JPH0350759B2 - - Google Patents
Info
- Publication number
- JPH0350759B2 JPH0350759B2 JP58001495A JP149583A JPH0350759B2 JP H0350759 B2 JPH0350759 B2 JP H0350759B2 JP 58001495 A JP58001495 A JP 58001495A JP 149583 A JP149583 A JP 149583A JP H0350759 B2 JPH0350759 B2 JP H0350759B2
- Authority
- JP
- Japan
- Prior art keywords
- peptide
- reaction
- acid
- antigen
- val
- 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.)
- Expired - Lifetime
Links
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- 229940009098 aspartate Drugs 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 description 1
- 229910000024 caesium carbonate Inorganic materials 0.000 description 1
- HUCVOHYBFXVBRW-UHFFFAOYSA-M caesium hydroxide Inorganic materials [OH-].[Cs+] HUCVOHYBFXVBRW-UHFFFAOYSA-M 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- VDQQXEISLMTGAB-UHFFFAOYSA-N chloramine T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 201000007241 cutaneous T cell lymphoma Diseases 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 150000001989 diazonium salts Chemical class 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-O diazynium Chemical compound [NH+]#N IJGRMHOSHXDMSA-UHFFFAOYSA-O 0.000 description 1
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229960000587 glutaral Drugs 0.000 description 1
- 229940015043 glyoxal Drugs 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 201000005962 mycosis fungoides Diseases 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 238000004810 partition chromatography Methods 0.000 description 1
- 229960003424 phenylacetic acid Drugs 0.000 description 1
- 239000003279 phenylacetic acid Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229960005235 piperonyl butoxide Drugs 0.000 description 1
- 229920002643 polyglutamic acid Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- LPNYRYFBWFDTMA-UHFFFAOYSA-N potassium tert-butoxide Chemical compound [K+].CC(C)(C)[O-] LPNYRYFBWFDTMA-UHFFFAOYSA-N 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000025638 primary cutaneous T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 230000001566 pro-viral effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000012475 sodium chloride buffer Substances 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- RUPAXCPQAAOIPB-UHFFFAOYSA-N tert-butyl formate Chemical group CC(C)(C)OC=O RUPAXCPQAAOIPB-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Description
本発明は、ヒト白血病ウイルス(Adult T−
cell leukemia Virus;ATLV又はHumanT−
cell leukemia Virus;HTLV)に関連する新規
なペプチドであり、これはかかるウイルス感染な
らびに成人T細胞白血病、皮膚型T細胞リンパ腫
などの成熟T細胞白血病・リンパ腫に関連するペ
プチドでもある。
本明細書において、アミノ酸、ペプチド、保護
基、活性基、核酸塩基、その他に関して略号で表
示する場合はIUPAC、IUBの規定或いは当該分
野における慣用記号に従うものとし、その例を次
に挙げる。またアミノ酸等に関して光学異性体が
ありうる場合は、特に明記しなければL体を示す
ものとする。
Ser;セリン Leu;ロイシン
Thr;スレオニン Asn;アスパラギン
Gln;グルタミン Glu;グルタミン酸
Lys;リジン Pro;プロリン
Val;バリン Trp;トリプトフアン
His;ヒスチジン Asp;アスパラギン酸
Gly;グリシン Ile;イソロイシン
Ala;アラニン Tyr;チロシン
A;アデニン T;チミン
G;グアニン C;シトシン
Tos;p−トルエンスルホニル基
Boc;第3級ブトキシカルボニル基
ONP;p−ニトロフエノキシ基
Bzl;ベンジル基
OBzl;ベンジルオキシ基
Cl2−Bzl;2,6−ジクロルベンジル基
Cl−Z;2−クロルベンジルオキシカルボニル基
ヒト白血病ウイルスは、成人T細胞白血病
(ATL)より分離され、該疾患との関連が注目さ
れているウイルスである。本発明者の吉田、菅野
は、遺伝子工学的手法をもちい、宿主細胞の
DNAに組込まれたプロウイルス遺伝子をクロー
ニング(cloning)し、その全塩基配列を決定し
た。これに基づいて該疾患ならびに該ウイルス感
染の診断・治療・予防の基礎を確立し、さきに特
許申請を行なつた。
本発明は、上記の基礎的な情報を基にし完成さ
れたものであり、該ウイルス感染の診断を目的と
した該ウイルス関連ペプチド、ならびにそれ等に
対する特異抗体の作製と測定法に関する。決定さ
れた上記ウイルス遺伝子のコア(ギヤグ)蛋白前
駆体をコードする塩基配列を下記第1表に示す。
The present invention relates to human leukemia virus (Adult T-
cell leukemia Virus; ATLV or HumanT−
This is a novel peptide related to cell leukemia virus (HTLV), and it is also a peptide related to such viral infections as well as mature T-cell leukemias and lymphomas such as adult T-cell leukemia and cutaneous T-cell lymphoma. In this specification, when amino acids, peptides, protecting groups, active groups, nucleobases, etc. are indicated by abbreviations, they shall follow the IUPAC and IUB regulations or common symbols in the field, and examples thereof are listed below. Furthermore, if an amino acid or the like can have optical isomers, the L-isomer is indicated unless otherwise specified. Ser; serine Leu; leucine Thr; threonine Asn; asparagine Gln; glutamine Glu; glutamate Lys; lysine Pro; proline Val; valine Trp; tryptophan His; histidine Asp; aspartate Gly; ; adenine T; thymine G; guanine C; cytosine Tos; p-toluenesulfonyl group Boc; tertiary butoxycarbonyl group ONP; p-nitrophenoxy group Bzl; benzyl group OBzl; benzyloxy group Cl 2 -Bzl; 2,6- Dichlorobenzyl group Cl-Z; 2-chlorobenzyloxycarbonyl group Human leukemia virus is a virus that has been isolated from adult T-cell leukemia (ATL) and is attracting attention for its association with this disease. The inventors, Yoshida and Kanno, used genetic engineering techniques to transform host cells into
The proviral gene integrated into the DNA was cloned and its entire base sequence was determined. Based on this, we established the basis for the diagnosis, treatment, and prevention of the disease and viral infection, and filed a patent application. The present invention was completed based on the above-mentioned basic information, and relates to a method for producing and measuring virus-related peptides for the purpose of diagnosing infection with the virus, as well as specific antibodies against them. The determined nucleotide sequence encoding the core (gear) protein precursor of the above viral gene is shown in Table 1 below.
【表】【table】
【表】【table】
A−Leu−OH (イ)
↓
A−Leu−R1 (ロ)
↓
H−Leu−R1 (ハ)
↓A−Val−OH (ニ)
A−Val−Leu−R1 (ホ)
↓↓↓
A−Tyr−Val−Glu−Pro−Thr−Ala
−Pro−Gln−Val−Leu−R1 (ヘ)
↓
H−Tyr−Val−Gll−Pro−Thr−Ala
Pro−Gln−Val−Leu−OH (1)
〔式中Aはアミノ基の保護基及びR1は不溶性担
体を示す。〕
上記において、Aの好ましいものとしては
Boc、ベンジルオキシカルボニル基、p−メトキ
シベンジルオキシカルボニル基等を、またR1の
好ましいもとしてはクロロメチル化ポリスチレン
等をそれぞれ例示することができる。
また、各反応において、使用するアミノ酸が反
応に関与しない側鎖官能基を有する場合は、常法
どうり、前述した保護基により、保護され、これ
は不溶性担体R1の脱離と同時に脱離される。
上記方法において、アミノ酸(イ)と不溶性担体
R1との反応は、常法に従いアミノ酸(イ)の反応体
カルボキシル基を利用してこれをR1と結合させ
ることによつて行なわれる。該反応は例えばクロ
ロメチル化ポリスチレンを使用する場合は適当な
溶媒中、例えばトリエチルアミン、カリウムtert
−ブトキシド、炭酸セシウム、水酸化セシウム等
の塩基性化合物の存在下に行なわれる。溶媒とし
ては例えばジメチルホルムアミド、ジメチルスル
ホキシド、ピリジン、クロロホルム、ジオキサ
ン、ジクロロメタン、テトラヒドロフラン、N−
メチルピロリドン、ヘキサメチルリン酸トリアミ
ド等又はこれらの混合溶媒等を例示することがで
きる。上記反応は、通常0〜85℃、好ましくは25
〜80℃程度、数分〜24時間程度で終了する。アミ
ノ酸と不溶性担体との使用割合は通常後者1当量
に対して前者を過剰量、一般に1〜3倍当量とす
るのがよい。
かくして得られる一般式(ロ)の固相化アミノ酸の
保護基Aの脱離反応は、常法により行なわれる。
該方法としては例えばパラジウム、パラジウム黒
等の触媒を用いる水素添加、液体アンモニア中金
属ナトリウムによる還元等の還元的方法、トリフ
ルオロ酢酸、塩化水素酸、弗化水素、メタンスル
ホン酸、臭化水素酸等の強酸によるアシドリシス
等を例示することができる。上記触媒を用いる水
素添加は、例えば水素圧1気圧、0〜40℃にて行
ない得る。触媒の使用量としては通常100mg〜1
g程度とするのがよく、一般に1〜48時間程度で
反応は終了する。また上記アシドリシスは、無溶
媒下、通常0〜30℃程度、好ましくは0〜20℃程
度で約15〜1時間程度を要して行なわれる。酸の
使用量は原料化合物に対し通常5〜10倍量程度と
するのがよい。該アシドリシスにおいて保護基A
のみを脱離する場合は酸としてトリフルオロ酢酸
又は塩化水素酸を使用するのが好ましい。更に上
記液体アンモニア中金属ナトリウムによる還元
は、反応液がパーマネントブルーに30秒〜10分間
程度呈色しているような量の金属ナトリウムを用
い、通常−40℃〜−70℃程度にて行ない得る。
次いで得られる一般式(ハ)の固相化アミノ酸とア
ミノ酸(ニ)(もしくはそのカルボキシル基の活性化
されたもの)との反応は、溶媒の存在下に行なわ
れる。該溶媒としては、ペプチド縮合反応に慣用
される公知の各種のもの、例えば無水ジメチルホ
ルムアミド、ジメチルスルホキシド(DMSO)、
ピリジン、クロロホルム、ジオキサン、ジクロロ
メタン、テトラヒドロフラン、酢酸エチル、N−
メチルピロリドン、ヘキサメチルリン酸トリアミ
ド或いはこれらの混合溶媒等を例示することがで
きる。また該反応は、必要に応じて、通常のペプ
チド結合形成反応に用いられる試薬、例えばN,
N−ジシクロヘキシルカルボジイミド(DCC)、
N−エチル−N′−ジメチルアミノカルボジイミ
ド、1−エチル−3−ジイソプロピルアミノカル
ボジイミド、1−シクロヘキシル−3−(2−モ
ルホリニル−4−エチル)カルボジイミド等のカ
ルボジイミド類等の脱水縮合剤の存在下に行なう
ことができる。アミノ酸(ハ)とアミノ酸(ニ)との使用
割合としては、特に限定はないが、通常前者に対
して後者を等モル量〜10倍モル量、好ましくは等
モル量〜5倍モル量とするのがよい。脱水縮合剤
の使用量も特に限定はなく、通常アミノ酸(ニ)に対
して、好ましくは等モル量程度使用される。反応
温度はペプチド結合形成反応に使用される通常の
範囲、一般には約−40℃〜約60℃、好ましくは約
−20℃〜約40℃の範囲から適宜選択される。反応
時間は一般に数分〜30時間程度とされる。
かくして得られる一般式(ホ)のペプチドは、上記
と同様に保護基Aの脱離後、一般式(1)で表わされ
るアミノ酸配列に従い、A−Gln−OH、A−Pro
−OH、A−Ala−OH、A−Thr−OH、A−
Pro−OH、A−Glu−OH、A−Val−OH、A−
Tyr−OHの各アミノ酸もしくはそのカルボキシ
基の活性化されたものと順次縮合反応させること
により行なわれ、斯くして一般式(ヘ)で表わされる
ペプチドに誘導することができる。これら縮合反
応及び保護基Aの脱離反応は、それぞれ前記した
方法と同様にして行なわれる。
また得られるペプチド(ヘ)は、同様にして保護基
Aの脱離、アミノ酸の側鎖官能基の保護基の脱離
及び不溶性担体R1の脱離により、式(1)で表わさ
れるペプチドに誘導される。ここで側鎖官能基の
保護基及び不溶性担体R1の脱離反応は、保護基
Aの脱離反応と同様に行ない得、この場合酸とし
て弗化水素又は臭化水素酸を用いるのが好まし
い。尚、上記方法において使用される各アミノ酸
は、いずれも公知の市販品でよい。
以上のようにして製造された式(1)の本発明ペプ
チドは、反応混合物からペプチドの分離手段例え
ば抽出、分配、カラムクロマトグラフイー等によ
り単離精製される。
かくして得られる本発明のペプチドは、これに
125I、131I等の放射性物質、パーオキシダーゼ
(POX)、キモトリプシノーゲン、プロカルボキ
シペプチダーゼ、グロセロルデヒド−3−リン酸
脱水素酵素、アラミーゼ、ホスホリラーゼ、D−
Nase、P−Nase、β−ガラクトシダーゼ、グル
コース−6−フオスフエートデハイドロゲナー
ゼ、オルニチンデカルボキシラーゼ等の各種酵素
試薬等を導入することにより、ラジオイムノアツ
セイ(RIA)法又はエンザイムイムノアツセイ
(EIA)法において用いられる標識抗原として利
用できる。上記放射性物質の導入は、通常の方法
により実施できる。例えば放射性ヨードは、クロ
ラミンTを用いる酸化的ヨード化法〔W.M.
Hunter and F.C.Greenwood;Nature,194、
495(1962)、Biochem J.89、144、(1963)参照〕
等により行なわれ、酵素試薬の導入は、通常のカ
ツプリング法例えばエルランガー(B.F.
Erlanger)らの方法〔Acta.Endocrinol.Suppl、
168、206(1972)〕及びカロール(M.H.Karol)
らの方法〔Proc.Natl.Acad.Sci.、U.S.A.、57、
713(1967)〕等の公知の方法によつて行なうこと
ができる。
以下、本発明のペプチドをハプテンとして利用
した抗原の製造方法につき詳述する。
上記抗原は本発明ペプチドをハプテンとし、こ
れをハプテン−担体結合試薬の存在下に、適当な
担体と反応させることにより製造される。上記に
おいてハプテンに結合される担体としては、通常
抗原の作成に当り慣用される高分子の天然もしく
は合成の蛋白質を広く使用できる。該担体として
は例えば馬血清アルブミン、牛血清アルブミン、
ウサギ血清アルブミン、人血清アルブミン、ヒツ
ジ血清アルブミン等の動物の血清アルブミン類;
馬血清グロブリン、牛血清グロブリン、ウサギ血
清グロブリン、人血清グロブリン、ヒツジ血清グ
ロブリン等の動物の血清グロブリン類;馬チログ
ロブリン、牛チログロブリン、ウサギチログロブ
リン、人チログロブリン、ヒツジチログロブリン
等の動物のチログロブリン類;馬ヘモグロブリ
ン、牛ヘモグロブリン、ウサギヘモグロブリン、
人ヘモグロブリン、ヒツジヘモグロブリン等の動
物のヘモグロブリン類;キーホールリンペツトヘ
モシアニン(KLH)等の動物のヘモシアニン
類;同虫より抽出された蛋白質(アスカーリス抽
出物、特絵昭56−16414号公報、J.Immun.、111、
260〜268(1973)、J.Immun.、122、302〜308
(1979)、J.Immun.、98、893〜900(1967)及び
Am.J.Physiol.、199、575〜578(1960)に記載さ
れたもの又はこれらを更に精製したもの);ポリ
リジン、ポリグルタミン酸、リジン−グルタミン
酸共重合体、リジン又はオルニチンを含む共重合
体等を挙げることができる。
ハプテン−担体結合試薬としては、通常抗原の
作成に当り慣用されているものを広く使用でき
る。具体的にはチロシン、ヒスチジン、トリプト
フアンを架橋結合させる、例えばビスジアゾタイ
ズドベンジジン(BDB)、ビスジアゾタイズド−
3,3′−ジアニシジン(BDD)等のジアゾニウ
ム化合物;アミノ基とアミノ基とを架橋結合させ
る、例えばグリオキサール、マロンジアルデヒ
ド、グルタールアルデヒド、スクシンアルデヒ
ド、アジポアルデヒド等の脂肪族ジアルデヒド
類;チオール基とチオール基とを架橋結合させ
る、例えばN,N′−o−フエニレンジマレイミ
ド、N,N′−m−フエニレンジマレイミド等の
ジマレイミド化合物;アミノ基とチオール基とを
架橋結合させる、例えばメタマレイミドベンゾイ
ル−N−ヒドロキシスクシンイミドエステル、4
−(マレイミドメチル)−シクロヘキサン−1−カ
ルボキシル−N′−ヒドロキシスクシンイミドエ
ステル等のマレイミドカルボキシル−N−ヒドロ
キシスクシンイミドエステル類;アミノ基とカル
ボキシル基とをアミド結合させる通常のペプチド
結合形成反応に用いられる試薬、例えばN,N−
ジシクロヘキシルカルボジイミド、N−エチル−
N′−ジメチルアミノカルボジイミド、1−エチ
ル−3−ジイソプロピルアミノカルボジイミド、
1−シクロヘキシル−3−(2−モリホリニル−
4−エチル)カルボジイミド等のカルボジイミド
類等の脱水縮合剤等を挙げることができる。また
上記ハプテン−担体結合試薬としては、p−ジア
ゾニウムフエニル酢酸等のジアゾニウムアリール
カルボン酸類と通常のペプチド結合形成反応試
薬、例えば上記脱水縮合剤とを組合せたものも使
用可能である。
上記抗原の製造反応は、例えば水溶液もしくは
PH7〜10の通常の緩衝液中、好ましくはPH8〜9
の緩衝液中、0〜40℃、好ましくは室温付近で行
なわれる。該反応は通常約1〜24時間、好ましく
は3〜5時間で完結する。上記において用いられ
る代表的緩衝液としては、次のものを例示でき
る。
0.2N水酸化ナトリウム−0.2Mホウ酸−0.2M塩
化カリウム緩衝液、
0.2M炭酸ナトリウム−0.2Mホウ酸−0.2M塩化
カリウム緩衝液、
0.05M四ホウ酸ナトリウム−0.2Mホウ酸−
0.05M塩化ナトリウム緩衝液、
0.1Mリン酸二水素カリウム−0.05M四ウ酸ナ
トリウム緩衝液
上記においてハプテン、ハプテン−担体結合試
薬及び担体の使用割合は、適宜に決定できるが、
通常ハプテンに対して担体を1〜6倍重量程度、
好ましくは1〜5倍重量程度、及びハプテン−担
体結合試薬を5〜10倍モル程度用いるのがよい。
上記反応によりハプテン−担体結合試薬を仲介さ
せて担体とハプテンとが結合したペプチド−担体
複合体からなる所望の抗原が収得される。
反応終了後得られる抗原は常法に従い、例えば
透析法、ゲル過法、分別沈澱法等により容易に
単離精製できる。
斯くして得られる抗原は、通常蛋白質1モルに
対してペプチドが平均5〜60モル結合したもので
あり、いずれも引き続き該抗原に対して特異性の
高い抗体の製造を可能とするものである。
該抗原による抗体の製造は、上記抗原を哺乳動
物に投与し、生体内に所望抗体を産生させ、これ
を採取することにより実施される。
抗体の製造に供せられる哺乳動物としては、特
に制限はないが、通常ウサギやモルモツトを用い
るのが好ましい。抗体の産生に当つては、上記に
より得られる抗原の所定量を生理食塩水で適当濃
度に希釈し、フロインドの補助液(Complete
Freund′s Ajuvant)と混合して懸濁液を調整し、
これを哺乳動物体に投与すればよい。例えばウサ
ギに上記懸濁液を圧内注射(抗原の量として0.5
〜5mg/回)し、以後2週間毎に2〜10ケ月、好
ましくは4〜6ケ月間投与し免疫化させればよ
い。抗体の採取は、上記懸濁液の最終投与の1〜
2週間経過後、免疫化された動物から採血し、こ
れを遠心分離後、血清を分離することにより行な
われる。上記によれば、用いる抗原に対して優れ
た特異性を有する抗体を収得でき、これはRIA
法、EIA法等に利用してヒト白血病ウイルス関連
蛋白の定量に用い得る。
以下本発明を更に詳しく説明するため、一般式
(1)で表わされる本発明ペプチドの製造例及びこれ
により得られるペプチドからの抗原及び抗体の製
造例を挙げるが、本発明はこれらに限定されるも
のではない。
尚、各製造例におけるRf値はシリカゲル上の
薄層クロマトグラフイーにて下記混合溶媒を用い
て測定したものである。
Rf1……n−ブタノール−酢酸−水
(4:1:5)
Rf2……n−ブタノール−酢酸−ピリジン−水
(15:3:10:12)
<ペプチドの製造>
製造例
カリウム tert−ブトキシド5.88ミリ当量の
DMSO溶液14mlにBoc−Leu−OH1.54gを溶
解し、クロロメチル化ポリスチレン樹脂(財団
法人蛋白質研究奨励会)5gを加えて、80℃で
30分反応させる。樹脂をDMSO、50%酢酸/
クロロホルム、塩化メチレンの順に、充分に洗
浄し、減圧乾燥して5.06gのBoc−Leu−樹脂
を得る。
一部を加水分解後アミノ酸分析を行なつた結
果アミノ酸0.30mmol/g樹脂であつた。
上記で得たBoc−Leu−樹脂2.17gをクロ
ロホルム30mlで3回洗浄後、50%トリフルオロ
酢酸(TFA)のクロロホルム溶液30mlに加え、
室温で20分間反応させる。樹脂をクロロホルム
30mlで1回、塩化メチレン30mlで5回、10%ト
リエチルアミンの塩化メチレン溶液30mlで3
回、次いで塩化メチレン30mlで6回それぞれ洗
浄してH−Leu−樹脂を得る。
Boc−Val−OHの0.35gを塩化メチレンに溶
かした溶液25mlに上記H−Leu−樹脂を加え、
次いでDCCの0.33gを塩化メチレンに溶かした
溶液5mlを加え室温で2時間反応させる。樹脂
を塩化メチレン30mlで6回洗浄後、Boc−Val
−OHの0.35g及び1−ヒドロキシベンゾトリ
アゾール0.55gの塩化メチレン溶液25mlに加
え、次いでDCCの0.33gを塩化メチレンに溶か
した溶液5mlを加えて再度同様に反応させる
(二重カツプリング法)。樹脂を塩化メチレンで
充分に洗浄してBoc−Val−Leu−樹脂を得る。
上記と同様にして、Boc−Val−Leu樹脂
の脱Boc化を行ない、次いで下記アミノ酸を順
次縮合及び脱Boc反応に付す。
Boc−Gln−ONP 0.59g
Boc−Pro−OH 0.35g
Boc−Ala−OH 0.31g
Boc−Thr(Bzl)−OH 0.50g
Boc−Pro−OH 0.35g
Boc−Glu(OBzl)−OH 0.55g
Boc−Val−OH 0.35g
Boc−Tyr(Cl2−Bzl)−OH 0.71g
斯くしてH−Tyr(Cl2−Bzl)−Val−Glu
(OBzl)−Pro−Thr(Bzl)−Ala−Pro−Gln−
Val−Leu−樹脂の2.65gを得る。このうち1.35
gをアニソール3ml及び弗化水素30mlに溶か
し、−20℃で30分、次いで0℃で30分反応させ
た後、弗化水素を留去し、残渣を10%酢酸にて
抽出し、エーテルにて洗浄する。水層を凍結乾
燥し、次いでセフアデツクスG−10(フアルマ
シア社、溶出液10%酢酸)によるゲル過、次
いでセフアデツクスG−25(フアルマシア社、
溶出液BuOH:AcOH:H2O=4:1:5)に
よるパーテイシヨンクロマトグラフイ、更に
LH−20(フアルマシア社、溶出液、1/1000N
−HCI)にて精製して、H−Tyr−Val−Glu
−Pro−Thr−Ala−Pro−Gln−Val−Leu−
OHを得る。以下このペプチドを「ペプチド
A」と呼ぶ。
Rf値:
Rf1:0.12 Rf2=0.58
元素分析値:
(C52H81N11O16・7H2Oとして)
C(%) H(%) N(%)
理論値 50.27 7.71 12.40
分析値 50.41 7.83 12.41
<抗原の製造>
製造例
ペプチドの製造例1で得たペプチドAの5mg及
びKLH(シグマ社)12mgを0.05Mリン酸塩緩衝液
(PH=7.0)3.0mlに加え、この溶液に2%グルタ
ルアルデヒド溶液0.2mlを滴下し、室温で3時間
撹拌する。その後反応混合物を一夜蒸溜水で4℃
で透析し、凍結乾燥して、目的抗原16.5mgを得
る。以下この抗原を「抗原I」と言う。
抗原IはKLH1モル(平均分子量を10万とした
場合)に対してペプチドAが平均10モル結合した
ものである。尚このペプチドAとKLHとの結合
率は、得られる抗原Iを更にセフアデツクスG−
50(溶出液:生理食塩水、検出:OD280nm、流出
速度:3ml/時間、分取量:1mlづつ)でゲル
過した際、未反応のKLH及びペプチドAの存在
は認められないことより、該ゲル過によつて
KLHに結合したペプチドAのフラクシヨンと他
の生成体(ペプチドAの2量体)のフラクシヨン
とを分離し、ペプチド2量体の標準濃度の検量線
を作成して、上記2量体の量を求め、これを出発
原料として用いたペプチドAの量から差し引いた
値がすべてKLHに結合しているとして求めたも
のである。
<抗体の製造>
製造例
抗原の製造例で得た抗原のそれぞれ100μg
を1.5mlの生理食塩水に溶解後、これにフロイン
ドの補助液1.5mlを加えて調整した懸濁液を、そ
れぞれ3羽のウサギ(New−Zealand white
rabbits)(2.5〜3.0Kg)に皮下投与し、2週間毎
に6回同量を投与する。更にその後1ケ月毎に3
回、最初に投与した量と同量を投与する。最終投
与後7日経過してのち試験動物から採血し、遠心
分離して抗血清を採取して、目的抗体を得る。
A-Leu-OH (A) ↓ A-Leu-R 1 (B) ↓ H-Leu-R 1 (C) ↓A-Val-OH (D) A-Val-Leu-R 1 (E) ↓↓ ↓ A−Tyr−Val−Glu−Pro−Thr−Ala −Pro−Gln−Val−Leu−R 1 (f) ↓ H−Tyr−Val−Gll−Pro−Thr−Ala Pro−Gln−Val−Leu− OH (1) [In the formula, A represents a protecting group for an amino group and R 1 represents an insoluble carrier. ] In the above, preferable A is
Boc, benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, etc., and preferred examples of R 1 include chloromethylated polystyrene and the like. In addition, in each reaction, if the amino acid used has a side chain functional group that does not participate in the reaction, it is protected by the above-mentioned protecting group as usual, and this is removed at the same time as the insoluble carrier R 1 is removed. It will be done. In the above method, the amino acid (a) and the insoluble carrier
The reaction with R 1 is carried out by utilizing the carboxyl group of the reactant of amino acid (a) and bonding it to R 1 according to a conventional method. For example, when using chloromethylated polystyrene, the reaction is carried out in a suitable solvent, such as triethylamine, potassium tert.
- carried out in the presence of basic compounds such as butoxide, cesium carbonate, cesium hydroxide, etc. Examples of the solvent include dimethylformamide, dimethylsulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, N-
Examples include methylpyrrolidone, hexamethylphosphoric acid triamide, and a mixed solvent thereof. The above reaction is usually carried out at 0 to 85°C, preferably at 25°C.
The process will be completed in a few minutes to 24 hours at a temperature of ~80℃. The ratio of the amino acid and the insoluble carrier to be used is usually such that the former is used in an excess amount, generally 1 to 3 times equivalent, to 1 equivalent of the latter. The elimination reaction of the protecting group A of the immobilized amino acid of the general formula (b) thus obtained is carried out by a conventional method.
Examples of such methods include hydrogenation using a catalyst such as palladium or palladium black, reductive methods such as reduction with metallic sodium in liquid ammonia, trifluoroacetic acid, hydrochloric acid, hydrogen fluoride, methanesulfonic acid, and hydrobromic acid. Examples include acidolysis using strong acids such as Hydrogenation using the above catalyst can be carried out, for example, at a hydrogen pressure of 1 atmosphere and at a temperature of 0 to 40°C. The amount of catalyst used is usually 100 mg to 1
The reaction is generally completed in about 1 to 48 hours. The above acidolysis is carried out in the absence of a solvent, usually at about 0 to 30°C, preferably at about 0 to 20°C, for about 15 to 1 hour. The amount of acid used is usually about 5 to 10 times the amount of the raw material compound. In the acidolysis, protecting group A
In the case of removing only trifluoroacetic acid or hydrochloric acid, it is preferable to use trifluoroacetic acid or hydrochloric acid as the acid. Further, the above reduction with metallic sodium in liquid ammonia can be carried out using an amount of metallic sodium such that the reaction solution is colored permanent blue for about 30 seconds to 10 minutes, usually at about -40°C to -70°C. . The reaction between the resulting immobilized amino acid of general formula (c) and amino acid (d) (or its carboxyl group activated) is carried out in the presence of a solvent. Examples of the solvent include various known solvents commonly used in peptide condensation reactions, such as anhydrous dimethylformamide, dimethyl sulfoxide (DMSO),
Pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, ethyl acetate, N-
Examples include methylpyrrolidone, hexamethylphosphoric acid triamide, and a mixed solvent thereof. In addition, the reaction may be carried out using reagents used in ordinary peptide bond forming reactions, such as N, N,
N-dicyclohexylcarbodiimide (DCC),
In the presence of a dehydration condensing agent such as carbodiimides such as N-ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide, and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide. can be done. The ratio of the amino acid (c) and the amino acid (d) to be used is not particularly limited, but the latter is usually used in an equimolar amount to 10 times the molar amount of the former, preferably an equimolar amount to 5 times the molar amount of the latter. It is better. There is no particular limitation on the amount of the dehydration condensing agent used, and it is usually used in an equimolar amount relative to the amino acid (d). The reaction temperature is appropriately selected from the usual range used for peptide bond-forming reactions, generally from about -40°C to about 60°C, preferably from about -20°C to about 40°C. The reaction time is generally about several minutes to 30 hours. The thus obtained peptide of the general formula (E), after removing the protecting group A in the same manner as above, is converted into A-Gln-OH, A-Pro, according to the amino acid sequence represented by the general formula (1).
-OH, A-Ala-OH, A-Thr-OH, A-
Pro-OH, A-Glu-OH, A-Val-OH, A-
This is carried out by sequential condensation reaction with each amino acid of Tyr-OH or an activated version of its carboxyl group, and thus a peptide represented by the general formula (f) can be derived. The condensation reaction and the elimination reaction of the protecting group A are carried out in the same manner as described above. In addition, the obtained peptide (F) can be converted into a peptide represented by formula (1) by removing the protecting group A, removing the protecting group of the side chain functional group of the amino acid, and removing the insoluble carrier R 1 in the same manner. be guided. Here, the elimination reaction of the protective group of the side chain functional group and the insoluble carrier R 1 can be carried out in the same manner as the elimination reaction of the protective group A, and in this case, it is preferable to use hydrogen fluoride or hydrobromic acid as the acid. . Note that each amino acid used in the above method may be a known commercially available product. The peptide of the present invention of formula (1) produced as described above is isolated and purified from the reaction mixture by peptide separation means such as extraction, partitioning, column chromatography, etc. The thus obtained peptide of the present invention is
Radioactive substances such as 125 I and 131 I, peroxidase (POX), chymotrypsinogen, procarboxypeptidase, glyceroldehyde-3-phosphate dehydrogenase, aramase, phosphorylase, D-
By introducing various enzyme reagents such as Nase, P-Nase, β-galactosidase, glucose-6-phosphate dehydrogenase, ornithine decarboxylase, radioimmunoassay (RIA) method or enzyme immunoassay can be performed. It can be used as a labeled antigen used in the (EIA) method. Introduction of the above-mentioned radioactive substance can be carried out by a conventional method. For example, radioactive iodine can be prepared by oxidative iodination using chloramine T [WM
Hunter and F.C. Greenwood; Nature, 194 ;
495 (1962), Biochem J. 89 , 144, (1963)]
The introduction of the enzyme reagent is carried out using a conventional coupling method such as Erlanger (BF
Erlanger et al.'s method [Acta.Endocrinol.Suppl,
168, 206 (1972)] and MHKarol
[Proc. Natl. Acad. Sci., USA, 57 ,
713 (1967)]. Hereinafter, a method for producing an antigen using the peptide of the present invention as a hapten will be described in detail. The above antigen is produced by using the peptide of the present invention as a hapten and reacting it with a suitable carrier in the presence of a hapten-carrier binding reagent. As the carrier bound to the hapten in the above, a wide range of high-molecular natural or synthetic proteins commonly used in the preparation of antigens can be used. Examples of the carrier include horse serum albumin, bovine serum albumin,
Animal serum albumins such as rabbit serum albumin, human serum albumin, and sheep serum albumin;
Animal serum globulins such as horse serum globulin, bovine serum globulin, rabbit serum globulin, human serum globulin, and sheep serum globulin; Thyroglobulins; horse hemoglobulin, bovine hemoglobulin, rabbit hemoglobulin,
Animal hemoglobulins such as human hemoglobulin and sheep hemoglobulin; Animal hemocyanins such as keyhole limpet hemocyanin (KLH); Proteins extracted from the same insect (Ascaris extract, special edition No. 16414/1986) , J.Immun., 111 ,
260–268 (1973), J.Immun., 122 , 302–308
(1979), J.Immun., 98 , 893-900 (1967) and
Am.J.Physiol., 199 , 575-578 (1960) or further purified versions of these); polylysine, polyglutamic acid, lysine-glutamic acid copolymers, copolymers containing lysine or ornithine, etc. can be mentioned. As the hapten-carrier binding reagent, a wide variety of those commonly used for preparing antigens can be used. Specifically, tyrosine, histidine, and tryptophan are cross-linked, such as bisdiazotized benzidine (BDB) and bisdiazotized benzidine (BDB).
Diazonium compounds such as 3,3'-dianisidine (BDD); aliphatic dialdehydes such as glyoxal, malondialdehyde, glutaraldehyde, succinaldehyde, and adipaldehyde that crosslink amino groups; ;Crosslinking between thiol groups, such as dimaleimide compounds such as N,N'-o-phenylene dimaleimide and N,N'-m-phenylene dimaleimide;Crosslinking between amino groups and thiol groups for example metamaleimidobenzoyl-N-hydroxysuccinimide ester, 4
Maleimidocarboxyl-N-hydroxysuccinimide esters such as -(maleimidomethyl)-cyclohexane-1-carboxyl-N'-hydroxysuccinimide ester; reagent used in the usual peptide bond-forming reaction to form an amide bond between an amino group and a carboxyl group. , for example N, N-
Dicyclohexylcarbodiimide, N-ethyl-
N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide,
1-Cyclohexyl-3-(2-morpholinyl-
Examples include dehydration condensation agents such as carbodiimides such as 4-ethyl)carbodiimide. As the hapten-carrier binding reagent, a combination of a diazonium arylcarboxylic acid such as p-diazonium phenylacetic acid and a conventional peptide bond forming reaction reagent, such as the dehydration condensation agent described above, can also be used. The above antigen production reaction can be carried out, for example, in an aqueous solution or
In a normal buffer solution of PH 7-10, preferably PH 8-9
The reaction is carried out in a buffer solution at 0 to 40°C, preferably around room temperature. The reaction is usually completed in about 1 to 24 hours, preferably 3 to 5 hours. Typical buffer solutions used in the above may include the following. 0.2N sodium hydroxide - 0.2M boric acid - 0.2M potassium chloride buffer, 0.2M sodium carbonate - 0.2M boric acid - 0.2M potassium chloride buffer, 0.05M sodium tetraborate - 0.2M boric acid -
0.05M sodium chloride buffer, 0.1M potassium dihydrogen phosphate-0.05M sodium tetraurate buffer In the above, the proportions of the hapten, the hapten-carrier binding reagent, and the carrier can be determined as appropriate;
Usually, the carrier is about 1 to 6 times the weight of the hapten.
It is preferable to use about 1 to 5 times the weight and about 5 to 10 times the mole of the hapten-carrier binding reagent.
Through the above reaction, a desired antigen consisting of a peptide-carrier complex in which a carrier and a hapten are bound is obtained through the mediation of a hapten-carrier binding reagent. The antigen obtained after completion of the reaction can be easily isolated and purified by conventional methods such as dialysis, gel filtration, and fractional precipitation. The antigen thus obtained usually has an average of 5 to 60 moles of peptide bound to 1 mole of protein, and any of these makes it possible to subsequently produce antibodies with high specificity for the antigen. . Production of antibodies using the antigen is carried out by administering the antigen to a mammal, causing the desired antibody to be produced in vivo, and collecting the antibody. There are no particular restrictions on the mammal used for antibody production, but rabbits and guinea pigs are usually preferably used. For antibody production, dilute the prescribed amount of the antigen obtained above with physiological saline to an appropriate concentration, and add Freund's auxiliary solution (Complete
Freund's Ajuvant) to prepare a suspension;
This may be administered to a mammal. For example, inject the above suspension into rabbits (0.5% as the amount of antigen).
~5 mg/dose) and then administered every two weeks for 2 to 10 months, preferably 4 to 6 months, for immunization. Antibodies were collected from 1 to 1 of the final administration of the above suspension.
After two weeks, blood is collected from the immunized animal, centrifuged, and serum is separated. According to the above, an antibody with excellent specificity for the antigen used can be obtained, and this is RIA
It can be used for the quantification of human leukemia virus-related proteins by using methods such as EIA method and EIA method. Below, in order to explain the present invention in more detail, general formula
Examples of production of the peptide of the present invention represented by (1) and production of antigens and antibodies from the peptides obtained thereby will be given, but the present invention is not limited thereto. The Rf value in each production example was measured by thin layer chromatography on silica gel using the following mixed solvent. Rf 1 ...n-butanol-acetic acid-water
(4:1:5) Rf 2 ... n-butanol-acetic acid-pyridine-water
(15:3:10:12) <Production of peptide> Production example Potassium tert-butoxide 5.88 milliequivalents
Dissolve 1.54 g of Boc-Leu-OH in 14 ml of DMSO solution, add 5 g of chloromethylated polystyrene resin (Protein Research Foundation), and heat at 80℃.
Incubate for 30 minutes. Resin in DMSO, 50% acetic acid/
Wash thoroughly with chloroform and methylene chloride in this order, and dry under reduced pressure to obtain 5.06 g of Boc-Leu resin. After hydrolyzing a portion, amino acid analysis was performed and the result was 0.30 mmol of amino acids/g resin. After washing 2.17 g of Boc-Leu resin obtained above three times with 30 ml of chloroform, it was added to 30 ml of a chloroform solution of 50% trifluoroacetic acid (TFA).
Incubate for 20 minutes at room temperature. Chloroform the resin
Once with 30 ml, 5 times with 30 ml of methylene chloride, and 3 times with 30 ml of 10% triethylamine in methylene chloride.
The H-Leu-resin is obtained by washing twice and then six times with 30 ml of methylene chloride each time. Add the above H-Leu-resin to 25 ml of a solution of 0.35 g of Boc-Val-OH dissolved in methylene chloride,
Next, 5 ml of a solution of 0.33 g of DCC dissolved in methylene chloride was added and reacted at room temperature for 2 hours. After washing the resin 6 times with 30 ml of methylene chloride, Boc-Val
0.35 g of -OH and 0.55 g of 1-hydroxybenzotriazole are added to 25 ml of a methylene chloride solution, and then 5 ml of a solution of 0.33 g of DCC in methylene chloride is added and the same reaction is carried out again (double coupling method). The resin is thoroughly washed with methylene chloride to obtain Boc-Val-Leu-resin. In the same manner as above, Boc-Val-Leu resin is de-Boc-reacted, and then the following amino acids are sequentially subjected to condensation and de-Boc reaction. Boc-Gln-ONP 0.59g Boc-Pro-OH 0.35g Boc-Ala-OH 0.31g Boc-Thr(Bzl)-OH 0.50g Boc-Pro-OH 0.35g Boc-Glu(OBzl)-OH 0.55g Boc- Val−OH 0.35g Boc−Tyr(Cl 2 −Bzl)−OH 0.71g Thus H−Tyr(Cl 2 −Bzl)−Val−Glu
(OBzl)−Pro−Thr(Bzl)−Ala−Pro−Gln−
2.65 g of Val-Leu-resin are obtained. 1.35 of these
g was dissolved in 3 ml of anisole and 30 ml of hydrogen fluoride and reacted at -20°C for 30 minutes and then at 0°C for 30 minutes. Hydrogen fluoride was distilled off, and the residue was extracted with 10% acetic acid and diluted with ether. Wash with water. The aqueous layer was freeze-dried, followed by gel filtration with Cephadex G-10 (Pharmacia, eluent 10% acetic acid), and then Cephadex G-25 (Pharmacia, 10% acetic acid).
Partition chromatography with eluent BuOH:AcOH: H2O =4:1:5), and
LH-20 (Pharmacia, eluent, 1/1000N
H-Tyr-Val-Glu
−Pro−Thr−Ala−Pro−Gln−Val−Leu−
Get OH. This peptide is hereinafter referred to as "peptide A." Rf value: Rf 1 : 0.12 Rf 2 = 0.58 Elemental analysis value: (as C 52 H 81 N 11 O 16・7H 2 O) C (%) H (%) N (%) Theoretical value 50.27 7.71 12.40 Analysis value 50.41 7.83 12.41 <Production of antigen> Production example Add 5 mg of Peptide A obtained in Peptide Production Example 1 and 12 mg of KLH (Sigma) to 3.0 ml of 0.05 M phosphate buffer (PH = 7.0), and add 2 mg to this solution. % glutaraldehyde solution was added dropwise and stirred at room temperature for 3 hours. The reaction mixture was then soaked in distilled water overnight at 4°C.
and lyophilize to obtain 16.5 mg of the target antigen. This antigen will hereinafter be referred to as "antigen I." Antigen I has an average of 10 moles of peptide A bound to 1 mole of KLH (assuming the average molecular weight is 100,000). In addition, the binding rate of this peptide A and KLH is determined by adding the obtained antigen I to Cephadex G-
50 (eluate: physiological saline, detection: OD280nm, flow rate: 3ml/hour, fractional volume: 1ml each), the presence of unreacted KLH and peptide A was not observed. By gel filtration
The fraction of peptide A bound to KLH and the fraction of other products (dimer of peptide A) are separated, and a calibration curve of the standard concentration of peptide dimer is created to calculate the amount of the dimer. This value was calculated by subtracting this value from the amount of peptide A used as a starting material, assuming that all of the value was bound to KLH. <Production of antibodies> Production example 100 μg of each antigen obtained in the antigen production example
was dissolved in 1.5 ml of physiological saline, and 1.5 ml of Freund's auxiliary solution was added to the suspension.
The same amount is administered subcutaneously to rabbits (2.5-3.0 kg), and the same amount is administered 6 times every 2 weeks. Furthermore, every month thereafter, 3
Administer the same amount as the first dose. Seven days after the final administration, blood is collected from the test animal and centrifuged to collect antiserum to obtain the target antibody.
Claims (1)
−Leu−OH で表わされるペプチドからなるヒト白血病ウイル
ス関連ペプチド。[Claims] 1 Formula H-Tyr-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val
A human leukemia virus-related peptide consisting of a peptide represented by -Leu-OH.
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP58001495A JPS59128366A (en) | 1983-01-07 | 1983-01-07 | Peptide relating to human leukemia virus |
CA000437427A CA1262014A (en) | 1983-01-07 | 1983-09-23 | Human leukemia virus-related peptides, antibodies of the peptides and a process for production of the antibodies |
DE8383109481T DE3380564D1 (en) | 1982-09-30 | 1983-09-23 | Human leukemia virus-related peptides, antibodies of the peptides and a process for production of the antibodies |
US06/535,115 US4525300A (en) | 1983-01-07 | 1983-09-23 | Human leukemia virus-related peptides, antibodies of the peptides and a process for production of the antibodies |
EP83109481A EP0107053B1 (en) | 1982-09-30 | 1983-09-23 | Human leukemia virus-related peptides, antibodies of the peptides and a process for production of the antibodies |
US06/713,659 US4804746A (en) | 1983-01-07 | 1985-03-19 | Antibodies to human leukemia virus-related peptides and a process for production of the antibodies |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP58001495A JPS59128366A (en) | 1983-01-07 | 1983-01-07 | Peptide relating to human leukemia virus |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS59128366A JPS59128366A (en) | 1984-07-24 |
JPH0350759B2 true JPH0350759B2 (en) | 1991-08-02 |
Family
ID=11503033
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP58001495A Granted JPS59128366A (en) | 1982-09-30 | 1983-01-07 | Peptide relating to human leukemia virus |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS59128366A (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SE8900721D0 (en) | 1989-03-02 | 1989-03-02 | Blomberg Jonas | METHODS FOR DETECTION OF ANTIBODIES TO |
-
1983
- 1983-01-07 JP JP58001495A patent/JPS59128366A/en active Granted
Also Published As
Publication number | Publication date |
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JPS59128366A (en) | 1984-07-24 |
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