JPH0350759B2 - - Google Patents

Description

[Detailed description of the invention]

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.

【table】

【table】

[Reaction formula-1]

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)

[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.