JPH0461879B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention is directed to human leukemia virus (Adult T-
cell Leukemia Virus: ATLV or Human T-
Concerning a novel peptide related to cell leukemia virus (HTLV). DISCLOSURE OF THE INVENTION In this specification, when amino acids, peptides, protective groups, active groups, etc. are indicated by abbreviations, they shall comply with the IUPAC and IUB regulations or common symbols in the field, and examples thereof are listed below.
In addition, when an optical isomer is possible for an amino acid, etc., the L-isomer is indicated unless otherwise specified. Ser; serine Glu; glutamate Lys; lysine His; histidine Arg; arginine Phe; phenylalanine Thr; threonine Val; valine Cys; cysteine Tyr; tyrosine Tos; p-toluenesulfonyl group Boc; tertiary butoxycarbonyl group ONP; p -Nitrophenoxy group Bzl; benzyl group MBzl; p-methoxybenzyl group OBzl; benzyloxy group Cl ₂ -Bzl; 2,6-dichlorobenzyl group Cl-Z; 2-chlorobenzyloxycarbonyl group Human leukemia virus is It is a virus isolated from cellular leukemia (ATL) and is attracting attention for its association with this disease [Proc. Natl. Acad. Sci.
USA., 78 , 6476-6480 (1980); 79 , 1653-1657 (1982); 77 , 7415-7419 (1980); 79 , 2031-2035 (1982); 81 , 2534-2537 (1984) ), etc.]. The present invention provides an antibody having specific reactivity to the virus-related protein (virus-related antigen) that enables the purification and measurement (detection) of the virus-related protein (virus-related antigen), and serves as papten for the production of the antibody. The purpose of the present invention is to provide specific virus-related peptides that can be obtained, and to provide a technique using these to measure the virus-related proteins or antibodies (serum antibodies observed in people infected with the virus). The above purpose is based on the general formula R-Ser-Glu-Lys-His-Phe-Arg-Glu-Thr-Glu-Val-OH (1) [wherein R is a hydrogen atom, H-Cys- group or H-Tyr]
- indicates a group. ] This is achieved by the human leukemia virus-related peptide represented by the following and its salt. The peptide of the present invention can be easily synthesized by simple operations using readily available commercially available amino acids. Furthermore, antibodies having specific reactivity to the above-mentioned virus-related proteins can be easily obtained in large quantities and stably from immunizing antigens prepared using the peptides of the present invention as haptens. The specific antibody can be used for the purification of virus-related proteins by, for example, binding it to a carrier for affinity chromatography and using it in the chromatography, and can also be used in various immunoassay methods for the virus-related proteins. Can be used as a specific antibody. Furthermore, the above-mentioned peptide of the present invention can be used as a labeled antigen specific to the above-mentioned virus-related protein by introducing various labeling substances into it. The specific antibody and labeled antigen can be used to diagnose human leukemia virus infection,
Furthermore, it is useful for diagnosis, research, etc. of mature T cell leukemia/lymphoma such as adult T cell leukemia and cutaneous T cell lymphoma, and related diseases. The method for chemically synthesizing the peptide of the present invention will be described in detail below. The peptide of the present invention can be synthesized using a conventional peptide synthesis method, specifically, "The Peptides" No. 1.
Volume (1966) [Schro¨der and Luhke,
Academic press, New York, USA] or "Peptide Synthesis" [Izumiya et al., Maruzen Co., Ltd. (1975)], for example, the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method (p-nitrophenyl ester method, N-hydroxysuccinimide ester method, dianomethyl ester method, etc.), method using Woodward's reagent K, carbodiimidazole method, redox method, DCC/ It can be manufactured by additive methods (HONB, HOBt, HOSu), etc. In the above method, both solid phase synthesis method and liquid phase synthesis method can be applied. The peptide of the present invention is usually synthesized according to 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 a method in which the amino acids are coupled into several fragments. Manufactured. More specifically, for example, when using solid phase synthesis, the C-terminal amino acid (protected amino acid)
is first 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, and examples thereof include halogenomethyl resins such as chloromethyl resin and bromomethyl resin, hydroxymethyl resin, phenol resin, and tert-alkyloxycarbonyl hydrazide. Resin etc. can be used. Next, after removing the amino protecting group, amino group-protected amino acids are sequentially bonded by a condensation reaction with the reactive amino group and the reactive carboxyl group according to the amino acid sequence represented by the above general formula (1), and the synthesis is performed step by step. After synthesizing the entire sequence, the peptide of the present invention can be obtained by removing the peptide from the insoluble carrier. In the above various methods, each amino acid having a side chain functional group, such as Arg, Tyr, Glu, Thr, Lys,
It is preferable to protect the side chain functional group of His, Cys, Ser, etc., which is protected by a conventional protecting group, and the protecting group is removed after the reaction is completed. Furthermore, the functional groups involved in the reaction are usually activated. Each of these reaction methods is known, and the reagents used therein are also appropriately selected from known methods. Examples of protecting groups for amino groups include benzyloxycarbonyl, Boc, tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, Cl-Z, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, Examples include o-nitrophenylsulfenyl and diphenylphosphinothioyl groups. Examples of protective groups for carboxyl groups include alkyl esters (alkyl esters such as methyl, ethyl, propyl, butyl, tert-butyl, etc.), Bzl ester, p-nitrobenzyl ester, MBzl ester, p-chlorobenzyl ester, benzhydryl ester. , carbobenzoxyhydrazide,
Examples include groups that can form tert-butyloxycarbonyl hydrazide, trityl hydrazide, and the like. Protecting groups for the guanidino group of Arg include Tos,
Examples include nitro, benzyloxycarbonyl, amyloxycarbonyl groups, and the like. The hydroxyl groups of Ser and Thr can be protected, for example by esterification or etherification, but
Doesn't necessarily need to be protected. Groups suitable for this esterification include lower alkanoyl groups such as acetyl, aroyl groups such as benzoyl groups, and groups derived from carbonic acid such as benzoyloxycarbonyl and ethyloxycarbonyl. Examples of groups suitable for etherification include benzyl, tetrahydropyranyl, and tert-butyl groups. As a protecting group for the hydroxyl group of Tyr, for example, Bzl,
Examples include Cl ₂ -Bzl, benzyloxycarbonyl, acetyl, and Tos group. Protecting groups for the amino group of Lys include benzyloxycarbonyl, Cl-Z, Cl2 _- Bzl, Boc, Tos
Examples include groups. Protecting groups for the imino group of His include Tos, Bzl
etc. Protecting groups for the thiol group of Cys include MBzl,
Examples include Bzl and p-methylbenzyl group. The carboxyl group of Glu is protected by esterification with benzyl alcohol, methanol, ethanol, tert-butanol, etc. Examples of activated carboxyl groups include:
For example, the corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenztriazole, N-hydroxy-5-norbornene-
esters with 2,3-dicarboximide, etc.), and the like. In the above method, the condensation reaction (peptide bond formation reaction) between the reactive amino group and the reactive carboxyl group can be carried out in the presence of a solvent. As solvents, there are various solvents known to be useful for peptide bond formation, such as anhydrous or hydrous dimethylformamide (DMF), dimethyl sulfoxide (DMSO), pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran (THF). , ethyl acetate, N-methylpyrrolidone,
Hexamethylphosphoric acid triamide (HMPA) or a mixed solvent thereof may be used. There is no particular limitation on the ratio of the two raw material compounds used, but it is usually an equimolar amount to 5 times the molar amount of one to the other, preferably an equimolar amount to 1.5 times the molar amount of the other. The reaction temperature is within the usual range used for peptide bond forming reactions, generally about -40°C to about 60°C, preferably about -
The temperature is appropriately selected from the range of 20°C to about 40°C. The reaction time is generally about several minutes to 30 hours. The mixed acid anhydride method is carried out using an alkylhalocarboxylic acid such as methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate, isobutyl chloroformate, etc. in a suitable solvent in the presence of a basic compound. Examples of basic compounds include triethylamine, trimethylamine, pyridine, dimethylaniline, N-methylmorpholine,
1,5-diazabicyclo[4,3,0]nonene-
5 (DBN), 1,5-diazabicyclo[5,4,
0] Organic bases such as undecene-5 (DBU) and 1,4-diazabicyclo[2,2,2]octane (DABCO) and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate can be used. . Examples of solvents include various solvents commonly used in the mixed acid anhydride method, specifically halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, aromatic hydrocarbons such as benzene, toluene, and xylene, diethyl ether, and THF. , ethers such as dimethoxyethane, esters such as methyl acetate and ethyl acetate, aprotic polar solvents such as DMF, DMSO, and HMPA, etc. can be used. The reaction is usually carried out at -20 to 100°C, preferably -20 to 50°C, and the reaction time is generally 5 minutes to 10 hours, preferably 5 to 2 hours. The azidation method is carried out by first reacting an activated carboxyl group with an alcohol such as methyl alcohol, ethyl alcohol, or benzyl alcohol with hydrazine water in an appropriate solvent. It will be done. Examples of solvents include dioxane, DMF,
DMSO or a mixed solvent thereof can be used.
The amount of hydrazine water to be used is generally 5 to 20 times, preferably 5 to 10 times, the amount of activated carboxyl groups. The reaction is usually
It is carried out at a temperature of 50°C or lower, preferably -20 to 30°C. In this way, a compound (hydrazine derivative) in which the carboxyl group moiety is substituted with hydrazine can be produced. A compound in which the carboxyl group moiety is substituted with azide is produced by reacting the hydrazine derivative obtained above with a nitrite compound in a suitable solvent in the presence of an acid. The acid is usually hydrochloric acid,
As the solvent, dioxane, DMF, DMSO, or a mixed solvent thereof can be used, and as the nitrite compound, sodium nitrite, isoamyl nitrite, nitrosyl chloride, etc. can be used. The nitrite compound is generally used in an equimolar to 2-fold molar amount, preferably in an equimolar to 1.5-fold molar amount, relative to the hydrazine derivative. The reaction is usually carried out at -20 to 0°C, preferably -20 to -10°C, and is generally completed in about 5 to 10 minutes. The peptide bond forming reaction can also be carried out in the presence of a condensing agent such as a carbodiimide reagent such as dicyclohexylcarbodiimide (DCC) or carbodiimidazole, or tetraethylpyrophosphine. In each of the above reaction steps and the final step, when it is necessary to remove the protecting group, this is carried out according to a conventional elimination reaction. Examples of such methods include reductive methods such as hydrogenation using a catalyst such as palladium or palladium black, reduction with metal sodium in liquid ammonia, trifluoroacetic acid, hydrochloric acid, hydrogen fluoride, methanesulfonic acid, hydrobromic acid, etc. Examples include acidolysis using a strong acid. 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 about 100 mg to 1 g, and generally 1 to 1 g.
The reaction completes in about 48 hours. The above acidolysis is carried out without 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 5% based on the raw material compound.
It is best to use ~10 times the amount. When only the protecting group of the amino group is removed in the acidolysis,
Preference is given to using trifluoroacetic acid or hydrochloric acid as acid. Furthermore, the above-mentioned reduction with metallic sodium in liquid ammonia is carried out using such amount of metallic sodium that the reaction solution becomes permanently blue for about 30 seconds to 10 minutes, and usually at -40°C to -70°C.
It can be done to a certain degree. The peptide of general 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. In this way, the human leukemia virus-related peptide of the present invention represented by general formula (1) is obtained. The peptide thus obtained has ¹²⁵ I, ¹³¹ I
radioactive substances such as peroxidase (POX), chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D-Nase,
By introducing various enzyme reagents such as P-Nase, β-galactosidase, glucose-6-phosphate dehydrogenase, ornithine decarboxylase, radioimmunoassay (RIA) method or enzyme immunoassay (EIA) can be performed. It can be used as a labeled antigen for use in methods.
Introduction of the above-mentioned radioactive substance can be carried out by a conventional method. For example, radioactive iodine can be obtained by oxidative iodination using chloramine T [WMHunter and F.
C. Greenwood; Nature, 194 , 495 (1962),
Biochem . ]
and the method of MH Karol et al. [Proc.
Natl.Acad.Sci., USA., 57 , 713 (1667)]. Hereinafter, a method for producing an immunizing 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 represented by general formula (1) 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, rabbit serum albumin,
Animal serum albumins such as 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; equine thyroglobulin, bovine thyroglobulin, Animal thyroglobulins such as rabbit thyroglobulin, human thyroglobulin, and ovine thyroglobulin; Animal hemoglobulins such as equine hemoglobulin, bovine hemoglobulin, rabbit hemoglobulin, human hemoglobulin, and ovine hemoglobulin; keyhole limpets Animal hemocyanins such as hemocyanin (KLH); proteins extracted from roundworms (Ascaris extract, JP-A-56
−16414 Publication, 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 thereof); polylysine,
Examples include polyglutamic acid, lysine-glutamic acid copolymers, and copolymers containing lysine or ornithine. As the hapten-carrier binding reagent, a wide variety of those commonly used for preparing antigens can be used. Specifically, diazonium compounds such as bisdiazotized benzidine (BDB) and bisdiazotized-3,3'-dianisidine (BDD) that crosslink Tyr, His, and Trp; cross-linking, for example, aliphatic dialdehydes such as glyoxal, malondialdehyde, glutaraldehyde, succinaldehyde, adipaldehyde; cross-linking thiol groups together, for example, N, N'-o
dimaleimide compounds such as -phenylene dimaleimide, N,N'-m-phenylene dimaleimide; maleimidomethyl)-cyclohexane-1-carboxyl-
Maleimidocarboxyl-N-hydroxysuccinimide esters such as N'-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 DCC, 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 reaction for producing the above-mentioned immunizing antigen is carried out, for example, in an aqueous solution or a normal buffer solution with a pH of 7 to 10, preferably with a pH of 8.
The reaction is carried out in a buffer solution of 0 to 40°C, preferably around room temperature. The reaction is usually completed in about 1 to 24 hours, preferably 2 to 5 hours. Typical buffer solutions used above 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 1 to 10 times the mole of the hapten-carrier binding reagent.
Through the above reaction, a desired immunizing 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 method for producing antibodies using the immunizing antigen obtained as described above 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 Adjuvant) to prepare a suspension, which may be administered to mammals. For example, intradermally inject the above suspension into rabbits (as the amount of antigen
0.1 to 5 mg/dose), then every 2 weeks for 2 to 10 months,
Preferably, immunization may be carried out by administering for 4 to 6 months. 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, it is possible to obtain an antibody with excellent specificity for the immunizing antigen used, which is
It can be used to quantify human leukemia virus-related proteins using RIA method, EIA method, etc. EXAMPLES In order to explain the present invention in more detail, examples of producing a peptide of the present invention, an example of producing an immunizing antigen from the peptide, and an example of producing an antibody from the antigen will be given below, 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-pyridine-acetic acid-water (1:1:1:1) Rf ² ... upper layer of n-butanol-pyridine-acetic acid-water (30:20:6:24) <Production of peptide> Production Example 1 350 milliequivalents of potassium tert-butoxide
Dissolve 1.41 g of Boc-Val-OH in 16.8 ml of DMSO solution, add 5 g of chloromethylated polystyrene resin (Protein Research Foundation, 2% dihinylbenzene, mesh 200-400), and heat at 80°C.
Incubate for 30 minutes. The resin was thoroughly washed with DMSO, ethanol, 50% acetic acid, water, ethanol, and methylene chloride in that order, dried under reduced pressure, and then washed with Boc-
5.3 g of Val-resin are obtained. After hydrolyzing a portion, amino acid analysis was performed and the result was 0.29 mmol of amino acids/g resin. After washing 2.0 g of Boc-Val-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-Val-resin is obtained by washing twice and then six times with 30 ml of methylene chloride each time. Add 25 ml of a solution of 0.49 g of Boc-Glu(OBzl)-OH dissolved in methylene chloride to the above H-Val-resin, then add 5 ml of a solution of 0.30 g of DCC dissolved in methylene chloride, and react for 2 hours at room temperature. let After washing the resin 6 times with 30ml of methylene chloride,
0.49 g of Boc-Glu(OBzl)-OH and 88 mg of 1-hydroxybenzotriazole in methylene chloride
Then, 5 ml of a solution of 0.30 g of DCC dissolved in methylene chloride was added and the same reaction was carried out again (double coupling method). Wash the resin thoroughly with methylene chloride and convert it to Boc−Glu(BOzl)−.
Val-resin is obtained. In the same way as above, Boc−Glu(OBzl)−
The Val-resin is de-Boc-ized, and then the above-mentioned amino acids, side chain functional group-protected amino acids, or amino acids with activated carboxyl groups are sequentially subjected to condensation and Boc-de-Boc reaction. Boc-Thr(Bzl)-OH 0.45g Boc-Glu(BOzl)-OH 0.49g Boc-Arg(Tos)-OH 0.62g Boc-Phe-OH 0.39g Boc-His(Tos)-OH) 0.59g Boc- Lys(Cl-Z)-OH 0.58g Boc-Glu(OBzl)-OH 0.49g Boc-Ser(Bzl)-OH 0.45g Thus H-Ser(Bzl)-Glu(OBzl)-Lys(Cl
−Z)−His(Tos)−Phe−Arg(Tos)−Glu
(OBzl) − Thr (Bzl) − Glu (OBzl) − Val − Resin
Obtain 2.9g. Of this, 0.50g is added to 1.5ml of anisole,
Methyl ethyl sulfide 0.25 ml and hydrogen fluoride 10
After incubating at -20℃ for 30 minutes and then at 0℃ for 30 minutes, excess hydrogen fluoride was distilled off under reduced pressure, and the residue was extracted with 10% acetic acid and washed with ether. . The aqueous layer was freeze-dried, then gel-filtered using Cephadex G-25 (Pharmacia, eluent 10% acetic acid), and further HPLC (eluent; 5% 1/1000N-HCl/CH ₃ CNB as solution A).
Linear gradient from A/B = 85/15 to A/B = 50/50 using 50% 1/1000N-HCl/CH ₃ CN as B solution; ODS-120T, 7.8 x 300 mm,
Toyo Soda Co., Ltd.) to purify the target peptide (H-Ser-Glu-Lys-His-Phe-Arg-
85 mg of Glu-Thr-Glu-Val-OH) are obtained. This peptide is hereinafter referred to as "peptide A." Rf value: Rf ¹ = 0.58 Rf ² = 0.08 Retention time in HPLC In the above HPLC, A/B = 90/10 for 0 to 5 minutes, and A/B = 90/10 for 5 to 25 minutes.
Gradient from 10 to 10/90, ODS-120T
The retention time measured using a column (4.6 x 250 mm, flow rate 1 mm min) was 7.65 min. Amino acid analysis value: (analyzed with Hitachi model 835) Analysis value Ser(1) 0.91 Glu(3) 3.05 Lys(1) 1.06 His(1) 0.95 Phe(1) 1.03 Arg(1) 0.90 Thr(1) 0.95 Val (1) 1.01 The above amino acid analysis values are the results measured after hydrolysis with 6N-hydrochloric acid, and the same applies to each of the following examples. Production Example 2 H-Ser(Bzl)-Glu(OBzl)- obtained in Production Example 1
Lys(Cl−Z)−His(Tos)−Phe−Arg(Tos)−
To 500 mg of Glu(OBzl)-Thr(Bzl)-Glu(OBzl)-Val- resin, Boc-Cys
123 mg of (MBzl)-OH was reacted, and then the protecting group and resin were removed and purified in the same manner.
Target peptide (H-Cys-Ser-Glu-Lrs-His
−Phe−Arg−Glu−Thr−Glu−Val−OH) 70
I got mg. This peptide will hereinafter be referred to as "peptide B". Rf value: Rf ¹ =0.62 Rf ² =0.12 Retention time in HPLC The retention time measured under the same conditions as in Peptide Production Example 1 was 13.81 minutes. Amino acid analysis value: (analyzed with Hitachi model 835) Analysis value Cys(1) Unmeasurable Ser(1) 0.95 Glu(3) 3.11 Lys(1) 1.04 His(1) 0.96 Phe(1) 1.05 Arg(1) 0.88 Thr(1) 0.98 Val(1) 1.00 Production Example 3 In Production Example 2, 123 mg of Boc-Tyr(Cl ₂ -Bzl)-OH was used instead of Boc-Cys(MBzl)-OH, and the desired Peptide (H-Tyr-Ser-
Glu−Lys−His−Phe−Arg−Glu−Thr−Glu−
Obtain 75 mg of Val−OH). This peptide will be referred to as "peptide C" hereinafter. Rf value: Rf ¹ =0.62 Rf ² =0.12 Retention time in HPLC The retention time measured under the same conditions as in Peptide Production Example 1 was 12.29 minutes. Amino acid analysis value: (analyzed with Hitachi model 835) Analysis value Tyr(1) 1.02 Ser(1) 0.92 Glu(3) 3.08 Lys(1) 1.06 His(1) 0.95 Phe(1) 1.06 Arg(1) 0.91 Thr (1) 0.97 Val(1) 1.02 <Production of immune antigen> Production example 1 Add 0.44 mg of MBSB to 1 ml of 0.1M borate buffer (PH7.5) containing 2.5 mg of Ascaris extract protein in DMF.
Add 10μ of the solution, stir for 20 minutes, remove excess MBS with dichloromethane, and add 3mg of peptide B dissolved in 0.1M phosphate buffer (PH7.2).
was added and stirred for 20 minutes, and it was confirmed by Ellman's method that peptide B had completely reacted. The reaction mixture is then dialyzed against distilled water for 3 days at 4°C and lyophilized to obtain 5.4 mg of immunizing antigen. This antigen will be referred to as "antigen B" hereinafter. Production example 2 3 mg of peptide A and Ascaris extract protein 6
Add 0.46 mg of 0.1% glutaraldehyde to 3.0 ml of 0.1M phosphate buffer (PH=7.2) and add 0.46 mg of 0.1% glutaraldehyde to this solution.
ml (4176 μmol) and stirred at room temperature for 2 hours.
After this, the reaction mixture is dialyzed for 3 days at 4° C. against distilled water and lyophilized to obtain 6.3 mg of immunizing antigen. This antigen will be referred to as "antigen A" hereinafter. Production example 3 5 mg of peptide C and Ascaris extract protein 10
mg of BDB was added to 5 ml of 0.16 M borate buffer (PH = 9.0) containing 0.13 M sodium chloride, and the BDB
Add 3.35 mg of the solution and stir at 4°C for 2 hours. the above
For the BDB solution, 83.25 mg of benzidine was added to a mixed solvent of 20 ml of 0.2N hydrochloric acid and 3 ml of DMF, and the mixture was stirred under ice cooling.
A solution of 87.03 mg of sodium nitrite in 2 ml of distilled water was gradually added thereto, and the mixture was stirred for 30 minutes. Thereafter, the reaction mixture was dialyzed against distilled water for 3 days at 4°C and lyophilized to obtain 13.0 mg of the immunizing antigen. This antigen will be referred to as "antigen C" hereinafter. <Production of antibody> Production example 1 500 μg of antigen B obtained in production example 1 of immune antigen
After dissolving in 1.5 ml of physiological saline, the suspension was prepared by adding 1.5 ml of Freund's auxiliary solution to 3 rabbits (New-Zealand white rabbits, 2.5 ~
3.0Kg) according to the procedure shown in the immunization schedule below.
A single antigen inoculation dose of 500 μg/body is administered subcutaneously, and thereafter, the same amount as the first dose is administered three times every month. Seven days after the final administration, whole blood is collected from the test animal and centrifuged to obtain antiserum (antibody). The antibodies obtained from each rabbit are referred to as "antibody B-1,""antibodyB-2," and "antibody B-3."

【table】

[Table] Production Example 2 Antigens A and C obtained in Immunization Antigen Production Examples 2 and 3
The target antibody is immunized using each of the antibodies according to the same immunization schedule as in Antibody Production Example 1, whole blood is collected after the fifth inoculation of the antigen, and the target antibody is obtained in the same manner. The antibody obtained using antigen A will be referred to as "antibody A" and the antibody obtained using antigen C will be referred to as "antibody C." Production of Γ-labeled peptide Peptide C obtained in Peptide Production Example 3 is labeled using chloramine T as follows. That is, 5 μg of the above peptide was added to 10 μg of 0.5 molar phosphate buffer (PH7.5) with Na[ ¹²⁵ I] (Carrier
Add 20μ of 0.5M phosphate buffer containing 1 milliCurie, then add 20μ of 0.5M phosphate buffer containing 20μ of Chloramine T. Stir for 25 seconds at room temperature and terminate the reaction by adding 100 μg of sodium metabisulfite (Na ₂ S ₂ O ₅ ) in 20 μg of 0.5 M phosphate buffer. Next, 10μ of a cold 10% sodium iodide aqueous solution was added to the reaction mixture, and the reaction mixture was passed through a Sephadex G-10 column (1.0 to 30 cm, eluent;
0.1% gelatin, 0.15M-NaCl and 0.02%
0.1 molar phosphate buffer containing _NaN3 , PH7.4)
to obtain ¹²⁵ I-labeled peptide C. The radioactivity of the labeled peptide is 1.598×
It was 10 ³ μCi/μg. Measurement of Γ titer The titer of the antibody obtained above is measured as follows. That is, each antibody was serially diluted with physiological saline, and ¹²⁵ I-labeled peptide (approximately
diluted to 9500 cpm) 0.1 ml and 0.05 molar phosphate buffer (PH = 7.4) [0.25%
BSA, 10mM EDTA, and 0.02% _NaN3 ] was added and incubated at ⁴ °C for 24 hours. ℃, 30 minutes, 3000 rpm) to separate from unreacted (unbound) ¹²⁵ I-labeled peptide, count the radiation, and measure the binding rate (%) of the antibody to the ¹²⁵ I-labeled peptide at each diluted concentration. The binding rate of the antibody to the ¹²⁵ I-labeled peptide (B/T%) is plotted on the vertical axis, and the dilution factor of the antibody is plotted on the horizontal axis, and the binding rate is plotted at each concentration. The results obtained for antibodies B-1 to B-3 are shown in FIG. In the figure, 1 is antibody B-1,
2 indicates antibody B-2, and 3 indicates antibody B-3. Table 1 below shows the results of determining the antibody dilution ratio at which binding was 50% in the above test, that is, the antibody titer.

[Table] Specificity test of Γ antibody against human leukemia virus-related proteins HTLV-producing cell line HUT-102 [Proc. Natl.
Acad.Sci.USA., 77 , 7415-7419 (1980)]
was metabolically labeled with ³⁵ S cysteine for 16 h and
Cells were soaked in RIPA buffer (150mM-NaCl, 1%).
Triton x 100, 1% deoxycholate, 0.1
50mM Tris-HCl buffer containing %SDS, PH
7.2) and centrifuged to obtain a cell extract. The extract containing 5 μg protein was added to (a) 2 μg of ATL patient serum, (b) 2 μg of normal adult serum, and (c) antibody B-
The immunoprecipitates obtained were subjected to 14% polyacrylamide gel electrophoresis and analyzed. The results are shown in Figure 2. In the figure, lane numbers a to d correspond to the above-mentioned samples used, and the numbers indicate size markers in kilodaltons (kd). From Figure 2, antibody B-1 (lane c) is
It was found that a 40kd human leukemia virus-related protein was precipitated. The protein is also precipitated by ATL patient serum (lane a), but does not react with normal adult serum (lane b) or normal rabbit serum (lane d). In the above test using antibody B-1,
2 μg peptide B (lane b), 20 μg peptide B (lane c) and 100 μg peptide B
(lane d) in the presence of each of the same tests (but using a 12% polyacrylamide gel).
repeated. The results are shown in Figure 3. In the figure, the lane numbers are as described above (lane a indicates no addition of peptide B), and the numbers are the same as in the test. From Figure 3, the specificity of the reaction between antibody B-1 and the 40kb protein seen in the test (Figure 2) is
This was confirmed by the competitive effect of peptide B. Cell extracts were prepared in the same manner as in the above test without radiolabeling using the following cells or normal adult lymphocytes. Using 10 μg of protein from each cell extract, the reactivity of these proteins with antibody B-1 was determined using a Western blotting analysis method using ¹²⁵ I-labeled donkey anti-rabbit Ig antibody [Proc. Natl. A cad. Sci. .
USA., 76 , 3116-3120 ( 1979 );
4350-4354 (1979)]. The results are shown in Figure 4. In the figure, lane numbers indicate the use of each of the following cells, and the numbers are the same for the tests. Lane number a; normal adult lymphocytes b; HUT-102 c; MT-2 [Nature, 294 , 770-771 (1981)] d; TARL [J.Exp.Med., 159 , 1105-1116
(1984)] e; HAYAI f: Molt 4F [J.Nat.Cancer Inst., 49 , 891
(1972)] g; HL60 [Nature, 270 , 347-349 (1977)] h; CEM [Cancer, 19 , 1725-1742 [1966]] However, the cells shown in lane numbers b to e are HTLV
These are infected cell lines, and the cells shown in lane numbers f to h are HTLV-uninfected cell lines. FIG. 4 shows that antibody B-1 has specific reactivity with human leukemia virus-related proteins.

[Brief explanation of the drawing]

Figure 1 is a graph showing the binding rate of antibodies obtained using the peptide of the present invention with labeled peptides at various dilutions, and Figures 2 to 4 show the specific reaction of the antibodies with human leukemia virus-related proteins. FIG. 2 is a diagram illustrating the results of electrophoretic analysis that reveal that the molecule has a specific property.

Claims (1)

[Claims] 1 General formula R-Ser-Glu-Lys-His-Phe -Arg-Glu-Thr-Glu-Val-OH [In the formula, R is a hydrogen atom, a H-Cys- group, or a H-Tyr
- indicates a group. ] A human leukemia virus-related peptide represented by and a salt thereof.