Method of obtaining of complex of antimicrobic peptides of insect

FIELD: biotechnologies.

SUBSTANCE: method of obtaining of a complex of antimicrobic peptides of an insect includes infecting of adipose body of an insect at a larval instar with Micrococcus luteus A270 and Escherichia coli D31 bacteria with the subsequent extraction of adipose body of an insect at a larval instar. The adipose body of an insect is placed into a nutrient medium containing water solution of sugars, inorganic salts and the antibiotic meropenem in pre-set ratio and incubated during a day with the subsequent elution of the complex of antimicrobic peptides of an insect from cultural liquid by the method of reverse-phase chromatography on the column Vydac C18 at the linear gradient of acetonitrile from 0% up to 50%.

EFFECT: invention allows to simplify a method of obtaining antimicrobic peptides.

5 dwg, 4 ex

 

The invention relates to biotechnology and can be used to produce antimicrobial proteins and peptides. Preferred area of application of the invention is the obtaining of natural complexes of peptides with antibacterial and antifungal activity intended for the treatment of bacterial and fungal infections of humans and animals, including antibiotic-resistant forms.

In medicine and veterinary medicine uses a large variety of antibiotics belonging to different classes of low molecular weight organic compounds (beta-lactams, macrolides, tetracyclines, fluoroquinolones, sulfonamides, aminoglycosides, imidazolov, etc.). In recent years, the situation in therapy of bacterial infections is considerably complicated due to the wide spread of bacteria resistant to most or all known antibiotics. Treatment of such infections with the help of modern Arsenal of antibiotics is ineffective or even impossible.

To solve this problem it is known to use as medicaments antimicrobial peptides of animal origin. In particular, it is known to use for this purpose of defensins [1-7], tetracyclines [8], Virginia, austerity oysters and gibbosity Scorpions [9], defensins annelid worms [10].�water melanocyte-stimulating hormone [11], derivatives ubiquitin [12], magainin [13], derived mucin [14], analogues of theta defensins [15], antimicrobial peptides crustaceans [16], cryptgine [17]. Furthermore, it is known the use of synthetic cationic peptides as antimicrobial agents [18, 19, 20]. In some cases, for the treatment of bacterial infections it is proposed to use antimicrobial peptides insect bacteriolytic peptide lepidopteran Hyalophora cecropia [21], antibacterial peptide gloverin [22], antibacterial peptides beetle Oryctes rhinoceros [23], defininately peptide of the dragonfly Aeschna cyanea [24], the complex antimicrobial peptides Calliphora vicina [25].

There are three primary methods for obtaining antimicrobial peptides:

1) Chemical synthesis of peptides,

2) Extraction from the host body,

3) Synthesis in cell culture-producers.

Each of the above methods has its field of use.

Technology chemical synthesis can be applied for the production of relatively short (preferably not more than 30 amino acids) peptides that retain antimicrobial activity of their natural prototypes. Its advantage lies in the possibility of obtaining the drug on an industrial scale and with a maximum degree of chemical purity is required, for example, in the manufacture of dosage forms for votive�tion introduction. The limitation of this technology is the technical difficulties and high cost of synthesis of long peptide chains, especially in the case of peptides with complex three-dimensional organization.

The technology of extraction from the host body can synthesize the whole natural complexes of antimicrobial peptides and provides the most accurate reproduction of the structure of the active components, however, presents considerable difficulties in terms of standardization of the method. As a source of antimicrobial peptides in this case is hemolymph (blood) of an insect or whole homogenate (crushed insects). This assumes that the synthesis of antimicrobial peptides occurs in vivo, i.e. the target product has accumulated in the body is the host to the time of allocation. Disadvantages of technology for antimicrobial peptides of the biomass of insects associated with a high content of ballast compounds in the resulting material (hemolymph, homogenate) and the risk of activation in it phenoloxidase system, which leads to irreversible degradation of the target products and their pollution melanin in the treatment stage.

Synthesis in cell culture-producers is usually to use genetic engineering methods. Genetic engineering allows to obtain peptides of any length, but in some cases allows you to quickl� accurately reproduce the spatial structure of the peptide and usually offers a substantial cost of the final product. To say with certainty that any peptide synthesized correctly, can in fact only when its receipt is in the culture of cells isolated directly from the host body and having a natural ability to synthesize this peptide. The problem is that the main source of antimicrobial peptides in the insect's body are the fat cells of the body, most of which terminal differentiated and unable to proliferate in vitro is a barrier to obtaining appropriate cell lines, and therefore excludes the possibility of creating long-term culture. One solution to this problem is to obtain antimicrobial peptides in short term primary culture of adipose cells of the body.

The claimed invention relates to the third method of obtaining antimicrobial peptides.

A method of producing a complex of antimicrobial peptides in the fat body culture the larvae of dipteran Calliphora vicina [26], the closest solution to the technical problem with the claimed invention and is selected as a prototype.

In the known method from the larvae of flies extract the fat body and transported it to the salt solution, containing additional antibiotics - penicillin and streptomycin to prevent bacterial contamination of the culture. About 30% of the volume of the culture medium is heat-treated plasma hemolymph of larvae, which serves as the activator of the synthesis of antimicrobial peptides by the fat body cells. The fat body is cultivated in this environment during the day, then the culture fluid was separated from the fat body and use it as a source of antimicrobial peptides.

The main disadvantage of the prototype is the low degree of purity of antimicrobial peptides due to high inert content in the fat added to the body of culture medium and, as a consequence, in the final product - the culture fluid. The second disadvantage is the inability to zoom the described method due to the complexity and duration of the process of obtaining the required quantities of hemolymph of larvae - activator of the synthesis of bactericidal molecules in this model. The third disadvantage is the difficulty of purification of the target product - complex antimicrobial peptides - from subsidiary antibiotics - penicillin and streptomycin - after the time of cultivation.

The claimed invention is directed to solving these problems.

The technical result of the invention consists in increasing the degree of purification obtained natural complexes of antimicrobial peptides, simplifying the process of obtaining their required quantities with simultaneous reduction of time of this process and it� cheaper, as well as a substantial simplification stage of treatment.

Said technical result is achieved by a method of obtaining complex antimicrobial peptides of the insect, which consists in removing from the insect in the larval fat body, the premises of the fat body in liquid culture medium with the addition of auxiliary antibiotics, the cultivation of body fat in one single day and the subsequent collection of the culture fluid, which is in accordance with the claimed invention, before removing the fat body of the insect larvae infect bacteria, the culture medium is an aqueous solution of sugars and inorganic salts, as a subsidiary of antibiotic in the culture make Meropenem in a concentration of at least 2 mg/l of culture medium, after the time of cultivation from the culture fluid by the method of reversed-phase chromatography of extracted complex antimicrobial peptides of the insect.

Said technical result is achieved by the fact that the bacteria to infect insect larvae use the coccus Micrococcus luteus A and coli Escherichia coli D31.

In addition, the specified technical result is achieved in that the complex of antimicrobial peptides extracted from the culture fluid by the method of reversed-phase chromatog�aafia on the C18 sorbent.

Said technical result is achieved in that the fat body of an insect cultured in a saline solution not containing an exogenous protein and a mixture of hydrophobic antibiotics. For the production of antimicrobial peptides fat body uses the stock plastic protein substances which have accumulated in it to the moment of removal from the host body. In turn, the synthesis of antimicrobial peptides the fat body and the absorption of proteins from hemolymph (at the stage of finding the body fat in the body the larvae) initiated by pre-infection of insect bacteria. For sterilization of culture of isolated fat body is not used a mixture of hydrophobic antibiotics - streptomycin and penicillin, and moderately hydrophobic antibiotic Meropenem exhibiting excellent antimicrobial peptides from the chromatographic mobility - this contributes to the simplification of the procedure of purification of the target product from the auxiliary antibiotic by HPLC.

The essence of the claimed method is illustrated in Fig.1 - Fig.5.

Fig.1 shows the daily dynamics of the synthesis of antimicrobial peptides fat body isolated from healthy and infected larvae of Calliphora vicina.

Fig.2 shows the chromatogram of a mixture of streptomycin and penicillin, obtained by the method OF HPLC.

Fig.3 presents� chromatogram of Meropenem, obtained by the method OF HPLC.

Fig.4 shows the chromatogram of the culture liquid of the fat body of larvae of Calliphora vicina, incubated in protein-free salt solution.

Fig.5 shows the chromatogram of the culture liquid of the fat body of larvae of Calliphora vicina, incubated in medium containing heat-treated plasma of larvae of the same species.

The claimed invention was tested in St. Petersburg state University, feasibility and technical level of the invention is confirmed by the following examples.

Example 1: obtaining a complex of antimicrobial peptides S. vicina.

The proposed method of producing antimicrobial peptides consists of seven stages:

Step 1. Get the primary biological material (larvae of C. vicina)

For initial biological material adult C. vicina are grown in short-day conditions (12 hours light: 12 hours dark) at a temperature of +20°C to 22°C, and larvae - in the dark with sequentially alternating temperatures: +20°C (in the first days after hatching), from 8°C to 12°C (under power), 6°C (at the stage of purgation from the remnants of food), 0°C to +2°C (at the stage of diapause). In experiments using diapausing larvae of C. vicina.

Step 2. Infection of insects

The immune response induced by puncture of the cuticle �of ICIEC thin needle (width: 25 μm), soaked in a concentrated suspension of cells of Escherichia coli strain D31 (2×1011cells/ml) and coccus Micrococcus luteus strain A (2×1011cells/ml). Infected insects stored at room temperature.

Step 3. Preparation of culture media

As a culture medium for cells of the fat body using the MM environment [27] devoid of lactalbumin hydrolysate and yeast extract, the final composition: 7 g NaCl, 0.2 g NaH2PO4, 0.2 g KCl, 0.2 g CaCl2x2H2O, 0.12 g NaHCO3, 0.1 g MgCl2x6H2O to 1 liter of distilled water. To prevent contamination of culture in the environment contribute antibiotic Meropenem at a concentration of 2 μg/ml of medium.

Step 4. Extract fat body

Before the release of body fat the body surface of the larvae are sterilized in 70% ethanol solution, washed with distilled water and dried on filter paper. The rear part of the body, the larvae cut off with scissors and remove the fat body in conjunction with the trachea, gut and malpighian vessels in a Petri dish filled with culture medium. Fat body clear of the trachea, gut, malpighian tubules and, after pre-washing, transferred to a culture medium of fixed volume. The volume of culture medium is 400-1000 ál in the fat body of one larva.

Step 5. Kul�iferouane fat body

Capacity (tablet or Petri dish) with the culture of fat body are mounted on a platform orbital shaker OS-10 (Vector-best) with a given frequency of rotation of the platform 50 to 90 Rev/min. the Culture was incubated at room temperature (acceptable range of temperatures - from +20°C to +28°C) during the day.

Step 6. Extraction of hydrophobic compounds from the culture fluid

The samples are acidified with an equal volume of 0.1% aqueous solution of acetic acid and centrifuged for 5 minutes at 10000 g to remove the insoluble precipitate. Extraction of hydrophobic compounds from the culture fluid produced in the cartridge Sep-Pak C18 Classic (Waters). First, activate the cartridge 5 ml of acetonitrile and washed with 5 ml of 0.05% aqueous solution of acetic acid. Then the cartridge consistently applied acidified (0.05% acetic acid) culture fluid and washed cratridge 5 ml of 0.05% aqueous solution of acetic acid for the removal of hydrophilic compounds. Hydrophobic extract-containing antimicrobial peptides eluted in a 50% solution of acetonitrile in 0.05% acid. The eluate is subjected to lyophilization in a vacuum dryer FreeZone 2,5 (Labconco).

Step 7. Extraction of complex antimicrobial peptides

For purification of the complex of antimicrobial peptides from hydrophobic impurities using the method OF HPLC column Vydac C18 5×250 mm, Waters). Column balance 0.1% solution of trifluoroacetic acid and put an acidified sample. The peptides eluted with a linear gradient of acetonitrile from 0 to 50% over 50 min, collecting the eluate at 1 ml vials at the rate of elution 1 ml/min, and recording the change in absorption of light with a wavelength of 220 nm. The eluate exiting the column when the concentration of acetonitrile 24%-39%, pooled, and dried in vacuum. Valium is a purified hydrophobic impurities complex antimicrobial peptides of the insect.

Example 2: Quantitative characteristics of the antimicrobial activity of the fat body of larvae of C. vicina.

In this example, comparative data characterizing the performance of organ culture of intact fat body and infected larvae.

Obtaining larvae and the infection was performed according to the method described in Example 1. The fat body was extracted after 2 hours after infection of larvae with bacteria and incubated for 3 days in a saline solution containing glucose and antibiotics - streptomycin and penicillin at concentrations of 100 μg and 100 U, respectively, in 1 ml of medium. Volume of medium was 400 µl in the fat body of one larva. The cultivation of body fat was performed according to method described in Example 1. The collection and replacement of the medium produced daily. The control variant was distinguished those�, before the release of the fat larvae not infected.

The titer of anti-microbial components in the culture fluid was assessed using the standard method agar plates [28], using as a test organism gram-negative bacterium Escherichia coli strain D31. According to the method, aliquots of the analyzed samples with a volume of 5 µl was applied on the surface of solidified agar medium containing bacterial cells of Escherichia coli strain D31 (5×107cells/ml). Petri dishes coated samples were incubated overnight in an incubator at 37°C. a fter incubation, the medium in the cups became opaque due to the appearance of visible bacterial colonies, with the exception of the area around the put samples in which an antimicrobial substance inhibited the growth of bacteria. Since the area free from bacteria the area is proportional to the content of anti-E. coli peptides in the sample described the method used to assess the quantitative content of the respective antimicrobial peptides in the material.

According to the test results, the antimicrobial activity of the culture liquid of the fat body of infected larvae (Fig.1, dark gray columns) is higher than the activity of the culture fluid body fat of intact larvae (Fig.1, light grey bars). This pattern persists for 3 with�current cultivation of body fat. This means that infection of larvae by bacteria stimulates the synthesis and release of antimicrobial peptides fat body, and therefore is an integral stage of the proposed method of producing antimicrobial peptides.

Example 3: Chromatographic mobility subsidiary of antibiotics.

In this example, comparative data characterizing the chromatographic mobility of a mixture of streptomycin and penicillin, chromatographic mobility of Meropenem and chromatographic mobility of antimicrobial peptides.

A mixture of streptomycin and penicillin (Biolot, Russia) was applied onto a chromatographic column Vydac C18 (5×250 mm, Waters) in the amount of 300 µg (streptomycin) and 300 IU (for penicillin). Meropenem (Sumitomo Pharmaceuticals, Japan) was applied to the column in an amount of 100 μg. Antibiotics were suirable a linear gradient of acetonitrile from 0 to 50% within 30-50 min, the Eluate was collected in 1 ml vials at the rate of elution 1 ml/min, registering the change in absorption of light with a wavelength of 220 nm.

According to the results of the experiment, streptomycin and penicillin come from the speakers when the content of acetonitrile 23%-42% (Fig.2), Meropenem - when the content of acetonitrile, 15%-17% (Fig.3).

Antimicrobial peptides in specified conditions alumroots 24%-39% acetonitrile (Fig.4). This means that the chromatographic mobility of antimicrob�s peptides partially coincides with that of a mixture of streptomycin and penicillin, and it complicates the purification of antimicrobial peptides. Meropenem same goes with speakers much earlier target components. In this case, as well as streptomycin and penicillin, Meropenem effectively inhibits the growth of bacteria-symbionts in the larvae of Calliphora vicina, preventing contamination of the culture of body fat (minimum inhibiting concentration is 2 µg/ml). These circumstances make Meropenem preferred sterilizing agent, which at the end of the extraction process antimicrobial components can be easily separated from the desired product.

Example 4: Chromatographic mapping of the culture fluid body fat

This example provides comparative data illustrating the degree of purity of the complex antimicrobial peptides produced by culturing the fat body of infected larvae in protein-free medium in the cultivation of intact fat body of larvae in a medium containing heat-treated plasma of intact larvae (compared with the prototype method).

In the first case, the fat body was extracted from 12 infected larvae. Infection of insects, the allocation of body fat, its cultivation and extraction of antimicrobial peptides from the culture fluid was performed according to the method described in Example 1. The difference was that received�record in the result chromatography fractions was pooled, and dried individually. Then each of the fractions was added to 30 μl of deionized water. The titer of anti-microbial components in each of the obtained chromatographic fractions was assessed using a standard method agar plates, using as test organisms gram-positive bacterium Micrococcus luteus strain A and gram-negative bacterium Escherichia coli strain D31 (see Example 2). Volume aliquots deposited on the surface of agar medium, was 5 µl.

According to the results of chromatographic mapping (Fig.4), anthropologically the peptides from the culture fluid alumroots column with 26%-39% acetonitrile, antigravitation peptides - 24%-39% of acetonitrile. "Peaks" anti-M. luteus activity correspond to the fractions Nos. 28 and 33, "spades" anti-E. coli activity of the fractions Nos. 26, 31 and 37.

In the second case, the fat body was extracted from 3 intact larvae and incubated in 30% solution of a heat-treated plasma of intact larvae (heat treatment for 1 minute at a temperature of +95°C) containing streptomycin and penicillin at the rate of 1 ml culture medium in the fat body of one larva. The cycle of cultivation of fat body are also restricted to one day. Extraction of hydrophobic compounds from the culture fluid was performed according to the method described in Example 1. Chromatographic fraction�El of hydrophobic compounds the culture fluid and further mapping was carried out according to the method described above.

According to the results of chromatographic mapping (Fig.5), anthropologically activity of culture liquid was concentrated in fractions Nos. 30-36, and antigravitational - in fractions Nos. 28-39. "Peaks" Akti-M. luteus activity correspond to the fractions Nos. 31 and 36, the "peaks" of anti-E. coli activity No. 28, 33 and 37.

Since the "peaks" of bactericidal activity (in the histogram) correspond to the main families of antimicrobial peptides presented in the culture fluid, based on the similar distribution of the antimicrobial activity of fractions in either cases, it can be concluded that the composition of peptide antibiotics in the samples is identical. In the first case, the chromatogram can be traced clear peaks-eluted column with antimicrobial peptides, while the second is dominated by "peaks" of the ballast components that do not have direct antibacterial activity (although the larvae in the experiment were used four times less). This means that the purity of antimicrobial peptides by culturing body fat in protein-free medium is higher than when it is cultured in a medium containing exogenous protein is heat-treated plasma.

Technical and economic efficiency of the claimed method consists in the possibility of the production of antibacterial drug�in local and systemic action, in which the effector components are antimicrobial peptides of the insect, which has become feasible due to the significant increase in the degree of purity of the natural complexes of antimicrobial peptides, the simplification and cheapening of the process of obtaining their required quantities while reducing its duration.

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The method of obtaining complex antimicrobial peptides of the insect, which consists in removing from the insect in the larval fat body, the premises of the fat body in liquid culture medium with the addition of auxiliary antibiotics, the cultivation of body fat in one single day and the subsequent collection of the culture fluid, characterized in that before the extraction of the fat body, the insect larvae infect bacteria Micrococcus luteus A and Escherichia coli D31, as a culture medium an aqueous solution of sugars and inorganic salts, as a subsidiary of antibiotic in the culture make Meropenem in a concentration of at least 2 mg/l of culture medium, after the time of cultivation from the culture fluid by the method of reversed-phase chromatography column (Vydac C18 eluted complex antimicrobial peptides of the insect with a linear gradient of acetonitrile from 0% to 50%.



 

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1 dwg, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is presented is a method for preparing a recombinant protein of type III interferon-like factor (ILF III) of the producing strain E. coli. The inclusion bodies E. coli are washed and dissolved with using 2% aqueous γ-cyclodextrin. That is followed by the sequential Ni-Sepharose, Q-Sepharose and SP-Sepharose chromatographic procedures. Refolding of a target protein is performed with using a mixture of cysteamine and cystamine at pH 10.5. The Amberchrome Profile XT20, Amberchrome Profile HPR10 and Kromasil 300-5C18 chromatographic procedures are sequentially performed.

EFFECT: invention enables optimising the ILF III purification environment at the stage of washing and dissolving the inclusion bodies Ecoli and provides 12% target protein yield.

3 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to compositions for intensive generation of a target protein in eucariotic cells, which includes a DNA vector with an insert of target protein gene and an agonist of cell receptors. Besides, the invention relates to methods for increasing generation of a target protein coded with a transgene in eucariotic cells by using the above compositions.

EFFECT: invention allows effective increase of generation of a target protein in eucariotic cells.

28 cl, 4 dwg, 7 tbl, 10 ex

FIELD: biotechnology.

SUBSTANCE: method provides culturing in the liquid culture medium of the virulent strain of bacteria Staphylococcus aureus No. 6 followed by separation of the culture medium from the bacteria by filtration through the filter PLASMAFILTER PLASMAFLUX PSu 2S with obtaining the filtrate. Ammonium sulphate to 80% saturation is added to the resulting filtrate to obtain the precipitate. The resulting precipitate is separated by centrifugation at 10000 g for 30 minutes and dissolved in phosphate buffer at pH 7.4 with subsequent microfiltration of the protein-containing fraction through the 0.22 mcm membrane and desalting on the column PD-10, purification and concentration by carrying out ion-exchange chromatography on the column of Q-sepharose, elution with 0.15 M NaCl and ultrafiltration on two filters of 100 and 30 kDa to obtain protein containing fraction with the molecular weight of 30-90 kDa and protein content of 0.5-1.0 mg/ml.

EFFECT: invention enables to obtain the products based on protective secreted protein-containing compound.

1 dwg, 5 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method involves enzymic hydrolysis of initial raw material, centrifugation with further sterilising filtration of the obtained substance through finely porous filters with pore diametre of 0.2 mcm. Donor blood erythromass is used as initial raw material, and first it is subject to hemolysis, virus inactivation, inactivation of a ferment after enzymic hydrolysis. Clarification is performed by means of hydrogen peroxide solution with final concentration of 0.83% with further exposure at 66°C during 15 minutes.

EFFECT: invention allows separating low-molecular peptides and increasing their biological activity.

2 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to chemical engineering and techniques for producing veterinary, medical and pharmaceutical preparations. The method of producing a novel antiviral substance based on 2,5-dihydroxybenzoic acid and gelatine includes oxidising 2,5-dihydroxybenzoic acid with laccase enzyme to intermediate phyenoxy radicals and semiquinones, which are then copolymerised with gelatine, and separating the obtained copolymer from low-molecular weight components by dialysis; optimum concentrations of components of the reaction mixture are as follows: 2,5-dihydroxybenzoic acid - 15-80 mM, gelatine - 1-13 mg/ml reaction mixture, laccase - 0.5-10 units of activity/ml reaction mixture.

EFFECT: obtained copolymer has antiviral activity on herpesvirus, particularly Aujeszky's disease virus.

2 tbl, 1 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biotechnology. What is described is a protein-polypeptide complex (PPC) possessing tissue specific regenerative-reparative and rejuvenating action on skin tissue, produced of extracted homogenate of nerve and skin tissues of farm hoofed embryos, a method for preparing it and a pharmaceutical composition thereof. The PPC contains tissue-specific negative faintly acid neutral proteins and peptides with molecular weight 0.5 to 200 kDa with min. 70% of total protein weight having molecular weight falling within the range of 20 to 180 kDa. The BPC is used as an active ingredient in pharmaceutical compositions applicable for preparing therapeutic and cosmetic products.

EFFECT: invention enables producing the preparations having high biological activity and applicable for treating autoimmune, cardiovascular, traumatic, toxic skin diseases, as well as used in aesthetic medicine and cosmetology.

5 cl, 14 dwg, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biotechnology. What is described is a protein-polypeptide complex (BPC) possessing antihypoxic, tissue-specific, reparative action on the central and peripheral nervous systems, prepared of extracted homogenate of nerve tissue of farm hoofed embryos of a gestation age from the middle of the first to the middle of the last trimester of pregnancy, a method for preparing it and a pharmaceutical composition thereof. The PPC contains tissue-specific negative faintly acid neutral proteins and peptides with molecular weight 0.5 to 200 kDa with min. 70% of total protein weight having molecular weight falling within the range of 20 to 160 kDa. The PPC is used as an active ingredient in pharmaceutical compositions applicable for preparing therapeutic products.

EFFECT: inventions enables producing the products having high biological activity and applicable for treating the diseases of the central and peripheral nervous systems, hypoxic conditions, and also used in sports medicine, emergency medicine.

5 cl, 14 dwg, 1 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to combinations of peptides in each case with the same sequence length (SEQL) which can be prepared in a stable reproducible quality and quantity of a mixture (A) containing a number of x amino acid with protected acid groups or a number of z peptides with the acid groups protected by the protective groups and the activated amino groups, with the amino acids in the mixture (A) found in a specific molar ratio, and a mixture (B), containing a number of y amino acids with the amino groups protected by the protective groups, with a molar ratio of the amino acids of the mixture (B) being the same as the molar ratio of the amino acids of the mixture (A), and the number x=y, and x is a figure from 11 to 18.

EFFECT: new combinations of the peptides are presented.

13 cl, 2 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biosensors and can be used in studying proteins via a luminescence technique. A protein luminescence-activating hybrid complex is obtained through ultrasonic treatment of a protein which contains aromatic amino acids in a physiological solution in the presence of phosphorus Y0.95Hr0.05VO4 or Y0.95Er0.05YO3Cl. The complex contains donor-acceptor pairs.

EFFECT: use of the invention increases luminescence of the investigated protein and reduces labour input of the protein labelling procedure, widens the range of proteins analysed using a luminescence technique, and lowers the technological level of the process of obtaining donor-acceptor pairs in the protein luminescence-activating hybrid complex.

2 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of obtaining and purifying human inhibin-A. Pieces of placenta are crushed and homogenised. A physiological solution is added in ratio of 1:2. Butyl alcohol is added to the obtained mass in ratio of 1:10 and then left for a day in a refrigerator at t +4°C. The mixture is then centrifuged with diethyl ether in ratio of 1:3 for 10-12 minutes at 5000 rpm in a cold centrifuge twice. Supernatant fluid containing inhibin is put into a flask which is then put into a refrigerator at t +4°C for a day. The supernatant fluid is centrifuged once more and over protein content therein is determined. The obtained solution is mixed with an equal volume of saturated ammonium sulphate solution and centrifuged in a cold centrifuge for 45-50 min at 10000 rpm. The obtained residue is dissolved in a tris-HCl buffer with pH 7.8, and then deposited on a lectin-sepharose column balanced with tris-HCl buffer. The entire volume of the dissolved residue obtained at the previous step is passed through. The column is washed with 1M sodium chloride solution buffered with tris-HCl buffer. The inhibin bound on the column is then eluted with 0.1M lactose solution in a borate buffer at pH 9.0. The eluate undergoes dialysis and concentration.

EFFECT: method enables to obtain inhibin-A with high output, high specific activity and high degree purification.

1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention refers to peptide chemistry and concerns producing tripeptide diacetate H-β-Ala-Pro-DabNHBzl referred to a biologically active compound used in cosmetic industry as an active component for cosmetic products, particularly for stimulating skin rejuvenation, tightening and prevention. The method is based on the 6-staged synthesis and is free from the stages of setting and releasing the protective groups. The method involves proline β-chlorpionyl chloride acylation followed by producing N-(3-chlorpropionyl)proline pentafluorphenyl ester in the presence of N,N'-dicyclohexyl carbodiimide. The above pentafluorphenyl ester is condensed thereafter with glumatic acid monomethyl ester to produce N-(β-chlorpropionyl)-Pro-Glu(δ-OMe)OH. That is followed by benzylamine amidation in a combination with ammonolysis and chlorine substitution by an amino group. That enables producing the tripeptide β-Ala-Pro-Glu(δ-NH2)NHBzl; Hofmann rearrangement is conducted with the use of iodo-benzene diacetate to produce a target product.

EFFECT: method is characterised by simplicity, effectiveness; it is cost-effective and uses more accessible and cheap agents.

6 ex

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