JPH05310785A - Hiv-related cyclic peptide and adsorption material obtained by immobilizing the same - Google Patents

Abstract

PURPOSE:To obtain an HIV-related cyclic peptide useful for diagnosing AIDS, searching a therapeutic agent for AIDS, etc., wherein amino acids at both ends are cystine, a human immune deficiency virus neutralized epitope domain, etc., are contained between the ends and both ends are subjected to intramolecular disulfide bond. CONSTITUTION:By a solid-phase synthetic method for peptide, a phenylacetamidomethyl resin bonded to C end amino acid is used as a solid-phase carrier, an amino acid containing protected alpha-amino group and side chain functional group is successively bonded with a condensation agent from the C end side according to amino acid sequence of peptide to synthesize a protected peptide resin. Deprotection and scission of peptide from a substrate resin are carried out by adding a solution containing trifluoroacetic acid to give a crudely purified peptide. Then the peptide is purified by a reversed phase column chromatography, oxidize with air for four days to give the objective cyclic peptide having an amino acid sequence of the formula wherein amino acids at both ends are cystine, a human immune deficiency virus neutralized epitope domain, etc., are contained between the ends and both ends are subjected to intramolecular disulfide bond.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cyclic peptide related to human immunodeficiency virus (HIV) which is a pathogenic virus of acquired immunodeficiency syndrome (AIDS), and an adsorbent on which the cyclic peptide is immobilized. INDUSTRIAL APPLICABILITY The peptides and adsorbents of the present invention are useful in the study of HIV and HIV-related cells, and in the search, development and production of AIDS preventive / therapeutic agents.

[0002]

BACKGROUND OF THE INVENTION The most effective neutralizing antibody that suppresses HIV infection in vitro is HIV.
Since the variable region of the envelope glycoprotein gp120 of -1 is recognized as an epitope (antigenic determinant), its neutralizing activity is effective only in a limited strain of virus.
It makes the development of IV vaccines difficult. Therefore, if a substance that is more specific than the neutralizing antibody and binds to the neutralizing epitope domain of all HIV mutants and suppresses the infection of the virus is found, the substance itself or the neutralizing epitope domain An anti-HIV effect due to a substance that inhibits the interaction of is expected.

Tryptase TL ₂ is known as a substance indirectly shown to interact with the neutralizing epitope domain (FEBS Lett. 286, 233-236 (1991)). TL ₂
Of the isolation method of HIV-1 by noting that the neutralizing epitope domain of HIV-1 has homology with trypstatin, and using trypstatin as an inhibitor of tryptase as an indicator of tryptase activity. Is the way
It requires complicated operations in multiple stages. Also, the anti-H of TL ₂
The IV effect has not yet been recognized.

As a neutralizing epitope of HIV, HIV-
1 (Human immunodeficiency virus type 1) HTLV
-III BH-10 strain, Sequence Listing, SEQ ID NO: 1 below
The 24 amino acid sequence shown by is known (Rusch
e, JR et al., Proc. Natl. Acad. Sci., USA, 85 , 31
98-3202 (1988)), a peptide fragment having the amino acid sequence of SEQ ID NO: 1 (hereinafter referred to as peptide (I))
Is already on the market (manufactured by Sigma, H789
4). However, peptide (I) does not have sufficient binding to a neutralizing antibody, and there is a problem in using it for searching a substance having affinity for a neutralizing epitope domain.

An object of the present invention is to provide a peptide similar to a neutralizing epitope, which has a high binding property with an HIV neutralizing antibody. Another object of the present invention is to provide a means for conveniently isolating and purifying a substance having affinity for a neutralizing epitope domain of HIV using the above peptide.

[0006]

The present inventors have found that HIV-
In the sequence of the envelope glycoprotein gp120 of No. 1, at all ends of the third variable region (V3 domain) containing the neutralizing epitope, all HIV-1s found so far were
It was noted that there are two cysteine residues conserved in the strain, and it was considered that the intramolecular disulfide bond by these cysteine residues is essential for the three-dimensional structure close to the natural neutralizing domain.

Therefore, an intramolecular disulfide-bridged cyclic peptide (V3 loop) having the amino acid sequence of the V3 domain was synthesized, and the cyclic peptide was more neutralizing antibody and HIV infectivity than conventional linear peptides. It has been found that it exhibits remarkably high binding properties to cells (host cells).
Furthermore, we succeeded in isolating and purifying neutralizing epitope domain affinity protein from cell membrane components of host cells using an adsorbent having the cyclic peptide immobilized thereon.

The gist of the present invention is as follows. A peptide having amino acids at both ends which are cysteines, and a neutralizing epitope domain of human immunodeficiency virus, or a virologically equivalent region between the both cysteines, wherein the cysteines at both ends are intramolecular disulfide bonds The cyclic peptide thus obtained. A cyclic peptide obtained by binding a linker to the above cyclic peptide. The above cyclic peptide labeled with a labeling substance. The above cyclic peptide bound to a protein. An adsorbent of a human immunodeficiency virus neutralizing epitope domain affinity substance, which is obtained by immobilizing the above cyclic peptide as a ligand on a support.

In the present invention, the neutralizing epitope domain of HIV is a region recognized by an antibody having a neutralizing activity against HIV, and the region contains an antigenic determinant in the induction of neutralizing antibody. Currently known neutralizing epitope domains are HIV envelope glycoproteins, especially HIV-
1 exists in gp120. Virus strains has been studied in detail neutralizing epitopes is HTLV-III _B-based, its amino acid sequence is already known (Rucshe,
JR et al., Proc. Natl. Acad. Sci., USA, 85 , 3198-
3202 (1988)).

The region corresponding to the virologically neutralizing epitope domain corresponds to the region on the amino acid sequence arranged so that the homologous amino acids are maximized in the virologically classified mutant strain group. Indicates the area to
Especially in HIV-1, HTLV-III BH-10
Since it is known that the neutralizing epitope domain of the strain is contained in the V3 region of gp120, and that the neutralizing monoclonal antibody of the MN strain is also present in the V3 region (S
cott, CF, Jr., et al., Proc. Natl. Acad. Sci., US
A, 87 , 8597-8601 (1990)), the neutralizing epitope domain of the HIV-1 strain in which neutralizing antibody has not been induced is considered to be contained in the V3 region.

In the cyclic peptide of the present invention, the cysteine linked by an intramolecular disulfide bond is preferably the cysteine located closest to both ends of the neutralizing epitope domain.

In the present invention, as long as the neutralizing epitope exists between the cysteines at both ends, the derived H
The IV mutant strain is not particularly limited. For example, H
TLV-III BH-10, MN, RF, Z3, LAV
V3 domains derived from HIV-1 strains such as _ELI . The amino acid sequences of the neutralizing epitope domain in various HIV-1 strains are shown in Table 1. In the present invention,
The neutralizing epitope domain includes these.

[0013]

[Table 1]

The blank in the table indicates that the amino acid residue corresponding to the HTLV-III BH10 strain does not exist, and "-"
Indicates that it has the same amino acid residue as that of the HTLV-III BH10 strain. The amino acid represented by each symbol is shown in Table 2.

[0015]

[Table 2]

The cyclic peptide of the present invention may be chemically modified, for example, one having one or more kinds of linkers additionally bound to the amino acid sequence of the neutralizing epitope domain. Particularly, those having a carboxyl group at both ends of the peptide, that is, those having an amino acid having a carboxyl group in the side chain (eg, glutamic acid, aspartic acid, etc.) bound to the amino terminal side are preferable for the convenience of chemical modification. ..

The cyclic peptide of the present invention may be labeled with a labeling substance. Examples of the labeled peptide include those labeled with a labeling substance such as a radioisotope, a fluorescent dye and biotin for specific labeling. Further, for the purpose of analysis and purification, it may be immobilized on an immobilization carrier such as a microplate, beads, or a chromatography carrier. Furthermore, a peptide or the like bound to a carrier protein for use as an antibody-producing antigen is exemplified.

The cyclic peptides of the present invention include not only free peptides but also salts thereof. Examples of salts of such peptides include acetates, citrates, organic acid salts such as succinates, hydrochlorides, inorganic acid salts such as phosphates, sodium salts,
Examples thereof include alkali metal salts such as potassium salts.

A preferred example of the cyclic peptide of the present invention is shown in FIG.
It is the cyclic peptide shown in (SEQ ID NO: 2) (provided that the two cysteines (Cys) at the 4th and 39th positions in SEQ ID NO: 2 are linked by an intramolecular disulfide bond. The peptide (I
I)). Peptide (II) has the same origin as peptide (I), gp120 of HTLV-III BH10 strain.
Amino acid sequence of the neutralizing domain of E. coli (Nature, 313, 277-284
(1985)) and cysteine residues located at both ends thereof and having an intramolecular disulfide bond, and further, three residues of linkers are respectively bonded from the amino terminal and the carboxyl terminal.

In peptide (II), an amino acid residue having a carboxyl group in the side chain is present in the amino acid sequence inside the cyclic structure formed by the intramolecular disulfide bond (from Thr at position 5 to His at position 38). do not do. Therefore, it is considered to be biologically important, by linking from the cysteine on the amino terminal side of the neutralization domain to the glutamic acid having a carboxyl group located on the amino terminal side of the three residues on the amino terminal side. These two carboxyl groups can be used as specific functional groups by extending the carboxyl terminal by 3 residues from the cysteine residue so that they can be chemically modified at a position some distance from the inside of the cyclic structure. As described above, the peptide (II) has a feature that it can be chemically modified by labeling or immobilization with the two carboxyl groups at both ends.

Another preferred example of the cyclic peptide of the present invention is
It is the cyclic peptide shown in FIG. 2 (SEQ ID NO: 3) (however, the two cysteines (Cys) at the 7th and 40th positions in SEQ ID NO: 3 are linked by an intramolecular disulfide bond. The cyclic peptide is referred to as peptide (III)). Peptide (III) is HIV-1 LA
Amino acid sequence of the neutralizing domain of gp120 of V _ELI strain
(Cell, 46 , 63-74 (1986)) and cysteine residues located at both ends thereof and having an intramolecular disulfide bond, and further having a linker bonded to the amino terminal and the carboxyl terminal.

The cyclic peptide of the present invention may be chemically one homogeneous peptide or a mixture thereof. If chemically one homogeneous peptide is desired,
A homogeneous peptide can be obtained by synthesizing by a chemical method. Such homogeneous peptides are particularly suitable for use as reagents.

The cyclic peptide of the present invention is produced by the decomposition of gp120, the peptide synthesis method and the modification method used in ordinary peptide chemistry. The peptide synthesis method may be either a solid phase method or a liquid phase method. The solid phase method is
For example, it is performed as follows. That is, first, a carboxyl-terminal amino acid whose amino group is protected is bound to an insoluble carrier by the carboxyl group. As the insoluble carrier, those known per se may be used. Then, after removing the amino-protecting group, amino acids protected with amino groups are sequentially peptide-bonded according to the amino acid sequence and reacted one step at a time to synthesize the entire amino acid sequence, and then the insoluble carrier is removed. At this time, the reactive functional group is preferably protected by a protecting group known in peptide synthesis.

The peptide thus produced can be purified to an arbitrary purity by a purification method usually used in peptide chemistry such as ion exchange resin, partition chromatography, gel filtration method, reverse phase chromatography and the like. .. An intramolecular disulfide bond is formed by subjecting the purified peptide to an oxidation reaction in a dilute solution. The oxidation reaction is preferably performed under mild conditions such as air oxidation and an enzymatic method. The formation of disulfide bonds can be confirmed by quantifying the remaining thiol groups. The peptide produced as described above can be converted into a salt by a method known per se, and a free peptide is produced by hydrolyzing the salt by a method known per se.

The above-mentioned cyclic peptide or a chemically modified form thereof is used as a ligand in the adsorbent for an HIV neutralizing epitope domain affinity substance of the present invention. Examples of the support for immobilizing the ligand include cellulose, dextran, agarose, polyacrylamide, and porous glass that are commonly used in affinity chromatography, or membrane filters such as ultrafiltration membranes and microfiltration membranes. ..

As a method for binding the ligand and the support, as a covalent bonding method, a method using a cyanogen bromide-activated or epoxide-activated support, or a ω-carboxylalkyl derivative, ω-aminoalkyl derivative, bromo is used. A method using a support having a spacer such as an acetyl derivative or a hydrazide derivative is exemplified, and a specific binding method is a method of binding a ligand bound to biotin to a support via avidin bound to the support. Are exemplified.

By using the adsorbent of the present invention, H
A substance having an affinity for the IV neutralizing epitope domain can be conveniently isolated and purified. Neutralizing epitope domains derived from different virus mutants may be used in combination of two or more adsorbents each having a cyclic peptide as a ligand for isolation and purification. Usually, neutralizing epitope domains derived from virus mutants with low homology It is desirable to purify by sequentially using an adsorbent having a cyclic peptide as a ligand. HI
The V-neutralizing epitope domain-binding substance can be purified to a desired purity by changing the adsorption condition to the adsorbent and the elution condition from the adsorbent. Factors that change the above conditions include ionic strength with sodium chloride and the like, pH of the buffer solution, and the like.

By combining the affinity chromatography using the adsorbent of the present invention with other separation methods usually used in the art, a substance having a higher degree of purification can be obtained. Other separation methods include salting out, treatment with an organic solvent, p
Fractionation method by solubility difference such as H treatment and water-soluble polymer treatment, fractionation method by chromatography such as ion exchange, hydrophobicity, gel filtration, density gradient, polyacrylamide gel, isoelectric point, two-dimensional electrophoresis, etc. The law is exemplified.

The crude materials used for the isolation and purification of the HIV neutralizing epitope domain affinity substance include human cell membrane-derived proteins, natural compounds derived from animals and plants, and microorganisms.

A preferred example of the purification method using the adsorbent of the present invention is a peptide (II) obtained by using a crude extract of a cell membrane-solubilized cell culture of a cultured cell confirmed to be an HIV host cell as a crude material. Alternatively, it is a method of purification using two kinds of affinity columns packed with an adsorbent on which peptide (III) is immobilized as a ligand. Specifically, the crude cell membrane extract of host cells is desalted, adsorbed on a peptide (III) -immobilized column, washed with a low salt concentration buffer solution, and then the bound protein is eluted with a high salt concentration buffer solution. Let Further, the eluate is desalted, adsorbed on a peptide (II) -immobilized column, washed and eluted in the same manner as the peptide (III) -immobilized column to obtain a neutralizing epitope domain binding protein.

In the present specification, amino acids, peptides,
When the abbreviations for protecting groups are indicated, they are IUPAC-
It is based on the abbreviations by the IUB Commission on Biological Nomenclature or the abbreviations commonly used in this field.

[0032]

The cyclic peptide of the present invention exhibits a higher binding ability to a neutralizing antibody or a substance that recognizes a neutralizing epitope domain than conventional linear peptides. By using this cyclic peptide, a neutralizing epitope domain affinity substance can be easily isolated and purified.

Neutralizing antibodies are specific to virus strains and are difficult to use as vaccines, but since infectivity is suppressed by binding of antibodies to neutralizing epitope domains, this region is infected with HIV. Anti-HIV effect is expected for a substance that plays an important role in the above and has affinity for the neutralizing epitope domain. The cyclic peptide-immobilized adsorbent of the present invention can be used for searching anti-HIV agents.

It is expected that there will be molecules on the host cell surface that have an affinity for the neutralizing epitope domain of all HIV strains. By preparing an affinity column on which the cyclic peptide of the present invention was immobilized and separating the membrane-solubilized extract of host cells with the column, a purified protein was obtained until one major band was confirmed. ..

In addition to the above, the cyclic peptide of the present invention is used in studies on HIV and HIV-related cells, and AID.
It is useful in the search, development, and production of a preventive / therapeutic drug for S, and is also expected to be used as a hapten for antibody production.

[0036]

EXAMPLES The present invention will be specifically described below with reference to examples, but these do not limit the present invention in any way. Example 1 Peptide (III) was synthesized. The amino acids used in the following synthesis examples are all L-forms. Peptide synthesis was performed by the solid phase method using Peptide Synthesizer 430A manufactured by Applied Biosystems. 0.
5 mmol of t-butyloxycarbonyl (t-Boc) -L-
To the serine-bonded phenylacetamidomethyl resin, 2 mmol of t-Boc amino acid whose amino terminal was protected with t-Boc was added.
Condensation was performed in N, N-dimethylformamide by the symmetrical amino acid anhydride method using 1 mmol N, N-dicyclohexylcarbodiimide, and the peptide bond was extended one by one. However, as for the condensation of Asn, Gln, and Arg, the activated ester method using 1 mmol of hydroxybenzotriazole in N, N-dimethylformamide was used to conduct the condensation reaction.
It condensed once. As a side chain protecting group of t-Boc amino acid,
Glu is benzyl ester group, Cys is 4-methoxybenzyl group, Thr and Ser is benzyl group, Arg is mesitylene-2-sulfonyl group, Lys is 2-chlorobenzyloxycarbonyl group, and His is 2,4-dinitrophenyl group. In addition, Met used a methionine sulfoxide group.

The standard peptide synthesis program is 60% trifluoroacetic acid / dichloromethane for 1 minute or 15 minutes for deprotection, 3 times for 1 minute with dichloromethane for washing, 10% N, N-diisopropylethylamine for neutralization. The symmetric amino acid anhydride method using N, N-dicyclohexylcarbodiimide in N, N-dimethylformamide as a condensation for 20 minutes and as a wash for 2 minutes Perform 1 minute twice with N, N-dimethylformamide and 4 times with dichloromethane. In these standard synthesis programs, the reaction temperature and the reaction time are precisely controlled by a microcomputer for each amino acid.

The peptide resin obtained by the peptide automatic synthesizer was deprotected from the amino acid side chain and cleaved from the support resin. Deprotection of the His side chain was carried out by adding 5 ml of N, N-dimethylformamide and 20 molar equivalents of thiophenol to 1 g of synthetic peptide resin at room temperature for 4 hours.
It was stirred for an hour. It was washed with N, N-dimethylformamide, distilled water, ethanol and dichloromethane in this order on a glass filter and dried. Furthermore, for deprotection of other side chains and cleavage of the synthetic peptide from the support resin,
The above dried resin was stirred in 1 ml of thioanisole and 0.5 ml of ethanedithiol at room temperature for 10 minutes, and 10 ml of trifluoroacetic acid was added while cooling the flask in ice.
After stirring for 0 minutes, 1 ml of trifluoromethanesulfonic acid was added, and the mixture was stirred at room temperature for 30 minutes. Cold diethyl ether was added to the flask until precipitation of the peptide stopped, and after stirring for 1 minute, the crystals were filtered with a Teflon filter, and the crystals were dissolved with a small amount of trifluoroacetic acid and recrystallized to obtain a crude purified peptide. ..

The crude peptide was used as a reverse phase column (diameter 2c) consisting of a silica gel packing having an octadecyl group bonded thereto.
m, length 25 cm; YMC R-ODS-5). Elution was carried out at a flow rate of 5 ml / min with 90% acetonitrile 30-80 using 0.1% trifluoroacetic acid as a mobile phase.
% Linear gradient.

About 1 nmole of the purified linear peptide was analyzed by a gas phase protein sequencer, and it was confirmed that the peptide had the desired amino acid sequence.
Intramolecular disulfide cross-linking by two Cys side chains present in the peptide was carried out by neutralizing the fraction purified by the reverse phase column with aqueous ammonia and air-oxidizing the mixture at 4 ° C. with stirring. The progress of the binding reaction was performed by quantifying the remaining thiol groups. The thiol group was quantified by a known amount of Cys as a standard by fluorescence analysis using 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid. Four days of air oxidation reduced the thiol groups to about 5% before oxidation. After freeze-drying, in order to remove contaminating ammonium salts and the like, repurification was carried out by a reverse-phase column by the method described above. It was confirmed that the retention time of the peak did not change before and after the oxidation and that no intermolecular crosslink was formed.

Example 2 The peptide (III) obtained in Example 1 was labeled with biotin. In peptide (III), since there is no amino acid residue having a carboxyl group in the side chain in the amino acid sequence inside the cyclic structure formed by the intramolecular disulfide bond (from Ala at position 8 to His at position 39), Peptide (III) was biotinylated using the Glu side chain at the amino terminal and the two carboxyl groups at the carboxyl terminal of the neutralization domain. That is, 10 mg of peptide (III) and 50 mg
Biotin (long arm) hydrazide (VECTOR
(Manufactured by the company) is dissolved in 3 ml of N, N-dimethylformamide, and 5
400 mg dissolved in 150 ml of 150 mM sodium chloride solution
1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added to adjust the pH to 5.5, and the mixture was reacted at room temperature for 18 hours. The reaction solution was filtered and then desalted and purified using a reverse phase column in the same manner as in the purification method of peptide (III) to obtain biotinylated peptide (III).

Example 3 Biotinylated peptide (III) was immobilized on a support to prepare a peptide (III) -immobilized column. About 1 ml of agarose gel with immobilized avidin (manufactured by PIERCE) was packed in a polypropylene mini column, washed with 5 ml of phosphate buffered saline (PBS), and dissolved in 5 ml of PBS to prepare 1 mg of biotinylated peptide. (III) was added, and the mixture was reacted with shaking at room temperature for 1 hour while the column was closed. Unreacted substances were washed with 10 ml of PBS to obtain a peptide (III) -immobilized column.

Example 4 Peptide (II) was synthesized in the same manner as in Example 1. However, since peptide (II) contained Met, deprotection of the side chain was added. That is, after deprotecting the His side chain, 1 ml of m-cresol, 3 ml of methyl sulfide, 5 ml of trifluoroacetic acid, and 1 ml of trifluoromethanesulfonic acid were added to the dried peptide resin,
The reaction was carried out for 3 hours while maintaining -5 to 0 ° C. The peptide resin was washed on a glass filter with cold diethyl ether and after drying, the other side chains were deprotected and the synthetic peptide was cleaved from the support resin. The change in relative content of residual thiol groups during air oxidation is shown in FIG. The chromatogram of the purified peptide (II) is shown in FIG. The yield of the purified sample was 80 mg.

Example 5 A peptide (II) -immobilized column was prepared in the same manner as in Examples 2 and 3. However, streptavidin-immobilized agarose gel (manufactured by PIERCE) was used as a support in order to minimize non-specific adsorption to the support.

Example 6 Peptide (II) was immobilized on a 96-well microplate,
It analyzed by the ELISA method. 0.31-10 μM peptide (II) was adsorbed on a microplate, and HTLV
-III BH-10 strain neutralizing monoclonal antibody 0.5β (J. Virol. 62 , 2107-2114, (1988)) was bound, and after binding with alkaline phosphatase-labeled anti-mouse IgG antibody, a substrate was added. The amount of enzyme reaction one hour after was determined by the absorbance at 405 nm (FIG. 5).

Example 7 Peptide (II) was bound to a carrier protein to serve as an antigen for preparing anti-peptide (II) antibody. Peptide (II)
And 3 mg of egg albumin are dissolved in 1 ml of distilled water, 30 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added, and the mixture is reacted overnight at room temperature. It was sufficiently dialyzed against physiological saline and used as an antibody-producing antigen.

[0047]

[Experimental example]

Experimental Example 1 According to Example 2, peptides (I), (II) and commercially available insulin B chain were each labeled with biotin to give HTLV-III B.
The antigenicity against the neutralizing monoclonal antibody 0.5β against the H-10 strain was compared using the ELISA method. A biotin-labeled peptide was bound to 5 μg / ml of 0.5β adsorbed on a 96-well microplate, and alkaline phosphatase-labeled avidin was further bound, and the enzyme reaction amount after 10 minutes from the addition of the substrate was 405 nm. Determined by absorbance. As shown in FIG. 6, the peptide (II)
0.5 compared to peptide (I) and insulin B chain
It showed extremely high antigenicity to β, and was confirmed to maintain a three-dimensional structure close to that of nature.

Experimental Example 2 Biotin-labeled peptides (I) and (II) used in Experimental Example 1
And the binding properties of insulin B chain to the T cell surface were compared. That is, M which is a T cell line highly infectious to HIV
olt-4 clone 8 cells (J. Virol. 57 , 1159-116
2 (1986)) to the membrane surface was analyzed by flow cytometry. 2 × 10 ⁵ cells were washed twice with a phosphate buffered saline containing 0.1% bovine serum albumin and 0.01% sodium azide (Solution A), and then suspended in 50 μl of Solution A. Add 10 μl of biotin-labeled peptide diluted with the solution, leave it on ice for 30 minutes, and then add 2 cells with solution A.
After washing twice, 50 μl of fluorescein isothiocyanate-labeled avidin was added, and the cells were allowed to stand on ice for 30 minutes.
The cells were washed twice with the liquid and subjected to flow cytometry analysis. As the negative control cells, cells obtained by reacting only the fluorescently labeled avidin were used. As shown in FIG. 7, the positive cell rate when the concentration of the biotin-labeled peptide was changed showed a sigmoid-type increase curve at low concentration in peptide (II), confirming high specificity.

Experimental Example 3 Using a peptide (III) -immobilized column and a peptide (II) -immobilized column, an HIV neutralizing epitope domain binding protein was purified from a cell membrane-solubilized crude extract of human lymphocyte-cultured cells.

The cell membrane fraction of lymphocytes was prepared by partially modifying the method of Klempner et al. (J. Cell Biol., 86 , 21-28 (1980)). That is, 1 × 10 8 Molt-4 clone 8 cells (described above), which is a T cell line highly infectious to HIV.
15 ml after washing ¹⁰ pieces twice with Hank's solution (pH 7.4)
Was suspended in Hank's solution (pH 7.4) containing 2.5 mM MgCl ₂ but containing no calcium and homogenized for 30 strokes with a Dounce homogenizer. EDTA was added to the homogenate so that the final concentration was 2.5 mM, and the mixture was centrifuged at 1.5 x 10 ⁴ x g-min at 4 ° C to remove precipitated nuclei and undisrupted cells. The supernatant is 4 x 10 ⁵ x g-
The mixture was centrifuged at 4 ° C. for 4 minutes to remove precipitated mitochondria and lysosomes. The supernatant is further added at 6 × 10 ⁷ × g-min at 4 ° C.
The supernatant was removed from the cytoplasm to obtain a fraction containing endoplasmic reticulum and cell membrane. The precipitate was separated by dextran density gradient centrifugation. The precipitate containing 0.1 mM MgCl ₂ 1
Resuspend in mMHEPES buffer (pH 8.2), overlay on dextran density gradient (specific gravity 1.00 to 1.09), and centrifuge at 10 ⁸ × g-min at 4 ° C during the continuous density gradient. The portion in which a layer was formed was collected as a cell membrane fraction. The obtained membrane fraction was added with 50 mM n-heptyl-β-D-thioglucoside, 10 mM EDTA, 10 mM EGT.
2.5 ml of a Tris-hydrochloric acid buffer solution (pH 7.5) containing A was added and stirred at 4 ° C. for 1 hour to solubilize the membrane protein. After centrifuging the sample at 4 x 10 ⁵ x g-min at 4 ° C, the supernatant was added to a 20 mM phosphate buffer solution (pH 7.3; P containing low concentration NaCl).
It was desalted with a gel filtration column (PD-10; manufactured by Pharmacia) equilibrated with B) to obtain a cell membrane-solubilized crude extract.

The crude extract was adsorbed to a peptide (III) -immobilized column which had been washed with PB containing a low concentration of NaCl in advance, and the non-adsorbed substance was washed with 10 ml of the same PB, and then 2.5 ml
The peptide (III) affinity protein was eluted with PB containing a high concentration of NaCl. Further, this eluate was desalted on a PD-10 column equilibrated with PB, and then adsorbed on a peptide (II) -immobilized column washed with PB, and washed in the same manner as the peptide (III) -immobilized column. -Elution was performed.

The thus obtained peptide (III) -immobilized column and peptide (II) -immobilized column eluate were subjected to SD.
When the protein was detected by silver staining after separation by S-PAGE (20% gel), one band commonly bound to two types of columns was confirmed as shown in FIG. Lane 1: Cell membrane solubilized crude extract Lane 2: Peptide (III) -immobilized column eluent Lane 3: Peptide (III) and peptide (II) immobilized column eluent Lane 4: Lane 3 Under non-reducing conditions Migration

[0053]

[Sequence list]

SEQ ID NO: 1 Sequence length: 24 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin organism name: HIV-1 (Human immunodeficiency virus t)
ype 1) Strain name: HTLV-III BH10 Tissue type: Envelope sequence Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala 1 5 10 15 Phe Val Thr Ile Gly Lys Ile Gly 20 SEQ ID NO: 2 Sequence length: 42 Sequence type: Amino acid Topology: Both forms Sequence type: Peptide Origin organism name: HIV-1 (Human immunodeficiency virus t
ype 1) Strain name: HTLV-III BH10 Tissue type: Coat sequence characteristics Cys 4th and 39th form a disulfide bond. Sequence Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg 1 5 10 15 Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys Ile Gly 20 25 30 Asn Met Arg Gln Ala His Cys Asn Ile Ser 35 40 SEQ ID NO: 3 Sequence length: 43 Sequence type: Amino acid Topology: Both forms Sequence type: Peptide Origin organism name: HIV-1 (Human immunodeficiency virus t
ype 1) Strain name: LAV ELI Tissue type: Coat sequence characteristics The 7th and 40th Cys form a disulfide bond. Sequence Glu Ser Val Lys Ile Thr Cys Ala Arg Pro Tyr Gln Asn Thr Arg Gln 1 5 10 15 Arg Thr Pro Ile Gly Leu Gly Gln Ser Leu Tyr Thr Thr Arg Ser Arg 20 25 30 Ser Ile Ile Gly Gln Ala His Cys Asn Ile Ser 35 40

[Brief description of drawings]

FIG. 1 is the amino acid sequence of peptide (II).

FIG. 2 is the amino acid sequence of peptide (III).

FIG. 3 is a graph showing the change over time in the relative content of thiol groups remaining in the peptide (II) precursor during air oxidation.

FIG. 4 is a chromatogram of a purified peptide (II) preparation.

FIG. 5: Peptide (II) immobilized on a microplate
It is a graph which shows the antigenicity of.

FIG. 6 is a graph showing the binding property to 0.5β measured by an ELISA method in Experimental Example 1.

FIG. 7 is a graph showing the binding properties to Molt-4 clone 8 cells measured by flow cytometry in Experimental Example 2.

FIG. 8: SDS-PAG of the protein purified in Experimental Example 3
It is the figure which showed the separation by E and the detection by silver staining.

[Procedure amendment]

[Submission date] April 2, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Figure 8

[Correction method] Change

[Correction content]

FIG. 8 is an electrophoretic photograph of the protein purified in Example 3.

Claims (6)

[Claims] 1. An amino acid at both ends is cysteine,
A cyclic peptide comprising a neutralizing epitope domain of human immunodeficiency virus or a virologically equivalent region between the cysteines at both ends, wherein the cysteines at both ends are intramolecularly disulfide-bonded. 2. The cyclic peptide according to claim 1, wherein the cysteine linked by an intramolecular disulfide bond is a cysteine closest to both ends of the neutralizing epitope domain of immunodeficiency virus. 3. A cyclic peptide obtained by binding a linker to the cyclic peptide according to claim 1 or 2. 4. The cyclic peptide according to claim 1, which is labeled with a labeling substance. 5. The cyclic peptide according to any one of claims 1 to 4, which is bound to a protein. 6. An adsorbent of a human immunodeficiency virus neutralizing epitope domain affinity substance, which comprises the cyclic peptide according to any one of claims 1 to 4 immobilized on a support as a ligand.