Method and reagents for detection of luciferase activity
SUBSTANCE: group of inventions relates to biochemistry. Disclosed is a method of detecting luciferase in biological samples using 3-hydroxy hispidin or 3-hydroxy bisnoriangonin as luciferin. Proposed compound for implementing said method represents a 3-hydroxy hispidin. Besides, disclosed is a method of detecting luciferase in the presence of hispidin-3-hydroxylase in biological samples using hispidin or bisnoriangonin as luciferin precursor and NADP. Also disclosed is a reagents kit for implementing said method, including hispidin or bisnoriangonin and NADP.
EFFECT: group of inventions enables detecting luciferase fungi in biological samples and can be used in a wide spectrum of the bioluminescent analyses in vivo and in vitro.
4 cl, 19 dwg, 1 tbl, 5 ex
SUBSTANCE: agent contains mutant luciferase of glowworms Luciola mingrelica (SEQ ID No:2), recovered from recombinant cells E.coli transformed by plasmid pETL7, luciferin, magnesium sulphate, tris-(oxymethyl)-aminomethane, acetic acid, sodium ethylene aminotetraacetate, dithiotreitol, bovine serum albumin, sucrose and water.
EFFECT: higher sensitivity of ATP test, lower enzyme consumption.
2 cl, 3 dwg, 1 tbl, 4 ex
SUBSTANCE: biomodule contains the following, immobilised into gel: nicotinamide adenine dinucleotide (NADN): flavine mononucleotide (FMN)-oxidoreductase, myristic aldehyde, NADN, enzyme stabiliser - dithiothreitol in concentration of 1·10-4 M, a FMN solution and a substrate. The method of preparing the biomodule involves preparation of 3-5% gel by boiling a starch suspension in a phosphate buffer or heating a gelatin suspension in a phosphate buffer to 60-80°C, cooling the gel to 24°-30°C, mixing the buffer solution of a bienzymatic system of luminescent bacteria NADN:FMN-oxidoreductase - luciferase, solutions of myristic aldehyde, reduced nicotinamide adenine dinucleotide, enzyme stabiliser - dithiothreitol in concentration of 1·10-4 M with gel, depositing onto the substrate and drying at 4-10°C. The biomodule is activated with a solution of flavin mononucleotide.
EFFECT: invention increases activity output and maximum luminous intensity of the biomodule by 1,3-3 times, increases the time for constant illumination level of the biomodule to 2-20 minutes, increases the storage time of the biomodule without loss of activity by 2 times, increases thermal stability and resistance of the biomodule to chemical factors of the environment.
2 cl, 12 dwg, 12 ex
SUBSTANCE: luminescent biocatalyst contains immobilised cells of luminescent bacteria which are included in a polyvinyl alcohol based cryogenic gel at given ratio of components.
EFFECT: prolonged possible use of the biocatalyst, simplification of its composition and production technology.
1 dwg, 2 tbl, 6 ex
FIELD: medicine; pharmacology.
SUBSTANCE: it is obtained a chimerous photo protein (photin), presented by amino-acid sequence of obeline protein, which part (from the rest in position 50 to the rest in position 94) is replaced by a homologous site of amino-acid sequence of clitine protein (the rests 53-97). The method of obtaining new chimerous photo protein by the method of recombinant DNA and vectors applied to it and cells is described. It is offered to use photo protein under the invention as the calcium indicator in various test systems in vitro and in vivo.
EFFECT: increased level of bioluminescence in comparison with natural protein.
11 cl, 6 dwg, 2 tbl, 3 ex
FIELD: biochemistry, analytical biochemistry.
SUBSTANCE: reagent for quantitative determination of adenosine 5'-triphosphate (ATP) by bioluminescent method comprises luciferase isolated from recombinant E. coli cells comprising plasmid pLR with native firefly luciferase gene, or luciferase isolated from recombinant E. coli cells comprising plasmid with m-pLR with the Luciola migrelica mutant luciferase gene wherein histidine residue at the position 433 is replaced for tyrosine residue. Also, the reagent comprises magnesium sulfate, mixture of tris-(hydroxymethyl)-aminomethane and acetic acid as a buffer, mixture of ethylenediaminetetraacetate sodium, dithiothreitol, bovine serum albumin and trehalose as stabilizing agents and water. In the assay method of adenosine 5'-triphosphate the reagent provides enhancing sensitivity and reproducibility in determination of ATP and reducing consumption of enzyme due to increase of specific activity of the reagent. The reagent provides carrying out determination of adenosine 5'-triphosphate by bioluminescence intensity at 560-570 nm in using firefly Luceola migrelica native luciferase, and by bioluminescence intensity at 606-610 nm in using luciferase wherein histidine residue at the position 433 is replaced for tyrosine residue.
EFFECT: improved assay method, valuable properties of reagent.
1 dwg, 1 tbl, 4 ex
FIELD: biochemistry, analytical biochemistry.
SUBSTANCE: reagent for determination of adenosine 5'-triphosphate (ATP) represents an aqueous solution of firefly luciferase containing luciferin, magnesium sulfate, tris-(hydroxymethylaminomethane), acetic acid, ethylenediaminetetraacetate sodium, dithiothreitol, bovine serum albumin and a stabilizing agent taken among the group including polybrene, polyvinylpyrrolidone, N-phthalylchitosan and sucrose. Using the invention provides enhancing precision and reproducibility of ATP assay. Invention can be used medicine, ecology, pharmacy and food industry.
EFFECT: improved assay method.
5 cl, 2 tbl, 3 ex
FIELD: biotechnology, microbiology.
SUBSTANCE: method involves sowing out samples of mixed cultures in liquid selective media, determination of cells number accumulating in media during microorganisms growth by bioluminescent method and mathematical treatment of kinetic data of the growth of individual bacterial cultures, determination of the parent concentration of microorganisms relating to different taxonomic groups. Invention allows carrying out the simultaneous identification of bacterial cells relating to different taxonomic groups and presenting in mixed cultures simultaneously, and to enhance precision in determination of cells number in the broad concentration range and to reduce the total analysis time significantly. In industry mixed cultures are used widely in different branches of food industry (dairy, meat, brewing and others) and in other biotechnological processes (biological treatment of sewage waters, bioremediation of soils, producing methane from waste in different manufactures and others). Invention can be used for differentiated determination of microorganisms number in mixed cultures that are widely distributed in nature: in air, soil, ponds, as components of natural microflora in higher organisms and among contaminants causing injury of different objects.
EFFECT: improved method for determination.
1 tbl, 3 dwg, 5 ex
FIELD: biochemistry, in particular production of immobilized enzymes useful on chemistry, biochemistry, medicine, histology, microbiology, ecology and agriculture for bioluminescence analysis.
SUBSTANCE: target reagent is obtained by production of 3-5 % gel by boiling of starch suspension in phosphate buffer, gel cooling to 24-30°C, mixing of buffer solution of luminescent bacterium bienzyme system NADH:FMH-oxydoreductase-lucirerase with starch gel, dosage on lavsan film and drying at 4-10°C. According the invention components are introduced in the next order: trimyristin aldehyde, nicotinamidadeninedinucleotide, bienzyme system NADH:FMH-oxydoreductase-lucirerase, and flavin mononucleotide. Substrate solution of nicotinamidadenine dinucleotide, flavin mononucleotide, and trimyristin aldehyde are prepared in phosphate buffer with pH 6.8-7.0.
EFFECT: reagent of improved quality due to decreased quantity of reaction mixture components (from four to one), reduced analysis period by two times and increased measurement accuracy.
2 cl, 1 dwg, 4 ex
FIELD: gene and protein engineering for various luminescent assays.
SUBSTANCE: new luciferase mutant forms have been obtained. Said mutant forms have increased thermostability and optionally different emission wave length in contrast with respective wild type enzymes. In all disclosed muteins natural amino acid residue in position equivalent to 357-position in Photinus pyralis luciferase sequence is replaced with other residue, preferably with uncharged polar amino acid (in particular tyrosine) residue. Mutant luciferases of present invention are useful in various analytical systems as reporter agent.
EFFECT: Mutant luciferases with new properties.
22 cl, 15 dwg, 6 tbl, 12 ex
SUBSTANCE: invention relates to biochemistry, particularly to an isolated nucleic acid molecule which codes a polypeptide, having delta-9-elongase activity, as well as a purified polypeptide having delta-9-elongase activity, which codes said isolated nucleic acid molecule. The invention also discloses an expression vector containing said isolated nucleic acid molecule, as well as a host cell, a transgenic plant and a transgenic seed containing same.
EFFECT: invention provides efficient production of polyunsaturated fatty acid-enriched oils.
18 cl, 9 dwg, 9 tbl, 6 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to biotechnology and represents a purified recombinant urate oxidase (uricase) characterised by the tetramer and octamer content of 95% and more of the total molecule count. The invention also discloses a purified preparation of the above urate oxidase possessing lower immunogenicity, a conjugate of the above urate oxidase and polyethylene glycol or polyethylene oxide used to reduce mammalian tissue or liquid uric acid, purified fragments of the above urate oxidase, a pharmaceutical composition containing the above urate oxidase and a method for purifying the above urate oxidase with maintaining its uricolytic activity, involving the separation and removal of uricase aggregates bigger than octamers.
EFFECT: present invention enables producing the preparations of urate oxidase of lower immunogenicity.
23 cl, 6 dwg, 6 ex
SUBSTANCE: expression vector is also provided, comprising the polynucleotide and a transformant obtained from S. cerevisiae, for production of the said protein, comprising the polynucleotide or vector.
EFFECT: efficient production of fatty acids with long-chain, having 18 carbon atoms.
9 cl, 3 dwg, 3 tbl
SUBSTANCE: invention relates to a novel method of obtaining fluorescent catecholamines selected from dopamine and adrenalin, and their metabolites, selected from homovanillic and vanillin-mandelic acids, by a method of derivation. The compounds can be used as highly sensitive and selective markers for the determination of various diseases. The method of derivation includes oxidation of the initial compounds and their interaction with amines that form condensed structures in a medium of the CAPS-buffer solution or glycin - KOH 0.2 mM hydrogen peroxide in the presence of horseradish peroxidase as a catalyst. The process in preferably carried out in a 0.1 M buffer solution with the concentration of horseradish peroxidase 0.01-1 mcM; concentration of hydrogen peroxide - 100 mcM, amine concentration - 0.1-33 mM; concentration of catecholamines and metabolites - 0.03-1 mcM.
EFFECT: method is simple and producible as it does not require higher temperature and is realised in a water solution.
2 cl, 2 dwg, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: group of inventions relates to biotechnology. Claimed is cell of mycelial fungus Penicillium canescens with removed catabolite repression and arabinose induction, producing xylanase and laccase. Cell is transformed with plasmid, containing promoter of gene bgaS, bgaS leader peptide, xylanase-coding DNA fragment, bgaS terminator, β-lactamase gene and pMB1 replicon, and plasmid, which contains promoter of gene bgaS, bgaS leader peptide, laccase-coding DNA fragment, bgaS terminator, β-lactamase gene and pMB1 replicon. Also claimed is method of enzyme preparation of xylanase and laccase with application of said cell of Penicillium canescens.
EFFECT: group of inventions provides increased output of target enzymes.
10 cl, 9 dwg, 3 tbl, 4 ex
SUBSTANCE: invention relates to the use of the concentrate of the culture liquid of the strain Trichoderma harzianum Rifai, deposited in the Russian National Collection of Industrial Microorganisms under the number of RNCIM F-180 as an inhibitor of Andis virus of potato mottling.
EFFECT: invention enables to reduce losses of potato from the plant infection with the Andis mottling virus.
SUBSTANCE: group of inventions relates to biotechnology and can be applied in creation of analytic methods with application of peroxidases. Method includes preparation of substrate mixture, introduction of peroxidases in substrate mixture with the following registration of intensity of formed luminescence. Substrate mixture includes the following components, given in final concentrations: buffer solution 10-125 mM, luminol 0.05-8 mM, hydrogen peroxide 0.05-8 mM, 4-aminopyridines 0.1-10 mM, N-carboxyphenothiazine, or N-(carboxymethyl)phenothiazine, or N-(2-carboxyethyl)phenothiazine 0.1-10 mM. And pH of buffer solution constitutes 7.9-9.0.
EFFECT: invention ensures obtaining of high intensity of chemiluminescence and, accordingly, high sensitivity of peroxidase activity determination.
4 cl, 8 ex
SUBSTANCE: invention relates to biotechnology and discloses isolated polynucleotides which encode Δ8 desaturase. The invention also relates to Δ8 desaturases themselves, which encode isolated polynucleotides, expression vectors containing isolated polynucleotides, host cells containing expression vectors and methods of producing Δ8 desaturase and polyunsaturated fatty acids selected from a group consisting of dihomo-gamma-linolenic acid (DGLA), ω3-eicosatetraenoic acid (ω3-ETA) and any combinations thereof.
EFFECT: invention widens the range of Δ8 desaturases used to produce polyunsaturated fatty acids.
12 cl, 12 dwg, 14 tbl, 6 ex
SUBSTANCE: invention relates to field of biochemistry, in particular to genetic construction for expression of threonine-insensitive aspartate kinase in plant, where construction contains promoter, specific for phase of aging, functionally connected with coding sequence, which codes polypeptide, possessing activity threonine-insensitive aspartate kinase, and in which specific to aging promoter is selected from group, consisting of SAG12, SAG13, SAG101, SAG21 and SAG18, or their functional variants or fragments. Described are vector, plant cell, transgenic plant, material for plant reproduction, collected leaves, smoking tobacco, which contain claimed genetic construction. Also described is method of obtaining transgenic plant, which possesses ability to accumulate threonine in fading leaves in larger amount than in leaves of corresponding plant of wild type, and method of increasing the level of threonine in leaves of aging plant to the level of threonine, higher than the content of threonine in leaves of plant of wild type without impairing plant adaptation with application of said genetic construction.
EFFECT: invention makes it possible to obtain transgenic plant, which possesses ability to accumulate threonine in fading leaves in larger amount than in leaves of plant of wild type.
22 cl, 16 dwg, 2 tbl, 4 ex
SUBSTANCE: invention is the truncated mutant luciferase MLM4 of Metridia longa with the size of -16 kDa with improved properties for use as a genetically encoded bioluminescent reporter for visualisation of molecular processes in living cells. The amino acid substitutions I69L, N74E, K125V, W139F are introduced in the sequence of the truncated luciferase MLM4 of Metridia longa with the size of -16 kDa.
EFFECT: invention enables to provide the luciferase with improved thermal stability and the spectrum shift to long-wave region, which determines an overall increase in sensitivity of the bioluminescent reporter when used in living cells and organisms.
4 dwg, 1 tbl, 3 ex
SUBSTANCE: invention relates to compounds of general formula I, having cytostatic or cytotoxic activity, its pharmaceutically acceptable salts, tautomers or stereoisomers, a pharmaceutical composition on their basis. Compounds may be used for treatment of cancerous diseases. In the general formula I
Y is selected from the group comprising -CHRay- and -CHRay-CRby-CRcy-; each Ray, Rby and Rcy are independently selected from hydrogen and non-substitute C1-C12-alkyl; each R1, R2, R3, R4 and R5 are independently selected from hydrogen and non-substitute C1-C12-alkyl; R6 is selected from NR8R9 and OR10; A means , W means NR7; R7 means hydrogen; R8 means hydrogen; R10 means non-substitute C2-C12-alkenyl; each dotted line means unnecessary additional link, but when there is a triple link between atoms of carbon, to which R1 and R2 are attached, then R1 and R2 are absent, and when there is a triple link between carbon atoms, to which R3 and R4 are attached, then R3 and R4 are absent; R9 is selected from substitute C2-C12-alkenyl and substitute C4-C|2-alkenylyl, where substitutes are selected from the group, consisting of: halogen, OR', OCONHR' and OH, protected with a simple silyl ether; where R' means hydrogen; provided that whenever Y means -CHRay-CRby=CRcy- and there is a single or double link between atoms of carbon, to which R3 and R4 are attached, then R9 means substitute C4-C12-alkenylyl; and each R16, R17 and R18 are independently selected from hydrogen and ORa; each Ra is selected from hydrogen or non-substitute C1-C12-alkyl.
EFFECT: higher efficiency of compound application.