Method of isolation of secretion inhibitor of siderophores synthesised by pgm-strains y. pestis and isolated inhibitor

FIELD: biotechnology.

SUBSTANCE: method of obtaining SSI comprises the following steps. The strain Yersinis pestis KM 1279 is grown on 1.5% agar LB, the bacteria are washed three times with cold buffered normal saline. The bacteria are pelleted by centrifugation, suspended in a solution of 5 mM NaOH, kept at 37°C for two hours and the cells are pelleted by centrifugation. The supernatant is selected and the procedure as repeated three times, three supernatants are combined and filtered through the nitrocellulose membrane. The filtrate is extracted three times with the mixture of chloroform-methanol-water in a ratio of 5:2:1. The chloroform fractions are separated by centrifugation, combined and freed from water-soluble impurities. The aqueous fraction is separated by centrifugation and removed, and the chloroform fraction is dried in a vacuum rotary evaporator and the dry preparation SSI is obtained. The proposed SSI is characterised with brown colouring of dry crystals, hydrophobic properties, fluorescence in ultraviolet, lipopeptide nature, the presence of iron ions, the molecular weight of 380.6 Da.

EFFECT: inventions enable to obtain the natural regulator of virulence of plague agent.

2 cl, 6 dwg, 6 ex

 

The invention relates to medical Microbiology and searching for new approaches to the development of antibacterial drugs that inhibit these essential for pathogenic bacteria properties as the secretion of siderophores, autoagglûtinaciâ and virulence for animals.

Currently in the strategy fight infections the attention of researchers is attracted by the process of assimilation of iron bacteria, in particular of siderophore - low molecular weight iron chelators used by pathogenic bacteria to extract iron from its complexes with proteins in the host organism. Siderophore are recognized virulence factors of many pathogenic bacteria. Is no exception and is the causative agent of plague (Yersinia pestis), which is siderophore yersiniabactin (Ybt) is required for growth in iron-deficient conditions at 37°C and the existence of high virulence for laboratory animals [1].

Known synthetic inhibitors of the biosynthesis yersiniabactin who possessed antibacterial activity against the causative agent of plague in iron-deficient conditions in vitro [2]. Evidence of review of the effectiveness of these drugs on animal models in the literature. The development of these drugs was based on the notion that mammals do not synthesize siderophore, and therefore, the process of inhibition of their synthesis �predstavlyalsya specific bacteria. However, in 2010 siderophore and enzymes of their biosynthesis, similar to those of the bacteria were discovered in mammals [3]. It was found that siderophore mammals form the so-called labile pool of intracellular iron required for the formation of reactive oxygen compounds of immunocompetent cells. The animals lack the ability to synthesize these siderophore led to disruption of iron metabolism and increased sensitivity to bacterial infections. These data indicate that therapeutic use of inhibitors acting on the synthesis stage of siderophores may have side effects.

Closest to the object of the invention is the work [4], in which the first data that attenuated bacteria with a high frequency are formed in the population of epidemic strains of Y. pestis, synthesize natural inhibitor siderophore activity. In this work, the authors showed that the Y. pestis mutants in which there was a deletion of the chromosomal pgm locus (pgm-strains), encoding proteins of the biosynthesis and transport of siderophore yersiniabactin, not only are not active on the indicator medium for the detection of siderophores, but also emit into the environment a substance inhibitory siderophore pgm activity+strains. However, this prophetic�tvo has not been isolated and characterized.

Conducted by the authors of the present invention to examine this inhibitor showed that it does not affect the synthesis and secretion of siderophores Wednesday by various strains of Y. pestis, and so he was named an inhibitor of the secretion of siderophores (siderophore secretion inhibitor, SSI).

The technical problem of the invention was to provide a method for the isolation of inhibitor secretion siderophores (SSI) as a drug that reduces the virulence of Y. pestis.

The task is achieved in that the method of allocation of inhibitor secretion siderophores synthesized pgm-the Y. pestis strains, comprising the following steps:

a) as a producer strain using strain Y. pestis 1279 km (deposited in the Public collections of pathogenic bacteria "Microbe"), which is obtained from the pgm-the vaccine strain Y. pestis EV76;

b) Y. pestis strain 1279 km loop seeded in 5 ml LB medium and grown in an incubator at 26°C for 48 h;

C) in 0.2 ml of culture was inoculated in 10 Petri dishes with 1.5% LB agar (pH of 7.2) and grown at 26°C for 48 h;

g) wash away bacteria from the surface of the agar in cold (4°C) phosphate-buffered saline ('s SGA) and precipitated them by centrifugation (8000 rpm) in cold (4°C) for 15 minutes.

e) baclass in an amount of 10 g twice washed from the remnants of the nutrient medium, suspending the bacteria in a's SGA and precipitating them by centrifugation (8000 rpm) n� cold (4°C) for 15 minutes;

(e) cellular precipitate suspended in 100 ml of 5 mm NaOH (pH 9,0), incubated with intermittent shaking, at 37°C for two hours and separated from the cells by centrifugation (8000 rpm) at 20°C for 15 minutes, supernatant is taken and the procedure was repeated thrice;

g) three of the supernatant in a volume of 300 ml were pooled and filtered through a nitrocellulose membrane with a pore diameter of 0.22 μm and get alkaline solution, which contains the inhibitor;

h) the alkaline solution is extracted three times with a mixture of chloroform-methanol-water (5:2:1) in an amount of 50 ml, separating the chloroform and aqueous methanol fraction by centrifugation at 8000 rpm for 10 min;

and) chloroform fraction are selected, pooled and freed from soluble impurities by extraction with water, the aqueous fraction was separated by centrifugation at 8000 rpm for 10 minutes and remove;

K) chloroform fraction was dried in a vacuum rotary evaporator and receive 10 mg of dry product.

In addition, an inhibitor of secretion of siderophores, which is located on the surface of Y. pestis bacteria and the presence of which correlates with the ability of the cells of Yersinia pestis to block siderophore activity and reduce the autoagglûtinacii bacteria, characterized by:

- molecular weight 380,6 Yes;

- the presence of ions of iron;

- fluorescence in ultraviolet;

- lipopeptide� nature;

- hydrophobic properties;

brown coloration of the dry crystals.

A method of separating SSI is as follows.

As the producer strain SSI used a strain of Y. pestis 1279 km (deposited by the applicants in the Public collections of pathogenic bacteria FGPs Russian research anti-plague Institute "Microbe" of the CPS). The strain obtained from pgm-strain Y. pestis EV76 by removing him three characteristic of the plague pathogen plasmid replicons. A method of separating SSI strain of Y. pestis km 1279 includes the following techniques:

I - obtaining a bacterial mass;

II - allocation of the SSI;

III - purification of the drug.

The first technique is to obtain a bacterial mass of the producer strain SSI. To this end, the strain Ypestis km 1279 pre-inoculated loop in 5 ml LB medium (Difco, USA) and grown in an incubator at 26°C for 48 h. After that, 0.2 ml of the culture was inoculated on 10 Petri dishes with 1.5% LB agar (pH of 7.2) and grown at 26°C for 48 hours. Then the bacteria are washed from the surface of the agar in cold (4°C) phosphate-buffered saline ('s SGA) and precipitated them by centrifugation (8000 rpm) in cold (4°C) for 15 minutes. Next, 10 g of Bakassi twice washed from the remnants of the nutrient medium, suspending the bacteria in a's SGA and precipitating them by centrifugation (8000 rpm) in cold (4°C) in the Techa�their 15 minutes.

The second method is to SSI flush with the surface of bacteria. To this end, the cell precipitate was suspended in 100 ml of 5 mm NaOH (pH 9,0), incubated with intermittent shaking, at 37°C for two hours. After that separate the cells by centrifugation (8000 rpm) at 20°C for 15 minutes, supernatant is taken and the procedure was repeated three times. Then three of the supernatant were pooled and filtered through a nitrocellulose membrane with a pore diameter of 0.22 μm and get alkaline solution, which contains SSI.

The third technique is a clean drug. To do this, to the resulting alkaline solution was added 50 ml of a mixture chloroform-methanol-water in the ratio (5:2:1) and extraction is carried out three times SSI. Chloroform and water-methanol fraction is separated by centrifugation at 8000 rpm for 10 min. then chloroform fractions are selected, pooled and freed from soluble impurities by extraction with water, the aqueous fraction was separated by centrifugation at 8000 rpm for 10 min and removed. Then the chloroform fraction was dried in a vacuum rotary evaporator and receive 10 mg of dry product SSI, representing crystals are brown.

Characterization of physicochemical properties of SSI

Derived drug SSI is characterized by the following properties: (a) the dry preparation SSI represents crystals to�ichnevogo color having hydrophobic characteristics, because it does not dissolve in water, but dissolve in organic solvents (ethanol, ethyl acetate, acetone, chloroform, benzene);

b) SSI is a low molecular weight fluorescent substance lipopeptide nature (example 1);

b) SSI has a molecular weight 380,6 Yes (example 2);

g) the composition of the SSI includes iron ions, which are detected in the product after processing of 30 mm Hcl (example 3).

Thus, the analysis of physico-chemical properties obtained using the above method of the drug showed that SSI is a low molecular weight, hydrophobic, fluorescent substance lipopeptide nature. The presence of iron ions, which are detected only after treatment of the drug acid, indicates that SSI is a associated with iron chelator.

Characterization of the functional properties of SSI

The use of the selected drug in microbiological experiments and in experiments on infection of laboratory animals with bacteria vysokovalentnogo strain of Y. pestis revealed SSI following functional properties:

(a) in the presence of bacteria SSI pgm+Y. pestis strains exhibiting siderophore activity on an indicator medium for the detection of siderophores, lose the ability to distinguish siderophore Wednesday (example 4);

b) preparation SSI snige� the autoagglûtinaciâ pgm +bacteria Y. pestis, characterized, unlike the pgm-bacteria, high aggregability even in distilled water (example 5);

C) introduction of the drug to mice with vysokovitaminnym strain Y. pestis 231 SSI reduces the ability of a strain to cause lethal disease of animals (example 6).

Therefore, analysis of the functional properties of SSI showed that it not only inhibits the secretion of Wednesday's siderophores, but also inhibits autoagglûtinaciâ bacteria and their virulence for mice.

Example 1. Indicating the purity of the drug and lipopeptides nature SSI

The study of the properties obtained by the above method of preparation SSI by gel electrophoresis and thin-layer chromatography was carried out using as a control drug siderophore yersiniabactin (Ych). The drug Ych is prepared from a strain of Y. pestis 1279 km, as described previously [5]. Analysis of drugs SSI and Ych by gel-electrophoresis in polyacrylamide gel showed that in low-molecular the field of SSI gives one lane that is heavily fluoresce when irradiated with ultraviolet light with a wavelength of 254 nm (see photo 1A, where 1 SSI TO mcg, 2 - Ych, 10 µg).

After staining gels universal dye "Stains-all" in preparation SSI also identified one lane, painted in typical lipids yellow. The prep�Rath Ych had no fluorescence and were not stained by the dye "Stains-all".

The lack of SSI in the preparation of admixtures was also confirmed by ascending thin-layer chromatography on plates of silica gel and using 80% ethanol as a mobile phase (see photo 1B, where 1 SSI, 1 μg, 2 - Ych, 1 µg). And in this analysis in samples containing SSI, on the chromatogram showed a single spot with Rf of 0.9, which was reacted in the vapor of iodine and fluoreszierende when irradiated with ultraviolet light. Thus the preparation Ych, which had a Rf of 0.7, were stained with iodine, but did not possess fluorescent properties. After spraying the plates with 0.2% ninhydrin solution in acetone (followed by heating at 110°C) SSI, but not Ych, was painted in violet color, characteristic of the α-amino acid containing peptides.

Conclusion: SSI is lipopeptide, containing in its composition of fluorophor.

Example 2. Illustrating the molecular weight of SSI

Molecular weight of SSI determined using time-of-flight mass spectrometry with ionization by laser desorption-assisted matrix-assisted laser desorption ionization-time of flight mass-spectrometry, MALDI-TOF-MS). With the aim of using the mass spectrometer Bruker Reflex III (Bruker Daltonics Coventry, UK). As the matrix used oxybenzone acid, which caused the SSI drug dissolved in ethyl acetate. Range registered in the form of positively charged ions in the range m/z 100-6000. Spectrum analysis (see graph�to Fig.1) showed what SSI molecular ion corresponds to m/z 380,6.

Conclusion: SSI is a low molecular weight substance having a molecular weight 380,6 Yes.

Example 3. Proving the presence of bound iron in the SSI

In preparation SSI, which is treated with 30 mm hydrochloric acid, in contrast to the untreated drug, iron ions were detected using a commercial chromogenic chelator of iron chromazurol S (CAS). Separating from SSI in the acidic environment of the iron ions were stained with CAS reagent in blue. Fractionation of the acid-treated drug using SSI high performance liquid chromatography (HPLC) showed that it contains two fractions (see graph in Fig.2).

On the chart (Fig.2): A. HPLC on a column of C-18-Nucleosil ODS. Eluent a solution of acetonitrile with a linear concentration gradient (0-60%) for 30 min. B. MALDI-TOF mass spectra of the two SSI fractions obtained by HPLC.

The first of them (1) had weak fluorescence and inhibited the secretion of siderophores, i.e. did not possess the properties of SSI. The second fraction (2) was kept intensely fluorescent in ultraviolet light substance, so its absorption at 215 nm was significantly lower than that of the substance from the first fraction.

When MALDI-TOF-MS (Fig.2B) substance 1 had m/z 302,63, and the substance 2 - 359,9. The substance with m/z 359,9, unlike substance with m/z 302,63, possessed all the properties of the SSI. The difference in m/z of the source p�of Ephrata SSI and its fractions in acidic medium, and identifying in the product treated with acid, iron ions allowed us to conclude that the value of m/z 380,6 corresponds to the sodium salt associated with iron substances (m/z 359,9), which in the acidic environment loses iron (m/z of 302.6).

Conclusion: SSI is associated with the iron chelator of iron.

Example 4. Confirming the ability of the drug SSI block the secretion of siderophores bacteria Y. Pestis

Siderophore-inhibiting activity of the drug was investigated when it is added to the indicator strain Y. pestis 336 (subspecies caucasica) and the seeding of bacteria on the test environment to identify siderophores. The medium contains chromogenic chelator of iron chromazurol S (CAS), which is at 30% saturation of iron has a blue-green color, changing to yellow after removing the iron ions siderophore. Strain Y. pestis 336, unlike most strains of Y. pestis, which with high frequency lose pgm locus, steadily maintains the pgm locus and siderophore activity on indicator medium. Adding SSI at a dose of 1-10 mcg to 10 μl of cell suspension 336 Y. pestis containing 109cells/ml, and the seeding suspension on CAS-agar revealed the ability of the drug to block the release of siderophores on Wednesday. In contrast to the control culture containing no SSI, culture with SSI does not form zones of enlightenment AS-reagent around the crops (see photo 2).

In the photo: 1. Y. pestis 336 - control without doba�ing SSI. 2. Y. pestis 336+1 µg SSI. 3. Y. pestis 336+5 µg SSI. 4. Y. pestis 336+10 µg SSI.

Conclusion: SSI inhibits the secretion of siderophores bacteria Y. pestis.

Example 5. Demonstrating the ability of the drug to reduce the SSI autoagglûtinaciâ Y. pestis, the Influence of SSI on the expression of the trait of autoagglûtinacii were identified when comparing isogenic pgm+and pgm-clones of Y. pestis strain TS. Both types of cells have this symptom, although it was expressed to them in varying degrees. Producing SSI pgrri cells that form flakes easily dispersed in a liquid medium, compared to pgm+cells have greater surface hydrophobicity and agglutinable only in salt solutions. More pronounced AA was not producing SSI pgm+cells, which form stable aggregates even in distilled water, unlike the pgm-cells. Analysis of the ability of pgm+bacteria to be autoagglûtinaciâ in water showed that in the presence of SSI, they lose their ability to agglutinate in the water (see Photo 3).

In the photo: 1. Y. pestis TS control without the addition of SSI. 2. Y. pestis TS+1 µg SSI. 3. Y. pestis TS+5 µg SSI. 4. Y. pestis TS+10 µg SSI.

After interaction with SSI pgm+bacteria agglutinant only in salt solutions, as pgm-bacteria.

Conclusion: SSI reduces the ability of Y. pestis to autoagglûtinacii.

Example 6. Revealing an inhibitory effect of SSI on the virulence of the causative agent of plague

The influence of SSI on the virulence of the plague pathogen analyze when using vysokovalentnogo strain Y. pestis 231 (LD502,5±1,4 CL.). In this experiment three groups of mice injected subcutaneously with 1000 bacteria (the first group), 1000 bacteria +10 µg SSI (the second group), and 1000 bacteria +10 µg SSI, inactivated by the removal from it of iron in acidic medium by extraction of the drug 8-oksikhinolinom (the third group). Analysis of the number of fallen in the past 21 days (observation period) after infection of animals showed that the drug SSI reduces the ability of the strain Y. pestis 231 to cause lethal infection in mice. In three independent experiments add to the bacteria active drug SSI for 30 min before infection resulted in survival of more than half of the animals. While the drug is inactivated SSI, lost bound iron and the ability to inhibit the secretion of siderophores and autoagglûtinaciâ did not affect virulence. All animals infected with Y. pestis along with this drug, was killed in the terms is not significantly different from those in the control group (see Fig.3).

Conclusion: SSI reduces the virulence of the plague pathogen.

Thus, selected using the method described above is the preparation SSI has an inhibitory action on such essential for pathogenic bacteria properties of Y. pestis as the secretion of siderophores, autoapply�inazia and virulence for laboratory animals.

The obtained results allow to conclude that the SSI produced attenuated pgm-cells and absent in virulent pgm+bacteria Y. pestis, is a natural regulator of virulence of the causative agent of plague. Further study the regulator will contribute to deciphering the molecular mechanisms of regulation of pathogenic properties of Y. pestis, as well as the development of a new generation of drugs for the pathogenetic treatment of plague. The inhibition of the virulent Y. pestis limiting the growth of bacteria in the host organism, will not only potentiate the effect of antibiotics, but will also provide the immune system is able to effectively eliminate the pathogen. The creation of such medicines can expand the Arsenal of tools to combat plague both when used alone and in combination with regulated etiotropic therapy.

Sources of information

1. Perry R., Fetherston J. Yersiniabactin iron uptake: mechanisms and role in Yersinia pestis pathogenesis. Microb. Infect. 2011, 13, 808-817.

2. Stirrett K. L, Ferreras, J. A., V. Jayaprakash, B. N. Sinha, Rene T., L. E. Quadri, N. Small molecules with structural similarities to siderophores as novel antimicrobials against Mycobacterium tuberculosis and Yersinia pestis. Bioorg. Med. Chem. Lett. 2008, 18, 2662-2668.

3. Devireddy, L. R., Hart D. O., Goetz, D. H. and Green, M. R. A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell. 2010, 141, 1006-1017.

4. Podladchikova, O. H., Ivanov, B. C., Eremenko H. C., C. A. Lebedev Characteristics�and mutants of the plague agent distinguished on the basis of pigmentable. Mol. the Genet., mikrobiol. and virusologii. 2003, 1, 26-31.

5. Podladchikova, O., Rykova V., Antonenka U., Rakin A. Yersinia pestis autoagglutination is also been other ideas where by Hep-like protein and siderophore yersiniachelin. Adv. Exp. Med. Biol. 2012, 954, 289-292.

1. A method of separating an inhibitor of secretion of siderophores synthesized pgm-the Y. pestis strains, comprising the following stages:
(a) as of the producer strain SSI used a strain of Y. pestis 1279 km (deposited in the Public collections of pathogenic bacteria "Microbe"), which is obtained from the pgm-the vaccine strain Y. pestis EV76;
b) Y. pestis strain 1279 km loop seeded in 5 ml LB medium and grown in an incubator at 26°C for 48 h;
C) in 0.2 ml of culture was inoculated in 10 Petri dishes with 1.5% LB agar (pH 7.2) and grown at 26°C for 48 h;
g) wash away bacteria from the surface of the agar in cold (4°C) phosphate-buffered saline ('s SGA) and precipitated them by centrifugation (8000 rpm) in cold (4°C) for 15 minutes;
e) backass (10 g), washed twice from the remnants of the nutrient medium, suspending the bacteria in a's SGA and precipitating them by centrifugation (8000 rpm) in cold (4°C) for 15 minutes;
(e) cellular precipitate suspended in 100 ml of 5 mm NaOH (pH 9,0), incubated with intermittent shaking, at 37°C for two hours and separated from the cells by centrifugation (8000 rpm) at 20°C for 15 minutes, supernatant is taken and the procedure was repeated thrice;
g) three soup�of mutant unite in a volume of 300 ml and filtered through a nitrocellulose membrane with a pore diameter of 0.22 μm and get alkaline solution, which contains the inhibitor;
h) the alkaline solution is extracted three times with a mixture of chloroform-methanol-water (5:2:1) in an amount of 50 ml, separating the chloroform and aqueous methanol fraction by centrifugation at 8000 rpm for 10 min;
and) chloroform fraction are selected, pooled and freed from soluble impurities by extraction with water, the aqueous fraction was separated by centrifugation at 8000 rpm for 10 min and removed;
K) chloroform fraction was dried in a vacuum rotary evaporator and receive 10 mg of dry product.

2. Inhibitor of secretion of siderophores, which is located on the surface of Y. pestis bacteria and the presence of which correlates with the ability of the cells of Yersinia pestis to block siderophore activity and reduce the autoagglûtinacii bacteria, characterized by:
- molecular weight 380,6 Yes;
- the presence of iron ions;
- fluorescence in the ultraviolet radiation;
- lipopeptide nature;
- hydrophobic properties;
brown coloration of the dry crystals,
obtained by the method according to claim 1.



 

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3 cl, 4 dwg, 4 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: microorganism, which codes antigens and protein toxins, contains a first component, which is at least one nucleotide sequence coding at least at least one complete or partial antigen of at least one wild-type or mutated protein, a second component which is at least one nucleotide sequence coding for at least one protein toxin and/or at least one protein toxin subunit, a third component consisting of at least a first subcomponent which is at least one nucleotide sequence coding at least one transport system which enables the expression of the first and second components on the outer surface of the microorganism and/or enables the secretion of the expression products of the first and second components, and/or coding at least one signal sequence which enables the secretion of the expression products of the first and second components, and/or optionally, from the second subcomponent, which is at least one nucleotide sequence coding at least one protein for lysing the microorganism in the cytosol of mammalian cells and for intracellularly releasing plasmids or expression vectors, which are contained in the lysed microorganism; and a fourth component which is at least one nucleotide sequence for at least one activation sequence for the expression of one or more of the first, second and third components, wherein said activation sequence can be activated in the microorganism and/or is tissue cell-specific, tumour cell-specific, macrophage-specific, dendrite-specific, lymphocyte-specific, function-specific or non-cell-specific, wherein any of the first, second, third or fourth components, present in the microorganism more than once, are independently identical or different, wherein the first and second components are different from each other. Also disclosed are a medicinal agent for stimulating immune response and a pharmaceutical composition based on said microorganism, methods of obtaining said microorganism, the corresponding expression plasmid and expression vector for obtaining said microorganism.

EFFECT: invention enables to obtain novel antitumour vaccines, which induce a strong systemic cellular immune system response, which increases effectiveness of antitumour therapy.

21 cl, 22 dwg, 8 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: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to immunostimulating compounds and may be used in medicine. An immunostimulating peptide of an amino acid sequence XLYDKGYTSKEQKDCVGI, where N-terminal X is N-acetylalanine, and may be covalently linked to fatty acids, selected from C2-C25, to form PDAG (peptidyl-2,3-diacylglycerides). The resulted compound may be included in pharmaceutical formulations for stimulating an immune response.

EFFECT: invention provides efficient stimulation of an immune response in subjects and may enhance the immunogenicity of the antigenic peptide when administered with PDAG.

29 cl, 10 dwg, 9 ex

FIELD: biotechnology.

SUBSTANCE: method of production of peptides is proposed. Yeast autolysis is carried out. The cell membranes are separated by centrifugation. The autolysate is purified on the gel sulphocationite in the hydrogen form, containing 12-16% divinylbenzene. The resulting peptide aqueous solution is passed sequentially through the gel anion-exchange material to obtain the solution at pH 2.0-2.6, and then through the gel cation-exchange material with the divinylbenzene content of 1-2% or macroporous cation-exchange material.

EFFECT: obtaining highly purified peptides that have biological activity.

11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to purification of various gamma-carboxylated olypeptide forms with application of ion-exchange chromatography. In particular, in accordance with invention, claimed is method of purification of polypeptide, which has desirable content of gamma-carboxyglutaminic acid, from sample, containing mixture of said polypeptide versions, which have different content of gamma-carboxyglutaminic acid, with the claimed method including stages: (a) loading said sample on anion-exchange chromatographic material; (b) elution of said polypeptide with application of solution with pH lower than 9.0, containing at least one salt, selected from ammonium acetate, ammonium chloride and sodium acetate; and (c) selection of fraction, obtained after said elution, with polypeptides in said fraction having desired content of gamma-carboxyglutaminic acids.

EFFECT: claimed is method of purifying polypeptide, which has desirable content of carboxyglutaminic acid.

8 cl, 13 dwg, 6 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is presented is a composition of an antibody for treating HER2-positive cancer containing an active antibody presented by an antibody characterised by the fact that it has a variable light and heavy chain domain, and its acidic variants, namely: glycosilated, deaminated variants, as well as a variant with a reduced disulphide bond, a syalylated variant and a irreducible variant. A number of acidic variants makes less than approximately 25%. There are described a pharmaceutical composition containing this composition, for treating HER2-positive cancer and a method for preparing the composition involving the evaluation of the acidic variants and verification of the fact that their number makes at least than approximately 25%.

EFFECT: using the invention provides the new composition, wherein the antibody and its acidic variants have the pharmacokinetic parameters that can find application in treating HER2-positive cancer.

14 cl, 17 dwg, 1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention refers to gene engineering, more specifically to producing the peptide GLP-1, modified by an oligosaccharide chain, and can be used in medicine for treating or preventing diseases associated with GLP-1. In the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3 two amino acid peptides are substituted by an amino acid modified by a complex bi-antennal oligosaccharide chain, and wherein each of the centres is specified in a group consisting of positions 18, 22, 26, 30, 34 and 36 in the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3. The above modified peptide GLP-1 can involve the deletion, substitution or attachment of 1-5 amino acids, except for the amino acids modified by the oligosaccharide chain.

EFFECT: invention enables producing the peptide GLP-1 modified by the oligosaccharide chain, which shows the stronger activity of blood glucose suppression and twice increased half lifetime as compared to GLP-1 with SEQ ID NO: 3.

24 cl, 5 dwg, 6 tbl, 16 ex

FIELD: chemistry.

SUBSTANCE: matrix can be used in purification of proteins, where protein represents antibody, fragment of antibody or antibody-containing fused protein. Ligand corresponds to the following formula (I): R1-R2-N(R3)-R4-R5, where R1 represents non-substituted phenyl group; R2 represents hydrocarbon chain, containing 0-4 carbon atoms, preferably 1-4 carbon atoms; R3 represents hydrocarbon chain, containing 1-3 carbon atoms; R4 represents hydrocarbon chain, containing 1-5 carbon atoms; and R5 represents OH or H. As base matrix contains particles, in fact representing spherical particles, or has membranous or porous structure. Method of obtaining separation matrix includes immobilisation of said ligand on base mainly through amine group. Obtained matrix is placed into chromatographic column and after that sterilised if necessary. In order to separate one or more antibodies from one or more other compounds in liquid sample mobile phase, containing said antibodies and compound(s), are brought into contact with separation matrix. Liquid sample can contain supernatant, obtained in cell fermentation or unprocessed nutritional substance. In the process of application of chromatographic column mobile phase passes through column under impact of gravity and/or rocking, and antibodies are obtained in flow liquid of column. Invention also described set for purification of antibodies from one or more other components in liquid, containing in separate compartments chromatographic column, filled with separation matrix, one or more than one buffer and written instructions.

EFFECT: claimed invention relates to novel separation matrix, containing ligand, bound to base.

20 cl, 6 dwg, 4 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: method includes steps of: (1) extracting fermentation broth containing a compound of formula I or a salt thereof to obtain an extract 1 after filtering and centrifuging; (2) diluting or concentrating the extract 1 in a vacuum with reduction of organic solvent content to obtain an extract 2; (3) feeding the extract 2 into a macroporous adsorption resin; (4) washing the macroporous adsorption resin with water or a mixture of water and an organic solvent as a washing solution and (5) eluting the compound of formula I from the macroporous adsorption resin with the mixture of water and organic solvent as an eluent.

EFFECT: method enables to use a smaller amount of organic solvent and improves purity of the collected compound of formula I.

8 cl, 2 dwg, 2 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns recombinant plasmid pPA-OPRFI DNA coding hybrid recombinant F-I protein of Pseudomonas aeruginosa outer membrane, of the bacterial strain E.coli PA-OPRFI producing this hybrid protein, and a method for producing this recombinant protein. The presented plasmid DNA contains a DNA fragment containing a sequence of modified promoter of bacteriophage T5 and two lactose operons; a DNA fragment containing a ribosome entry site, an initiation ATG-codon and a sequence coding six histidines; a DNA fragment containing the full-size sequences of oprF and opri genes of P.aeruginosa; a DNA fragment containing a ribosome entry site, and a DNA fragment containing a lambda t0 transcription stop region.

EFFECT: presented inventions enables producing the hybrid recombinant F-I protein of Paeruginosa outer membrane for carrying out an immunobiological assay in developing a Pseudomonas aeruginosa vaccine, and also for producing donor immunoglobulins for therapy of active forms of Paeruginosa infection.

3 cl, 3 dwg, 1 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutically acceptable crystalline or amorphous salts of D-isoglutamyl-D-tryptophan, methods of their obtaining, pharmaceutical compositions, containing them, and their application for obtaining pharmaceutical compositions for treatment of different conditions and/or diseases. In particular claimed invention relates to potassium salt of D-isoglutamyl-D-tryptophan (1:1) and magnesium salt of D-isoglutamyl-D-tryptophan (2:1).

EFFECT: obtaining pharmaceutically acceptable crystalline or amorphous salts of D-isoglutamyl-D-tryptophan.

22 cl, 15 dwg, 13 ex

FIELD: biotechnology, biochemistry.

SUBSTANCE: invention relates to extracts prepared from vegetable somatic embryos for the cell-free translation system and/or the coupled transcription-translation system. Method involves preparing embryonic callus from the primary material and the embryonic suspension culture. After induction of the secondary somatic embryogenesis extract is prepared from somatic embryos. Based on the extract the diagnostic system is developed for detection of biologically active compounds. Invention provides overcoming the species limitations and strain specificity and to attain the high effectiveness of the cell-free translation system and the coupled transcription-translation system also.

EFFECT: improved preparing method, valuable biological and biochemical properties of system.

49 cl, 5 dwg, 2 tbl, 9 ex

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