ−Ｃｙｓ−Ｖａ ｌ −Ｐｈ ｅ−Ｌｅｕ
−Ｇ １ ｎ −Ｖａ １−Ｔｒｐ −Ｐｈ ｅ−Ｌ
ｅｕ −Ｔｙ ｒ−Ｌｙｓ−間ａ ｔ−Ｃｙｓ−Ｖａ
ｌ−Ａｒｇ−Ｖａ １’−５ｅ ｒ＝ＬｅｕＧｌｙ−Ｐ
ｈｅ−５ｅ ｒ−Ｌｅｕ−Ａ １ ａ −Ｐ ｒ ｏ−
５ｓ ｒ−Ｌｅｕ−Ｍｅ ｔ−Ｔｒｐ−Ａｓｎ−Ｐ ｒ
ｏ −Ｌｙ ｓ−Ｍｅ ｔ−Ｇ １ ｙ−Ｐ ｒ 。Figure 1 shows the base sequence of mYSB-1. Figure 2 shows the base sequence of mYSB-2. FIG. 3 shows the amino acid sequence translated from mYSB-1. Patent 1! ) Nouto Sanwa Kagaku Institute Co., Ltd. Figure 3 Leu (rp-Ala-Thr-Met-Gln-Va
l-Lys-Tyr-Gly-Met-Leu-Ha
1a-Asp-Cys to t-Pro-Tyr-Arg-
-Pro-Ser-Pr. Thr-Pro-Ala-Pro-Asn-Pro-H
l 5-Asn-Gln-Thr-Thr-5er-81a-Gin-Thr-Glu-Val-Thr-Gl
y-Lys-Val-PheLeu-Ph e-3e
r-Ph a-Cys-Val-Ph e-Leu
-G 1 n -Va 1-Trp -Ph e-L
eu -Tyr-Lys-at-Cys-Va
l-Arg-Va 1'-5e r=LeuGly-P
he-5e r-Leu-A 1 a -P r o-
5s r-Leu-Me t-Trp-Asn-P r
o-Lys-Met-G1y-Pr.
Claims (8)
略す）のアミノ酸配列をコードしている約５．４Ｋｂの
ヌクレオチドを含む以下のｃＤＮＡクローン。 （ａ）血清中から単離した粒子画分に含まれる精製ＲＮ
Ａを鋳型として作製した２重鎖ｃＤＮＡ断片にＥｃｏＲ
Ｉリンカーを付加し、ラムダｇｔ１０ベクターのＥｃｏ
ＲＩ部位に置換挿入して作製したファージクローンＹＳ
１。 （クローンＹＳ１はＮＡＮＢ由来のＥｃｏＲＩ制限酵素
消化で約１．８Ｋｂ、約１．４Ｋｂ、約１、２Ｋｂ、約
１．０Ｋｂからなる約５．４ＫｂのｃＤＮＡ断片を含む
） （ｂ）血漿中単離した粒子画分に含まれる精製ＲＮＡを
鋳型として作製した２重鎖ｃＤＮＡ断片にＥｃｏＲＩリ
ンカーを付加し、ラムダｇｔ１０ベクタ−のＥｃｏＲＩ
部位に置換挿入して作製したファージクローンＹＳ２。 （クローンＹＳ２はＮＡＮＢ由来のＥｃｏＲＩ制限酵素
消化で約０．８ＫｂＣのｃＤＮＡ断片を含む）(1) The following cDNA clone containing approximately 5.4 Kb of nucleotides encoding the amino acid sequence of post-transfusion non-A, non-B hepatitis virus (hereinafter abbreviated as NANB). (a) Purified RN contained in particle fraction isolated from serum
EcoR was added to the double-stranded cDNA fragment prepared using A as a template.
Eco of lambda gt10 vector by adding I linker
Phage clone YS created by substitution insertion into the RI site
1. (Clone YS1 contains a cDNA fragment of approximately 5.4 Kb consisting of approximately 1.8 Kb, approximately 1.4 Kb, approximately 1, 2 Kb, and approximately 1.0 Kb after digestion with EcoRI restriction enzyme derived from NANB) (b) Isolation in plasma An EcoRI linker was added to a double-stranded cDNA fragment prepared using the purified RNA contained in the purified particle fraction as a template, and EcoRI linker was added to the lambda gt10 vector.
Phage clone YS2 was created by inserting a substitution at the site. (Clone YS2 contains a cDNA fragment of approximately 0.8 KbC after digestion with EcoRI restriction enzyme derived from NANB)
下のｃＤＮＡサブクローン。 （ａ）ＹＳ１のＮＡＮＢＤＮＡの一部を含む１．８ｋｂ
ＤＮＡ断片をｐＵＣ１１８及びｐＵＣ１１９に組込んだ
プラスミドクローンｐＹＳＡ−１及びｐＹＳＡ−２。 （ｂ）ＹＳ２に含まれるＮＡＮＢＤＮＡ断片をＭ１３ｍ
ｐ１８及びＭ１３ｍｐ１９に挿入したｍＹＳＢ−１及び
ｍＹＳＢ−２。 （ｃ）塩基配列が以下式で示される事を特徴とする請求
項（２）（ｂ）記載のｃＤＮＡクローン。 ｍＹＳＢ−１ 【遺伝子配列があります】 ｍＹＳＢ−２ 【遺伝子配列があります】 （式中Ａ、Ｃ、Ｇ及びＴはアデニン、シトシングアニン
及びチミン塩基を意味し且つ上記の式はアミノ酸に対応
するコドン毎の配列として示されている）』 （ｄ）アミノ酸配列が以下式で示される事を特徴とする
請求項（２）（ｂ）記載のｃＤＮＡクローン。 【遺伝子配列があります】(2) The following cDNA subclones derived from YS1 and YS2 according to claim 1. (a) 1.8kb containing part of the NANB DNA of YS1
Plasmid clones pYSA-1 and pYSA-2 in which the DNA fragment was integrated into pUC118 and pUC119. (b) M13m NANB DNA fragment contained in YS2
mYSB-1 and mYSB-2 inserted into p18 and M13mp19. (c) The cDNA clone according to claim (2)(b), wherein the base sequence is represented by the following formula. mYSB-1 [Gene sequence available] mYSB-2 [Gene sequence available] (d) The cDNA clone according to claim (2)(b), wherein the amino acid sequence is represented by the following formula. [There is a gene sequence]
グリコール（４０００）溶液（ＴＥＮ緩衝液：Ｔｒｉｓ
−ＨＣｌｐＨ８．０、５０ｍＭ、２ｍＭＥＤＴＡ、１５
０ｍＭＮａＣｌ、１ｍＭＰＭＳＦ）を加え、攪拌、約３
０分放置、高速遠心にて沈澱画分を分取する行程。 ［２］沈澱の５倍量のＴＥＮ緩衝液により、沈澱の可溶
化、高速遠心により不溶画分を除去する行程。 ［３］１５％、６０％ｓｕｃｒｏｓｅｉｎＴＥＮのｃｕ
ｓｈｉｏｎに重層し、ＳＷ２８ローターを用いて、２５
０００回転１２時間超遠心により、沈澱画分を得てＲＮ
Ａ画分とする行程。 ［４］ＴＥＮにより希釈しｃ）の操作を繰返す行程。 ［５］粒子画分を１５％ｓｕｃｒｏｓｅｉｎＴＥＮのｃ
ｕｓｈｉｏｎに重層し、ＳＷ２８ローターを用いて、２
５０００回転１２時間超遠心により、沈澱画分を得ＴＥ
Ｎにより沈澱を可溶化する行程。 ［６］該試料にＧＩＴ液を加え、ＣｓＣｌ（密度１．７
）ｉｎＴＥＮに重層し、ＳＷ４０ローターを用いて、３
５０００回転１２時間超遠心により、沈殿部分を得て、
ＲＮＡ画分とする工程。 ［７］Ｃｈｌｏｒｏｆｏｒｍ−ｐｈｅｎｏｌ、ついでＣ
ｈｌｏｒｏｆｏｒｍによる有機溶媒による抽出後、水溶
液層にグリコーゲン（最終濃度；２０μｇ／ｍｌをキャ
リヤーとして加え、エタノールにより核酸を沈殿し最終
製品とする工程。(3) A virus preparation method characterized by the following steps. [1] 20% polyethylene glycol (4000) solution (TEN buffer: Tris
-HCl pH 8.0, 50mM, 2mM EDTA, 15
Add 0mM NaCl, 1mM PMSF) and stir, approx.
Step of separating the precipitate fraction by leaving it for 0 minutes and using high-speed centrifugation. [2] A step of solubilizing the precipitate with TEN buffer in an amount 5 times the amount of the precipitate, and removing the insoluble fraction by high-speed centrifugation. [3] 15%, 60% sucrose in TEN cu
layered on shion and using a SW28 rotor, 25
The precipitate fraction was obtained by ultracentrifugation at 000 rpm for 12 hours and RN
Step to make A fraction. [4] Step of diluting with TEN and repeating the operation of c). [5] Convert the particle fraction to 15% sucrose in TEN c
ushion and using a SW28 rotor, 2
The precipitated fraction was obtained by ultracentrifugation at 5,000 rpm for 12 hours.
Step of solubilizing the precipitate with N. [6] Add GIT solution to the sample and add CsCl (density 1.7
) inTEN and using a SW40 rotor, 3
Obtain a precipitate by ultracentrifugation at 5000 rpm for 12 hours,
Step of obtaining RNA fraction. [7] Chloroform-phenol, then C
After extraction with an organic solvent using hloroform, glycogen (final concentration: 20 μg/ml) is added as a carrier to the aqueous solution layer, and the nucleic acid is precipitated with ethanol to obtain a final product.
応性を測定するための、クローン化ＤＮＡ酵母等組込発
現性蛋白質性抗原もしくは合成蛋白性抗原を主成分とす
る、非Ａ非Ｂ肝炎診断用組成物。(4) To measure the reactivity with non-A, non-B hepatitis virus antibodies in human serum, a non-A, cloned DNA yeast, etc. whose main component is an integratively expressed protein antigen or a synthetic protein antigen. A composition for diagnosing non-B hepatitis.
測定するための、クローン化ＤＮＡ酵母等組込発現性蛋
白質もくしは合成蛋白質を抗原とし任意的に得られた抗
体を主成分とする、非Ａ非Ｂ型肝炎診断用組成物。(5) Cloned DNA for measuring reactivity with non-AB hepatitis virus antigens in human serum The main component is an optionally obtained antibody using an antigen that can be expressed in yeast, etc. or a synthetic protein. A composition for diagnosing non-A, non-B hepatitis.
は合成蛋白質を抗原とし任意的に得られた抗体を主成分
とする、ヒト血清中の非Ａ非Ｂ型肝炎ウィルス抗原除去
剤。(6) An agent for removing non-A, non-B hepatitis virus antigens in human serum, the main component of which is an optionally obtained antibody using cloned DNA, yeast, etc., as an antigen, an integrally expressed protein, or a synthetic protein.
は合成蛋白質を抗原とし任意的に得られた抗体を主成分
とする、非Ａ非Ｂ型肝炎の治療剤。(7) A therapeutic agent for non-A, non-B hepatitis, whose main component is an optionally obtained antibody using cloned DNA yeast, etc., as an antigen, an expressible protein, or a synthetic protein.
くしは合成蛋白性抗原からなる非Ａ非Ｂ型肝炎用ワクチ
ン。(8) A vaccine for non-A, non-B hepatitis consisting of a protein antigen expressed in cloned DNA yeast or a synthetic protein antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1004059A JPH02186990A (en) | 1989-01-10 | 1989-01-10 | Cdna clone of post-transfusion non-a non-b hepatitis virus (nanb) and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1004059A JPH02186990A (en) | 1989-01-10 | 1989-01-10 | Cdna clone of post-transfusion non-a non-b hepatitis virus (nanb) and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02186990A true JPH02186990A (en) | 1990-07-23 |
Family
ID=11574295
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1004059A Pending JPH02186990A (en) | 1989-01-10 | 1989-01-10 | Cdna clone of post-transfusion non-a non-b hepatitis virus (nanb) and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02186990A (en) |
-
1989
- 1989-01-10 JP JP1004059A patent/JPH02186990A/en active Pending
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