Method of production of cytochrome c
SUBSTANCE: method comprises degreasing of hearts of pigs, cattle or horses. The prepared raw material is ground to obtain minced meat, then extracted with 2.5% trichloroacetic acid solution at the temperature of 10-15°C and pH of 3.9-4.2. The extract is separated and its sorption is carried out on the cation exchanger Nuvia™ S, equilibrated with distilled water. After completion of sorption the cation exchanger Nuvia™ S is washed with distilled water and ammonia-ammonium buffer solution. Then cytochrome C is eluted with from the cation exchanger Nuvia™ S with ammonium sulphate solution followed by its purification by diafiltration until complete removal of sulphate ammonium ions and concentration of cytochrome C by ultrafiltration.
EFFECT: invention enables to increase the yield of product, increase its purity and reduce the technological process.
3 cl, 1 tbl, 3 ex
The present invention relates to biotechnology, the field of production of medicinal protein preparations, namely, the method of production of cytochrome C from heart muscles mammals - pigs, cattle and horses.
Cytochrome C is an enzyme with a molecular weight of about 12700 Yes, as a prosthetic group contains hematomas iron catalyzes oxidation-reduction reactions and serves as a carrier of electrons (Y.A. Ovchinnikov. Bioorganic chemistry. M.: Chemistry, 1987, s).
Cytochrome C is widely used in medicine and veterinary medicine as a medicinal product, and as a reagent in biochemistry and biotechnology.
The invention may be used upon receipt of cytochrome C on an industrial scale.
Cytochrome C can be selected from various kinds of natural raw materials: yeast, rice, heart muscles mammals (horses, cattle, pigs, dogs, rabbits), birds (pigeons, penguins, hearts, salmon and others. Known methods for producing cytochrome C, including the allocation of the heart muscle mammal, preferably cattle, by solvent extraction and sorption purification of the extract by using a magnesium aluminosilicate (F. Okumura and al. Chem. Soc. Japan. V.49, No.1, p.65, 1975) or carboxylic cation exchangers of the type Amberlite HEH - 64 (B. Hagihara and al. J. Biochem V. 45, No. 8, p.551, 1975) and CMDM (L.K. Shataeva, H.H. Kuznetsova, GA the El'kin Yury Evgen'evich. Carboxylic cation exchangers in biology. Leningrad: Nauka, 1979, s.285).
These sorbents are characterized by small size and complexity of industrial applications, especially carboxylic cation exchange resin due to the difference in the degree of swelling in the H+and Na+form.
Closest to the proposed invention is a method for cytochrome C from heart muscles mammals - pigs, cattle and horses (Patent RU 2435597, publ. 10.12.2011, bull. No. 34), including degreasing hearts of pigs, cattle or horses, the removal of ligaments and blood vessels, grinding and extraction of a 2.5% solution of trichloroacetic acid at a temperature of 10-15°C, at a pH of 3.9 to 4.2, and the separation of the extract from the stuffing. Sorption of the target product are conducted directly from the filtered extract at a pH of extract on the cation exchanger UNOsphere™ S balanced starting buffer solution containing 2 mm of citric acid and 1.5 mm Tris (Tris (hydroxymethyl) aminomethane) at pH 3.9 to 4.2, and after the sorption of the cation exchanger is washed with the starting buffer solution, ballast proteins are removed from the cation exchanger UNOsphere™ S 80 mm Tris solution and then spend the elution of the cytochrome C solution of ammonium sulfate with a concentration of 0.3 M, after the stage of elution carry out the deposition of ballast proteins by adding dry with whom lipata ammonium based 0,270 gml
In the prototype using a cation exchanger UNOsphere™ S (UNOsphere Polymer Technology, the production of "Bio-Rad USA) - having as functional groups - SO3group.
The disadvantage of this method of obtaining cytochrome C is a low degree of purity of cytochrome C, an indicator of purity (E550280) cytochrome C in the eluate (the ratio of the extinction of the solution of cytochrome C at a wavelength of 550 nm and a solution of cytochrome C at a wavelength of 280 nm) is 0.92-0,93 that requires deposition of ballast proteins from the eluate using the increased amount of ammonium sulfate (0.27 gml), also significant disadvantages of the above method of obtaining cytochrome C is to use solutions, which include expensive reagents, such as citric acid and Tris(hydroxymethyl) aminomethan. These shortcomings significantly reduce the efficiency and effectiveness of the process.
Thus, the present invention is to provide a method of producing cytochrome C from the extract using ion exchangers of the new generation, providing a high sorption capacity and reversibility of sorption with the use of inexpensive reagents, providing a high degree of purity of the target product, which makes them preferable for use in an industrial scale.
The technical is a mere result of the present invention is to increase the capacity of the target product, the increase in the purity of cytochrome C reduction process.
According to the present invention is proposed sorption method of producing cytochrome C from the extract using cation of a new generation Nuvia™ S (Nigh - Capacity Cation Exchange Media, the production of "Bio-Rad USA) containing as functional groups - sulfopropyl. During the final stages of obtaining cytochrome C used diafiltration to remove ions of ammonium sulfate) and ultrafiltration (for concentration of cytochrome C) in a tangential flow membranes with a limit exceptions molecular weight 5 kDa firm Millipore or Sartorius.
To achieve the technical result of the proposed method, which includes the following stages:
From the hearts of pigs, cattle or horses after degreasing and removal of ligaments, blood vessels, by grinding receive the stuffing. The resulting meat is extracted with a 2.5% solution of trichloroacetic acid at a temperature of 10-15°C, at a pH of 3.9 to 4.2. After separation from the meat directly lead sorption of the target product from the filtered extract by cation Nuvia™ S cation exchange resin pre-balance distilled water. After sorption, cation Nuvia™ S washed with distilled water. Removal of ballast proteins with columns hold of 0.075 M ammonium Ammon the emergency buffer solution pH of 10.5. The elution of cytochrome C from the cation exchanger Nuvia™ S conduct with a solution of ammonium sulfate with a concentration of 0.25 M For the Department of cytochrome C from ammonium sulfate are diafiltration in tangential flow membranes with a limit exceptions 5 kDa, the same ultrafiltration membranes provide a concentration of cytochrome C.
For equilibration and washing the column, the best option is distilled water. To remove the ballast protein from the cation exchanger is optimal 0,075 M ammonia / ammonium buffer solution pH=10,5, at lower concentrations and pH of the buffer solution is not completely washed out of the ballast proteins, higher concentrations and pH does not have a significant impact on the number of remote ballast proteins. For elution of cytochrome C from the cation exchanger is the most optimal solution of ammonium sulfate with a concentration of 0.25 M, a low concentration of ammonium sulfate is not completely elute the target product with the cation exchanger, at higher concentrations of the cation exchange resin partially suiryudan impurity proteins, which significantly reduces the degree of purity of the target product.
The use of cation Nuvia™ S, you can directly lead sorption of cytochrome C from the extract (at a pH of extract), use a smaller amount of the cation due to the greater its capacity to significantly increase the degree of purity citoh the OMA C. The increase in the purity of cytochrome C at a stage of desorption from the cation exchanger Nuvia™ S makes it possible exception stage of deposition of ballast proteins that significantly increases the effectiveness and efficiency of the process by reducing the consumption of reagents and by reducing the time of the process.
The present invention is illustrated by the following examples.
2.6 kg of meat derived from cardiac muscle of pigs after removal of blood vessels, ligaments and grinding, washed with water from the remnants of blood, extracted with 2.5% trichloroacetic acid. Extraction was carried out for 2 h at 10-15°C. the Extract is separated from the feedstock by filtration through a cotton-gauze filter with subsequent centrifugation at n=3000 rpm / min for 10 minutes the pH of the resulting extract to 4.1. Just get 2750 ml of the extract. The extract obtained is passed through a cation exchanger Nuvia™ S balanced distilled water. The column volume 49 ml (diameter 2.5 cm, height of the layer of sorbent 3.8 cm, the amount of sorbent 19 ml). Sorption is carried out at a throughput rate of solution 2240 ml/h Time of sorption of about 1.2 hours and Then the cation Nuvia™ S washed with 200 ml of distilled water. Washing time 0,09 hours For removal of ballast proteins from the column is passed 400 ml of 0.075 M ammonium ammonium buffer solution pH=10.5V at a speed of 750 ml / HR. Washing time of 0.53 hours
The elution is of akroma spend With a solution of ammonium sulfate with a concentration of 0.25 M. The feed rate of eluting solution of 75 ml/h elution 0.5 hours the Eluate selected fractions. Faction, red, unite. The volume fractions of about 25 ml. Indicator of the purity of cytochrome C (E550280) amounts to 1.15. Yield 245 mg of cytochrome C or 94.6 mgkg of raw material.
Is performed under the conditions of example 1. For the Department of cytochrome C from the ions of ammonium sulfate are diafiltration. For this purpose a solution of cytochrome C in the amount of 125 ml (combined fractions 5xdownload pass by ultrafiltration installation mode diafiltration in tangential flow across the membrane of the company Millipore (cassette type Pellicon XL) or Sartorius (cassette type Vivaflow 50) with a limit of exclusion molecular weight of 5 kDa. Collect 135 ml of a solution containing the target product.
The indicator of the purity of cytochrome C (E550280) - 1,24. The output 232 mg of cytochrome C or 89.2 mgkg of initial raw material (in terms of per download).
Is performed under the conditions of example 1 and 2. Cytochrome C after diafiltration concentrated by ultrafiltration. 135 ml of a solution of cytochrome C (combined fractions 5xdownload after diafiltration) is passed to the ultrafiltration installation in a tangential flow across the membrane of the company Millipore (cassette type Pellicon XL) or Sartorius (cassette type Vivaflow 50) with a limit of exclusion molecular weight of 5 to the and. Get 45 ml of a solution containing the target product.
The indicator of the purity of cytochrome C (E550280) - 1,26. Output 225,7 mg of cytochrome C or 86,8 mgkg of initial raw material (in terms of per download).
Examples 4-9 in stages made in the conditions of example 1, 2 and 3. All experimental data presented in the table.
The index And mean raw materials of the heart muscle pig in example 1, 2 and 3;
B - raw from the heart muscle of cattle; raw material of the cardiac muscles of the horse.
1. The method of producing cytochrome C, including degreasing hearts of pigs, cattle or horses, the removal of ligaments and blood vessels, grinding and extraction of a 2.5% solution of trichloroacetic acid at a temperature of 10-15°C, at a pH of 3.9 to 4.2, and the separation of the extract from beef, sorption and elution of the cation exchange resin, removing the ballast proteins, concentration and purification by diafiltration and ultrafiltration to remove ions of ammonium sulfate, characterized in that the sorption of the target product are conducted directly from the filtered extract at a pH of extract on the cation exchanger Nuvia™ S balanced distilled water, after sorption, cation washed with distilled water, ballast proteins are removed from the cation exchanger Nuvia™ S 0,075 M ammonia / ammonium buffer solution of pH 10.5 and then spend is linked to cytochrome C from the cation exchanger Nuvia™ S solution of ammonium sulfate with a concentration of 0.25 M
2. The method according to claim 1, characterized in that the purification of cytochrome C from the ions of ammonium sulfate spend diafiltrate in tangential flow through the membrane to limit the exclusion molecular weight of 5 kDa.
3. The method according to claim 1, characterized in that the concentration of cytochrome C was carried out by ultrafiltration in a tangential flow through the membrane to limit the exclusion molecular weight of 5 kDa.
SUBSTANCE: invention relates to biotechnology and a recombinant strain of Escherichia coli bacteria - a producer of biologically active flagellin. The described strain is obtained by transformation of an E. coli BL21[DE3] cell culture with recombinant plasmid DNA pET151FliC, which is obtained based on a pET151FliC vector in which was embedded a fliC gene which codes biologically active flagellin, having a nucleotide sequence represented in Seq ID No 3. The strain is deposited in the Russian National Collection of Industrial Microorganisms (RCIM) of the Research Institute for Genetics and Selection of Industrial Microorganisms under No B-11369.
EFFECT: present solution has higher production capacity with respect to recombinant flagellin, which is an effective adjuvant.
1 dwg, 2 tbl, 3 ex
SUBSTANCE: invention relates to the field of biotechnology and can be used for obtaining a nanostructured material based on recombinant flagella of archea H. salinarum, bound with metal ions or nanoparticles. Transformed with a recombinant plasmid cells of archea are grown, flagella, containing peptide inserts for binding with metal ions or nanoparticles, are separated. The surface of flagella is modified by binding peptide inserts with the said ions or nanoparticles with further washing, drying and packaging of the obtained material. The plasmid construction contains recombinant genes for synthesis of A1 and A2 flagella-forming flagellins. A sequence of flagellin A1 and/or flagellin A2 contains a peptide insert for selective binding metal ions or nanoparticles, where the location of the peptide insert is determined in the region between the first and second sites of glycosylation, located in flagellin A1 between position 86 and position 96 SEQ ID NO: 2, and in flagllin A2 between position 82 and position 92 SEQ ID NO: 3.
EFFECT: invention makes it possible to obtain the nanostructured material with improved adhesive properties, resistant with respect to impact of high temperatures.
10 cl, 7 dwg, 7 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.
EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.
11 cl, 32 dwg, 14 ex
SUBSTANCE: hybrid proteins GFN80 and GFN100 are formed based on recombinant human interferon alpha-2 fused on the N-terminus with the amino acid sequence of polypeptide S(G4S)16 or S(G4S)20, respectively. The strains of producer Saccharomyces cerevisiae RNCIM Y-3927 and Saccharomyces cerevisiae RNCIM Y-3928 are produced by recombinant method. The strains are used in the method of production of the hybrid protein GFN80 and GFN100, which comprises culturing under suitable conditions of yeast cells transformed by the expression vector, which contains the region of replication initiation of endogenous 2-micron plasmid of yeast Saccharomyces cerevisiae, and the promoter of yeast GAL1 controlling the expression of the gene comprising the DNA sequence SEQ ID NO:1 or SEQ ID NO:2, respectively, followed by isolation of the hybrid protein from the culture fluid.
EFFECT: invention enables to produce the hybrid recombinant human interferon alpha-2 with the prolonged action in the body of animals.
5 cl, 7 tbl, 15 ex
SUBSTANCE: invention relates to field of biotechnology. Claimed is separated chimeric polynucleotide for amplification of production of heterologous protein of interest, which contains polynucleotide sequence of promoter SigA or SigH, functionally connected with polynucleotide, coding protein YmaH, with chimeric polynucleotide connecting sequence, which by, at least, 90% is identical to SEQ ID NO: 1, 2, 3 or 13. Also described are: expression vector, containing claimed nucleotide structure, and host cell Bacillus for production of heterologous protein of interest, which contains said vector. Claimed is method of obtaining modified Bacillus cell, including transformation of host cell of Bacillus-producent of heterologous protein of interest with said vector; and growing said modified cell in optimal conditions. Described is method of obtaining protein of interest in modified Bacillus cell, where method includes cultivation of said host cell; and growing said modified Bacillus cell in optimal conditions. Also described is method of amplification of expression of heterologous protein from Bacillus of interest includes obtaining said modified Bacillus cell; growing modified Bacillus cell in optimal conditions; and expression of said protein of interest in modified Bacillus cell, where expression of said heterologous protein of interest in modified Bacillus cell is amplified in comparison with expression of said protein of interest in said parent Bacillus host-cell.
EFFECT: invention makes it possible to increase output of target protein due to superexpression of protein YmaH.
30 cl, 4 dwg, 3 ex
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: invention relates to biotechnology. Disclosed is a purified preparation of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme, where said enzyme includes an amino acid sequence which is at least 95% identical to amino acids 27-522 SEQ ID NO:4, which is suitable for treating a subject suffering from a lysosomal storage disease associated with GALNS, where: (a) said GALNS enzyme preparation has purity of at least about 95% as determined by Coomassie Blue staining when subjected to SDS-PAGE under non-reducing conditions; and (b) the cysteine residue at position 79 of at least 50% of molecules of the GALNS enzyme in said GALNS enzyme preparation is converted to Cα-formylglycine (FGly); where said GALNS enzyme is N-linked glycosylated at the asparagine residues at positions 204 and 423, wherein at least about 50% of the oligomannose chains attached to the asparagine residue at position 204 are bis-phosphorylated. Disclosed is a method of treating a subject suffering from mucopolysaccharidosis type IVa (MPS IVa), Morquio A syndrome or multiple sulfatase deficiency (MSD), which involves administering a therapeutically effective amount of said purified preparation of recombinant human GALNS to the subject.
EFFECT: invention enables to obtain a pharmaceutical preparation of recombinant highly phosphorylated human GALNS, having a high content of molecules with a cysteine residue at position 79 converted to Cα-formylglycine, owing to which it is highly absorbed through the mannose-6-phosphate receptor (MPR) and has high activity.
29 cl, 13 dwg, 15 tbl, 11 ex
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
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: invention refers to medicine, particularly to transfusion medicine, namely to a method for preparing polymer modified haemoglobin by the step multifunctional condensation of purified haemoglobin recovered from concentrated red cells in the mixed oxy/deoxy form with a synthetised multifunctional cross-linking agent that is glutaric aldehyde with glutamic acid and sodium glutamate, wherein the first stage of the reaction involves the intramolecular chemical cross-linking of a haemoglobin molecule labile in an aqueous solution; the second stage involves the intramolecular cross-linking of the ready modified haemoglobin molecules to form polymer modified haemoglobin.
EFFECT: invention provides preparing more effective polymer modified haemoglobin in the form of the multifunctional blood substitute having the effective oxygen transfer function, anti-shock and haemodynamic action able to initiate own blood formation.
3 cl, 1 ex, 2 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to pharmacology, namely a method for producing cytochrome C. The method for producing cytochrome C involving degreasing of pig, cattle and horse hearts, removal of ligaments and vessels, grinding and extraction in a trichloracetic acid solution under certain conditions, separation of the extract from stuffing, sorption of the filtered extract at extract pH in the UNOsphere™ S cation exchanger balanced with a starter buffer solution at pH 3.9-4.2, after completion of the sorption process, the cation exchanger is washed with the starter buffer solution; ballast proteins are removed from the UNOsphere™ S cation exchanger by a tris solution, and it is followed with elution of cytochrome C from the UNOsphere™ S cation exchanger with an ammonium sulphate solution, concentration and dialysis purification to remove ammonium sulphate ions completely.
EFFECT: method allows increasing product yield, providing higher purity and reducing production process.
4 cl, 1 tbl, 5 ex
SUBSTANCE: invention relates to novel hemin derivatives of general formula pharmaceutically acceptable salts thereof, synthesis method, pharmaceutical and disinfection compositions.
EFFECT: compounds have decomposition effect on lipid membrane, antimicrobial properties, antibacterial and antifungal, and simultaneously virucidal and antimicrobial properties.
15 cl, 2 dwg, 20 ex
SUBSTANCE: invention relates to novel hemin derivatives of general formula I
where R1=R2 and represent β-alanyl histamine or γ-gutamyl histamine, or β-alanyl histidine, or R1 represents γ-gutamyl histamine; Y represents CI-; Me represents Fen+, where n=2, 3; and where hemin carboxyl group can be modified with methyl or other C1-8 ether their pharmaceutically acceptable salts; method of their obtaining and pharmaceutical compositions.
EFFECT: hemin derivatives of general formula I can be used as artificial nucleases, means, possessing peroxidase activity, catalysts of methyl linoleate oxidation and disinfecting, antiseptic composition, possessing virucidal activity.
9 cl, 1 dwg, 7 tbl, 10 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.
EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.
3 dwg, 5 ex
SUBSTANCE: method can be used for quantitative evaluation of superoxide in membrane preparations (microsomes, mitochondria) expressing cytochrome C reductase and cytochrome C oxydase activity. Besides its can be used in medical biochemistry to study the functions of superoxide in pathogenesis of neurodegenerative diseases. The method includes preparation of a measuring medium, mixing of a superoxide source and said measuring medium, addition to the prepared mixture of an oxidation substratum and mutant cytochrome with K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K7K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K. It is followed with spectrophotometric measurement of cytochrome C derivative reduction rate and determination of superoxide generation rate.
EFFECT: method is simpler in comparison with common analogues and allows for high-accurate determination of superoxide generation rate.
1 tbl, 4 dwg, 1 ex
SUBSTANCE: group of inventions concerns medicine and pharmacology, namely to blood substitutes on the basis of polyhemoglobin. The blood substitute with function of oxygen transfer which basis is made of polymerised glutaric aldehyde haemoglobin received from blood of animals is offered, thus it represents a dry substance and contains not less than 90% of polymerised haemoglobin with molecular mass in a range 192000-320000Da, and methemoglobin content in a blood substitute makes not more than 5%. The pharmaceutical compositions containing the specified blood substitute, containing either polyvinylpyrolidone, or polyoxydin, or physiological saline, or solution of sodium chloride, potassium chloride and magnesium chloride and sodium fumarate are offered. The invention provides blood substitute creation, comparable by efficiency of oxygen transfer with erythrocytes of human blood.
EFFECT: creation of blood substitute, comparable by efficiency of oxygen transfer with erythrocytes of human blood.
10 cl, 1 ex, 1 tbl
SUBSTANCE: offered is method of blood substitute production and related installation for method implementation. Method of blood substitute production includes production of deoxygenated haemoglobin, its polymerisation and purification. Production of deoxygenated haemoglobin includes haemolysis of water addition to erythrocytic mass, stroma separation, non-heme protein precipitation and removal from produced haemoglobin solution. Polymerisation includes processing of produced deoxygenated haemoglobin with modified glutaric aldehyde and restoration with sodium borane, with purification including ultra filtration. Deoxygenated haemoglobin is produced using leukocyte-free erythrocytic mixture. Non-heme proteins are precipitated by concentrated sodium chloride solution added to haemoglobin solution. Removal of non-heme proteins is followed with ultra filtration concentration of haemoglobin solution. Haemoglobin is produced in polymeric disposable containers, while deoxygenation and polymerisation are carried out in gas vortex reactor with nitrogen atmosphere within 1-6 hours each. Diafiltration purification is performed in polymeric disposable containers on shutoff dampers to produce end product molecular weight within 100 kDa to 450 kDa. Method allows for simplified production of polyhaemoglobin with lowered cost and higher outcome. Related installation for method implementation includes series haemoglobin production area, haemoglobin polymerisation reactor and end-product purification system. Haemoglobin production area contains series haemolysis tank with filtration manifold for stroma separation, non-heme protein precipitation tank with filtration manifold for removal of precipitated non-heme proteins. End product purification system contains ultra filtration tanks and units with shutoff dampers. All tanks within haemoglobin production area are polymeric disposable containers. Non-heme protein precipitation tank is connected to the tank for concentrated solution of sodium chloride. Polymerisation reactor is designed as gas vortex unit. End product purification system tanks are polymeric disposable containers. Haemoglobin production area, haemoglobin polymerisation reactor and end product purification system, as well as all tanks and units are interconnected by means of sterile rapid-action coupling.
EFFECT: allows for reduced material consumption of installation with higher productivity, sterile conditions of technological process.
6 cl, 1 dwg
SUBSTANCE: perform blood-substituting solutions comparable by efficiency of gas transport on oxygen transfer with erythrocytes of blood of the person. The blood substitute with function of oxygen transfer includes polymerised haemoglobin with modified glutaric aldehyde, thus polyhemoglobin consists exclusively from oligomers, containing from 2 to 6 molecules of haemoglobin.
EFFECT: improvement of quality of a blood substitute at the expense of an exception of undesirable by-effects.
SUBSTANCE: group of inventions refers to stabilised oxygen carrying. More specifically it concerns haemoglobin solutions in which after associated processing stability of polymeric communications is raised while content of formed tetramer is decreased. Offered is production method of haemoglobin solution inherently not containing tetramer including haemoglobin polymerisation in solution, thermal processing polymerised haemoglobin in solution at temperature more that approximately 45°C during at least approximately 24 hours with partial degradation of polymer to tetramer and removal of tetramer from haemoglobin solution. Offered is haemoglobin solution produced by specified method. Offered is method of stabilisation of polymerized haemoglobin solution inherently not containing tetramer. Offered are production method of stabilised polymerised haemoglobin solution and production method of haemoglobin solution inherently not containing tetramer.
EFFECT: content of microbic and virus antigens and pathogens are decreases to non detectable level.
23 cl, 15 ex
FIELD: biotechnology, genetics.
SUBSTANCE: invention relates to methods used for detecting low frequency mutations occurrence in gene encoding cytochrome b. Method involves isolation of DNA from known fungi for constructing oligonucleotide probe or primer. Then polymerase chain reaction (PCR) is carried out for assay of binding the nucleotide probe with amplicon generated by this reaction, or the presence of amplicon is detected that is generated as result of PCR using indicated primers. Invention provides rapid and precise detection of mutations conferring resistance of fungus against fungicide.
EFFECT: improved diagnostic methods for detecting mutations.
24 cl, 18 dwg, 14 tbl, 18 ex