Strain of filamentous fungus aspergillus oryzae - producer of maltogenic alpha-amylase
SUBSTANCE: strain of fungus Aspergillus oryzae Amy T-52-3-21 produces maltogenic α-amylase, and it is deposited in the All-Russian Collection of Microorganisms Institute of Biochemistry and Physiology of Microorganisms n.a. GK Scriabin RAS under the number F-4476D. The strain is made on the basis of strain Aspergillus oryzae of All-Russian Collection of Microorganisms F-3927D using the genetic engineering methods. Activity of α-amylase at 120 h of growth of the strain is 600-640 units/ml.
EFFECT: higher level of activity of maltogenic α-amylase.
1 tbl, 2 ex
The invention relates to the field of biotechnology and can be used in microbiological industry in the production of acidic α-amylase used in various fields of industry and agriculture for saccharification (hydrolysis) of starch-containing raw materials, namely in baking, distilling, brewing, confectionery and starch industry (production of patok, syrups, glucose), in the production of cereal products, vegetable products, fruit (juices, syrups, extracts, jams, pectin), baby food, textile, paper industry, in the manufacture of detergents, as well as in medicine.
The invention relates to the creation of highly active recombinant strain of the fungus Aspergillus oryzae producer maltogenic α-amylase.
The strain created on the basis of patented industrial producer acidic α-amylase - strain Asp.oryzae VKM F-3927 D, using genetic engineering techniques (1).
The invention provides for obtaining a highly active enzyme preparations of α-amylase in the submerged culture of a new recombinant strain the flour fermentation environments.
α-Amylase (α-1,4-glucan-glucanohydrolase, 18.104.22.168) is an amylolytic enzyme action, belongs to the class of hydrolases, subclass field of glycosidase inhibition, hydrolyzes internal α-1,4-glucose the data communication starch, the glycogen and related poly - and oligosaccharides, resulting in the formation of maltodextrins of various molecular weights, maltotriose, isomaltose and glucose.
Maltogenase α-amylase .oryzae (Taka-amylase) effectively hydrolyzes amylose and amylopectin with the formation of mainly maltose. Role maltogenic α-amylase in the process of bioconversion of starch-containing raw materials currently significantly increased in connection with the development and implementation of technologies for production of fuel ethanol from starch-containing raw materials, involving the use of low-temperature water-heat treatment with the use of highly active acid and enzyme preparations of α-amylase and glucoamylase. Joint use of enzymes helps to increase the speed and depth of the degradation of starch, the degree of hydrolysis of starch reaches 98-99%, while the efficiency of the process of the enzymatic treatment can be greatly enhanced through the use of highly active enzyme preparations.
In the world practice for industrial production of products of hydrolysis of starch is used mainly amylase produced by fungi belonging to the genus Aspergilllus, with yellow-green pigmentation: Asp.flavus Asp.oryzae, Asp.glauscus, Asp.ochraceusi, Asp.fumigatm.
Known for such producers acidic α-amylase, kastama Asp.oryzae 740-A2 (2), Asp.oryzae NRW-169 (collection of the Central Museum of industrial microorganisms "Vniigenetika", collection number CMPM F-257) (3). Also known strains Asp.oryzae 387 (VKPM F-683) (4), Asp.oryzae VKPM F-369 (5), Asp.awamori M-2002 (VKM F-3771D) (6, 7), synthesizing complex hydrolytic enzymes, including acid α-amylase.
The disadvantages of all these producers are the low productivity of the strain applied to the acidic α-amylase. Highly active producers is not available for the organization of domestic production, as are owned by foreign manufacturers.
Closest to the claimed object on the essential features and the achieved result is a strain Asp.oryzae VKM F-3927 D (1)that is under cultivation in deep conditions in flasks for 120 h in culture medium containing as a source of flour of cereals, corn extract, and salt, synthesize α-amylase activity 400-500 IU/ml
However, to increase the profitability of production of enzyme preparations of α-amylase requires the creation of more productive strains.
The problem to which the invention is directed, is getting a new producer strain maltogenic α-amylase capable under cultivation for cheap technological environments to ensure a high level of activity C is left of the enzyme.
The technical result from the creation and use of a new recombinant strain Asp.oryzae ATU T-52-3-21, containing additional copy of the gene maltogenic α-amylase, is to obtain a highly active enzyme preparations sour maltogenic α-amylase for use in various sectors of agriculture.
In order to increase the level of biosynthesis of target enzymes by fungal strains currently successfully applied recombinant DNA technology. Increased expression of secreted enzymes reach by obtaining strains with an increased number of gene copies of the target enzymes (so-called multicopying strains) or directional changes in the mechanism of regulation of the synthesis of these enzymes.
The essence of the object of the invention is a new recombinant strain of Aspergillus oryzae Amy T-52-3-21 - producer maltogenic α-amylase.
The creation of genetically engineered strain based on a mutant strain Asp.oryzae VKM F-3927 D conducted in several stages. In the first stage using induced mutagenesis was obtained auxotrophic recipient strain Aspergillus oryzae 52-3 niaD-defective in nitroreductase, for selection of transformants by their ability to grow on minimal medium with sodium nitrate as the sole nitrogen source. In the second stage, the recipient strain Aspergillus oryzae 52-3 niaD-was cotransformation plasma the Oh frame-Amu, carrying a gene homologous amylase under amylase promoter, plasmid pSTA10, carrier selective marker gene for nitrate reductase (8). In the subsequent breeding was obtained transformant with the level of activity by 20-30% higher than the original strain.
Comparative characterization of the level of activity of α-amylase claimed recombinant strain and initial strain Asp.oryzae VKM F-3927 D. the Activity of α-amylase activity was determined according to GOST 20264.4-89 (9). Table 1 presents data on the biosynthesis of α-amylase original mutant strain (prototype) and recombinant strain Asp.oryzae.
Thus, the proposed strain of Aspergillus oryzae Amu T-52-3-21 under cultivation on a nutrient medium based on wheat and soy flour, provides high activity maltogenic α-amylase activity in the culture fluid, which would greatly increase the yield of the target enzyme with one of the substrate and accordingly reduce the cost of the process of production of enzyme preparations derived based on it.
A strain of Aspergillus oryzae Amu T-52-3-21 deposited in the all-Russian collection of microorganisms at the Institute of biochemistry and physiology of microorganisms them. Gscreen RAS under the number VKM F-4476D.
Culiural-morphological characteristics of the strain Aspergillus oryzae Amu T-52-3-21
Macroscopic characteristics: Colonies on agar of čapek with yeast extract (CA) at 25°C have a diameter of 65-70 mm/7 days greenish-yellow, the surface until fluffy flaky, edge smooth; mycelium dense, white, bleed and have exudate absent, the reverse side unpainted.
Colonies on agar of čapek with yeast extract (CYA) at 37°C have a diameter of 60-65 mm/7 days, olive-yellow, fluffy surface, edge smooth; mycelium dense, white, bleed and have exudate absent, the reverse side unpainted.
Colonies on agar of čapek with yeast extract and 20% sucrose (CY20S) at 25°C have a diameter of colony 67-71 mm/7 days, olive-yellow, fluffy surface to ragged, edge smooth; mycelium dense, white, bleed and have exudate absent, the reverse side unpainted.
Colonies on the best-agar (MEA) at 25°C have a diameter of 65-70 mm/7 days, greenish-yellow, ragged surface, edge smooth; the mycelium is not very dense, white, bleed and have exudate absent, reverse yellowish-gray.
Microscopic characteristics: Conidial heads globose to loosely kolosovidnyh. Conidiophores are not painted, smooth to faintly rough, 500-1000×10-25 μm. Vesicles (apical extension) semi-spherical to pear-shaped, 22-50 µm wide; sterigma single and bunk in one colony, cover 1/2 to 2/3 of the vesicle, Metulla - 8-12×4-6 μm, Fieldy - 8-12×3-5 µm. Conidia globose, about 4.0 to 8.5 microns in diameter, smooth to faintly rough stare at the AI.
Physiological-biochemical properties of strain:
Culture strains metabolizes glucose, sucrose, arabinose, raffinose and poorly - maltose, lactose, galactose and rhamnose. Forms of the enzyme system that allows you to grow at a corresponding complex substrates: starch, β-glucane, pectin and galactomannan.
Well assimilates ammonium salts of inorganic acids. Consumes peptone, casein, amino acids.
Temperature optimum growth 34-35°C, pH of 4.5-6.0. Aerobe.
This filamentous fungus is not listed as pathogenic in the "Regulations on the procedure for accounting, storage, handling, dispensing and delivery of cultures of bacteria, viruses, Rickettsia, fungi, protozoa, Mycoplasma, bacterial toxins, poisons of biological origin".
The obtained recombinant strain differs from the original by the presence in the genome of additional copies of a gene Amu encoding multigene α-amylase (Taka-amylase), which provides an increased ability of a producer to the biosynthesis of α-amylase in submerged cultivation in liquid media and stability transfers.
The strain can be stored in a lyophilized state for several years or jambs with agar medium Saburo or the best agar at 4°C with mandatory replanting at least once in 3 months.
The cultivation of recombinant strains Aspergllus oryzae Amy T-52-3-21 carried out under aerobic conditions at 30°C for 120 h, pH 5.8 and 6.2. For culture growth and biosynthesis of acidic α-amylase source of carbon and nitrogen can serve as starch, hydrolyzed starch, soy, corn or other grain flour, or their extracts, ammonia nitrogen, corn extract.
When the cultivation of the strain Aspergillus oryzae Amy 7-52-3-21 for 120 h in flasks in a medium containing wheat and soy flour, corn extract and inorganic salts, the activity of α-amylase activity in the culture fluid ranges from 580 to 640 IU/ml
The enzyme preparations obtained using the proposed strain, can be used in the form of cultural liquid, concentrated liquid preparations, obtained by ultrafiltration or evaporation of the culture fluid, or in the form of dry preparations (Amiloride GH, GH), obtained by precipitation with ethanol or other organic solvents from ultrafiltrate culture liquid, and drying or granulation.
α-Amylase Asp.oryzae active at a temperature of from 30° to 70°C with optimum action at 58-60°C; stable in wide range of pH values from 3.5 to 8.0, with an optimum activity when pH of 5.0-6.0; more acidic zone, characteristic for flour mixtures (pH 4.5), shows activity at 85-95%; is maltogenic α-amylase, limit the degree of hydrolysis of soluble and insoluble starch is 38 is 34%, respectively.
|The biosynthesis of α-amylase source and recombinantly strain A.oryzae in the fermentation medium based on wheat flour|
|No.||Strain||AC 120 h of growth, IU/ml|
|1||Asp.oryzae VKM F-3927 D (the original strain, prototype)||480|
|2||Asp.oryzae Amu T-52-3-21 (recombinant strain)||620|
The possibility of using the invention is illustrated by examples which do not limit the scope and nature of the claims associated with them.
Example 1. Seed material (AMF inoculum) is produced by cultivation of a strain on liquid nutrient medium composition: wheat flour - 6%, soy flour 4%, corn extract - 1%, NH4NO3to 0.3%, KN2RHO4- a 0.02%, caso31.2%and distilled water, pH 5.8 and 6.2, in flasks containing 50 ml sterile environment, on the rocking chair with 240 rpm at 30°C for 48 hours
Received seed in the amount of 2% by volume of protection is made in Catalogne flask 750 ml containing 50 ml of medium of the following composition (wt.%): wheat flour - 6%, soy flour 4%, corn extract - 1%, NH4NO3to 0.3%, KN2RHO4- a 0.02%, caso3- 1,2%, tap water, pH 5.8 and 6.2. Wednesday pre-treated with the enzyme preparation of bacterial α-amylase at the rate of 2 units/g starch at 70°C for 20 minutes
The cultivation is carried out in aerobic conditions on the rocking chair (240 rpm) at 30°C.
The maximum enzymatic activity of α-amylase (120 h of growth) is 640 IU/ml
Example 2. The cultivation is carried out in katalozhnyh flasks as described in example 1, using liquid nutrient medium of the following composition (wt.%): cornstarch - 10%, soybean flour - 4%, corn extract - 1%, NH4NO3to 0.3%, KN2RHO4- a 0.02%, caso3- 1,2%, tap water, pH 5.8 and 6.2. The maximum enzymatic activity of thermostable α-amylase (120 h of growth) is 600 IU/ml
1. RF patent №2245364 from 27.01.2005.
2. Kalunian K.A., Holger LI Microbial enzyme preparations. Feet, Food industry, 1979, p.8-9.
3. Inventor's certificate SU # 1158579.
4. RF patent №2070921 from 27.12.1996.
5. Inventor's certificate SU # 1440922.
6. RF patent 2196821 from 20.01.2003.
7. Semenov, M.V. and other Microbial biocatalysts for processing agricultural industries/ - M: VNIIBT, 2006. 304 S.
8. Gomi et al. Molecular cloning and characterization of a transcriptional activator gene, amyR, involved n the amylolytic gene expression in Aspergillus oryzae. // Biosci. Biotechnol. Biochem. 2000. V. 64(4). P. 816-827.
9. The enzyme preparations. GOST 20264-4 .89.-M.:Izd. The USSR state Committee for standards, 1995. P.70.
Similar patents of the Russian Federation
1. RF patent №2196821 from 20.01.2003.
2. RF patent №2245364 from 27.01.2005.
3. RF patent №2354697 from 10.05.2009.
The strain of the fungus Aspergillus oryzae Amu T-52-3-21 - producer maltogenic α-amylase, deposited in the all-Russian collection of microorganisms under the number VKM F-4476D.
SUBSTANCE: strain of bacteria Bacillus vallismortis VKPM V-11017 is proposed - destructor of oil and oil products. Strain may within short period of time in the wide range of temperatures from +8 to +37°C degrade oil by 78.3%.
EFFECT: strain may be used to clean soil and water contaminated by oil and oil products, such as diesel fuel, motor oil, gas condensate.
3 tbl, 5 ex
SUBSTANCE: strain of bacteria Bacillus atrophaeus VKPM V-10592 is grown, and suspension is prepared from it, which is introduced into permafrost soil and water environment. Maintained at the specified parameters from 7 to 60 days, and then they determine quantity content of oil and oil products in permafrost soil and water environment.
EFFECT: invention makes it possible to reduce time for oil and oil product denaturation and to reduce their concentration in permafrost soil and water environment.
5 tbl, 6 ex
SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining androst-4,9(11)-dien-3,17-dione from phytosterol. Microbiological oxidative elimination of side chain at atom C17 with formation of 9α-hydroxyandrost-4-en-3,17-dione is performed. Biomass is separated. 9α-hydroxyandrost-4-en-3,17-dione is extracted from clarified cultural liquid with aprotic organic solvent, selected from aromatic hydrocarbons or organochlorine hydrocarbons. After that, reaction of 9α-hydroxygroup of 9α- hydroxyandrost-4-en-3,17-dione dehydration is carried out in obtained extract. As dehydration agent applied is mineral acid, which contains water and is selected from group, which includes orthophosphoric, pyrophosphoric and chloric acids. Mineral acid is applied in quantity from 1 to 10 mol per 1 mol of 9α- hydroxyandrost-4-en-3,17-dione. In the process of dehydration reaction removal of excessive water is carried out either in presence of effective quantity of pyrophosphoric acid or by azeotropic distillation.
EFFECT: invention makes it possible to intensify dehydration process with application of smaller quantity of mineral acid and exclude side product formation.
11 cl, 1 tbl, 1 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, microbiology, and concerns the recovery and identification of pseudotuberculosis and intestinal yersiniosis agents (Y. Pseudotuberculosis and Y. Enterocolytica). A nutrient medium contains microbiological agar (dry), lactose, glucose, urea, calcium chloride, 1% alcoholic phenol red, 1% alcoholic methylene blue and distilled water in specific proportions.
EFFECT: invention enables reducing the length of studies.
3 dwg, 2 tbl
SUBSTANCE: invention relates to biotechnology. Claimed is strain of Fusarium sambucinum, deposited in VKPM collection under number F-1161. Claimed strain is producent of protein food biomass.
EFFECT: invention makes it possible to accumulate biomass with high protein content with higher quantity of valuable unsaturated fatty acids, complete in composition.
2 tbl, 3 ex
SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining food fungal biomass with high protein content. Multicycle deep cultivation of Fusarium sambucinum All-Russian collection of industrial microorganisms F-1161 on liquid nutritional medium, containing sources of carbon, nitrogen, mineral salts, separation and drying of wet fungus biomass are carried out. Cultivation is performed at pH from 3.5 to 7.0 under conditions of air aeration from 0.5 to 2.0 l/l/min. Temperature mode in each cycle of fermentation is supported from the beginning of the cycle to the point of switch at the level from 26 to 30°C, and further to the end of the cycle at the level from 22 to 25°C. Point of switch is determined by accumulation of biomass to concentration from 45 to 60% from maximally achievable in fermentation apparatus, or point of switch is determined by concentration of dissolved oxygen by its reduction to the value from 20 to 40% of saturation ( calculated per atmospheric air pressure).
EFFECT: invention makes it possible to obtain the largest accumulation of biomass with high protein content with specified quantity of nucleic acids.
SUBSTANCE: growing of Staphylococcus aureus is carried out in a nutrient medium containing yolk-salt agar. At a stage of preparation for analysis the growth stimulators of Staphylococcus aureus are introduced into the nutrient medium in the form of aqueous solutions at concentrations of 10-4-10-6 wt %. The following compounds are used as growth stimulators: tris(2-hydroxyethyl)ammonium 4-chlorophenyl-sulfanylacetate or tris(2-hydroxyethyl)ammonium 2-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 2-methyl-4-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 1-benzylindol-3-yl-sulfanylacetate.
EFFECT: invention enables to accelerate growing of Staphylococcus aureus for diagnostics of infections, by reducing the time of growing from 48 to 6-9 hours in comparison with the control in a standard nutrient medium.
2 tbl, 7 ex
SUBSTANCE: invention relates to biotechnology and deals with recombinant strain of E. coli TG1(pRVMoscow3253G-L) for obtaining PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253".Recombinant strain is created on the basis of strain of E. coli TG1 by transformation with plasmid pRVMoscow3253G-L. Plasmid is obtained by ligation of fragment G-L of the region of genome of fixed rabies virus of strain "Moskva 3253", which has sequence SEQ ID NO1, into plasmid pGem-T. Also claimed is set of PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253" in rabies antigen. Set contains solutions of plasmid pRVMoscow3253G-L DNA in concentrations 108, 107, 105, 103 GE/ml. Concentration is determined by method of polymerase chain reaction, with hybridisation-fluorescence account of results.
EFFECT: invention contributes to standardisation of stage of preparation of rabies antigen in production of heterological anti-rabies immunoglobulin.
2 cl, 3 dwg, 2 ex
SUBSTANCE: invention relates to biochemistry and molecular biology. Conservation of cells of Escherichia coli in presence of buffered 80-90% glycerol is performed. Cell envelopes are removed with 3% triton X-100. Cell supramolecular structures are successively extracted with increasing concentrations of salts: 0.14 M (bacterioplasm), 0.35 M (loosely linked with cell residue), 2 M NaCl (strongly linked with cell residue), and 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol (cell residue). Acid hydrolysis is carried out in said fractions. Anthrone method is carried out, with preliminary purification of anthrone preparation. Calibration graph is built and quantity of hexoses is determined by means of calculation formula.
EFFECT: invention makes it possible to determine quantity of hexoses in supramolecular structures of bacterial cell of Escherichia coli.
3 dwg, 3 tbl, 1 ex
SUBSTANCE: invention is production of the nutrient medium, which creates optimal conditions for growing legionella, comprising: enzymatic hydrolyzate of pig lung, enzymatic hydrolyzate of chicken egg yolk, potassium monophosphate, trihydrate disubstituted potassium phosphate, L-cysteine hydrochloride, activated carbon, microbiological agar and distilled water at a predetermined ratio of ingredients.
EFFECT: invention enables to produce the high-quality, easy-to-prepare nutrient medium, to reduce the time of growing legionella.
SUBSTANCE: strain Aspergillus oryzae RCAM01135 is a producer of proteolytic and amylolytic ferments, which has ability to produce proteolytic and amylolytic ferments. It was deposited at GNU VNIISKHM with the following registration number: RCAM01135. It can be used at production of different food products (fermented spices, additives, and drinks).
EFFECT: invention allows increasing the growth speed and intensity of spore formation.
4 tbl, 2 ex
SUBSTANCE: method provides the hydrolysis of starch-containing raw materials, following adding of the mineral salts to the obtained hydrolysate and medium fermentation with acid-forming fungi Aspergillus niger. The hydrolysis is carried out with ferment preparate of bacterial α-amilase at increased temperature and excessive pressure. The starch-containing raw material are rice flour allowed beforehand in the water at 20-25°C during 10-12 hrs. in amount of 205.3-235.7 g and rye flour in amount of 232.0-290.6 g. The carbon and nitrogen ratio in the obtained rice flour suspension is provided respectively equal (20.0-23.0):1, in rye flour suspension - (14.0-16.0):1. The ferment preparate is added up to achieving of carbon source dextrose equivalent value 35-40%. The sugars conversion to citric acid is 86.7-93.2%. α-amilase and glucoamylase activity is respectively 4.5-6.2 units A,C/cm3 and 160-180 units Gl.C/cm3.
EFFECT: increase of α-amilase and glucoamylase yield while maintaining the high yield of citric acid.
1 tbl, 7 ex
FIELD: biotechnology, microbiology, biochemistry.
SUBSTANCE: invention relates to the strain Aspergillus oryzae-4150 obtained by selection from the known strain Aspergillus oryzae-387 (VKPM F-683) by multistep selection using effective methods of mutagenesis. The strain is stored as lyophilic dried culture and on slants with wort-agar in section of biotechnology of enzyme preparation, in food processing industry department of the State Scientific Institute of VNII food processing technology in Moscow. Invention provides preparing acid α-amylase showing high activity that exceeds activity in analogue by 2-3 times and reducing the process culturing time.
EFFECT: improved and valuable properties of strain.
2 tbl, 5 ex
FIELD: biotechnology, microbiology, organic chemistry.
SUBSTANCE: invention relates to a method for preparing organic acids, in particular, citric acid. Method involves isolation of calcium citrate and acid stable amylolytic enzymes from the solution cultural fluid after fermentation with fungus Aspergillus niger. The isolation process is carried out at temperature 10-50°C and pH 3.2-5.9. Method provides increasing yield of calcium citrate and complex of acid stable amylolytic enzymes: α-amylase and glucoamylase.
EFFECT: improved preparing method.
1 tbl, 6 ex
SUBSTANCE: invention proposes a method for obtaining yeast library including incubation of yeast cells in a solution with 0.01-1 M of lithium acetate and 1-100 mM of dithiothreitol, obtaining a suspension containing a linearised DNA vector and a DNA insert in the ratio of 1:0.5-1:10, sorbite and CaCl2 or MgCl2 at the ratio of 4 mcg of the DNA vector to 400 mcl of 1.6×109 yeast cells/ml, and electroporation of the solution of yeast cells with a suspension at voltage of 0.5-12.5 kV/cm with capacity of 10-50 mcF.
EFFECT: invention can be used for production in yeast cells of recombinant products and creation of libraries.
15 cl, 4 dwg, 8 tbl, 9 ex
SUBSTANCE: invention relates to biotechnology, specifically a method of producing artificial oligonucleotides that are potentially capable of forming non-canonical structures that stable in physiological conditions and conditions close to physiological, said structures being imperfect G-quadruplexes (lmGQ) which include one nucleotide substitution in the G4 plane in the G-quadruplexes (GQ). Said method includes using an algorithm describing nucleotide sequences in form of a defined set of formulae for further synthesis of selected oligonucleotides.
EFFECT: invention enables to use bioinformation analysis to obtain artificial oligonucleotides that are potentially capable of forming a new conformation - imperfect G-quadruplexes.
4 dwg, 2 tbl, 2 ex
SUBSTANCE: proposed method involves the following: obtaining EML4-ALK cDNA by means of a polymerase chain reaction with reverse transcription (RT-PCR) on RNA matrix of EML4-ALK gene using specific primers; amplification of fragments of EML4-ALK gene by means of a multiplex PCR method on cDNA matrix obtained at the first stage of RT-PCR by means of high-specific primers; obtaining fluorescent-marked PCR-product at the second stage of RT-PCR; creation of a biochip for analysis of EML4-ALK translocations, which contains a set of immobilised probes; hybridisation of fluorescent-marked PCR-product with probes in gel cells on a plastic substrate of the biochip; recording and interpretation of hybridisation results. The method provides for use of a technology of DNA biochips designed for the purpose of determining 6 versions of EML4-ALK translocations (V2, V3, V5, V4, V7, V1).
EFFECT: method has high sensitivity and specificity; it is general-purpose, and its implementation is possible with operating material and tumour tissue enclosed in paraffin units; recording of results is performed on a single occasion at the end of the investigation; the method's implementation lasts for less than 24 hours.
3 dwg, 3 tbl, 3 cl
SUBSTANCE: invention refers to creation of recombinant plasmids providing expression of poly-epitopic tumour-associated antigens in dendritic cells capable of stimulation of specific cytocidal cells, and it may be used in medicine. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumour-associated antigens of colorectal cancer, its size is 6 355 n. p. and it expresses the following amino acid sequence: DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGlTEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE. Mature dendritic cells obtained by adding to immature dendritic cells of pro-inflammatory TNF-α (tumour necrosis factor) cytokine are transfected by constructed plasmid DNA pCl-UB-POLYEPI thus activating them. Then activated dendritic cells are cultured together with peripheral mononuclear blood cells of people sick with colorectal cancer for generation of antigen-specific antitumour cytocidal cells.
EFFECT: invention allows efficient generation of antigen-specific cytocidal cell with antitumour activity in vitro, required for immune response by the 1-st type T-helper to colorectal cancer antigens.
2 cl, 1 dwg, 4 ex
SUBSTANCE: invention represents plasmide determining synthesis of α-galactosidase α-PsGal, which includes Ncol/Sall - fragment of plasmid pET-40b(+) (Novagen) and DNA fragment, with the size of 2142 pairs of bases, which contains a chimeric gen consisting of structural part of gene α-PsGal, which is adapted on N-end for expression in E. coli cells, and a nucleotide coding a specific sequence for enteropeptidase. The invention also refers to strain E. coli Rosetta (DE3) transformed with the above plasmid - producer of chimeric protein containing amino-acid sequence of recombinant α-galactosidase α-PsGal and sequence specific for enteropeptidase. The invention also refers to a method for obtaining recombinant α-galactosidase α-PsGal, which involves the following stages: incubation of the above strain-producer in liquid nutritional medium LB during 12 hours at 16°C, deposition of bacterial cells by centrifugation, disintegration of a suspension of cells in a buffer, centrifugation of an extract, chromatography of above-deposit liquid on a column with metal affine resin, elution of protein, concentration of active fractions by means of ion-exchange resin, incubation with enteropeptidase and separation of a target product by gel-filtration.
EFFECT: invention allows obtaining more active and stable recombinant alpha-galactosidase with high efficiency degree.
3 cl, 1 dwg, 3 ex