RU2304146C2 - Method for stabilizing loop-shaped structure of synthetic peptides using n- and c-terminal modification - Google Patents

Abstract

FIELD: organic chemistry, peptides.

SUBSTANCE: invention relates to technology used for stabilization of loop-shaped structure of synthetic peptides. Invention proposes a method for stabilization of loop-shaped structure of synthetic peptides by noncovalent interaction of hydrophobic hydrocarbons or their radicals between them wherein hydrophobic hydrocarbons or their radicals are arranged at opposite N- and C-ends of peptide, or in immediate vicinity from their, and these hydrophobic hydrocarbons or their radicals are bound with a hydrophobic carrier noncovalently also. Invention provides stabilizing loop-shaped peptide structures without changes of amino acid sequence of peptide and possibility for noncovalent binding the stabilized synthetic peptide antigen with a carrier. Proposed method shows universality and can be used for stabilizing different structures. Invention can be used in pharmacology for preparing preparations of a novel generation and designated for diagnosis, therapy and vaccine prophylaxis of infectious diseases and pathological states in humans and animals.

EFFECT: improved stabilizing method of peptides.

9 cl, 3 dwg, 2 tbl, 6 ex

Description

The invention relates to stabilization technologies for bends and loop-like structures of synthetic peptides and can be used in pharmacology for the production of a new generation of drugs intended for the diagnosis, therapy and vaccination of infectious diseases and pathological conditions of humans and animals.

Synthetic peptide antigens are increasingly being used as key components of qualitatively new drugs for diagnosis, therapy and vaccination, since they lack the disadvantages characteristic of traditionally used recombinant and natural antigens, such as pathogenicity, residual virulence, incomplete inactivation, pyrogenicity, concomitant non-specific antigens and etc. Nevertheless, synthetic peptides have a number of specific features that impede their successful use in the diagnosis, therapy and vaccination, namely:

1. Low molecular weight synthetic peptides that are not conjugated to a carrier are generally not able to elicit an immune response [Hermanson GT, Mallia AK, Smith PK. Immobilized Affinity Ligand Techniques San Diego: Academic Press; 1992].

2. Low molecular weight synthetic peptides require special sorption conditions on the surface of a carrier used in immunodiagnostic systems [Gomara MJ, Riedemann S., Vega I., Ibarra H., Ercilla G., Haro I. “Use of linear and multiple antigenic peptides in the immunodiagnosis of acute hepatitis A virus infection "// J. Immunological Methods 234, 23-34; 2000].

3. Low molecular weight synthetic peptides, as a rule, are not able to form a stable conformation similar to that of fragments of a native antigen imitated by peptides [Rizo J, Gierasch LM. Constrained Peptides: Models of Bioactive Peptides and Protein Substructures. // Annual Review of Biochemistry Vol. 61: 387-416; 1992].

To improve the immunological characteristics of peptide antigens, they are conjugated to any high molecular weight carrier. Natural and recombinant proteins are used as conjugates [Wong SS: Chemistry of Protein Conjugation and Crosslinking. Boca Raton: CRC Press; 1991], and non-protein carriers: microparticles, liposomes, virus-like particles, etc. [Tam JP, Spetzler JC: Multiple Antigen Peptide System. Methods Enzymol. 289: 612-636; 1997]. Peptide antigens are conjugated to a carrier through the formation of both covalent bonds and non-covalent interactions. In order to increase the efficiency of conjugation based on non-covalent interactions with modern carriers (microparticles, liposomes), synthetic peptides are modified with hydrophobic hydrocarbons having a high affinity for the hydrophobic parts of microparticles and liposomes [Haro I, Perez S, Garcia M, Chan WC, Ercilla G. “Liposome entrapment and immunogenic studies of a synthetic lipophilic multiple antigenic peptide bearing VP1 and VP3 domains of the hepatitis A virus: a robust method for vaccine design” // FEBS Lett. Apr. 10; 540 (1-3): 133-40; 2003].

In order to increase the efficiency of sorption of peptide antigens on the surfaces of carriers used in immunodiagnostic systems, the surfaces of hydrophobic polymers are modified in various ways. Either they modify the peptide antigen itself, binding it covalently with the surface of the carrier, or with a high molecular weight structure that can efficiently adsorb on the surfaces of carriers used in immunodiagnostic systems [Gomara MJ, Riedemann S, Vega I, Ibarra H, Ercilla G, Haro I. “Use of linear and multiple antigenic peptides in the immunodiagnosis of acute hepatitis A virus infection "// J. Immunological Methods 234, 23-34; 2000].

Synthetic peptides used in pharmacology typically mimic surface fragments of native antigens. The surface of native protein antigens contains a large number of bends and loop-shaped areas [Kuntz ID. "Protein-Folding" // J. Amer. Chem. Soc. 94 4009 (1972)]. Synthetic peptides, as a rule, are not able to form any stable conformation in solution, which leads to a loss in the affinity of binding of anti-peptide antibodies to the native antigen and antibodies from native antigens to synthetic peptides and, in turn, greatly reduces the activity of peptide-based substances antigens.

A known method of stabilizing a secondary loop-like structure using a disulfide bond [Broun JR. "Struktural origins of mammalian albumin" // Fed. Proc 35 2141 (1976)]. In this case, by including the N- and C-terminal cysteines in the peptide, followed by closure of the disulfide bond, stable loop-like structures can be obtained. To implement this method, the amino acid cysteine is included in the sequence of the target peptide at the N and C ends. The amino acid cysteine can be included in the sequence, either directly terminal or localized in the immediate vicinity of the terminal amino acids. At the final stage of the formation of the cyclic peptide, thiol groups of cysteines are oxidized, which leads to the formation of an intramolecular disulfide bond.

This stabilization method has the following disadvantages: there is a high probability of the formation of non-target intermolecular disulfide bonds; restrictions are imposed on the amino acid composition of the target peptide, since the structure of the peptide should not contain internal cysteines, otherwise the formation of non-target intramolecular disulfide bonds is possible; the peptide thus obtained is not a ready-made precursor for binding to the carrier.

There is also a method for stabilizing beta bends of short peptides, which is based on the tendency of amino acid sequences to form certain secondary structures of the peptide chain stabilized by intramolecular non-covalent interactions (hydrogen bonds, hydrophobic interactions, salt bridges). Hydrophobic interactions under normal conditions in the aquatic environment are the strongest of all non-covalent interactions. One example of stabilization of beta bends in short peptides is the tryptophan zipper [A.G. Cochran, N.J. Skelton, M.A. Starovasnik "Triptophan zippers: stable, monomeric β-hairpins". PNAS, 98, (10), p. 5578-5583; 2001]. The stabilization of beta bending is caused by hydrophobic interactions of the side groups of pairs of tryptophan molecules located symmetrically with respect to the center of bending. This method is adopted as a prototype of the invention.

The prototype has the following disadvantages:

- stabilization of the bend of the peptide chain often requires intervention in the amino acid sequence of the peptide, which can lead to loss of affinity of antigen-antibody complexes;

- the resulting peptide is not a ready-made precursor for binding to a carrier;

- the method is suitable only for stabilization of beta bends.

The invention solves the problem of creating a method for stabilizing the bends and loop-like structures of peptides, which does not affect the amino acid sequence of the peptide, as well as creating peptides that are a ready-made precursor for binding them to carriers, while being universal, suitable for stabilizing the bends and loop-like structures of peptides having an arbitrary amino acid sequence.

The problem is solved in that a method is proposed for stabilizing the secondary loop-like structure of synthetic peptides by non-covalent interaction of hydrophobic hydrocarbons or their radicals with each other, according to which hydrophobic hydrocarbons or their radicals are located on opposite N and C ends of the peptide, wherein said hydrophobic hydrocarbons or their radicals are also non-covalently bound to a hydrophobic carrier.

When covalently bonds at least two strongly hydrophobic hydrocarbon radicals located at opposite N and C ends of the peptide chain, the peptide chain bends due to the convergence of its N and C ends and the formation of non-covalent interaction between the N and C terminal hydrophobic hydrocarbon radicals, as well as non-covalent interaction of the aforementioned hydrophobic hydrocarbon radicals with a hydrophobic carrier.

Hydrophobic hydrocarbon radicals can be covalently linked to or in close proximity to the reactive groups of the peptide contained at its opposite N and C ends, the reactive groups of the peptide being carboxyl groups, or amide groups, or thiol groups, or other reactive groups capable of to provide selective covalent binding of hydrophobic hydrocarbon radicals to the peptide molecule.

The carrier can be made of polymer, it can be a high molecular weight structure of amphipathic molecules, and other solutions are also possible.

The hydrophobic hydrocarbon radicals attached to the ends of the peptides may be aliphatic hydrocarbons or aromatic hydrocarbons.

On figa shows the amino acid sequence of the peptide, designated here as peptide No. 1, corresponding to a fragment of 89-119 protein E of tick-borne encephalitis virus (strain 205). The Cys amino acids at positions 92 and 116 are marked with an asterisk, and the peptide was modified according to their thiol groups.

On figb shows a schematic representation of the spatial structure of peptide No. 1.

Figure 1B shows the spatial structure of a fragment of 89-119 protein E of tick-borne encephalitis virus, corresponding to the native one obtained by X-ray diffraction analysis of a single crystal protein [Rey FA, Heinz FX, Mandl C, Kunz C, Harrison SC. “The envelope glycoprotein from tick-bome encephalitis virus at 2 A resolution.” // Nature. May 25; 375 (6529): 291-8; 1995].

On figa shows the primary structure of the peptide designated here No. 1-M - modified palmitic acid peptide No. 1. Asterisks mark Cys amino acids modified at palmitic acid at positions 92 and 116.

On figb schematically shows the spatial structure of the peptide No. 1-M.

Figure 3 schematically shows the spontaneous process of stabilization of the loop-like structure of peptide No. 1-M, similar to the native structure of fragment 89-119 of protein E of tick-borne encephalitis virus. The spontaneity of the process is provided by reducing the contact area of hydrophobic radicals with water [Chothia C. "Hydrophobic bonding and accessible surface area in proteins." // Nature. Mar 22; 248 (446): 338-9; 1974].

The synthetic peptide is modified by covalent binding of derivatives of hydrophobic hydrocarbons to carboxyl, amide or thiol groups contained at the N and C ends of the peptide (Example 1). A prerequisite for such a modification is the formation of a pair of highly hydrophobic hydrocarbon radicals at opposite ends of the peptide chain, as shown in FIG. Such a modification, on the one hand, leads to the formation of a non-covalent intramolecular bond between the N- and C-ends of the peptide, and, on the other hand, allows non-covalent binding of the obtained modified peptide to a hydrophobic carrier, as shown in FIG. 3. In the proposed method, stabilization of the bends or loop-like structures of the peptide occurs due to the introduction of additional terminal molecules capable of making an intramolecular bond, which is confirmed by the high affinity of the antipeptide No. 1-M antibodies to the native antigen (Example 6), and the synthetic peptide is bound to hydrophobic carriers behind account of non-covalent interactions (example 3).

Thus, the proposed method has the following advantages:

- the possibility of stabilization of both the bends of the peptide chain and loop-like structures due to non-covalent interactions;

- the possibility of binding the peptide to hydrophobic carriers and surfaces;

- the ability to form high molecular weight structures (micelles) without the participation of additional high molecular weight carriers.

The following are examples revealing the essence of the invention.

Example 1. Solid-phase synthesis of peptide No. 1.

The amino acid sequence of peptide No. 1 is shown in FIG. 1A - it corresponds to a fragment of 89-119 protein E of tick-borne encephalitis virus. This fragment 89-119 in the native protein E of tick-borne encephalitis virus is a loop-shaped bend, as shown in figv. The spatial structure of native protein E of tick-borne encephalitis virus was obtained using X-ray diffraction analysis of a protein single crystal [Rey FA, Heinz FX, Mandl C, Kunz C, Harrison SC. “The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution” // Nature. May 25; 375 (6529): 291-8; 1995]. A schematic representation of the spatial structure of peptide No. 1 is shown in FIG. 1B.

The synthesis of the peptide, the amino acid sequence of which simulates a loop-like fragment 89-119 of the surface of the protein E virus of tick-borne encephalitis, was carried out by manual solid-phase method on 4-hydroxymethylphenoxymethyl styrene-divinylbenzene (1%) copolymer (Wang resin) [Albericio F, Barany G. “Application of N , N-dimethylformamide dineopentyl acetal for efficient anchoring of N alpha-9-fluorenylmethyloxycarbonylamino acids as p-alkoxybenzyl esters in solid-phase peptide synthesis ”// Int J Pept Protein Res. Apr; 23 (4): 342-9; 1984]. A standard 10 ml polypropylene syringe equipped with a porous polypropylene plate was used as a reactor. The peptide was synthesized by the Nsc / tBu method [Sabirov AN, Kirn YD, Kim HJ, Samukov VV. “FMOC- and NSC-groups as a base labile N (alpha) -amino protection: A comparative study in the automated SPPS.” // Protein Peptide Lett. 4, 307-312; 1997]. Nγ-Trt for Asn and Gln were used as permanent protecting groups; Nim-Trt for His; S-Trt for Cys; tBu for Thr and Ser; tBu ether for Asp; NG-Pbf for Arg; Nε-Boc for Lys. To add the first amino acid, 200 mg of the polymer, 6 ml of dichloroethane, 2 ml of N-methylpyrrolidone, 0.4 mmol of Nsc-Ala-OH, 1.0 mmol of dicyclohexylcarbodiimide and 0.1 mmol of 4-dimethylaminopyridine were stirred for 24 hours. The resulting aminoacyl polymer was washed DMF, methanol, ether and chloroform were dried in vacuo. Yield 220 mg, amino group content 500 μmol / g in the picric acid test [Gisin BF. “The monitoring of reactions in solid-phase peptide synthesis with picric acid.” // Anal Chim Acta. 1972 Jan; 58 (1): 248-9]. Peptide chain growth was performed by single condensations. At the end of the assembly, the N-terminal Nsc-protection was removed from the peptide, the peptidyl polymer (1.4 g) was washed with chloroform, methanol, ether and dried in vacuum. The resulting peptidyl polymer was treated for 60 min with a mixture of 4 ml of TFA, 0.4 ml of thioanisole, 0.1 ml of ethanedithiol and 0.1 ml of meta-cresol, the polymer was separated by filtration. The peptide was precipitated from the filtrate with a large volume of cold ether. The crude peptide precipitate was filtered off, washed with ether and dried in vacuo. The yield of crude peptide is 150 mg. The peptide was purified by semi-preparative reverse-phase HPLC using a Pharmacia-LKB 2249 chromatograph (Sweden): analytical - on a 4 × 150 mm column with a Lichrosorb RP18 sorbent, 5 μm (Merck) at a flow rate of 1 ml / min in a linear gradient of acetonitrile concentration ( 0-80%) in water with 0.5% H ₃ PO ₄ ; preparative - on a 10 × 250 mm column with a SynChropak RPP 100 phase, 10 μm (Eichrom Ind.), at a flow rate of 4 ml / min in a linear gradient of acetonitrile concentration (0-66%) in water with 0.1% trifluoroacetic acid. Detection was carried out spectrophotometrically by absorbance at 226 nm with processing of the results using the MultiChrom system (Ampersand). The fractions containing the basic substance were evaporated in vacuo with isopropanol and lyophilized from water containing 10% acetic acid. Yield 42 mg (13%, for TFA salt with mv 3250). The content of SH-groups by reaction with 2,2'-dipyridyldisulfide 0.58 μmol / mg (94.5%). Using preparative HPLC, a major product with a purity of about 85% was isolated, the identity of which was proved by amino acid analysis. Elemental analysis was performed on a Perkin-Elmer CHN analyzer, and amino acid analysis on a Biotronik LC5001 instrument after hydrolysis of the sample in 6 N HCl, 110 °, 24 h. Quantitative determination of sulfhydryl groups was carried out using a test with 2,2'-dipyridyldisulfide (Carlsson et al., 1978), qualitatively by reaction with sodium nitroprusside.

Thus, a peptide was prepared whose amino acid sequence corresponds to a fragment of 89-119 protein E of tick-borne encephalitis virus. This peptide is a precursor for the preparation of a palmitic amino acid modified peptide capable of forming a stable loop-like structure.

Example 2. The synthesis of the modified peptide No. 1-M.

Peptide No. 1-M, as already mentioned, is a peptide No. 1 modified by palmitic acids. The primary structure of peptide No. 1-M is depicted in figa. Modification of peptide No. 1 was carried out according to the thiol groups of Cys amino acids at positions 92 and 116, these positions in Fig. 1A are marked with asterisks.

To prepare the modified peptide No. 1-M, a mixture of 1.0 g (10 mmol) of maleic anhydride and 2.4 g (10 mmol) of cetylamine in 20 ml of dioxane was boiled over 2 g of molecular sieves (4Å) for 5 h, cooled to room temperature and the precipitated precipitate of N-hexadecylmaleamide was filtered off. Yield 2.7 g (80%), mp. 58-59 ° C. A part of the obtained product (1 g) was heated in an open tube in an argon flow for 1 h at 120 ° С, cooled, dissolved in 10 ml of hexane, and filtered from insoluble residue. The filtrate was evaporated and dried in vacuo. Yield 0.8 g (85%). Found: C, 74.02; H 11.02; N, 4.18. C ₂₀ H ₃₅ NO ₂ . Calculated: C 74.12; H 10.97; N, 4.36. 20 mg of peptide No. 1 was dissolved in a mixture of 4 ml of ethanol and 0.5 ml of water, a solution of 10 mg of N-hexadecylmaleimide in 0.2 ml of ethanol was added and kept at 35 ° C until a negative test for SH-groups with sodium nitroprusside (5 h) ) The reaction mass was evaporated in vacuo, washed with ether, dry isopropanol, lyophilized from water with the addition of 10% acetic acid. The output of the modified peptide No. 1-M 18 mg

Thus, peptide No. 1-M was made containing, in the immediate vicinity of the N and C ends, hydrophobic radicals representing palmitic acid residues. A schematic representation of the spatial structure of peptide No. 1-M is shown in Fig.2B. Hydrophobic molecules (radicals) in a hydrophilic medium (water) tend to form tight contacts, eliminating the presence of molecules of a hydrophilic medium (water) in the contact area. The formation of tight contacts between hydrophobic radicals leads to a decrease in the area of contact with the hydrophilic medium (water), this process is called "hydrophobic interactions", it is energetically favorable and proceeds spontaneously. The hydrophobic interaction is strong enough to stabilize the structures of even large protein molecules [Chothia C. “Hydrophobic bonding and accessible surface area in proteins” // Nature. Mar 22; 248 (446): 338-9; 1974]. In the manufactured peptide No. 1-M, hydrophobic radicals are located in close proximity to the N and C ends of the peptide chain of FIG. 2B. The hydrophobic interaction between the N and C ends of the peptide tends to bring them closer, thereby bending the peptide chain and stabilizing the bending of the synthetic peptide, as shown in FIG. 3. It should be noted that the amino acid sequence of the peptide makes an insignificant contribution to the formation of the bend of the peptide chain, in other words, this method of stabilization of bends is acceptable for any synthetic peptides.

Example 3. Conjugation of the modified peptide No. 1-M.

In addition to the fact that the hydrophobic radicals that make up the peptide No. 1-M provide stabilization of the bend of the peptide chain, they also provide non-covalent binding of the molecules of the modified peptide to other hydrophobic media. So at physiological pH values after 1-2 days in a solution of peptide No. 1-M high molecular weight structures (emulsion) are formed. These high molecular weight structures are aggregated molecules of peptide No. 1-M due to the non-covalent interaction of hydrophobic radicals that make up neighboring peptide molecules. Peptide No. 1-M possessed a higher peptide No. 1 ability to adsorb on the surface of a hydrophobic polymer, polystyrene, compared to unmodified palmitic acids.

In addition, the hydrophobic radicals included in the peptide No. 1-M are able to fix the peptide on the surface of hydrophobic microparticles, as shown in Fig.3.

The microparticles, which are small cationic micelles, were made on the basis of the commercial cationic amphiphile DDAB - / Dimethyl-dioctadecyi - ammonium bromide (ICN) / and non-ionic amphiphile Tween 80. According to spectroturbidimetric analysis, the microparticles were 200 nm in size. Conjugation of the modified peptide No. 1-M with microparticles was carried out by an injected mixture of the peptide and microparticles through a porous membrane with a pore diameter of 0.5 μm. The completeness of conjugation of peptide No. 1-M with microparticles was confirmed by electrophoresis on the content of free peptide. A schematic representation of the spontaneous stabilization process of the loop-like structure of peptide No. 1-M, similar to the native structure of fragment 89-119 of protein E of tick-borne encephalitis virus, is presented in Fig.3.

Thus, peptide No. 1-M was conjugated both with cationic microparticles and the hydrophobic surface of polystyrene, as well as molecules of peptide No. 1-M with each other with the formation of emulsions. The ability of peptide No. 1-M to form high molecular weight structures (emulsions) and to spontaneously bind to hydrophobic microparticles makes it easy to produce substances containing a peptide antigen and capable of eliciting an immune response.

Example 4. The preparation of polyclonal antipeptide antibodies.

The affinity of the antigen-antibody interaction is very sensitive to the structure of the antigen. This property of antigen-antibody interaction was used to confirm that the proposed stabilization method really allows to stabilize the bend or loop-like structures of the peptide, similar to the native one. For this, antipeptide antibodies of mice were obtained.

BALB / c mice aged 5-6 weeks were immunized intraperitoneally with triply prepared peptides in cationic microparticles diluted in phosphate buffered saline. Mice were injected with 0.2 ml of a solution of peptides at a dose of 50 μg / goal, the second and third immunizations were carried out after 2 and 4 weeks, respectively, after the first immunization. The control group of animals was injected with a solution of a peptide that mimics the antigenic portion of the human immunodeficiency virus gp120 protein, according to the same scheme and in the same dosages. 10 days after the last immunization, a blood sample was taken from the retroorbital vein in animals to determine the titer of specific antibodies in serum. To determine the titer of specific antibodies in the wells of microplates for enzyme-linked immunosorbent assay (Medpolymer, Moscow), the following antigens were immobilized: 1) peptide No. 1 or 2) peptide No. 1-M. Peptide antigens were dissolved in 0.05 M carbonate-bicarbonate buffer solution, pH 9.6, to a final concentration of 10 μg / ml, 0.1 ml of solution was added to each well of the microplate and incubated overnight at 37 ° C. Wells were washed 3 times with Tween 20 phosphate-buffered saline and blocked non-specific binding with 0.5% bovine serum albumin. Twice dilutions of blood serum samples of mice in Tris-HCl buffer solution were applied 0.1 ml per well with immobilized antigen and incubated at 37 ° C for 30 min, after which the wells were washed 5 times with phosphate buffer solution. The resulting antigen-antibody complexes were detected by applying 0.1 ml of a phosphate buffer solution containing horseradish peroxidase conjugated goat anti-mouse class G immunoglobulins (Sigma) to the microplate wells, incubating at 37 ° C for 30 min and adding a substrate mixture containing ortho-phenylenediamine. The enzyme reaction was stopped by the addition of a solution of sulfuric acid, the result of the reaction was taken into account by the optical density measured using a Multiscan EX plate reader at a wavelength of 492 nm. Serum titer was considered the maximum dilution at which the optical density was 2 times higher than the corresponding signal in the well with a negative control. Serum from the control group was used as a control.

The results of determining the titer of antipeptide antibodies in the blood serum of mice are presented in table 1.

Table 1 Immobilized antigen Title specific antipeptide No. 1 antibodies The titer of specific antipeptide No. 1-M antibodies Peptide No. 1 1: 800 1: 800 Peptide No. 1-M 1: 400 1: 3200 Human immunodeficiency virus protein peptide gp120 <1: 100 <1: 100

Thus, triple intraperitoneal immunization of mice with peptides mimicking the TBE protein E fragment causes the formation of serum antibodies specific for the peptides (immunizing antigen).

Example 5. Preparation of native tick-borne encephalitis virus antigen.

To obtain the native KE virus antigen, strain 205, the clarified virus suspension was purified by ultracentrifugation in a combined gradient of glycerol and potassium tartrate, in which the upper part forms a 30% (w / w) solution of glycerol in STE buffer (50 mM Tris-HCl, 1 mM EDTA (pH 7.5)), and the lower (denser) 40% (w / w) potassium tartrate solution in STE buffer (pH 8.5). A viral suspension was layered onto the prepared gradient and centrifuged at 100,000 g for 3 hours at a temperature of + 4 ° C in a L8-70 centrifuge (Beckman). The virus-containing fraction (“band”) was diluted in an equal volume of STE buffer (pH 7.5) and centrifuged for 2 hours at 100,000 g at a temperature of + 4 ° C. The virus pellet was dissolved in borate buffer (0.15 M NaCl, 50 mM H ₃ BO ₃ , pH 9.0) and stored at -70 ° C. The concentration of total protein was evaluated using the Bio-Rad Protein Assay Kit. The purity of virus isolation was checked by electrophoresis in 12% polyacrylamide gel with 0.1% sodium dodecyl sulfate (Saminsky, 1966). The gels were stained with Coomassie G-250 dye. Two bands on the electrophoregram corresponded to proteins E (54 kD) and NS1 (41 kD) of the TBE virus. To obtain TB virus antigens, the purified viral suspension was treated with 0.2% sodium dodecyl sulfate solution.

Example 6. Determination of the affinity of antipeptide antibodies to the native antigen.

The affinity of the antigen-antibody interaction is very sensitive to the structure of the antigen. High antibody affinities clearly indicate that the antigen is present in the spatial structure to which the antibodies were obtained. In other words, the structure of the peptide antigen that will cause the formation of the antiserum with the highest affinity for the native antigen has a spatial structure that is most similar to the native one.

To analyze the interaction of specific antipeptide antisera, 1 μg of native tick-borne encephalitis virus antigen in 100 μl of a 0.05 M solution of bisubstituted phosphate buffer solution, pH 8 was applied to polystyrene plates and incubated at 37 ° C for 1 hour. Non-specific binding sites were saturated with a 0.5% casein solution at 20 ° C for 60 minutes. Then incubated with specific antipeptide antisera for 1 hour at 37 ° C. The resulting antigen-antibody complexes were detected by applying 0.1 ml of a phosphate buffer solution containing horseradish peroxidase conjugated goat anti-mouse class G immunoglobulins (Sigma) to the microplate wells, incubating at 37 ° C for 30 min. The enzyme reaction was stopped by the addition of a solution of sulfuric acid, the result of the reaction was taken into account by the optical density measured using a Multiscan EX plate reader at a wavelength of 492 nm.

The affinity antigen-antibody interaction is characterized by the value of the dissociation constant of the antigen-antibody (Kd) complex - the lower the Kd, the higher the affinity. To determine Kd, the results of measuring optical densities at various dilutions of the serum were presented in Scatchard coordinates (A, A / C), where A is the optical density and C is the concentration of antibodies (serum). It was assumed that there is an interaction of antibodies with two types of binding sites: specific binding of antibodies to an antigen and non-specific interaction of an antibody with plastic. The tangents of the slope of the tangents to the Scatchard curve correspond to the values of Kd ₁ ^{-1 of the} specific antigen-antibody interaction and (Kd ₁ ^-1 + Kd ₁ ^-2 ) of the specific and non-specific interaction. For the two types of binding centers, provided that one of them is highly specific and the second non-specific, the equation of interaction in the coordinates of Scatchard has the form [S. Vorfolomeev, S. Zaitsev. "Kinetic methods in biochemical research." - Publishing house of Moscow University, 1982]:

y = (((R1-x) / K1 + (R2-x) / K2 + (((R1-x) / K1 + (R2-x) / K2) ^ 2 + 4 * x * (R1 + R2-x) / (K1 * K2) ^ 0.5) / 2),

where y is the number of antigen-antibody complexes / antibody concentration;

x is the number of highly specific complexes and the number of low specific complexes;

K1, K2 — dissociation constants of highly specific and low-specific complexes;

R1, R2 - the number of highly specific and low specific binding sites.

The values of the dissociation constants of the highly specific interaction of antipeptide antisera with the native antigen calculated using the Scatchard equation are presented in table 2.

table 2 Antipeptide serum Kd dissociation constants of antipeptide antibodies with native antigen Antipeptide No. 1 10 ^-6 Antipeptide No. 1-M 10 ^-8

Thus, antisera obtained on peptide No. 1-M have less Kd, or greater affinity for the native antigen. Therefore, the N and C terminal modifications lead to the formation of a spatial structure similar to the native loop-like structure of region 89-119 of tick-borne encephalitis virus E protein. In other words, the modification of the N and C ends, leading to the formation of hydrophobic radicals at the ends of the peptide, provides stabilization of the loop-like spatial structure of the peptide, similar to the native loop-like structure of the mimicking region.

From the above examples it is seen that the proposed method for stabilizing the bends and loop-like structures of peptides allows not affecting the amino acid sequence of the peptide, as well as creating peptides that are a ready-made precursor for binding to carriers. Moreover, the method is universal, suitable for stabilizing the bends and loop-like structures of peptides having an arbitrary amino acid sequence.

Claims (9)

1. The method of stabilization of the loop-like structure of synthetic peptides by non-covalent interaction of hydrophobic hydrocarbons or their radicals with each other, characterized in that the hydrophobic hydrocarbons or their radicals are located on opposite N and C ends of the peptide or in close proximity to them, moreover, these hydrophobic hydrocarbons or their radicals also non-covalently bound to a hydrophobic carrier. 2. The method according to claim 1, characterized in that the hydrophobic hydrocarbons or their radicals are covalently linked to the reactive active groups of the peptide contained at its opposite N and C ends or in close proximity to them. 3. The method according to claim 2, characterized in that the reactive active groups of the peptide to which hydrocarbons or their radicals are bound are carboxyl groups. 4. The method according to claim 2, characterized in that the reactive active groups of the peptide to which hydrocarbons or their radicals are bound are amide groups. 5. The method according to claim 2, characterized in that the reactive active groups of the peptide to which hydrocarbons or their radicals are bound are thiol groups. 6. The method according to claim 1, characterized in that the carrier is made of a hydrophobic polymer. 7. The method according to claim 1, characterized in that the carrier is a high molecular weight structure of amphipathic molecules. 8. The method according to claim 1, characterized in that the hydrocarbons or their radicals are aliphatic. 9. The method according to claim 1, characterized in that the hydrocarbons or their radicals are aromatic.

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