Method of production of protective antigen and protein of s-layer ea1of asporogenous recombinant strain in anthracis 55 Δtpa-1spo-

FIELD: biotechnology.

SUBSTANCE: method includes cultivation of previously prepared culture of the recombinant strain B. anthracis 55ΔTPA-1Spo-. The cell mass is separated using the filtration module with a membrane having a pore diameter of 0.2 mcm. Protein EA1 is extracted from the washed cell mass using a buffer with 1% sodium dodecyl sulfate, and purified by diafiltration using membrane filters and two-stage ion-exchange chromatography on hydroxyapatite. The protective antigen is isolated from the culture filtrate and purified by successive steps of concentration and diafiltration.

EFFECT: use of the invention enables to obtain in one processing chain the highly purified antigens of anthrax microbe - protective antigen and protein EA1 needed to create chemical vaccines.

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The invention relates to biotechnology, in particular to a technology for immunogenic anthrax antigens - protective antigen and protein EA1, and can be used in the production of specific prophylaxis of anthrax.

Century anthracis is the etiological agent of especially dangerous infectious disease anthrax. The high virulence of the causative agent of anthrax is caused by the genes determining the synthesis of the capsule and exotoxin. The capsule protects the microbial cell from phagocytosis and exotoxin composed of protective antigen, edema and lethal factors, causes abnormal reaction of the microorganism. One of the components of the toxin protective antigen, has a strong immunogenic potential and is the current top of all the anthrax vaccine [1]. Despite the dominant role of protective antigen in the formation of the immune response, no doubt part in the immunogenesis and other antigens, including protein S-layer - EA1 [2]. The introduction of the vaccine is more immunogenic antigens is one of the approaches to increase their immunological efficiency.

Russia produces a combined vaccine, which is the composition dispute vaccine strain Century anthracis STI-1 and isolated from his protective antigen is, adsorbed on aluminium hydroxide. In the UK licensed chemical vaccine consisting of alum precipitated protective antigen selected from the culture filtrate vaccine strain Century anthracis Sterne 34F2. Licensed in the U.S. chemical vaccine contains adsorbed on aluminium hydroxide protective antigen, obtained from the culture filtrate proteocephalidea derived Century anthracis Sterne 34F2- Century anthracis V770-NP1-R.

Known methods for producing protective antigen of the vaccine strains Century anthracis STI-1, B. anthracis Sterne 34F2, B. anthracis V770-NP1-R [3, 4, 5]. A method of obtaining protective antigen from strain Century anthracis 55 Vniivvim, used in veterinary medicine [6]. In all these ways the producers of the protective antigen of Bacillus anthracis strains are similar according to the method of obtaining the elimination of the pXO2 plasmids, and plasmid composition - Rho+pXO2-. Due to the lack of plasmid pXO2 they lack the ability to synthesize the capsule and, consequently, to cause infection when injected into susceptible organism. However, these strains are characterized by relatively high reactogenicity, due to, among other things, synthesized their pathogenicity factors - edema and lethal factors. In addition, they, like all typical prospect who some members of the Swiss of the genus Bacillus, form spores that are resistant to most disinfecting agents. Using spore-forming microorganisms in the industrial production of vaccines is not without risk of bacterial contamination of equipment and premises.

Another disadvantage of ways using vaccine strains-producers is their inability to provide a high level of product protective antigen due to genetic characteristics, namely the presence in the genome of C. anthracis complex system of regulation, limiting its synthesis [7, 8]. The level of production of protective antigen attenuated strains depends on the content of CO2in the atmosphere and is not more than 15-30 µg/ml To increase the production of protective antigen developed the method lies in the deep cultivation of the strain Century anthracis STI-1 in nutrient medium with sodium bicarbonate with continuous aeration of the culture liquid of a mixture of several gases - nitrogen, argon, helium [9]. However, the use of mixtures of different gases for aeration unnecessarily complicates the technology of cultivation. High concentration of CO2in an environment of growing is achieved by adding sodium bicarbonate. However, it is established that under these conditions in the microbial population increases the proportion of cells with mutations in the gene pag, encoding it sintesizer way offering added on Wednesday sodium bicarbonate at a certain stage of growth of the organism [10]. This leads to more stable products protective antigen, but does not change the possible limit of its synthesis, due to the peculiarities of the genotype.

All of the above methods of obtaining protective antigen of the vaccine strains involve the preparation of master seed culture of the strain Century anthracis; deep anaerobic cultivation on a nutrient medium containing sodium bicarbonate; concentrated and sterilized culture filtrate; precipitation is present in the filtrate protective antigen on the gel aluminium hydroxide or similar Freund. However, they do not include the stages of thin chromatographic purification, whereupon the resulting product has an indefinite composition and contains a large amount of ballast substances that do not meet modern requirements, submitted to prophylactic drugs. In addition, get ways protective antigen, even when phase chromatography to contain impurities edema and lethal factors, due to the complexity of the chromatographic separation of proteins with very similar values of molecular masses. The presence of chemical vaccines irregular amounts of reactive components NIB is reasonale toxin can lead to the development of post-vaccination complications.

Today, priority is given to methods of obtaining protective antigen of genetic engineering producers with the cloned gene pag determining its synthesis.

Known methods for producing protective antigen from a recombinant strains on the basis of bacillary strains Century anthracis and B. subtilis [11, 12]. These methods use genetic engineering producers containing hybrid plasmids with included gene pag. These strains do not synthesize the factors of pathogenicity of Bacillus anthracis capsule, edema and lethal factors, resulting in the safe. In addition, they have low activity of proteolytic enzymes, the levels of production of protective antigen reach high values of 100-120 µg/ml. Methods of obtaining protective antigen from a recombinant bacillary strains include the preparation of seed culture; growing under normal conditions in a nutrient medium with intensive stirring for aeration; concentrated and sterilized culture filtrate; isolation and chromatographic purification of protective antigen. However, the drawback of the proposed technology education is in the process of cultivation of microbial spores that are resistant to UV radiation, disinfectants and persisting in the environment in those who tell many decades.

Known methods for producing the anthrax protective antigen from Escherichia coli containing the cloned gene pag [13, 14]. However, unlike the representatives of the genus Bacillus, with the ability to exogenous protein synthesis, for E. coli characteristic intracellular mechanism of secretion of protective antigen, which has an adverse effect on the amount of the target product, and also complicates its isolation and purification. For the production of non-toxic protivoseborainey vaccine was offered the procedure for obtaining protective antigen from strain E. coli DH5α with the cloned gene pag [15]. To increase the yield of protein antigen was achieved through improvement of culturing in the fermenter, as supplements to the standard environment used polyols, carbohydrates, organic acids and mineral salts. However, changing technology by introducing significant amendments to the composition of the medium cultivation reduces its profitability.

A known method of creating a vaccine of the anthrax protective antigen [16]. This patent describes a method for protective antigen from a recombinant asporogenic smear-positive strain. The method includes preparing a seed culture of a producer; growing at a constant temperature of 37±0.1°C until they reach the Stazione the nuclear biological chemical (NBC phase growth; filtration through a bacterial filter (0.2 μm); concentrated culture filtrate and diafiltration; isolation and purification of protective antigen to a chromatographic system high pressure using ion-exchange media. However, in the proposed method as a producer of protective antigen used strain Century anthracis DELTA Sterne-1(pPA102)CR4 derived from besplodnogo derived vaccine strain Century anthracis Sterne, highly active proteolytic enzymes [17], which only partially stopped due to the ingredients of the growing environment [18]. Proteolytic action of producing microorganism is a serious problem for the organization of technological lines for production of biologically-relevant antigens. In addition, scalable cultivation of recombinant strains of C. anthracis DELTA Sterne-1(pPA102)CR4 is carried out in a nutrient medium with the addition of selective antibiotic in a relatively high concentration of 40 µg/ml Use of the antibiotic in the technology of getting a bacterial antigen, a part of the preparation for vaccination of people undesirable. Besides all the above, the basis of how the producer is not available to Russian manufacturers.

A method of obtaining protein S-layer of Bacillus anthracis EA1[19]. The way in which incorporates both of the following stages: growing strain Century anthracis V770-NP-1R (Rho+pXO2-in liquid nutrient medium (R medium) for 18-20 hours, the deposition cell mass by centrifugation, disintegration of cell membranes and incubated with guanidine-hydrochloride for 2 hours at room temperature, precipitation of the guanidine extract alcohol with subsequent washing with distilled water. However, the information derived from this method, the final product contains in addition to EA1 numerous associated proteins that do not meet the requirements of modern chemical vaccine.

The analysis of the prior art, including the search for patents and scientific and technical information sources showed that information about the way that unites obtaining protective antigen and protein EA1 in one process, not detected.

The objective of the invention is to develop a biologically safe and effective way to obtain preparative amounts of highly purified antigens of Bacillus anthracis - protective antigen and protein EA1 needed to create chemical vaccines.

The technical result consists in the possibility of obtaining in a process chain preparative quantities of highly immunogenic preparations protective antigen and protein EA1 Bacillus anthracis, effectively protects against the contamination of vazbu what ielem anthrax, interacting with structures of innate immunity and is not toxic to the host.

The technical result is achieved by a method of obtaining a protective antigen and protein EA1 Bacillus anthracis, including the preparation of seed culture of the strain-producer; cultivation of recombinant asporogenic strain Century anthracis 55Δ-1Spo-at a temperature of 37±0.1°C for 16 hours in a liquid nutrient medium containing triptan with a concentration of not less than 20 mg/ml) with constant stirring at 150 rpm; Department of cell mass with the use of the filtration module with a membrane having a pore diameter of 0.2 μm; the selection of protein EA1 of the washed cell mass and purified using successive stages of incubation containing 1% sodium dodecyl sulphate extracting buffer, diafiltration using membrane filters with a limit exceptions proteins with a molecular mass of 30 kDa and two-step ion exchange chromatography on hydroxyapatite; allocation of protective antigen from the culture filtrate and purified using successive stages of concentration and diafiltration using membrane filters with a limit exceptions proteins with a molecular mass of 30 kDa and a two-step chromatography on ion-exchange carrier and the carrier for gel filtration.

In the implemented method as producer protective antigen and protein S-layer (EA1) Bacillus anthracis use asporogenic and avirulent genetically engineered strain Century anthracis 55Δ-1Spo-. The strain developed FCUS Russian scientific-research Institute "Microbe" and is protected by RF patent No. 2321629. The strain created genetically engineered by on the basis of besplodnogo derived vaccine strain Century anthracis 55, he retains the biological properties during cultivation on nutrient media and storage, there are no determinants of synthesis of the main virulence factors of the pathogen anthrax, and the presence of chromosomal mutations determines asporogenic phenotype. The use of asporogenic avirulent strain of B. anthracis 55Δ-1Spo-to obtain a protective antigen and protein Bacillus anthracis EA1 provides biological safety and eliminates the risk of contamination of premises and equipment spores of anthrax.

High level production of protective antigen of Bacillus anthracis is ensured by the functioning of the cloned gene pag in the composition multicopying hybrid plasmids. Strain Century anthracis 55Δ-1Spo-synthesizes 10-20 times more protective antigen than vaccine strain Century anthracis STI-1, due to the lack of a negative regulator of the synthesis of protective antigen pagR and increase the number of copies of the gene encoding pag by multicabinet vector plasmids. For the effectiveness of the procedure, the separation of the deposits and cleaning of protective antigen and protein EA1 is extremely important what strain Century anthracis 55Δ-1Spo-characterized by sharply reduced activity of proteolytic enzymes. The proposed producer of anthrax antigens grows well in both traditional domestic environments, for example, the broth of Hottinger and standardized semi-synthetic media with a high content of tryptone. Add in the broth of Hottinger of tryptone in concentrations up to 20 mg/ml increases the production of protective antigen in 1,5-2 times. Established experimentally by the applicant, the duration of cultivation 16 hours leads to an increase of approximately 5 times the output of a protective antigen. The essential point to create vaccines is that the cultivation of genetically engineered strain Century anthracis 55Δ-1Spo-within 16 hours can be performed without adding selective antibiotic. It is established that during this period the elimination of hybrid plasmids. In addition, the lack of strain Century anthracis 55Δ-1Spo-plasmid positive regulation of AtxA allows you to receive the maximum amount of protective antigen without additional material costs and efforts to build a high concentration of carbon dioxide in the atmosphere. Consequently, the use of strain Century anthracis 55Δ-1Spo-to obtain a protective antigen and protein EA1 NIB is reasonale microbe significantly increases the profitability of the production process of chemical anthrax vaccine.

The concentration and diafiltration drugs protective antigen and protein EA1 using membrane filters with a limit exceptions proteins with a molecular mass of 30 kDa, and two-stage procedures chromatographic purification on different carriers provide highly purified antigens, effectively interacting with structures of innate and adaptive immunity, not reactive and not toxic to the host.

The possibility of carrying out the invention is confirmed by the example 1.

Example 1. Isolation and purification of protective antigen and protein S-layer of Bacillus anthracis from a strain of B. anthracis 55Δ-1Spo-

Pilot fermenter with a working volume of 14 liters fill 5 liters of broth of Hottinger (pH 7,2-7,4), the medium is sterilized. After sterilization additionally make a sterile solution of triptone to a final concentration of at least 20 mg/ml

Preparation of culture of the producer strain to obtain sufficient amounts of biomass at a certain stage of growth is produced as follows. One full loop 18-hour agar culture of the producer strain inoculated in tubes with beveled agar of Hottinger containing kanamycin at a concentration of 25 μg/ml, and incubated at 37°C for 6 hours. After a period of incubation, the biomass was washed by adding 1 m is cooled to 0.85% of sodium chloride solution (pH of 7.2), resus-ponderous and transferred into vials with beveled agar of Hottinger containing selective antibiotic in the same concentration. Crops incubated at 37°C for 18 hours. Microbial mass from the surface of the agar wash, adding to each bottle 3 ml of chilled of 0.85% sodium chloride solution (pH of 7.2).

For scalable cultivation 50 ml of cell suspension of strain-producer Century anthracis 55Δ-1Spo-with the concentration of microbial cells is not less than 50 billion M.K./ml make ready a sterile environment. The cultivation is carried out at a temperature of 37±0.1°C for 16 hours with constant stirring at 150 rpm After 16 hours the level of production of protective antigen according to the immunoassay is 640 µg/ml, which is 20 times greater than the values determined for vaccine strain Century anthracis STI-1 (figure 1).

After the end of the cultivation period add 2 mm EDTA. Department of cell mass and sterilization is carried out at room temperature and operating pressure of about 2 bar on the unit for concentration and purification of proteins (Sartorius, Germany), using the module tangential filtration Vivaflow 200 (Sartorius, Germany), containing a membrane filter with a pore diameter of 0.2 μm. After sowing 0.1 ml of the culture filtrate to plates with agar of Hottinger and incubation at 37°C for 10 suto is not noted characteristic of Bacillus anthracis colony morphology.

For selection of protective antigen after sterilization culture filtrate diluted in a volume ratio of 1:1 with buffer containing 25 mm diethanolamine, 50 mm NaCl, 2 mm EDTA (pH 8,9). Then concentrated approximately 20-fold at the facility for concentration and purification of proteins by filtration tangential flow Vivaflow (operational pressure of about 2.5 bar), using the module Vivaflow 200 (Sartorius, Germany) with a limit exceptions proteins with a molecular mass of 30 kDa. The next step are diafiltration 10-fold volume of the same buffer on the same module at room temperature. Concentrated and definitavely preparation containing protective antigen, purified through column chromatography with Macro Prep 50Q (Bio-Rad, USA). Carrier pre-Tegaserod and washed with the same volume of buffer containing 1M NaCl. Then balance column, passing successively with 10 volumes of buffer containing 25 mm diethanolamine, 50 mm NaCl, 2 mm EDTA (pH 8.9) and 1 volume of the same buffer with the addition of 30 mm KCl (pH 8,9). Proteins of the culture filtrate elute when the velocity of the flow 10 ml/min, using one free volume of the column, then the column is washed with the last buffer in a volume corresponding to the volume of the media. The resulting sample is combined with the eluate. The drug protective antigen concentrate 10 times on the module Vivaflow 200 (30 to the a) when the operational pressure of about 2 bar and room temperature. Diafiltration carried out on the same module with serial 10-fold volume of buffer containing 25 mm diethanolamine, 50 mm NaCl, 2 mm EDTA (pH 8.9) and 10-fold volume of buffer containing 145 mm ammonium acetate, 2 mm EDTA (pH of 10.0). Before the final stage of the treatment drug protective antigen filtered through membrane filters with a pore diameter of 0.2 μm (Sartorius, Germany), poured into aliquot and store at a temperature of minus 70°C. Further purification is performed on a chromatographic system BioLogic Duo Flow(BioRad, USA). As a carrier for gel filtration using Sephacryl-HR300 (BioRad, USA). The corresponding column is filled with media and balance 3 volumes of buffer solution containing 0.1 M Triton, 1 mm EDTA, 50 mm NaCl (pH 8.0). Cooled to 4°C. the preparation protective antigen (protein concentration of 2.5 mg/ml) is applied onto the column portions in the amount of not more than 3% of the volume of the gel. Chromatographic system programming of the flow speed of 2 ml/min. Output proteins is controlled by spectrophotometry at a wavelength of 280 nm. Samples corresponding to the maximum peak absorption, select and after several such cycles of cleaning unite. Given what is happening during gel filtration dilution of the preparation of the consolidated samples concentrated 10 times using hubs "Vivaspin-20" (Sartorius, G is Romania). The degree of purification protective antigen defined by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate, was 90%. The results of cleaning of protective antigen presented on figure 2, where: 1 - culture filtrate of C. anthracis 55Δ-1Spo-; 2 - after drug concentration on the module Vivaflow 200 (30 kDa); 3 - the product after purification on a column of Macro Prep 50Q; 4 - the drug after diafiltration; 5 - the drug after a secondary concentration on the module Vivaflow 200 (30 kDa); 6 - molecular mass markers(260, 135, 95, 72, 52, 42, 34, 26, 17, 10 kDa); 7 - the final drug protective antigen after chromatography on a column of Sephacryl-HR300. The drug protective antigen poured into aliquot and store at a temperature of minus 70°C., and after drying is at a temperature of 8°C.

To highlight EA1 precipitated by centrifugation of cell mass were washed off the remnants of the environment with 0.9% sodium chloride solution. 500 ml of biomass mixed with 50 ml of extracting buffer containing 5 mm Tris-HCl, 1% sodium dodecyl sulfate, 5 mm 2-mercaptoethanol. The mixture is heated on a water bath at 70°C for 30 minutes, then cooled to room temperature and centrifuged for 30 minutes at 10,000 rpm and 4°C. Cell extract is sterilized by passing through a module tangential filtration Vivaflow 200 (0.2 μm), when the current having the drop of about 2 bar. In the next step cell extract diafiltrate 10 volumes of buffer containing 0.1 M Tris-HCl, 2 mm EDTA (pH 8.0)at room temperature using module Vivaflow 200 (30 kDa) under a pressure of about 2 bar. To stage cleaning preparation containing EA1, maintained at a temperature of minus 70°C. Purification is carried out on a chromatographic system BioLogic Duo Flow(Bio-Rad, USA). The column is filled pre dekotirovaniem hydroxyapatite and balance 10 free volumes of buffer containing 5 mm K2HPO4·KH2PO4(pH 6.8), when the velocity of the flow 10 ml/min to obtain a stable baseline. The product containing EA1, put on a column and create a linear gradient of potassium phosphate (0 to 1 M K2HPO4·KH2PO4using 3 free volume of the column. Fractions corresponding to the absorption peaks at 280 nm, collect and obyedinayutsya conduct the second stage of the treatment on the same media, creating a gradient of potassium phosphate as described above. Purified protein EA1 diafiltrate 10 volumes of bidistilled water at a temperature of 4°C using a module Vivaflow 200 (30 kDa) under a pressure of about 2 bar. The degree of purification protein EA1 determined by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate, was 95%. Results protein purification EA1 presented on figure 3, where 8 is leocrystal after incubation in buffer with 1% sodium dodecyl sulfate; 9, 10 - the drug after the first stage of purification on hydroxyapatite; 11 - end the drug protein EA1 after the second round of purification on hydroxyapatite; 12 - markers molecular mass(116,0; 97,0; 66,0; 45,0; 29,0 kDa). If necessary, the drug protein EA1 concentrated, poured into aliquot and store at a temperature of minus 70°C., and after drying is at a temperature of 8°C.

Thus obtained preparations protective antigen and protein EA1 suitable for use as the primary and secondary components of the anthrax chemical vaccine, which is confirmed by the following examples.

Example 2. Determining the ability of drugs based on protective antigen and protein EA1 to protect laboratory animals when infected the test strain of anthrax

Laboratory animals subjected to immunization protective antigen and protein EA1, obtained as described above (example 1). Separate groups of mice BALB/c mice (20 animals per group) twice impose protective antigen in a dose of 10 µg or complex preparation containing the protective antigen in the same dose and protein EA1 in a dose of 5 μg. Immunobiological restructuring assess indicators LD50the test strain Century anthracis 71/12 and indexes of immunity. In the result, the values of LD50infecting strain for non-immune individuals is 5×102 (100÷2512) dispute; for immunized protective antigen of 7.9×104(15849÷501187) the dispute; for immensively comprehensive drug is 1.9×105(39811÷1000000) dispute. Accordingly, the indices of immunity following: 158,5 for protective antigen, 398,2 for protective antigen with EA1. Thus, adding EA1 in immunizing preparation based on protective antigen increases the resistance line of mice to infection with the test strain of anthrax 2.5 times.

Equal groups of Guinea pigs twice impose protective antigen combined with adjuvant-blockers or protective antigen in combination with protein EA1 and adjuvant. Immunizing dose of protective antigen for Guinea pigs is 25 µg/ml, protein EA1 - 10 μg. Immunization vaccine strain Century anthracis STI-1 (drug comparison) carry out once subcutaneously at a dose of 5×107a dispute. As a result, LD50test-infecting strain Century anthracis 71/12 for control animals is 3.1×102dispute; for Guinea pigs immunized with protective antigen, protective antigen with EA1 and vaccine strain Century anthracis STI-1 1,9·107a dispute. Accordingly, the index of immunity in all cases 100072,0. That is twice the immunization of biomodels protective antigen or a protective antigen with EA1 provides protection from C the expressions of the test strain Century anthracis 71/12, comparable with produktivnost live anthrax vaccine Century anthracis STI-1. Thus, the proposed method allows to obtain antigenic preparations, effectively protecting them from infection by anthrax.

Example 3. Determining the ability of purified protective antigen preparations and protein EA1 to interact with structures of innate immunity toll-like receptors

Mice of BALB/c mice subjected to immunization subcutaneously once the protective antigen (10 μg) or protein EA1 (10 μg)obtained as described above (example 1). Animals killed after 4 hours, 1, 3, 7 and 14 days after immunization and take the material from the organs of the Central and peripheral immunogenesis (thymus and spleen). Isolation of RNA from cells of the spleen and thymus carried out using the kit reagents "RIBO-Sorb" (Interlabservice, Russia) according to the instructions of the manufacturer. Reverse transcription was carried out using a set Reverta" (Interlabservice, Russia) according to the manufacturer's instructions. With the obtained cDNA carry out PCR with specific primers to toll-like receptors (TLRs) 2 and type 6. Positive control is amplificatory fragment of the gene of the ' household ' β-actin. Amplification of gene fragments carried out on a programmable amplifier T is the person" (DNA-technology, Russia) according to the scheme: one cycle at 95°C for 5 min, 30 cycles at 95°C - 1 min, 60°C - 1 min, a temperature of 72°C for 1 min and a final cycle of 5 min at a temperature of 72°C. Electrophoretic analysis of PCR products was carried out in a horizontal chamber (Bio-Rad, USA) in 2% agarose gel. To determine the size of the amplicons used commercial markers molecular masses Of' GeneRuler 100 bp DNA Ladder Plus ready-to-use (Fermentas, Lithuania). Visualization of DNA bands is performed using a source of ultraviolet light and is recorded by a system gel documentation (Bio-Rad, USA). As a result, all samples taken from 4 hours and up to 14 days, the immunized animals in contrast to the control noted a significant increase in the expression of TLRs 2 and type 6 in the cells of the spleen and thymus.

Example 4. Determination of the toxicity of purified protective antigen preparations and protein EA1 in vivo and in vitro

Guinea pigs (10 animals per group) once subcutaneously injected protective antigen or protein EA1 in doses of 25, 50 and 100 µg. During the first two weeks with a frequency of 1 every two days carry out the weighing and inspection of the animal. In result within 21 days of observation in Guinea pigs visual and palpatory changes at the injection site is not marked, the body weight of animals is not reduced.

For assessment of the damaging effect of in vitro protective antigen and protein EA1 doses of 10 µg incubated with 3 ml of whole defibrinating blood of healthy donors at 37°C for 24 hours. Color samples of leukocytes spend mixture mithramycin (125 µg/ml) and ethidium bromide (25 µg/ml). The cytotoxic effect of antigenic preparations is controlled by the number of cells in apoptosis using the method of flow cytofluorimetry. In the purified recombinant PA Bacillus anthracis does not increase apoptotic activity and has no damaging effect on the blood leukocytes of man.

For the study of the proliferative activity of lymphocytes bodies of the Central and peripheral immunogenesis mice of BALB/c mice (6 animals per group) subjected to immunization subcutaneously protective antigen or protein EA1 doses of 10 µg. The controls are intact animals. Samples of thymus and spleen take over 4 hours, 1, 3, 7 and 14 days after immunization. The percentage of thymocytes and splenocytes in the phases of cell division is recorded by a flow cytometer. Apoptosis assessed by accumulation hypodiploidy cells at the peak, which is located to the left of the peak, corresponding diploid cells. Activation of immunocompetent cells reflects their number in the stage of proliferation. Also determine the proportion of cells undergoing apoptosis and proliferation. The value of this index must be less than unity

The result is that in all study periods apoptotic and prolifer the effective activity of the cells of the thymus is not significantly different from those in the control group. The balance of apoptosis and proliferation does not exceed 1.0, indicating the absence of the damaging effect of study drug on immunocompetent cells. In spleen samples taken after 4 hours, at 1 and 3 days, the number of proliferating splenocytes match the control values. At 7 and 14 days was observed the increase of the proliferative activity of cells of the spleen, which may be caused by the proliferation of b cells and antibody production. Administration to mice of a protective antigen and protein EA1 does not affect the apoptotic activity of splenocytes. The index is the ratio of spleen cells under apoptosis and proliferation does not exceed 1.0.

Thus obtained with the use of the method described above preparations protective antigen and protein EA1 not have a damaging effect on immunocompetent cells of the Central and peripheral immune system of the host.

Example 5. Pathomorphological and histological examination of the organs of laboratory animals immunized with protective antigen and protein EA1

Guinea pigs once the drug is administered protective antigen in a dose of 50 mg in combination with protein EA1 dose of 20 μg. The organs for histological studies carried out on 1, 3, 7, 21, 28 days after immunization.

Subcutaneous administration of the anthrax Antiga is s cavies does not cause the death of animals and does not affect their overall condition. In the introduction after immunization celebrate minimal changes in the form of focal moderate edema of the subcutaneous fat and slight lymphocytic infiltration of the dermis, completely disappearing after 3 days. Changes in the adrenal glands, observed during the first 7 days, indicate mild stress reaction. From parenchymatous organs with macroscopic study for the entire period of observation do not celebrate gross degenerative changes, infiltrative processes, sharp infringements of blood vessels. In the period from 1 to 7 day (s) histological examination register signs of moderate functional voltage parenchyma cells of the liver, kidneys, cardiomyocytes against the background of the focal phenomenon of hyperemia of the vessels. Changes in the glomerular apparatus of the kidneys characterized by moderate plethora of capillary loops vascular glomeruli of kidney cells. The number of renal Taurus with changes less than 50%. In the liver of Guinea pigs in the early stages (1-3 days) see moderate stagnation in the circulation system (vnutritrekovye sinusoidal emocapella) and outflow (Central vein) blood, combined with functional voltage light hepatocytes center of the hepatic lobules. However, by day 7 the functional state of the organ no significant difference is between the observed in animals from the control group.

In lymphoid organs registering an increase of activity from 3 to 21 days, with a maximum of 27 days, indicating that the activation of the immune system. From the Central organ of the immune system - thymus, the increase in the mass of body 2 times in relation to the mass of body control animals recorded at 21 days of observation. In the spleen register the increase in the mass of body 2 times compared to the same indicator in the intact animals and moderate hyperplasia of the follicular structures (grain) at 21 days of observation. Marked hyperplasia of regional lymph nodes note 21 days. The mass of distant lymph nodes reaches maximum values at 21 days, during this period it 30 times the value of the reference index. However, by 27 days of the dimensions and mass of lymph nodes returned to control values. The histological examination of the thymus in normal ratio of cortical and medullary substance moderate hyperplastic processes of lymphoid elements and the relative activation of mitotic activity in the medulla begin after 7 days, gradually decreasing to 27-th day. The functional status of the peripheral organs of the immune system fits into the picture of immunogenesis that manifested placentas is consistent activation of T - and b-zones in them. Thus, the changes identified by histological examination of the internal organs of Guinea pigs immunized with protective antigen in combination with protein EA1, are valid and reversible. From immunocompetent organs register manifestations of immunogenesis.

Thus, the inventive method can effectively and without risk of contamination of premises and equipment spores of anthrax to obtain preparative amounts vysokomanevrennyh and non-toxic drugs protective antigen and protein EA1 necessary to create means of specific prophylaxis of anthrax.

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12. Baillie L. Vaccine production of the Bacillus anthracis protective antigen // United States Patent No. US6267966 (C12N 15/31; A61K 39/07; C12R 1/125) - 2001.

13. Bhatnagar R., Batra, S., Chauhan C., Singh, A., Ahuja N., Kumar P., Gupta P. a method of obtaining a non-toxic protivoseborainey vaccine // Patent RF №2287581 (C12N 15/31, C07K 2/00, C12P 21/00, A61K 39/07) - publ. 20.11.2006.

14. Brehm J., Mcentee I., Vincent P., Allison N., R. Brehm, Jack G., Herbert M., Solow Century, Arpoyo J., Lapcevich R. Preparatio of protective antigen from Bacillus anthracis // KR 20070114263 (A) (A61K 39/07; C07K 14/32) - publ. 30.11.2007.

15. Chauhan V., Singh, A., Waheed S.M., Singh, S., Bhatnagar R. Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli // Biochem. Biophys. Res. Commun. - 2001. - Vol.283. - N 2. - P.308-315.

16. Ivins Century, Worsham, P., Friedlander, A., Farchaus, J., S. Welkos Method of making a vaccine // United States Patent No. US2002034512 (C12N 15/75; C12N 15/74). - 21.03.2002.

17. Farchaus, J., Ribot W., Jendrek, S., Little, S. Fermentation, purification, and characterization of a protective antigen from a recombinant, avirulent strain of Bacillus anthracis // Applied and Environmental Environ. - 1998. - Vol.64. No. 3. - P.982-991.

18. Ezzell J., T. Abshire Immunological analysis of cell-associated antigens of Bacillus anthracis // Infect. Immun. - 1988. - V.56. No. 2. - P.349-356.

19. Mikshis NI, Popov, Y.A., Shulepov A.I recombinant Asporogenic strain Century anthracis 55Δ-1 Spo- (pUB110PA-1) - producer of protective antigen Bacillus anthracis // Patent RF №2321629 (C12N 1/21, C12P 21/00, C12R 1/07). - 10.04.2008.

A method of obtaining a protective antigen and protein from strain EA1 Century anthracis, including the preparation of seed culture of the strain-producer; cultivation of recombinant asporogenic strain Century anthracis 55Δ-1Spo-at a temperature of 37±0.1°C for 16 h in liquid nutrient medium containing triptan with a concentration of not less than 20 mg/ml) with constant stirring at 150 rpm; Department of cell mass with the use of the filtration module with a membrane having a pore diameter of 0.2 μm; the selection of protein EA1 of the washed cell mass and purified using successive stages of incubation containing 1% sodium dodecyl sulphate is xtraverse buffer, diafiltration using membrane filters with a limit exceptions proteins with a molecular mass of 30 kDa and two-step ion exchange chromatography on hydroxyapatite, and the identification of a protective antigen from the culture filtrate and purified using successive stages of concentration and diafiltration using membrane filters with a limit exceptions proteins with a molecular mass of 30 kDa and a two-step chromatography on ion-exchange carrier and the carrier for gel filtration.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: alcohol (8-C) strain Saccharomyces cerevisiae No 8 having a high generative activity was deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number RNCIM B-3855 and can be used in production of alcohol.

EFFECT: invention enables to increase the alcohol yield and to reduce the formation of byproducts.

3 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of isolating and purifying recombinant human growth hormone which is secreted by Saccharomyces cerevisiae yeast during fermentation thereof in suitable conditions. The target protein is precipitated in biomass-free culture fluid by either acidification to pH 2.9-4.0 or adding polyethylene glycol with molecular weight of 3000-6000 Da. The obtained precipitate is then dissolved in a suitable solvent. Preliminary purification of the target protein is carried out either by anion-exchange chromatography at pH 5.6 or by diafiltration in the presence of 0.1-0.5 M sodium chloride. Main purification of the target protein is then carried out by anion-exchange chromatography at pH not below 7.3 and gel filtration.

EFFECT: invention enables to obtain a growth hormone which is free from parent proteins, host-producer protein and other impurities such as pigments, with output of up to 60%.

8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to use of the Lactobacillus paracasei CNCM I-2116 strain to treat irritable bowel syndrome. A probiotic includes dead Lactobacillus paracasei CNCM I-2116 bacteria, a fermentation substrate and/or material made from Lactobacillus paracasei CNCM I-2116.

EFFECT: invention provides the capacity to normalise post-infection hyper-contractible state of intestinal muscles.

2 cl, 4 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to Saccharomyces cerevisiae CNCM I-3856 and Saccharomyces cerevisiae var. boulardii CNCM I-3799 yeast strains that are used as probiotic which is suitable for preparing food or pharmaceutical compositions. Also disclosed is a composition which contains yeast strains Saccharomyces cerevisiae CNCM I-3856 and/or Saccharomyces cerevisiae var. boulardii CNCM I-3799 and/or at least one parietal mannoproteins EL 05 and EL 06 of the Saccharomyces cerevisiae CNCM I-3856 yeast strain.

EFFECT: invention enables to reduce relieve paint in the intestines, induction of anti-inflammatory action without pro-inflammatory action, difficult and reduced adhesion and population of the gastrointestinal tract with bacteria that are pathogenic and/or invasive in nature.

12 cl, 30 dwg, 6 tbl, 9 ex

FIELD: process engineering.

SUBSTANCE: invention relates to biochemistry. Effluents are cleaned of suspended substances, oil products, phenols and chlorides for water to be discharged into pool. Inner surface of filtration dam is processed by bacterial culture Pseudomonas fluorescens "ВКГ" RCAM 00538 with titre of 10-13-10-11 in amount of 30 mg/dm3 in dry weight to obtain biofilm. Said filtration dam is filled with water to be cleaned and kept therein for at least 3 days. Effluents are forced through filtration dam consisting of the following rocks: Crushed stone or sand-gravel mix, or mix of mudstone with siltstone.

EFFECT: efficient cleaning to MPC acceptable for water discharge into pool.

1 dwg, 14 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of altering immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which involves obtaining preparations of lipopolysaccharides (LPS) and mouse toxin (MT) Yersinia pestis with subsequent formation of a LPS-MT complex thereof. A modified form of LPS-MT is used as an inducer of synthesis of cytotoxins TNF-α and IFN-γ. To this end, a test sample is prepared, to which LPS is added in amount of 5 mcg (50 mcl from working dilution of 100 mcl/ml) and MT is added in amount of 50 ng (5 mcl from working dilution of 10 mcg/ml); the sample is then incubated for 30 min at 37°C. The volume of the sample in eppendorfs is then brought to 100 mcl with sterile buffered physiological solution of NaCl and the obtained mixture is added a tray dimple containing a culture of human monocyte cell line U-937 (1×106 cells in a dimple); the latter is cultured in a medium of PRMI 1640 with simultaneous double control. Further, 1, 4, 20 hours after the beginning of combined incubation of the preparations of LPS with monocytes, quantitative accounting of the synthesised cytotoxins is carried out, wherein change in the immunomodulating properties of LPS of plague bacteria in vitro is determined from the amount of cytotoxins produced and the dynamics of their synthesis.

EFFECT: invention enables to alter immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which enables to realise toxic potential of the endotoxin of plague bacteria.

2 cl, 8 dwg, 2 ex

FIELD: biotechnologies.

SUBSTANCE: strain Rhodococcus erythropolis 1-KP, extracted from podzol soil contaminated with oil from the territory of the Kolsky peninsula, having high speed of oil utilisation, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VNIISHM) under the number RCAM01142 and may be used for treatment of contaminated soils from oil.

EFFECT: improved quality of soil cleaning from oil.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: strain pseudomonas citronellolis 48-U, having high speed of oil and diesel fuel recycling, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VIISHM) under the registration number RCAM 01441 and may be used for treatment of contaminated soils from oil and diesel fuel.

EFFECT: improved quality of soil cleaning from oil and diesel fuel.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: strain Rhodococcus fascians 4-G, extracted from soil contaminated with black oil and sampled from the territory of a boiler plant located in the settlement Gorelovo, Leningrad region, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VNIISHM) under the number RCAM01140 and may be used for treatment of contaminated soils from oil.

EFFECT: invention makes it possible to increase quality of soil treatment from oil.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: strain Pseudomonas aeruginosa RCAM01139 is proposed for decomposition of oil and diesel fuel.

EFFECT: strain is characterised by high efficiency in processes of oil and diesel fuel utilisation, which are the only sources of carbon and energy.

3 tbl

FIELD: biotechnology.

SUBSTANCE: culture fluid of producer strain is concentrated on hollow fibers truncating macromolecules with a molecular weight higher than 15 kDa. The cell concentrate is added to dry NaCl in a final concentration of 0.5 M. Mixed on a shaker for 20 min. The suspension is centrifuged for 15 min at 10000 g and the supernatant is separated, the supernatant pH is made up to 3.0 by four-molar solution of HCl. The suspension is centrifuged. The residue is added to water in a volume of 0.1% of the initial volume of the culture fluid, the residue is suspended and alcohol is added in amount of 0.1% of the initial volume of the culture fluid. Ethanol or propanol or isopropanol is used as alcohol. Exposed for 30 minutes at 0°C and centrifuged. The alcohol is removed from the solution by evaporation. The peptide fraction is purified. Water is added to 0.1% of the initial volume of the culture fluid, active carbon is added in an amount of 0.5% w/v (v - water volume). Centrifuged removing impurities adsorbed on the carbon. The aqueous solution is passed through the membrane truncating macromolecules with a molecular weight more than 10 kDa.

EFFECT: invention enables to accelerate preparation of bacteriocin and to increase the degree of purification of the resulting product in a yield of 90% of the total activity in the culture fluid.

1 dwg, 1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of isolating and purifying recombinant human growth hormone which is secreted by Saccharomyces cerevisiae yeast during fermentation thereof in suitable conditions. The target protein is precipitated in biomass-free culture fluid by either acidification to pH 2.9-4.0 or adding polyethylene glycol with molecular weight of 3000-6000 Da. The obtained precipitate is then dissolved in a suitable solvent. Preliminary purification of the target protein is carried out either by anion-exchange chromatography at pH 5.6 or by diafiltration in the presence of 0.1-0.5 M sodium chloride. Main purification of the target protein is then carried out by anion-exchange chromatography at pH not below 7.3 and gel filtration.

EFFECT: invention enables to obtain a growth hormone which is free from parent proteins, host-producer protein and other impurities such as pigments, with output of up to 60%.

8 ex

FIELD: biotechnology.

SUBSTANCE: method provides for culturing mammalian cells expressing the recombinant protein of interest in a culture medium containing anti-aging compound selected from carnosine, acetyl-carnosine, homo-carnosine, anserine, and K-alanine and their combinations, under conditions and for a time sufficient for expression of the protein.

EFFECT: invention enables to improve the performance of the cell culture selected from higher titer, of increased specific cell productivity, increased cell viability, increased integrated viable cell density, decreased accumulation of high molecular aggregates, decreased accumulation of acidic molecules, and their combinations.

44 cl, 14 dwg, 3 ex

Novel antibodies // 2490277

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed is an anti-α5β1 antibody, which is described through amino acid sequences of six hypervariable regions and an antigen-binding moiety thereof. Described are conjugates of the disclosed antibodies with a medicinal agent or a label, a pharmaceutical composition, use of the disclosed antibodies to prepare a medicinal agent, methods and an industrial product for inhibiting angiogenesis and/or vascular permeability in a subject, and for treating cancer, an ophthalmic disease and an autoimmune disease in a subject. The invention describes an isolated nucleic acid, an expression vector, a cell and a method of producing an antibody or an antigen-binding moiety thereof, as well as a method of detecting α5β1 protein in a sample.

EFFECT: present invention can find further use in therapy and diagnosis of α5β1-mediated diseases.

52 cl, 11 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: method consists in expression of a gene of a human plasminogen fragment from 453 to 543 amino acid within E.coli cells with subsequent extraction and treatment of a finished product from cell periplasm. Expression is carried out in cells E.coli BL21 (DE3), transformed by plasmid DNA pEK5 or pEK5H with physial maps represented in figure 2, containing a gene of target polypeptide fused with a gene of signal peptide OmpA, origin of replication pUC ori, a gene of resistance to kanamycin and a gene coding lac-repressor under control of T7-promotor. At the same time the recombinant plasmid DNA pEK5H additionally contains between genes OmpA and target polypeptide the sections coding amino acid sequences HHHHHH and DDDDK.

EFFECT: invention provides for secretion of target polypeptide into cell periplasm with high yield.

4 cl, 5 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: fusion peptide is presented for neutralisation and destruction of organophosphorous compounds, comprising a signal peptide TAT3 with amino acid sequence SEQ ID NO: 5, presented in the description, functionally connected to the sequence of organophosphate hydrolase SEQ ID NO: 18, classified as protein EC 3.1.8. The following components are described: extracted polynucleotide, which codes the specified fusion protein; a vector containing the specified polynucleotide; and a procaryotic host cell containing the specified vector and expressing the specified fusion protein. The method is proposed to produce a ferment, which destroys organophosphorous compounds, including expression of the specified polynucleotide in the procaryotic host cell and production of the specified ferment.

EFFECT: invention makes it possible to increase expression of organophosphate hydrolase in a host cell.

13 cl, 1 tbl, 9 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: disclosed are versions of human IL13 specific antibodies and a producing hybridoma cell line deposited in ATCC under number PTA-5657. Described are: versions of encoding polynucleotides; an antibody expression vector; a host cell for antibody expression, as well as versions of a method of producing an antibody using a vector, polynucleotide, hybridoma or host cell. The invention discloses a pharmaceutical composition for treating IL13-mediated diseases and methods of treating allergic, inflammatory and other diseases, which employ an anti-IL13 antibody.

EFFECT: providing antibodies which do not bind with target IL13 and neutralise activity of human IL13.

39 cl, 29 dwg, 11 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. There are presented versions (aCt1 and aCt2) of one-domain antibodies specifically binding the Chlamydia trachomatis antigen. There are described versions of the method of inhibiting an infection caused by Chlamydia wherein the method involves the preparation of elementary bodies C.trachomatis by a therapeutically effective amount of the nanoantibody aCt1 or aCt2 before being attached to infected target cells.

EFFECT: use of the invention provides the antibodies to detect and block the infections Chlamydia trachomatis that can find application in medicine.

6 cl, 4 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of preparing a pharmaceutical composition, CHO cell to obtain the desired protein, the CHO cell - DNA recipient encoding the desired polypeptide, the method of production of the desired polypeptide. The method of production of the desired polypeptide comprises cultivation of CHO cell which is transformed DNA encoding alanine aminotransferase and DNA encoding the desired polypeptide. In the particular case the CHO cell is cultured in α-ketoglutarat-containing medium. The method of preparing of the pharmaceutical composition comprises preparing of the desired polypeptide with the method described above. The polypeptide is mixed with pharmaceutically acceptable carriers or additives. The preparation is prepared. The CHO cell for preparing of the desired protein has DNA transferred into it, encoding alanine aminotransferase, and DNA transferred into it, encoding the desired polypeptide.

EFFECT: invention enables to prepare a desired polypeptide with a high yield.

10 cl, 22 dwg, 3 ex

FIELD: biotechnology.

SUBSTANCE: method of highly effective production of methionine-containing form of staphylokinase (SAK-1) and some of its biologically active analogs in a host cell, providing culturing a bacterial cell transformed by the expression vector which comprises the DNA sequence encoding SAK-1 at a high degree of aeration and reducing the level of oxygen by 5% of atmospheric level with achievement the exponential growth phase.

EFFECT: use of the invention provides a significant increase in the yield of the target product.

11 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to genetic engineering and biotechnology, and may be used in food and pharmaceutical industries. A microorganisms of the genus Corynebacterium producing 5'-inosinic acid is with provided with a capability to overexpress the genes encoding enzymes for the purine biosynthetic pathway. These enzymes include ribosephosphate-pyrophosphokinase, phosphoribosylglycinamide-formyltransferase, phosphoribosylformylglycinamidine synthetase, phosphoribosylformylglycinamidine synthetase II, phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase, inosinic acid cyclohydrolase, phosphoribosylpyrophosphate-amidotransferase and phosphoribosylaminoimidazole synthetase. A method for preparing 5'-inosinic acid involves culturing this microorganism and recovering an end product from the culture medium.

EFFECT: use of the invention provides a high yield of 5'-inosinic acid.

12 cl, 7 dwg, 1 tbl, 2 ex

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