Glarea lozoyensis mutant strain and its application
SUBSTANCE: mutant strain is obtained by impact on Glarea lozoyensis ATCC 20957 strain by nitrosoguanidine and it is deposited in CGMCC with number CGMCC 2933.
EFFECT: fungi strain has stable genetic and productive properties, produces low quantity of impurities during fermentation and is acceptable for commercial production of antibiotic.
6 cl, 2 dwg, 2 tbl, 3 ex
The technical field
The invention relates to the production of antibiotics, in particular it refers to a strain that produces an antibiotic with a high yield, the method of its production and application.
The level of technology
In the last few decades as the number of cases and the number of types of fungal infections that cause significant harm to the health of the people is gradually increasing, especially in patients with weakened immune systems. At the same time, the clinical application of some widely used antifungal agent such as amphotericin, imidazoles and triazoles, which are widely used clinically, is limited because of significant neurotoxicity, drug resistance, etc. Echinocandin as the type of new antifungal substances are a group of natural products, opened in 1970 agricultural Structural echinocandin have similar cyclic polypeptide KOR, but different side chains of fatty acids. Echinocandin can competitively inhibit the synthesis of β-D-glucan in the cell walls of fungi. Advantages of echinocandins are low toxicity, strong fungicidal activity and excellent pharmacokinetic properties.
Family echinocandins includes the following echinocandin: cilofungin, pneumocandin, aculeoside, mountainmen and WF 11899A. Echino andini and pneumocandin actively investigated and are currently being used clinically.
Caspofungin is a water-soluble semi-synthetic derivative pneumocandin. Merck has developed caspofungin as antifungal/ protivopedikuleznogo substances with a wide range. In phase II clinical trials with control experiments it was found that in patients with weakened immune systems, suffer from invasive pulmonary aspergillosis, the use of caspofungin (intravenous injection, 50-70 mg/d) showed good efficiency, while the use of amphotericin b and azoles containing nitrogen, has not had any noticeable impact. In the case of 128 patients infected with HIV, the effectiveness of caspofungin when moniliales esophagitis reached 85%, while the effectiveness of amphotericin b was only 66.7 per cent. These types of medicines can be applied for the effective destruction of fungi resistant to azoles containing nitrogen, and amphotericin C. moreover, these types of drugs are much better than traditional antifungal substances due to the lack of hemolytic toxicity and less drug interactions.
|Pneumocandin is a class of natural against the fungal medicines produced Glarea lozoyensis. They can be divided mainly into three types in accordance with different substituents on the Proline in their structure: A0(3-hydroxyl-4-methylpropyl)0(3-hydroxyproline) and (C0(4-hydroxyproline). Moreover, in accordance with various substituents on the cyclic polypeptides pneumocandin And0can be subdivided into 6 subtypes: A0, A1And2And3And4and a5;0can be subdivided into 6 subtypes:0In1In2In3In4In5; where a0And1And3And4and In0produced by the strain ATS||20868 wild type, with A0is the main product. Through MMOs-mutagenesis (N-Nitroso-N-metalmachine) ATS 20868 was obtained mutant strain was ATSS 20957, capable of producing simultaneously And0and In0. However, ATSS 20957 has a relatively low ability to produce In0and0is produced in the form of an impurity in a relatively high number.|
Thus, to meet the requirements of industrial production it is necessary to find high-yielding strain with stable genetic properties, which can produce more In0and less And0.
Brief description of the invention
The purpose of the present invention is to propose a new mutant strain based on ATSC 20957.
Another objective of the present invention is to propose a method of obtaining mentioned a new strain.
Another objective of the present invention is to provide said mutant strain.
The fourth objective of the present invention is to propose a method of obtaining the compounds of formula I with help from the new strain.
In the first aspect of the present invention proposed a mutated strain of Glarea lozoyensis deposited in the China center collection of microbial cultures under number CGMCC 2933.
In the second aspect of the present invention, a method for obtaining a mutant strain, previously mentioned, which includes the following steps:
(a) mixing seed liquid Glarea lozoyensis No. ATS 20957 with nitrosoguanidine to obtain a mixture A;
(b) mixing the aforementioned mixture with an enzyme that destroys the cell wall, to obtain protoplasts;
(c) mentioned regeneration of protoplasts to obtain isolated colonies;
(d) culturing the aforementioned single colonies to obtain a mutant strain.
In a preferred example, the concentration of nitrosoguanidine on the stage (a) is 10-20 mcg/ml based on the total amount of the aforementioned mixture, and the concentration of the enzyme that destroys the cell with the evaluation, at stage (b) is 10-50 mg/ml based on the total volume after mixing the aforementioned mixture with an enzyme that destroys the cell wall.
In another preferred embodiment of the above-mentioned enzyme that destroys the wall, contains one or more of the following compounds: livolsi, enzyme snails and cellulose.
In another preferred embodiment, each enzyme is present at a concentration of 10-40 mg/ml
In another preferred embodiment of the mycelium in the seed liquid phase (a) is in logarithmic growth phase.
In another preferred embodiment of the dry weight of the cells is 5-10 g/l based on the total amount referred to sowing the liquid phase (a).
In the third aspect of the present invention proposed the application of the mentioned mutant strain to obtain the compounds of formula I:
In the fourth aspect of the present invention, a method for obtaining compounds of formula I, involving the following stages:
Cultivation mentioned mutant strain in a fermentation medium at a temperature of 15-35°C to obtain the compounds of formula I.
In another preferred embodiment mentioned fermentation medium contains the following components based on the total volume of fermentation medium: 15-50 g/l L-Proline, 6-20 g/l of sodium glutamate, 6-20g/l yeast extract, 4-20 g/l fructose, 1.5 to 7 g/l inorganic salts and 10-50 g/l of trace elements.
In another preferred embodiment mentioned inorganic salt selected from a phosphate, or sulfate, or combinations thereof.
In another preferred embodiment mentioned fermentation medium also contains 10-100 g/l mannitol in the process of cultivation.
In another preferred embodiment the amount of inoculum mentioned mutant strain is 4-10% vol. based on the total volume of fermentation medium.
In another preferred embodiment the source is mentioned pH of fermentation medium is 5.3 to 6.0.
In summary, the present invention offers a highly productive strain with stable genetic properties, which can produce more In0and less And0to best meet the requirements of industrial production.
Description of the drawings
Fig. 1 depicts the scheme of the manufacturing process for the production of a new strain CGMCC 2933 proposed in the present invention.
Fig. 2 depicts the structure of pneumocandin where pneumocandin0is a compound of formula I according to the present invention.
Ways of carrying out the invention
The authors of the present invention unexpectedly discovered highly productive mutant strain (No. CGMCC 2933), which may be the floor of the Chan through mutation engine strain Glarea lozoyensis ADS 20957 using nitrosoguanidine (IGT) in the application of leveltime for preparation of protoplasts and subsequent screening regeneriruyuschim protoplasts. Mentioned mutant strain can produce the compound of formula I in high yield in the fermentation process. Thus, the inventors have achieved the goals of the present invention.
A new strain
The present invention provides a new strain producing the compound of formula I. Taxonomically referred to a new strain refers to Glarea lozoyensis and deposited in the China center collection of microbial cultures under number CGMCC 2933.
The method of obtaining a new strain
The present invention provides a method of obtaining a new strain No. CGMCC 2933, and the above-mentioned method can be performed according to the following sequence:
The original strain → sowing liquid → mutagenesis by treatment with IGT → destruction of the cell wall by means of leveltime to obtain protoplasts → dilution and inoculation of protoplasts of the Cup → selection of single colonies and planting it on a beveled environment ® primary screening in shake flasks → selection of highly productive strain → sowing strain on the crop environment → secondary screening in shake flasks → selection of highly productive strain, verification in a fermentation VAT and execution of the experiment on the stability → conserver is of the strain.
In particular, the method proposed in the present invention, includes the following steps:
(a) mixing seed liquid Glarea lozoyensis No. ATS 20957 with nitrosoguanidine to obtain a mixture A;
(b) mixing the aforementioned mixture with an enzyme that destroys the cell wall, to obtain protoplasts;
(c) mentioned regeneration of protoplasts to obtain isolated colonies;
(d) culturing the aforementioned single colonies to obtain a new strain.
In one example of the present invention a new strain can be obtained through the following procedure:
cultivation of ATSS 20957 within 1-3 days in shake flasks to obtain seed liquid (dry cell mass, SCM=5-10 g/l), adding the appropriate quantity of IGT to seed liquid culturing for another 1-2 days and subsequent centrifugation seed liquid, laundering and resuspendable sediment and destruction of cell walls by means of leveltime to obtain protoplasts; dilution of protoplasts and subsequent inoculation of diluted protoplasts of the Cup with hypertensive PDA (potato agar with dextrose), cultivation of protoplasts to obtain single colonies of recombinant cells to obtain a new mutated strain.
In addition, the present invention offers the pic is b obtain the compounds of formula I by fermentation of a new strain obtained by mutagenesis.
In the example of the present invention to generate a new strain through mutagenesis and fermentation of a new strain for the production of compounds of formula I includes:
(1) Initial strain: Glarea lozoyensis ADS 20957.
(2) Sowing culture of the original strain.
Preserved in glycerol strain ADS 20957 defrost, sow in seed medium (insertion amount of 50 ml/250 ml), cultured on a shaker at 200 to 300 rpm and a temperature of 25-30°C for 1 to 3 days before until dry weight of the mycelium reaches approximately 5-10 g/L.
The composition of the seed medium: 10-20 g/l sucrose, 4-10 g/l yeast extract, 10-20 g/l of soybean tryptone, 1.5-2 g/l KN2RHO4, 0.4 to 1 g/l MgSO4·7H2Oh, 10-50 g/l of trace elements, the initial pH of 5.3 to 6.0. The medium is sterilized at 121°C for 20 minutes. Trace elements: 10-20 g/l FeSO4·7H2O, 10-20 g/l MnSO4·H2O 2-10 g/l ZnSO4·7H2O, 0.7 to 2.0 g/l l2, 0.56 to 2.0 g/l H3IN3, 0.25 to 2.0 g/l ul2·2H2O, 0,19-2.0 g/l (NH4)6Mo7O24·7H2O, concentrated hydrochloric acid to 500 ml/L.
(3) Selection of single colonies
At the beginning of the cultivation liquid initial strain is subjected to mutagenesis using the IGT and then treated with the help of LiveTime for the destruction of the cell wall. The resulting about oplasty regenerate to obtain a mutant strain.
(4) Screening of mutant strain
The protoplasts are seeded in a hypertonic environment of the PDA. Single colonies grown for 10-12 days, seeding in stubble medium for further cultivation, respectively. After 8-10 days the sowing medium inoculant (insertion amount of 25 ml/250 ml) using a lawn grown on stubble medium, and cultured on a shaker at 280 rpm and a temperature of 25-30°C for 6-10 days. The sowing medium sow in a fermentation medium (insertion amount of 25 ml/250 ml) and cultured on a shaker at 200 to 300 rpm and a temperature of 25-30°C for 6-12 days. After completion of cultivation, the fermentation liquid is extracted with methanol and the content of the compounds of formula I in the fermentation liquid was measured by high-performance liquid chromatography.
The composition of the medium can be found in the "Pneumocandins from Zalerion arboricola', Journal of antibiotics, T.45, H.12, December 1992, S-1874.
Hypertonic environment of PDA Petri dishes consists of 300 g/l of potato, 20 g/l glucose, 15 g/l agar, 273, 6 g/l sucrose and sterilized at 121°C for 20 minutes
The content of the compounds of formula I in the fermentation liquid was measured by high-performance liquid chromatography:
chromatographic column: Phenomex C18 (4.6 mm × 250 mm, 5 μm),
mobile phase: acetonitrile:water = 50:50,
the column temperature: 35°C,<> gradient elution, flow rate: 1.0 ml/min,
the volume of injected sample: 5 μl; wavelength detection: 210 nm.
(5) fermenting a mutant strain
Appropriate technical solutions described in the literature. For more detail see Biotechnology and Bioengineering, 78 So, H.3, 5 May, 2002; and Journal of industrial microbiology, 11 (1993), 95-103.
Characteristics of the present invention mentioned above, or specifications referred to in the examples, if desired, can be combined. Any of the features disclosed in the present description, can be applied in combination with any other features, and each feature disclosed in the description may be replaced by an alternative feature, which may be identical, equivalent or similar purpose. Thus, the characteristics disclosed in this document represent only a General illustrative examples of equivalent or similar characteristics, except where specified otherwise.
The main advantages of the present invention include the following.
1. Getting a new high-yielding mutant strain with stable genetic properties.
2. High genetic stability, and low contamination of the production of a new strain, contributing to the separation of the product and its purification in the process of obtaining the compounds of formula I, as well as the scale of the repression production, what makes the new strain is acceptable for industrial production.
3. The output of the compounds of formula I can be up to 5 g/l in the optimized fermentation conditions.
The present invention will be further illustrated below by reference to specific examples. It should be understood that these examples are only illustrative for the present invention and not limit its scope. The experimental methods described in the following examples, without specifying conditions, usually performed under conventional conditions or according to the manufacturer's instructions. Unless otherwise stated, all percentages, relationships, proportion or part of the calculated mass.
The unit of measure, expressed as a percentage of the mass to the volume used in the present invention, well-known specialists in the field of technology, for example, it refers to the weight of a solution in 100 milliliters of solution.
If not defined otherwise, all technical and scientific terms used in the present description have the meanings commonly understood by experts in the field of technology. In addition, any methods and materials similar or equivalent content, opened in this document can be used in these ways. The preferred methods and materials for implementation of these ways disclosed in this document, which can be found only as examples.
In the examples of the present invention proposed the following conditions of high performance liquid chromatography, used for measurement of the content of the compounds of formula I in a fermentation liquid:
chromatographic column: Phenomex C18 (4.6 mm × 250 mm, 5 μm),
mobile phase: acetonitrile: water=50:50,
the column temperature: 35°C,
gradient elution, flow rate: 1.0 ml/min,
the amount of insertion of the sample: 5 μl; wavelength detection: 210 nm.
Mutagenesis with the aim of obtaining a new strain CGMCC 2933
Preserved in glycerol strain ADS 20957 were thawed, seeded in the sowing medium with inoculum in a quantity of 4% (insertion amount of 50 ml/250 ml), then were cultured on a shaker at 280 rpm and a temperature of 25°C for 2 days before until dry weight of the mycelium will not constitute approximately 5-10 g/L. Mutagen IGT was added to the seed liquid at a concentration of 10 μg/ml, and cultivation fluid were cultured for another day. Then take 10 ml of the cultivated liquid containing IGT, centrifuged at 5000 rpm for 10 minutes, the precipitate was washed twice with two volumes of 0.6 M NaCl to remove protection and IGT.
The sowing medium: 10 g/l sucrose, 5 g/l yeast extract, 10 g/l of soybean tryptone, 1.5 g/l KN2RHO4, 0.4 g/l MgSO4·7H2O, 10 g/l m is croisements, the initial pH of 5.3. The sowing medium was sterilized at 121°C for 20 minutes
Micronutrients: 10 g/l FeSO4·7H2O, 10 g/l MnSO4·H2O, 2 g/l ZnSO4·7H2O, 0.7 g/l l2, 0.56 g/l H3IN3, 0.25 g/l ul2·2H2O, 0,19 g/l (NH4)6Mo7O24·7H2O, concentrated hydrochloric acid to 500 ml/L.
2. Preparation of protoplasts and selection of single colonies
It washed the mycelium was added 10 ml of enzyme mixture (in buffer containing disubstituted sodium phosphate and citric acid (pH of 6.0) with 0.5 M NaCl), which contains 20 mg/ml of leveltime (2000 units/mg), 10 mg/ml enzyme snails (5 units/mg) and 10 mg/ml cellulose (15 units/mg). The resulting mixture was shaken at 80 rpm and 30°C for 5 h for enzymatic lysis. Animalities the reaction mixture was filtered through cotton to remove mycelium and get unicellular suspension containing only the protoplasts. One milliliter of this solution was taken and centrifuged at 14000 rpm for 10 minutes the Precipitate was dissolved in 1 ml of buffer (pH 6,0)containing disubstituted sodium phosphate, citric acid and 0.5 M NaCl. Then did a series of dilutions of this solution with different concentrations, evenly sown on hypertonic environment PDA with 0.8 M sucrose and cultured at 25°C for 8-10 days in order to obtain single colonies.p> 3. The process of screening of high-yielding strain CGMCC 2933
After culturing for 10 days took a single colonies and planted in the crop environment for further cultivation. After 8 days was collected lawn culture area of 0.5-1 cm2sowing in seed medium (insertion amount of 25 ml / 250 ml) and cultured on a shaker at 280 rpm and 25°C for 8 days. Sowing liquid sowed in a fermentation medium with inoculum volume of 4% (insertion amount of 25 ml / 250 ml)were cultured on a shaker at 280 rpm and 25°C for 14 days (on the 7th day of cultivation) was added 5% mannitol and 0.5% Proline).
Obtaining the compounds of formula I using a new strain CGMCC 2933
A new strain CGMCC 2933 obtained in example 1 in the planting medium, seeded in a fermentation medium with inoculum in a quantity of 4% and were cultivated in shake flasks at 25°C. After culturing for 14 days yield the compounds of formula I reached 5 g/l (7-day cultivation was added 5% mannitol and 0.5% Proline).
Fermentation medium: 15 g/l L-Proline, 6 g/l of sodium glutamate, 6 g/l yeast extract (obtained from the company Oxiod), 4 g/l fructose, 1.5 g/l KN2RHO4, 0.4 g/l MgSO4·7H2O, 50 g/l mannitol, 10 ml/l trace elements, the initial pH of 5.3. Fermentation medium was sterilized at 121°C for 20 minutes
Micro is a separate estimate: 10 g/l FeSO 4·7H2O, 10 g/l MnSO4·H2O, 2 g/l ZnSO4·7H2O, 0.7 g/l l2, 0.56 g/l H3IN3, 0.25 g/l ul2·2H2O, 0,19 g/l (NH4)6Mo7O24·7H2O, concentrated hydrochloric acid to 500 ml/L.
The ability of the original strain of ATSS 20957 to produce the compound of formula I is compared with that of the mutant strain CGMCC 2933 using the following methods: the original strain and the mutant strain were cultured using the method of cultivation described in example 2, respectively. After completion of cultivation, the fermentation liquid was extracted with two volumes of methanol, and the content of the compounds of formula I in the fermentation liquid was measured by high-performance liquid chromatography. The results are shown in table 1.
|The number of strain||The output of the compounds of formula I (g·l-1)|
Applied environment below.
Environment for screening consists of 300 g/l of potato, 20 g/l glucose, 15 g/l agar, 273,6 g/l sucrose and sterilized PR is 121°C for 20 minutes
Beveled environment consists of 300 g/l of potato, 20 g/l glucose, 15 g/l agar and sterilized at 121°C for 20 minutes
The sowing medium consists of 10 g/l sucrose, 5 g/l yeast extract, 10 g/l of soybean tryptone, 1.5 g/l KN2RHO4, 0.4 g/l MgSO4·7H2O, 10 g/l of trace elements, the initial pH of 5.3 and sterilized at 121°C for 20 minutes
Fermentation medium consists of 15 g/l L-Proline, 6 g/l of sodium glutamate, 6 g/l yeast extract (obtained from the company Oxiod), 4 g/l fructose, 1.5 g/l KN2RHO4, 0.4 g/l MgSO4·7H2O, 50 g/l mannitol, 10 ml/l trace elements, the initial pH of 5.3, and sterilized at 121°C for 20 minutes
Micronutrients: 10 g/l FeSO4·7H2O, 10 g/l MnSO4·H2O, 2 g/l ZnSO4·7H2O, 0.7 g/l l2, 0.56 g/l H3IN3, 0.25 g/l CuCl2·2H2O, 0,19 g/l (NH4)6Mo7O24·7H2O, concentrated hydrochloric acid to 500 ml/L.
The stability of the new strain CGMCC 2933
Subcultivation performed using the same medium and culture conditions as in example 2.
The results are shown in table 2.
|The stability of the new strain in the passages|
|The number p is Saga||F1||F2||F6|
|The output of the compounds of formula I (g/l)||5,2||5,0||5,3|
The results show that the new strain has excellent stability.
The above description is only preferred examples of the present invention and is not intended to limit the scope of meaning of the technical content of the present invention. Meaning of the technical content of the present invention is widely defined in the scope of the claims attached to the present invention. Any technical object or method, performed in a different way, shall be deemed to refer to the volume formula of the present invention, if the object or method completely identical to those defined in the claims of this application, or their equivalent changes or modifications.
1. Mutant strain Glarea lozoyensis to obtain the compounds of formula I
deposited in the China center collection of microbial cultures under the number CGMCC 2933.
2. The use of a mutant strain according to claim 1 for obtaining the compounds of formula I.
3. The method of obtaining the compounds of formula I, comprising the following stages:
(1) the cult of the cultivation of the mutant strain according to claim 1 in a fermentation medium at a temperature of 15-35 °C to obtain the compounds of formula I, where fermentation environment includes the following components based on the total volume of fermentation medium: 15-50 g/l L-Proline, 6-20 g/l of sodium glutamate, 6-20 g/l yeast extract, 4-20 g/l fructose, 1.5 to 7 g/l inorganic salts and 10-50 g/l of trace elements, and the initial pH of fermentation medium is 5.3 to 6.0.
4. The method of receiving according to claim 3, where the inorganic salt is selected from phosphate or sulfate, or combinations thereof.
5. The method of receiving according to claim 3, where the fermentation medium further comprises 10-100 g/l mannitol in the process of cultivation.
6. The method of receiving according to claim 3, where the volume of inoculum mutant strain according to claim 1 4-10% vol. based on the total volume of fermentation medium.
SUBSTANCE: inoculum of microalgae Desmodesmus sp. strain 2C166E is introduced into mineral medium BG-11 till final concentration of chlorophyll in mixture of 4-6 mcg/ml. Incubation is performed under constant lighting and environment barbotage by atmospheric air during 12-16 days under temperature of 25-27°C with consequent separation of microalgae biomass from nutritional medium with obtainment of microalgae biomass containing 33-35% of fatty acids from dry weight of cells.
EFFECT: invention allows increasing content of fatty acids in microalgae biomass.
1 dwg, 4 ex
SUBSTANCE: invention relates to microbiology and biotechnology. Material to be investigated - pure culture of rod-like, gram-negative, glucose-fermenting, oxidase-positive or oxidase-negaive bacteria - is collected first. The investigated daily bacterial culture is seeded on the surface of nonselective nutrient agar (GRM-agar) with 1% sodium chloride. A paper disc is then placed seeded surface, said disc containing vibriostatic substance niclosamide (2,5-dichloro-4-nitrosalicylanilide) in amount of 10 mcg or 16 mcg per disc. The seeded material is incubated in aerobic conditions at 35°C for 24 hours. Vibrio bacteria are indicated a zone of inhibited bacterial growth around the disc.
EFFECT: invention increases diagnostic sensitivity of the method of identifying vibrio bacteria.
2 tbl, 4 ex
SUBSTANCE: invention relates to microbiology and biotechnology. Disclosed is a consortium of Lactobacillus rhamnosus VKM B-2726D and Lactobacillus plantarum VKM B-2725D strains.
EFFECT: consortium is used to produce a bacterial preparation and a direct administration ferment for producing fermented milk and fermented beet juice as functional nutrition products.
15 tbl, 13 ex
SUBSTANCE: strain Lactococcus casei VKPM V-8730 is obtained at available growth-supporting media and is used as starter material in cultured milk products production.
EFFECT: invention allows reducing the time of technical process and increasing cultured product shelf life.
SUBSTANCE: strain Enterococcus durans VKPM B-8731 is obtained at available growth-supporting medium and is used as starter material in cultured milk products production.
EFFECT: invention allows reducing final acidity of cultured milk product, reducing the time of technical process and increasing cultured product shelf life.
SUBSTANCE: group of inventions refers to microbiology and biotechnology. Proposed strains Lactobacillus crispatus "ВКМ" B-2727D, Lactobacillus gasseri "ВКМ" B-2728D and Lactobacillus plantarum "ВКМ" B-2731D have antagonistic activity in relation to pathogenic and semi-pathogenic microorganisms. Besides, the invention proposes a consortium based on the above strains for production of a bacterial preparation, a biologically active additive, and direct inoculation for obtaining fermented milk product of functional nutrition.
EFFECT: consortium has higher antagonistic activity against pathogenic and semi-pathogenic microorganisms in relation to individual strains of lactobacilli, use of a consortium will allow enlarging the range of devices intended for prophylaxis and treatment of urogenital infections of females.
4 cl, 1 dwg, 7 tbl, 12 ex
SUBSTANCE: invention represents a method for obtaining recombinant DNAse I of a human or its mutein, as well as their conjugates with polyethylene glycol, using a bacterium belonging to Escherichia class, transformed with expression plasmid, containing a promoter functioning in a bacterial cell, DNA fragment coding a hexahistidine cluster, a fragment coding enterokinase recognition sequence amalgamated in frame with human DNAse I or its functionally active mutein containing replacements of asparagine with cysteine, transcription termination section, vector pET28a(+) fragment containing initiation section of replication of bacteriophage fl, sequence coding aminoglycoside-3'-phosphotransferase, area of beginning of plasmid pBR322 replication, gene RNA-organising protein Rop, sequence coding lactose operon repressor.
EFFECT: invention allows obtaining recombinant human DNAse I or its mutein with high yield.
18 cl, 7 dwg, 1 tbl, 12 ex
SUBSTANCE: preparation containing biodestructor of oil contamination represents centrate of cultural liquid of microbial mass of consortium of oil-oxidising microorganisms immobilised on peat carrier. Consortium composition includes the following: Rhodococcus eqvi SRI MCC B-1115 bacteria strain, Rhodotorula glutinis SRI MCC Y-1113 yeast and Rhodotorula glutinis ARSRI MCC Y-1114 yeast strain in the ratio of 1:1:1 respectively, which have been grown jointly.
EFFECT: invention allows improving quality of soil cleaning from oil and oil products under conditions with reduced temperatures.
SUBSTANCE: Bacillus licheniformis strain has antagonism in relation to Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes and resistance to the following antibiotics: streptomycin and nalidixic acid. The strain is deposited with number BKM B-2712D in the All- Russian collection of microorganisms of the Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Science (IBFM RAN).
EFFECT: strain can be used for creation of probiotic bacterial preparations in veterinary.
SUBSTANCE: strain is deposited in CNCM and has number 1-3689. Strain has ability to inhibit the growth of pathogenic microorganisms in culture, and namely Escherichia coli, Salmonella enteritidis and Listeria monocytogenes. Strain has anti-inflammatory properties at the ratio of IL-10/IL-12 equal to 11.8.
EFFECT: food product containing the above living strain has antimicrobial or immunomodulating properties.
6 cl, 3 dwg, 4 tbl, 3 ex
SUBSTANCE: there proposed is an antibody specific to TENB2 containing light and heavy chains. Heavy chain contains substitution for free (reaction capable) cysteine A121C that corresponds to A114C (Kabat numbering) or A118C (Eu numbering). Conjugate versions are proposed for prostate cancer treatment containing antibody covalently bound to auristatin, also by means of linker. The following is described: pharmaceutical composition for prostate cancer treatment that uses as active beginning the antibody or its conjugate; product for prostate cancer treatment on the basis of such composition. The invention proposes: method for defining protein TENB2 in the sample - on the basis of antibody as well as analysis for revealing prostate cancer cells at mammal and method for cell proliferation inhibiting on the base of antibody conjugate and auristatin. There described is the method for obtaining antibody conjugate (Ab) and auristatin (D) with expression Ab-(L-D)p, where p is equal from 1 to 4, and L is linker.
EFFECT: invention application provides conjugates with increased stability in serum in comparison with the same conjugates without A121C substitution in antibody that can be used in medicine.
33 cl, 18 dwg, 2 tbl, 4 ex
SUBSTANCE: expression vector includes: (a) replication origin OriP obtained from Epstein-Barr virus (EBV), where replication origin contains: 1) symmetry element of the second order (DS); and 2) duplication section (FR) that contains fixation point EBNA; (b) replication origin SV40; (c) insertion section for inserting a gene of concern; (d) promoter EF-1b functionally bound to the insertion section; (e) poly-A signal; (f) bacterial replication origin; (g) selected marker; and unnecessarily containing (h) sequence of nucleic acid, which codes constant area of heavy or light chain of antibody, which is functionally bound to the insertion section. With that, replication origin OriP is bound to an initiation factor of replication EBNA 1, which acts from outside and is not coded with an expression vector.
EFFECT: use of an expression vector in an extracted host cell, a set and a method for obtaining recombinant protein provides production of abundant protein expression.
26 cl, 25 dwg, 3 tbl, 4 ex
SUBSTANCE: invention proposes a method for obtaining recombinant core protein of hepatitis E virus (rtHEV-ORF2) and recombinant vaccine for prophylaxis of hepatitis E virus. Core protein is obtained by cultivation of recombinant yeast strain Hansenula polymorpha "КБТ"-11/pHEV-001, which contains DNA sequence integrated into genom of yeast cell and coding the fragment of amino-acid sequence from position 86 to 607 of core protein of hepatitis E virus of genotype 3 (rtHEV-ORF2) under control of promoter of MOX gene. The method allows obtaining immunogenic antigene of hepatitis E virus, which has properties of natural protein. Based on the obtained antigene there created is recombinant vaccine for prophylaxis of hepatitis E virus. Vaccine includes effective amount of rtHEV-ORF2 protein, adjuvant and a physically acceptable diluter.
EFFECT: vaccine is immunodominant and non-toxic and has no by-effects.
3 cl, 5 ex
SUBSTANCE: renatured membrane protein obtaining method is proposed. The above method involves production of a homogeneous solution containing a denatured membrane protein, a detergent mixture, phospholipide or phospholipide mixture and apolipoprotein or its equivalent with: further removal of detergents and formation of lipid-protein nanodiscs (LPND) containing renatured protein.
EFFECT: method provides production of renatured membrane proteins with high yield, which are built into LPND, which can be used at development of new medical products, biocatalysts, biosensors and biophotonic devices.
8 dwg, 2 ex
SUBSTANCE: invention proposes a production method of recombinant protein through its hybrid precursor substance with natural decomposition site with enteropeptidase. The result is achieved by replacement in natural decomposition site with enteropeptidase Asp-Asp-Asp-Asp-Lys of amino-acid residue of lysine (Lys) with amino-acid residue of arginine (Arg) and further decomposition of hybrid precursor substance with light catalytic subunit of enteropeptidase of a human being or a bull.
EFFECT: improving quality and yield of target product under conditions when hybrid protein detects additional sites of decomposition with enteropeptidase.
3 tbl, 3 dwg, 4 ex
SUBSTANCE: present inventions relate to protein engineering, plant molecular biology and pest control, as well as a hybrid insecticide protein and use thereof. Described is a hybrid insecticide protein which includes from the N-end to the C-end an N-end portion of Cry3A protein which is fused with the C-end portion of Cry1Ab protein, wherein the position of the crossover of the Cry3A protein and the Cry1Ab protein is located in a conservative block 2, in a conservative block 3 or in a conservative block 4 and has anti-western corn rootworm activity. Also disclosed are nucleic acid molecules which code the novel proteins, methods of producing proteins, methods for use thereof, as well as transgenic plants and seeds thereof which contain such proteins.
EFFECT: inventions enable to obtain cheap means of controlling Diabrotica worms.
39 cl, 8 dwg, 9 tbl, 46 ex
SUBSTANCE: method is characterised in that the DNA of the structure RNAb indicated on Figure 1, which encodes the fused protein of three parts, where N-terminal position is green fluorescent protein GFP, central - peptide of 73 amino acid residues with the amino acid sequence of SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and C-terminal - light chain of double-stranded protein Kunitz-type inhibitor from potato tubers (PKPI-BI), are introduced into cells of E. coli. The cells transformed by this construction are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifugation. The product of expression in the form of inclusion bodies is solubilised with the denaturant. Chromatography is carried out under denaturing conditions. The resulting product is used for detection of specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.
EFFECT: invention enables to obtain the recombinant antigen G2 of Hantavirus Dobrava with increased yield.
6 dwg, 1 ex
SUBSTANCE: method is proposed to produce a polypeptide, including cell cultivation, which intensely expresses a bicarbonate carrier and has a transferred DNA, which codes the desired polypeptide.
EFFECT: invention makes it possible for the cell to produce the specified polypeptide and the appropriate cell.
12 cl, 15 dwg, 6 ex
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
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.
3 dwg, 5 ex
SUBSTANCE: detection method of microfungi Coccidioides posadasii 36 S and Coccidioides immitis C-5 in vitro involves pre-growth of culture in mycelial phase, preparation of a suspension corresponding to 5 units of activity of a standard opacity sample, possibility of spherules formation and detection of spherules filled with endospores. Culture in mycelial phase is grown during 3 days. Possibility of spherules formation is provided by infection of the one-day culture of cells of murine splenocytes, which is obtained on RPMI-1640, and further cultivation during 5 days at the temperature of 37°C, with content of CO2 in atmosphere of 5%. In order to detect spherules in the form of round double-outline formations filled with endospores, a sample is taken, deactivated with formalin and investigated by means of a light microscopy method.
EFFECT: invention allows simplifying the method and reducing the investigation period.