JPS59155347A - Peptide relating to human leukemia virus - Google Patents

Abstract

NEW MATERIAL:The peptide of formula I -III(R is H or Tyr). USE:Diagnosis of the infection with human leukemia virus, and diagnosis and research of mature T-cell leukemia, lymphoma, and relating diseases such as adult T-cell leukemia, cutaneous T-cell lymphoma, etc. An antigen can be produced by using each peptide as a hapten, and an antibody having specific reactivity with virus-relating protein can be obtained from the antigen. The specific antibody is bonded e.g. with a carrier for affinity chromatography, and used as a specific antibody for the purification of virus-relating protein or the immunoassay of virus-relating protein. PREPARATION:The objective peptide can be prepared either by condensing amino acids one by one to the terminal amino acid by conventional peptide synthesis (e.g. azide process, chloride process, DCC process, etc.) or by coupling several number of fragments constituting the peptide. If necessary, the side chain functional groups are protected before the reaction, and removed from the obtained reaction product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human leukemia virus (Δdult T-
cell leukemia V 1rus:
ATLV or l-1umanT-cell l
This is a novel peptide related to eukemia Virus (HTLV), and it is a peptide related to such viral infections as well as mature T cell leukemia/lymphoma such as adult hypocellular leukemia and cutaneous T111 cell lymphoma.

In Suimei M, when amino acids, peptides, protective groups, active groups, nucleobases, etc. are indicated by abbreviations, I
The UPACLILIB regulations or common symbols in the field shall be followed, examples of which 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 A sn; asparagine Gln; glutamine GII and glutamic acid Lys: lysine
pro; proline Va1; valine Trp nitributophane 11is; histidine AS+): aspartate Gly nigericin Ile; isoleucine Al minialanine Tyr; tyrosine Met; methionine Phe: phenylalanine Δrg; arginine Cys: cystine Δ: adenine D; thymine G nigeanine C; C]・synTos: p-toluenesulfonyl group B oc: tertiary monotoxycarbonyl group ONP: p-
21-lophenoxy group Bzl; benzyl group 0Bzl; benzyloxy group Clp~Bzl; 2,6-dichlorobenzyl group CI-Z
:2-chlorobenzyloxycarbonyl human leukemia virus is a virus isolated from adult T-cell leukemia (ATL), and its relationship with this disease has attracted attention. The inventors, Yoshihichi and Kanno, used genetic engineering techniques to clone the provirus gene integrated into the DNA of host cells, and determined its entire base sequence. Based on this, we established the basis for the diagnosis, treatment, and prevention of the disease and viral infection, and filed a patent application (Japanese Patent Application No. 1987-214287).

The present invention was completed based on the basic information in Section F, and relates to virus-related peptides for the purpose of diagnosing infection with the virus, as well as methods for producing and measuring In antibodies against them. The determined nucleotide sequence encoding the core (Gag) protein precursor of the above virus gene is shown in Table 1 below.

Table 1 1-cu Gln Ala Ala Tyr
Aro l-, cu Glu'Pr.

Leu Lys 1-ys Phe I-cu
t-ys Tle Ala l, euGA
A ACA CCG GCT CGG AT'CTGT
'CCCATI-Glu T'llr Pro
Ala Ara lie Cys Pro
flaΔ△C to C1”CCCTCG'T'A G
CCACCCTA CT'CAsn Tyr 3
er 1. ,eu I-cu Ala ser
Ieu teuCCA ΔAA GGA TA
CCCCGGCCGG GTG AATPro 1
-ys GIV Tyr PrOGly A
r(] Vat AsnGAA ATI-T
T'A CACATA CTCATCCAA A
CCGlu Tle Ieu His Ile
leu lle Gln Tl1rCAA
GCCCAG'ATCC'; CG TCCCGT C
CCGCGGln Ala Gln lle
Pro 3er ΔrgPro Δ1aCCA
CCG CCG CCG T'CA TCCCCCAC
CCAC Pro Pro Pro Pro S
er Ser Pro Thr fitsGA
G CCCCCG GAT-TCT GAI-CCA
CAA Al-CΔSp pro pro As
p Ser ΔSrl P"OG111 71e
CCCCCI' CCCTAT GTT' CAG G
CT' ACG GCCPI'OPro PrO'i
yr Val GILI pro Tl1l'
Δ1accc cΔA GTCCT'T C
CA GTCAT'G CAT' CCAPro
Gin Val I-etl Pro V
at Met His Pr.

CΔl' GG 1' GCT CC1' C
C1AACCAT CGCCCAf-1is GIV
Ala Pro Pro Asn His
Ara Pr.

TGG CAA ATCΔΔΔ GACCTA C
ACGCO△TTT+'p Gln ~vet
1. VS ASD' I O(I Gln
Ala rle△ΔGC△△ GΔΔ GTCT
CC(', ΔΔ G(CΔ GCCCCTlys G
ln Glu Val Set' G11
lAlaΔIaPr.

GGG ACCCOCCAG T-T"'I △
'T-G CACACCATCGIV Ser
Pro Gln PIIG Met
Gin T cityr l1eCGG CTl
-GCG GTG CΔGCΔG 4T'l-G
ACCCCΔrg l eu Ala V
at Gln Gln Phe As
p Pr.

△CT GCCΔ△Δ CACCTCCAA GAC
CTCCT-0 1+r Ala 1-ys
ASD 1-eu Gln Asp
1-eu L-euCΔG 1ΔCCTl-T
GCTCCTCCCTCGTG
Tyr 1. , ell Cys Ser
Ser leu Vat Δ1aTC
CCTCCAT CACCAG CAG CT-
Δ GAT AGC8er 1-eu HI
s His Gln Gln 1-c
u ASD 3erCTT ATA TC
Δ GAG GCCGΔΔ ACCCGA GGT
l-eu Ile Ser Glu
Ala GILI Dinghr Aro
GlyATT ACA GGT TAT A
ACCCA T'T-A GCCGGTlie
Thr Gly Tyr Asn
pro leu Ala GIVCCCC
TCCGT GTCCAA GCC△Δ0 △AT
CCAPro Leu ArG Va
l Gln Ala Asn Asn
pr.

C△△ O△△ 0△△ GGΔ TT−Δ AGCO
('iΔ 0ΔΔ ]ΔCG11l G11l G
in Gly i, eLI Δrg △r
(l Glu Tyr(CACO△8 CT(C
l'GG CT-CGCCGCCT'l'CGCCG
ln Gln +,,eu Trp I-eu
Ala Ala Phe Δ1aGCCCT
G CCG GGG AGT GCC△△Δ
CACCCT△fil l elf P rO
G ly Ser Ala Ivs
ΔSpP “0TCC-T-GG GCCI C1△DingCCTCCΔΔ GGCCTG3er -rrll
△1aSnl' rle l, elf
Gln Gl! / 1-euGΔG GAG
CC'T lΔ(CCACGCC'; TTCG
l△ GΔΔGlu Glu Pro T
VrHis △IaPleOVal GluC
GCCT'C△△C△1'Δ GCT CTT C
ACΔΔT' GGGΔrg leu As
n Ile Ala 1-eu As
n Asn Gly(〕T'G CCA GΔ
△ GGCΔCG CCCAΔ△ CACCCC1-
eu Pro Qlu GIV Th
r Pro Lys ASD pr.

ΔTCCTTA CGT' TCCTTA GCCT
ΔCTCCCCΔATr Ie l,,eu
Ara Ser l,,eu Ala
'T'yr Ser AsnGCΔ △Δ
CΔΔΔ GAΔ TGCCΔΔ ΔΔΔ TTA C
TAAla, ASn 1. ,ys Gl
u CVS Gln Lys l-,
eu jeuCΔG GCCCCA GGΔC
ACACT ΔΔT AGCCCTGln A
la Aro Gly His Th
rAsn, ScrPr.

Tbr Trp Thr Pro Lys
ASD LVS T'hr 1ysGT
'G T-TA Gl-T GTCCAG CCI
△ΔΔΔΔΔ CCCVal l, eu Vat
Val Gln pro 1-ys I-y
s Pr.

CCCCCAΔΔT CAG CCG TGC1'-1
-CCGG TGCpro pro ASn G
ln Pro C; VS Phe Ara
CysGGG △△ΔGCΔGGCCACTGGΔG'
T' CGG GΔCGly lys Ala
Qly His Trp Ser Δrg
ASI) TGCACT CAG CC'r CG'T-
CCCCCCCCCCGGGCys Thr Gln
Pro Ara Pro Pro Pro Pro
GIV)-Its Trp I ys Δ
“g Asn cvs Pro Δr(]
l, ellΔΔG CCCACT Δ1℃ C
CA GΔ△ CCA CAG CCAlys P
ro Tl1r Ila Pro Glu
Pro Glu pr.

GAG G△Δ G△1 GCCCT to CTA
TT8 CACCICGlu GILI Asn
Ala I-eu 1. -et+ l, eu
Asn 1euCCCGCT GACAAl'C
CCA CACCCA ΔΔΔ ΔΔCPro
Ala Asp Ile Pro His
Pro Iys ΔsnT'CCATΔ GGG
GGG CAG GTTSer rle
Gly GIV GILI Vat From Table 1 above, it is shown that the core protein precursor consists of 429 amino acids, and the previously reported ρ-24
The terminal structure (P roc, N atl, △cad.

Sci, U, S, A, Vol, 79, flD
l29i~1294 (1982)], the precursor is further cleaved to form p-14, p-24.
It was predicted that this protein would be the core protein of p-10.

Based on the above-mentioned viewpoints, the present inventors have discovered a specific bebudid that can act as a hubten for the human leukemia virus-related protein (core protein), and have hereby completed the present invention.

That is, the present invention provides a compound represented by the formula (1), the general formula RMej-GIV-GllllIlepHe-
3er −△ rg −S er −Δ Ia −3
er-Pro-0to1 (2) [In the formula, R represents a hydrogen atom or an I-1-Tyr group. ] and a human leukemia virus-related bebudide selected from the group consisting of a bebudide represented by the formula % and a bebudide represented by the formula % ((3).

The peptides represented by the above general formulas (1) to (3) of the present invention can be easily synthesized by simple operations using easily available commercially available amino F11i. These can be used as hubtains to prepare antigens, and antibodies that have specific reactivity to virus-related proteins can be obtained from the antigens thus obtained. The specific antibody can be used for purifying virus-related proteins by, for example, binding it to a carrier for affinity chromatography and using it in the chromatography.
In addition, it can be used as a specific antibody in various immunoassays for the virus-related protein, and can be used for the diagnosis of human 1 to Shiromen virus infection, as well as (J, mature T cell lymphoma, adult T cell lymphoma, etc.). cellular leukemia, lymphocytes and phyla)
It is useful for the diagnosis and research of widespread diseases. .

The peptides represented by the general formulas (1) to (3) of the present invention can be synthesized using conventional peptide synthesis methods [The Peptides, Vol. 1 (1)].
966) (3chroner and lubk
e￥, Δacademic prcss, N cw
Y ork, L) SΔ] or “Peptide Synthesis 1 [
Azumaya et al., Maruzen Co., Ltd. (1975)], for example, the azide method, chloride method, acid anhydride method, warm stone anhydride method, DCC method, active ester method (p-21 - lophenyl ester method, N-hydroxysuccinimide J Stell method, cyazmedel J Stell method, etc.)
, Woodward test) method using MK, carbodiimidazole method, redox method, DCC/additive (HON
B, HORt, HO8u) method, etc.

For the F-opening method J3, both solid phase synthesis method and liquid phase synthesis method can be applied. Usually, the peptide of the present invention is prepared by following the general polypeptide synthesis method described above, for example, by the so-called stepwise method in which amino acids are sequentially condensed one by one to the terminal amino acid, or by coupling it into several fragments. It is manufactured by several methods. More specifically, for example, when solid phase synthesis is employed, the C-terminal amino acid is bound to an insoluble carrier via its carboxyl group. The insoluble carrier is not particularly limited as long as it has a bonding property with a reactive carboxyl group, such as chloromethyl resin, bromomethyl resin, hydroxymethyl resin, phenol resin, tert.
-Alkyloxycarbonyl hydrazide resins and the like can be used.

Then, after removing the amino protecting group, general formulas (1) to (3
), amino group-protected amino acids are sequentially bonded by condensation reaction with their reactive amino groups and reactive carboxyl groups, and the synthesis is performed step by step. After synthesizing the entire sequence, the peptide is transferred from an insoluble carrier. Manufactured by removing.

J3 C arginine, tyrosine, glutamic acid,
The side chain functional groups of the amino acids threonine, lysine, aspartic acid and serine are preferably protected, and this is protected by a conventional protecting group, and the mature protecting group is removed after the reaction is completed. , and the functional groups involved in the reaction are usually activated.

Each of these reaction methods is publicly known, and the sample mulberry used therein is also appropriately selected from known methods.

Examples of protecting groups for amino groups include benzyloxycarbonyl, Boc, tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyldamandeloxycarbonyl, trifluoroacetyl,
Examples include phthalyl, formyl, 0-nitrophenylsulfenyl, diphenylphosphinothioyl groups, and the like.

Protective groups for arginine include Tos, 21-stituent, benzyloxycarbonyl, and the like.

As a protecting group for the water FFI group of serine and threonine,
Examples include Rzl, tert-methyl, acetyl, terahydrobinyl group, and the like.

As the rs group of tyrosine for 1 g of water, for example, Bzl,
Examples include Clp-3zl, benzyloxycarbonyl, acetyl, Tos group, and the like.

Examples of protecting groups for the amine group of lysine include benzyloxycarbonyl, C)-7, CI2 Bzl, f3oc
, Tos group, etc.

As a protecting group for the carboxyl group of glutamic acid and aspartic acid, for example, esterification with benzyl alcohol, methanol, ethanol, tert-butanol, etc. is carried out.

Examples of activated carboxyl groups include: acid chloride, acid anhydride or mixed acid anhydride, azide, active Nisdel (pentachlor[-1 phenol, p
-nitrophenol, N-human [ester with 1-xysuccinimide, N-hydroxybenztriazole, N-hydroxy-5-norbornene-2,3-dicarboximide, etc.]. The peptide bond forming reaction may be carried out in the presence of a condensing agent such as a carbodiimide reagent such as dicyclohexylcarbodiimide or carpodiimidazole, or a tertiary pyrophosphine.

Hereinafter, an example of the production of the peptide of the present invention will be specifically explained using a reaction scheme.

[Reaction formula-1] Δ-TVr-OH (a) ↓ △-Try-R' (b) ↓ 1l-Try-R' (c) ↓ Δ-Pro ro-〇 H (d) △-Pro -Try-R' (E)A -Val-
Val-G ln-Pro-1-ys-1 vs-Pr.

-Pro-Pro-Tyr-R' (A,)↓ H-Val-Val-G In-Pro -cys-L
ys-Pr.

-Pro-Pro-Tyr-〇H (1) (In the formula, △ indicates a protecting group for an amino group and an insoluble carrier for RIG.) In the above, preferable examples of Δ include 1aBOci,
Preferred examples of R1 include benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, and chloromethylated polystyrene.

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 R1 is removed. .

In the above method, amino Fin (a) and insoluble carrier R
One reaction with 1 is carried out by utilizing the reactive carboxyl group of amino acid (a) and bonding it to R1 according to a conventional method. For example, when using chloromethylated polystyrene, the reaction is carried out in a suitable solvent, for example 1-
ethylamine, potassium tert-butoxide, charcoal I
5! It is carried out in the presence of a basic compound such as cesium or cesium hydroxide.

As a solvent, for example, dimethylformamide (DM
F), Shi) (chill), Ruho * Shito (DMSO),
Examples of the solvent include pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, N-medylpyrrolidone, hexylymethylphosphoric acid triamide, and a mixed solvent thereof. The above reaction is usually completed at 0 to 85° C., preferably 25 to 80° C., and within a few minutes to 24 R. The ratio of amino acid and insoluble carrier used is usually the latter 1.
It is preferable to use an excessive amount of pre-balance, generally 1 to 3 times the amount of pre-balance.

The elimination reaction of the homomorphized amino acid of general formula (b) thus obtained to the protecting group 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 sodium chloride 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 is 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 + 11 (
The amount is preferably about 1 to 1 g, and the reaction is generally completed in about 1 to 48 hours. The acidolysis is carried out in the absence of a solvent, usually at about 0 to 30°C, preferably about 0 to 20°C, for about 15 minutes to 1 hour. The amount of acid used is usually about 5 to 10 times the amount of the raw material compound. When only the protecting group A is removed in the acidolysis, it is preferable to use trifluoroacetic acid or hydrochloric acid as the acid. Furthermore, reduction with metallic sodium in liquid ammonia was performed until the reaction solution changed from Permanage 1 to 30.
It is usually carried out at -40°C to -70°C using hanging metal sodium that remains colored for about 1 q to 10 minutes.
Ru.

Next, the reaction between the immobilized amino acid of the general formula (c) and the 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, pyridine, chloroform, dioxinane,
Examples include dichloro[1methane, tetrahydrofuran, ethyl acetate, N-methylpyrrolidone, hexamethylphosphoric acid i-lyamide, 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-dicyclohexylcarbodiimide (DCC>, N-ethyl-N'), if necessary.
-dimethylaminocarbodiimide, 1-ethyl-3-
It can be carried out in the presence of a dehydration condensing agent such as diisopropylaminocarbodiimide, 1-cyclohexyl-3-(2-morpholinyl-rubodiimide). Then, although there is no particular limitation, the latter should be used in an amount equal to m - 10 times more molar than the former, preferably equal in molar amount to 5 times the former.
It is better to cover it twice as much as possible.

There are no particular limitations on the use of dehydration condensation agents, and usually amino acids (
For 2), preferably equimolar culmality is used. 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 -20C to about 40C. Reaction times generally range from a few minutes to 30 hours.

The peptide of the general formula (B) obtained in this way is obtained by removing the protecting group Δ in the same manner as above! After +, according to the amino acid sequence represented by general formula (1), Δ-P l'o - 0 ) 1,
8-Pro-O t-1, Δ-1-. ys-O
H, Δ-1-1/S-OH, Δ-p
ro-OH, Δ-GIn-0
1-1, 8-Vat-OH, Δ-Vat-O
H(7) is carried out by sequential condensation reaction with each 7mi/W or those with protected side chain functional groups or those with activated carboxy groups, thus giving the general formula (Kube)
It can be induced into a peptide represented by These condensation reactions and elimination reactions to protect groups are carried out in the same manner as described above.

In addition, the iq peptide (to) is similarly removed from the protecting group (, nH 1! of the protecting group of the side chain functional group of the amino acid).
Elimination of II and insoluble carrier R1 leads to the peptide vI represented by formula (1). Here, the elimination reaction of the protective group of the side chain functional group and the insoluble carrier R1 is performed in the same manner as the elimination reaction to the protective group in step f. 1. In this case, it is preferable to use hydrogen fluoride or Ll water bromide as the precursor. Furthermore, ToW
f) Each amino acid used in the method is
) Known commercial product C is good.

The present defense of the formula (1) can be obtained by constructing the peptide as described in 1-1 below, and then extracting the peptide from the reaction mixture, distributing it, distributing it, etc. Manufactured.

Moreover, each peptide represented by general formulas (2) and (3) is also produced according to 1-.

The peptide of the present invention thus 19 has a 12'
Radioactive substances such as 5J, +”llj, peroxidase (
POX>, key trypsinogen, broboxypebudidase, glyceraldehyde-3-phosphorus hydrogenase, amylase, phosphorylase, I')-Na
By introducing 8 scales of enzymes such as sa, P-Nasc, β-galactosidase, gululose-6-phosphatate dehydrogenase, ornidine rupoxylase, etc., the RrΔ method or enzyme immunoassay can be used. It can be used as a labeled antigen for use in the F.IΔ method. ”-The introduction of radioactive materials can be carried out by conventional methods. For example, radioactive iodine can be prepared by the oxidative iodination method using Ramine T (W. M. Hunter and F.
,C. Greenwood; Nature;
194, 495 (1962), Bioc
hemJ. 89, 144, (1963))
The introduction of the M elementary reagent is carried out by a conventional coupling method such as the B.F. Erlana method.
er) et al.'s method [△cta. Endocrino
l. suppl. , 1 6 8.

206 (1972)) and Karol (M.H.

Karol et al.'s method (Proc, Na.tl.
Δcad. sci. .

(SA..57 Near 13 <1967>) and other known methods.

Hereinafter, the method for producing antigens 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 carriers to be 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 animal serum albumins such as horse serum albumin, bovine serum albumin, horse serum albumin, human serum albumin, and sheep serum albumin; horse serum globulin, bovine serum globulin, and horse serum. Animal face globulins such as globulin, human TM globulin, and ovine serum globulin; Animal diroglobulins such as equine thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin, and ovine thyroglobulin; equine hemoglobulin, bovine hemoglobulin , animal hemoglobulins such as rabbit hemoglobulin, human hemoglobulin, and sheep hemoglobulin; animal hemocyanins such as keyhole rimbet hemocyanin (KIH); proteins extracted from roundworms (Ascaris extract, JP Showa 56-164
14th Bulletin, J, r i+mun,.

111. 260-268 (1973), J.

Immun., 122. 302-30B (1979
), J, rmmun,, 98. 893~900 (1
967) and Am, J. r'hysio1. ．． 19
9. 575-578 (1960) or further purified versions thereof): polylysine, polyglutamic acid, lysine-glutamic acid copolymers, and copolymer caps containing lysine or ornithine.

As the hubten-carrier binding reagent, a wide variety of those commonly used in the preparation of antigens can be used.

Specifically, diazonium compounds such as bisdiazotized benzidine (BDB>, bisdiazotized 3°3'-dianisidine (BDD)) that crosslink tyrosine, histidine, and 1 to liptophan; Crosslinking with amino groups, such as glyokylyl, 7[-1 dialdezide, geltaraldehyde,
Aliphatic dialdehydes such as succinoaldezide and adibaldehyde: thiol groups, etc. Crosslinking with thiol groups, such as NNT O-phenylene dimaleimide,
A simatonimide compound such as N,N'-m-phenylene sima 1 neuimide; for example, metamaleimide, which crosslinks an amino group and a thiol group. Benzoyl-N-human 1-xysuccinimide ester, 4-(maleimidomedyl)-cyclohexane-1-carboxyl-N'-
Maleimidocarboxyl-N-hydroxysuccinimide esters such as hydroxysuccinimide ester;
Reagents used in the usual peptide bond-forming reaction to form an amide bond between an amino group and a carboxyl group, such as N
, N-disyc1'hexylcarbodiimide, N-ethyl-N'-dimedylaminojJ carbodiimide, 1-ethyl-3-di'isopropylaminocarbodiimide, 1-
Examples include dehydration condensation agents such as carbodiimides such as Schiff's hexyl-3-(2-morpholinyl-4-ethyl)carbodiimide. Furthermore, as the hubten-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 at pH 7 to
10 in a normal buffer, preferably at pH 8-9.
The reaction is carried out in il at 0 to 10°C, preferably around room temperature. The reaction is usually completed in about 1 to 24 hours, preferably 3 to 5 hours. The following are examples of typical M solutions used in the above.

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 tetraate buffer In the above, the proportions of habten, habten-carrier binding reagent and carrier Q can be determined as appropriate, but usually It is preferable to use the carrier in an amount of about 1 to 6 times IJI, preferably about 1 to 5 times IJI, and to use the Habten-carrier binding reagent in an amount of about 5 to 10 times IJI.

Through the above reaction, a desired antigen consisting of a peptide-carrier complex in which a carrier and habten are bound is harvested through the mediation of a habten-carrier binding reagent.

After the reaction is complete, the antigen can be collected using conventional methods, such as dialysis,
It can be easily isolated and purified by gel filtration, fractional precipitation, etc.

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 mammals used for antibody production, but rabbits and guinea pigs are usually used! It is preferable to use . For antibody production, a given antigen obtained as described above is diluted with physiological saline to an appropriate concentration, and then added to F.
Mix with AdJuvant)! l! A suspension may be prepared and administered to a mammal. For example, there is a V note for rabbits! I! Intradermal injection of cloudy liquid! in (0 as ω of antigen.
1 to 51nllz' times), and thereafter every 2311 times 2 to 1
Immunization can be achieved by administering for 0 months, preferably 4 to 6 months. Antibodies are collected by collecting specimens from the immunized animals 1 to 2 weeks after the final administration of the suspension, centrifuging the specimens, and separating the self-extinguished specimens. According to the above method, an antibody having excellent specificity for the antigen used can be obtained, which can be used in RIA method, ElA method, etc. to determine human leukemia virus-related proteins.

In order to explain the present invention in more detail below, general formula (1),
Examples of manufacturing the peptides of the present invention represented by (2) and (3) and examples of manufacturing antigens and antibodies from the resulting peptides will be given, but the present invention is not limited thereto.

In addition, Rf@ in each production example was measured using a thin layer chroma on silica gel and a Togelafy using the following mixed solvent.

Rf'/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 1 ■ Add 3oc-Tyr (Cl p
-Bzl)-OH 7.53111 is dissolved, 10q of chloromethylated borisdylene IL1 fat (Protein Research Foundation) is added, and the mixture is reacted at 80°C for 30 minutes. The resin was thoroughly washed with r) MSO15o96 acetic acid/chloro [1 form and methylene chloride in that order, and dried under reduced pressure to give 12 g.
13oc Tyr (C12Bzl)-resin is obtained.

After hydrolyzing a portion, amino acid analysis was performed and the results were 0 amino acids, 3111111101/(I resin tear ivy).

■ Boa TVr ((/I 2
After washing the resin 1.70(+ with 30 mQ of chloroform three times, add it to 30 mQ of a loloform solution containing 50% 1-helifluoroacetic acid (TEA) and react for 20 minutes at room temperature. 1 time with methylene chloride, 5 times with 30 mQ of methylene chloride, 10%]-ethylamine dissolved in methylene chloride, 3 times with 30 mQ of methylene chloride, and then 6 times with 30 mQ of methylene chloride.
12Bzl)-resin is obtained.

Add H-Tyr (C12-
Bzl)-resin is added, and then a solution of 0.27° DCC dissolved in methylene chloride 5111111 is added and reacted at room temperature for 2 hours. After washing the resin 6 times with 30 g of methylene chloride, 0.28 g of Boc-Pro-OH and 0.55 g of 1-human Oxybenzotriazole were added to 25 g of methylene chloride, followed by 0.27 g of DCC. Add solution 5- dissolved in methylene and react in the same manner again (double coupling method). The resin was thoroughly washed with methylene chloride and 3oc-Pro-Tyr (C12Bzl)
- Obtain resin.

■ In the same way as above ■, Boc -pro -T
The yr(Cl 2-, B zl )-resin is de-Boc-formed, and then the following amino acids, side-chain functional group-protected amino acids, or amino acids with activated carboxyl groups are sequentially subjected to condensation and de-3oc reaction.

Boa-Pro-OHO, 28a Boa-Pro-OH0, 280Boa-Lys
(CI-Z)-OHO, 55Ω13oa-LyS(CI
-Z)-OH0,55gBoc-Pro-OH0,28
0 Roe-Gln-ONP O,48
0Boc-Vat-OH0,29g Boa-Val-OH0,29++ Thus t-1-Vat-Vat-Gln-Pro-L
VS(CI-Z)-Lys(CI-Z)-Pro-Pr
o -Pro-Tyr(C12-BZI)-resin 2.
Obtain 57g. Of this, 1.20a is Anisole 2II
I12 and hydrogen fluoride were dissolved in 20% of hydrogen fluoride and incubated at -20°C for 30 minutes and then at 0°C for 30 minutes. Excess hydrogen fluoride was distilled off under reduced pressure, and the residue was extracted with 10% acetic acid and diluted with ether. Wash at The aqueous layer was freeze-dried, followed by gel filtration using Sephadex G-25 (Pharmacia, eluent 1M acetic acid), and further CM-23 cellulose (Wara I-Hun, 0.04M ammonium acetate,
16211 g of the target peptide was purified using pH 7.2).
get. This peptide is hereinafter referred to as "peptide A."

Rf value: Rfl-0,01Rf2=0.27 Elemental analysis value: (CF, e He e OH3N+ 3 ・3CH3
As CO9H・4H20) C (%) H (%) N (%) Theoretical value 53.02 7.82 12.96 Analytical value
52.94 8.06 12.74 Amino acid analysis value:
(Analyzed with Hitachi Model 835) Analysis value Gln(1) 1.05 Ll(2>2.17 Pro(4) 4.02 Tvr(1) 1.05 Vat(2,>1.69 Manufacturing example 2 Manufacturing BOC-ρr〇-resin (
0.44mw+O110 resin) 1.20g, Production Example 1
In the same manner as (1) and (2) above, each of the following amino acids or derivatives thereof is sequentially subjected to condensation and de-13oc reaction.

Boa-8er (Bzl)-OH0,39aBoa-A
la-OH0,25゜Boc-8er(Bzl) -OH0,39CIBo
c-Δrg(Tos)-OH0,56<IBoa-8e
r(Bzl)-OH0,39CIF3oc-P
he -0to1
0.35QBoa-11e-OH1/2H20 0,3,2! ;I Boa-Gin-ONP 0. 49
gBoa-Gly-01-10,230 Boa-Met-OH0,33Q Thus, H-Met -G IV -G In -I
Ia P he-S, er (B zl) −Δ
ra(Tos) -8et(Bzl)-Δ1a-8e
1.58 (+) of r(Bzl)-Pro-resin is obtained, of which 0.40 (l) is mixed with 10 ml of hydrogen fluoride, 12.1° 0.5 mG of 2-ethanedithiol and 1 tbsp of anisole,
After incubation at -20°C for 30 minutes and then at 0°C for 30 minutes, excess hydrogen fluoride was distilled off under reduced pressure to give a 10%
Extract with acetic acid, wash with ether, and freeze-dry.

Next, gel filtration was performed using UFadex G-25 (Pharmacia, eluent 1M acetic acid), and further CM-cell [1-
Purify by S23 (Whatman, 0.1→0.5M ammonium acetate, linear gradient o t-1=5.0) and add 32 mg of the target peptide. Hereinafter, this peptide will be referred to as "1 peptide B."

[value: Rfl-0,01Rf2-0.50 Elemental analysis value: (CsoHe+0+eN+sS.

CH3CO2H・5H (as O) C (%) H (%) N (%) Theoretical value, 46.94 7.20 15.79 Analysis value 47.01 7.32 15.66 Amino acid analysis value: (Hitachi type 835 Analysis value Ala (1) 1.07 Ara (1) 1.07 Gln (1) 1.00 Gly (1) 1.00 11e (1) 0.91 Met (1
) 0.94r'l+a(1)
0. 93Pro (1)
1.07Ser(3) 2.
98 Production Example 3 HM et-G ly GI obtained in Production Example 2 above
n -1le -Phe-8er (Bzl) -Aro
(Tos) -8er(Bzl) -Ala-8ep
(Bzl) -0.41g of -Pro- resin, Boa-
0.47o of TVr(C12Bztri-○H) is subjected to a double coupling reaction in the same manner as in Preparation Example 1-2.

The trifluoroacetic acid r s oc group is then removed to form H
-Tyr(C12-Bzl) -Met-Gly-Gl
n-11e-Phe-8er (Bzl) -ArlTo
s) -8er(Bzl) -Ala-8er(Bzl
)-Pro-resin is obtained. This was mixed with 10 mG of hydrogen fluoride, 1 ml of anisole, and 0.51 g of 1,2-ethanedithiol.
10 and at -30°C for 30 min, then at 0°C for 30 min.
After incubating for a minute and removing excess hydrogen fluoride under reduced pressure, the mixture was extracted with 10% acetic acid, washed with ether, and lyophilized. Next, gel filtration was performed using Fadex G-25 (Pharmacia, eluent: 1 M acetic acid), and 1-(PLO (Dam 3, eluent: 0) using a Deverosil force ram.
, IM Na H2PO4:7cell-2-lilu8
After purification by 0:20), Sephadex G
-25 (Pharmacia, eluent 1M acetic acid) to desalt the target peptide, 9.231+l! Get II. This peptide will hereinafter be referred to as "peptide C".

Rf value: Rf I -0,01Rf 2-0.52 Elemental analysis value: (as C59j19oO+eN+eSa CH3CO1!H・6H20) C (%) H (%) N (%) Theoretical value 48.47 7.07 14.82 Analysis value
48.63 7.01 14.77 Amino acid analysis W1
: (Analyzed with Hitachi Model 835) Analysis value Ala (111,03 Met(1) 1.00Arg(1)
1.07Phe(1)
1.09Gin(1) 1.03P
ro(1) 0.99Gly(1)
1.14Ser(3)
2.6811e(1) 1.03
Tyr (1) 1.03 Production Example 4 0,27 m1io110 m of 3oc-1eu-resin was produced in the same manner as in Production Example 1 (■), and 2 g of the resin was added in the same manner as in Production Example 1 (■) and ■. Then, the following amino acids or their derivatives were sequentially subjected to double coupling reaction and acute B
Perform OA reaction.

Boc-8er-OHo, 40Q Boa-Arc(Tos) -OHO,58(]Boc
-1-eu-OH-H200,34QBoc-1ie
-OH・1/2H200, 32gBoa-Pro-0)
-IO, 29°Boa-Asp(B
zl)-OHO,44gBoc-1,ys(CI-Z)
-OHO,56Ω3oc-Pro-OH0,29Boc-Thr(Bzl) -OHO,39(]]B
oa-Gly-OH0,24゜Boc-Glu(Bzl)-OH0,46+IBoa-
Pro-OH0,290 Boa-T'Vr(CI2 Bzl) OH0
,57(] Thus Boa-Dyr(CI 2-BZI) -Pr
o-Glu(Bzl) -Gly-TI+r(Bzl
) -Pro-Lys(CI-Z) -Asp
(Bzl)-Pro-11e-1eu-Ar(J (T
pS) -S0r-L00-Resin 2.62o is obtained.

Dissolve the 1,620 in hydrogen fluoride 20+112 and anisole 211112, heat at -20℃ for 30 minutes, and then
℃ for 30 minutes, excess hydrogen fluoride was distilled off under reduced pressure,
Extract with 10% acetic acid, wash with ether, and freeze-dry. Then, gel tF' was filtered with Sephadex G-25 (1M acetic acid), and HPI using a Deverosil column was performed.
Purification using C (eluent, O, 1M phosphite 1-buffer:aretonitrile 80:20)
T Vr -P ro-G Iu -G 1y-Thr
-Pro -Lys-ΔSD-Pro-1le
-l-, eu-Δrg -5er-L eu-OH peptide 104n+o is obtained. Hereinafter, this will be referred to as "Peptide D"
It is called.

Rf value: Rf blind = 0. 01 [2=0
．． 42 elemental analysis value: (Cr2H++g022N+ 8'CH3GO2H
・As 7H20) C (%) H (%) N (%) Theoretical value 50.16 7.62 14.23 Analysis value
49.87 7.60 14.29 Amino acid analysis value:
(Analyzed with Hitachi Model 835) Analysis value Asp (1) 0.96 Lys (1) 1 , 13 Δrg (1) 1.02 Pro (3) 2.79 Glu (1-) 1.05 Ser (1)
0゜92Gly(1,,)
1.03Thr(1) 0.
99Tle(1) 0.99Tyr(
1) 0.97 Leu(2)
2. 1 Production of 11 antigen> Production example 1 Peptides B5.09■ and K obtained in Peptide production example 1
L)-1 (Sigma) 25.10a+a, 0.05M
IJ phosphate 1111i solution (EI H-7,0) 3.01
In addition to this solution, add 0.2% of 2% glutaraldehyde solution.
Add mQ dropwise and stir at room temperature for 3 hours. The reaction mixture is then dialyzed against distilled water overnight at 4°C and lyophilized to obtain the target antigen 26.49aQ.

Hereinafter, this antigen will be referred to as "antigen engineering."

Antigen (1) has 10 moles of peptide B bound on average to 11 moles of KLI (assuming the average molecular weight is 100,000). The binding rate between this peptide B and K L H is
1q antigen ■ was further added to Fadex G50 (eluent: physiological saline, detection: O[)280 nm, efflux rate:
Unreacted K1. , H and peptide B were not observed, the gel filtration revealed that KL
)-1 and the fraction of peptide B bound to other products (2),
A standard concentration test mta of the peptide dimer was prepared, the suspension of the above 2jiI body was determined, and the value obtained by subtracting this from the drunkenness of peptide B used as a starting material was calculated as the amount of binding to Kl)-1. This is what I was looking for. The same applies to the following antigen production examples.

Production Example 2 ■ Add benzidine 83.25H to a mixed solvent of 0.2N-HCQ 20WIJ and DMF, stir under water cooling, and add 870,030 g of sodium nitrite and distilled water to the solution. The BDB solution was prepared by adding gradually and stirring for 30 minutes.

■ He7chit'D5.13no and HiK1-H8,10n
a to 0.13M Na CG! 0.16M borate buffer (pH-9,0) and stir gently at 4°C. 0.5 liters of the BDB solution (①) above was gradually added dropwise to the solution. The reaction solution was pH adjusted with 0.5N NaOH.
= 9.0, and further reacted at 4°C for 2 hours. Thereafter, the reaction mixture was dialyzed overnight at 4° C. against distilled water and lyophilized to obtain 12.76 u of the target antigen. This antigenic infarction is described below.
Antigen■. Antigen ■ has an average of 18 moles of peptide D bound to 1 mole of K L H.

Production example 3 Peptide A5.1711+a and K1-H8,03R1
(Target antigen 1 in the same manner as in Production Example 2 above using
Obtain 2.741B. Hereinafter, this antigen will be referred to as "antigen ■." Antigen ■ has an average of 25 moles of peptide Δ bound to 1 mole of K L H.

Production of antibodies> Production example 1 1100L each of antigens 1 to m obtained in antigen production example 1
After dissolving 1 in 1.5% physiological saline, 1.5mQ of Freund's auxiliary solution was added to the suspension.
e rabblts) (2.5~3.0k
o), administered subcutaneously, and the same dose administered 6 times every 2 weeks. Furthermore, the first dose was administered to the brain and immediately after administration three times every month. Seven days after the final administration, blood is collected from the test animal and centrifuged to collect the anti-supernatant to obtain each desired antibody.

(hereinafter -E)

Claims (1)

[Claims] A peptide represented by the formula 0, formula %, general formula R-Met-GIV-Gln -11e -Pl+e-
8er-Ara-8er-Δta-Ser-
Pro-011 [In the formula, R is a hydrogen atom or H-Tyr
Indicates the group. A human leukemia virus interconjugate selected from nY consisting of a peptide represented by the formula % and a peptide represented by the formula % ).