Escherichia coli bacteria strain - producer of recombinant flagellin
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.
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The invention relates to biotechnology, in particular to a technology for production of recombinant flagellin using a strain of Escherichia coli bacteria.
Bacterial protein flagellin is an effective stimulator of the immune system. Interacting with the cellular receptor TLR-5, which is expressed on the surface of epithelial cells, vascular endothelium, neutrophils, monocytes, dendritic cells and T-lymphocytes, flagellin induces rapid production of cells of inflammatory cytokines and chemokines, activating immune cells and attract them to the place of introduction of the pathogen. A special role in the development of the immune response plays an interaction of flagellin with TLR-5 dendritic cells that stimulate the maturation of dendritic cells, expressed, in particular, in the expression on their surface CD80, CD86, and MHCII. Stimulated flagellin dendritic cells strenuously produce TNF-α, IL-8, IL-1β, MCP-1, MIP-1α, MIP-1β, RANTES. It is shown that dendritic cells stimulated flagellin, in turn, stimulate the proliferation of T-cells and the production by them of interferon-γ. Activation flagellin monocytes and macrophages also enhances the immune response by stimulating phagocytosis.
Immunostimulatory effects of flagellin appears as if its systemic administration, and the effects on the mucous membranes. Interaction with Retz what porom TLR-5 is peculiar and occurs at low concentrations of flagellin - to 8.5×10-10M. in Addition, since TLR-5 is located on the surface of cells, interaction with him flagellin does not require pre-phagocytosis, which explains the responsiveness of cells to flagellin.
A number of patents, where flagellin is used as an adjuvant for immunization against a number of pathogens, including protein, fused to a target antigen in a single polypeptide chain [WO 2006078657, WO 2011028875], in the form of a mixture with the target immunogen [EA 201001820] or in the form of virus-like particles containing flagellin and the target antigen [US 201205082, WO 2009128949].
Known patent, where flagellin can be used as immunostimulant or immunomodulator for the treatment of diseases associated with chronic bacterial infection [WO 2007098371].
It is also known antiapoptotic action of flagellin, which allowed us to use it as a radioprotector and homeprotector [EA 010291, WO 2009102818].
The disadvantage of this technology is the lack of efficiency of recombinant flagellin, as well as the high cost of flagellin associated with its low output.
Known technology for producing recombinant human flagellin, where the proposed receipt of Salmonella Flic entericf Serovar Typhimurium ATCC 14028 [application EA 201001820, 2010]. The disadvantage of this method of obtaining flagellin is the need to cool the dependent Salmonella, that requires increased security measures, increases the amount of financial costs and time for receipt of flagellin.
The closest to the technical nature of the claimed invention is the development of the authors [Matyunina E.A. and other "preparation of recombinant protein FliC in the cells of E. coli<URL: http://shmain.ru/nauchnye-stati/matyunina-e-a-al-shexadat-r-i-duxovlinov-i-v-poluchenie-rekombinantnogo-belka-flic-v-kletkax-e-coli.html>where it is shown the possibility of obtaining flagellin from E. coli, however, further authors conducted tests have shown that it is denatured and so inactive that caused the need for further work leading to the establishment of the present invention.
To eliminate all these disadvantages, there is a need in bacterial producers, providing a high level of production high level of flagellin.
The task of the authors was to create a technology that allows to obtain active recombinant flagellin (with a specific activity of 106 units of activity/mg), and increase output.
The technical result was achieved by the creation of an Escherichia coli strain BL21[DE3]pET151FliC - producer of highly active recombinant human flagellin obtained by transformation of the cell culture of E. coli BL21[DE3] (Invitrogen, USA, genotype F - ompT hsdSB (rB-mB-) gal dcm rne131 (DE3) recombinant plasmid DNA pET151FliC created in vtro and containing the recombinant gene of flagellin fliC, vector pET151FliC that encodes a biologically active flagellin of Salmonella typhymurium T10 for Seq ID No 3.
The resulting strain E. coli BL21[DE3]pET151FliC deposited in Russian national collection of industrial microorganisms (VKPM) FSUE gosniigenetika no In-11369 on 30.10.2012.
The E. coli strain VKPM No-11369 characterized by the following cultural-morphological and physico-biochemical properties:
Cultural and morphological characteristics of strain: gram-negative straight rods, the amount of 1.1 to 1.5×2.0 to 3.0 μm, solitary, spores and capsules do not form. Catalanopolonesa. Oxidatively. Facultative anaerobes. Cells grow well on simple nutrient media containing and not containing ampicillin, for example, LB medium. On agar medium - colonies smooth, round, slightly convex, with smooth edge. In liquid media to form a uniform light-diffusing suspension, when stored without stirring settle to the bottom. Cells grow in the temperature range from 8°C to 43°C, the interval for cultivation - 28-38°C, with optimum growth at 37°C. the Interval of pH 5-7. Catalyze D-glucose and other carbohydrates with the production of acid and gas, not fermented galactose. Reaction Voges-Proskauer negative, do not form H2S, hydrolyzing urea.
Characteristics of useful substances produced by strain: Recombinant protein flagellin, a length of 528 and inability residues, composed of flagellin Salmonella typhimurium FliC T10 (495 AMK) and service sequence length of 33 amino acids
The productivity of the strain recombinant flagellin is at least 37% of the protein in the cell lysate under cultivation in the conditions of induction.
Cryopreservation. In a sealed ampoule, strain, freeze-dried in a medium containing 30 ml of water and 5 g of sucrose, 1.5 g of gelatin kept at room temperature for 20 years.
Cultivation of strain: cultivating at a temperature 28-38°C in thermostat or the rocking chair, agar (2% agar) or liquid LB-medium, respectively. Selective conditions - add 100 ág/ml ampicillin.
Fermentation: fermentation of lead in liquid medium PYP-5052 containing 0.2% lactose, with the addition of 100 μg/ml ampicillin, with stirring and aeration at a temperature of 28-38°C for 18 hours.
Figure 1 shows electrophoregrams lysates of cells of Escherichia coli VKPM No-11369 under cultivation in the conditions of induction of protein synthesis of lactose and without induction, where 1 is the protein marker of molecular weight, kDa; 2-cell lysate of E. coli strain VKPM No-11369 induction of expression of 0.2% lactose; 3-cell lysate of E. coli strain VKPM No-11369 without induction of expression of 0.2% lactose.
The invention is illustrated by the following specific examples of the use of E. coli strain VKPM No-11369.
Example 1. The creation of genetic structure, providing a synthesis of recombinant flagellin in E. coli cells.
By polymerase chain reaction using specific primers FliC-For (SEQ ID NO 1) and FliC-Rev (SEQ ID NO 2) on the matrix of genomic DNA vaccine strain Salmonella typhimurium T10 (RU2192886) was amplified fliC gene encoding flagellin, which was then sewn with vector RET. The nucleotide sequence of the obtained gene coincides with the sequence of flagellin FliC (flagellin phase 1) Salmonella enterica serovar Typhimurium, available in GeneBank (BAA02846.1) Obtained gene was inserted in the expression vector RET obtaining expression plasmids pET151FliC.
Plasmid RET FliC provides synthesis in Escherichia coli cells full of bacterial flagellin, optionally containing N-end service sequence length of 33 amino acids, which ensures highly efficient initiation of protein synthesis by ribosomes of E. coli, contains a plot consisting of 6 histidine residues (his-tag tag)that is used for subsequent purification of recombinant flagellin using metallogenetic chromatography, website recognition V5 antibody, and is also the site of hydrolysis by the protease TEV, allowing, if necessary, to remove most part of the service sequence processing purified recombinant human flagellin above asanoi a proteinase with subsequent re-cycle metallogenetic chromatography. The nucleotide sequence synthetic gene encoding the recombinant flagellin, is presented in SEQ ID No 3.
Example 2. Obtaining strain-producer of recombinant flagellin and research productivity.
The obtained plasmid pET151FliC were transformed cells of Escherichia coli strain BL21[DE3]Star, containing in its genome a gene that encodes a polymerase of phage T7 under the control of a bacterial promoter, induced by lactose. In addition, they do not contain protease Ion and carry the mutation in the gene encoding the protease OmpT. The absence of these two proteases reduces the degradation of heterologous proteins. This strain also contains a mutation in the rne gene encoding the ribonuclease E, which leads to increased stability of mRNA in the cell and, consequently, to increase the production of cells of this strain of recombinant protein.
The result was the resulting strain E. coli VKPM No-11369 - producing bacterial protein FliC.
To maintain the obtained strain-producer of protein FliC used dense agar LB-medium containing 100 μg/ml ampicillin and 1% glucose.
The productivity obtained producer strain was studied by culturing cells in medium PYP-5052 with 0.2% lactose, in a temperature-controlled shaker rotary type at a temperature of 37°C, the speed of rotation of the platform 250 rpm for 18 hours. The optical density (OD
The result is shown in figure 1.
As can be seen from figure 1, the induction by lactose cell culture of E. coli VKPM No-11369 leads to the synthesis of a protein with a molecular weight of approximately 57 kilodaltons, which corresponds to the expected molecular weight for recombinant human flagellin. Analysis of densitogram polyacrylamide gel presented in figure 1, is made using TotalLab showed that recombinant flagellin is 37% of the total protein in the cell lysate.
Example 3. Purification of recombinant flagellin and study of its biological activity.
Recombinant flagellin was purified from cell lysates method metallogenetic chromatography followed by dialysis. Purity dedicated flagellin amounted to not less than 97% according to the results of polyacrylamide gel electrophoresis followed by densitometry.
Biological activity of recombinant flagellin was assessed in the test, based on the ability of the cell line A-549 to secrete interleukin-8 by adding to the culture medium of flagellin. Telepostanovki test cell line a-549 was made in the amount of 5×10 4per well in 96-well flat-bottomed culture Board ("Costar") in 100 μl of medium DMEM/F12 with 10% fetal serum. Cost incubated for 24 hours in an incubator at 37°C in an atmosphere of 5% CO2in terms of absolute humidity. During incubation of the cells in the Board formed a dense monolayer. Next, the cells were added to the study drugs flagellin in various concentrations in a volume of 100 μl in the culture medium (at least 3 parallel wells for each concentration). Then charge incubated another 24 hours in CO2-the incubator and collected supernatant, which was determined by the concentration of IL-8 using a commercial kit based on enzyme-linked immunosorbent assay (LLC "Cytokine").
The results of two experiments to determine the biological activity of recombinant flagellin presented in tables 1 and 2.
|Determination of the activity of recombinant flagellin in experiment 1|
|The concentration of recombinant flagellin, ng/ml||The concentration of IL-8 in supernatant, PG/ml (mean ± STD. deviation)|
|Determination of the activity of recombinant flagellin in experiment 2|
|The concentration of recombinant flagellin, ng/ml||The concentration of IL-8 in supernatant, PG/ml (mean ± STD. deviation)|
From tables 1 and 2 that the recombinant flagellin produced by cells of the strain E. coli VKPM No-11369, has biological activity and dose-dependently enhances the production of IL-8 cells A, 50% effective dose of recombinant flagellin is approximately 10 ng/ml.
The advantage of the strain E. coli VKPM No-11369 is a high biological activity in adjuvant.
The bacterial strain Escherichia coli BL21[DE3]pET151FliC producing biologically active recombinant human flagellin obtained by transformation of the cell culture of E. coli BL21[DE3] recombinant plasmid DNA 151FliC obtained on the basis of the vector 151FliC, which was built fliC gene that encodes a biologically active flagellin having the nucleotide sequence represented in Seq ID No 3, deposited in Russian national collection of industrial microorganisms (VKPM) FSUE gosniigenetika no In-11369.
SUBSTANCE: strain Lactobacillus acidophilus No. 9-PS has biochemical activity and high acidity. The strain is deposited in the Departmental collection of beneficial microorganisms for agricultural purposes of Russian Agricultural Academy (RCAM) under the registration number of RCAM01850. The strain may find application in prevention and correction of disorders of microbiocenosis of the gastrointestinal tract.
EFFECT: invention enables to increase growth of mucous cultures of lactic acid bacteria and accelerates the process of colonisation of intestinal with beneficial microflora.
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SUBSTANCE: strain Gluconacetobacter sucrofermentans H - 110 is a producer of bacterial cellulose. The strain is deposited in the Russian National Collection of Industrial Microorganisms under the registration number of strain Gluconacetobacter sucrofermentans RNCIM B-11267 and can be used in medicine, food and pharmaceutical industries.
EFFECT: invention enables to increase the yield of bacterial cellulose.
2 dwg, 6 ex
FIELD: food industry.
SUBSTANCE: inventions group relates to the field of microbiology and may be used in food industry. One proposes Lactobacillus sanfranciscensis DSM 22063 strain and Lactobacillus plantarum DSM 22064 strain; the both strains are able to perform complete gluten decomposition in flour. The strains may be used for production of a mixture for complete gluten decomposition in flour. Additionally, one proposes a method for preparation of dough of flour with completely decomposed gluten. The produced gluten- detoxified flour dough may be used for production of a baking mixture with completely decomposed gluten. The said dough may be used for yeast bakery goods production. Additionally, the baking mixture and gluten- detoxified dough may be used for manufacture of food products suitable for recovery of nutritional imbalance being the consequence of gluten-free food ration.
EFFECT: inventions group allows to manufacture a product with residual gluten concentration lower than 20 ppm.
24 cl, 7 dwg, 2 tbl, 5 ex
SUBSTANCE: bacterial strain Exiguobacterium mexicanum RNCIM B-11011 is proposed, having the ability to dispose quickly of oil, diesel fuel, motor oil, gas condensate.
EFFECT: strain can be used to clean soil and water reservoirs contaminated by crude oil and petroleum products in a wide temperature range.
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SUBSTANCE: strain of bacteria Exguobacterium mexicanum RNCIM V-11011 is grown, and the suspension is made from it, which is applied in the cryomorphic soil and water environment. It is exposed under the specified parameters from 7 to 60 days and the quantitative content of oil and petroleum products in the test soil and water environment is determined.
EFFECT: invention enables to reduce the time of denaturation of oil and petroleum products and to reduce the concentration of oil and petroleum products in the soil and water environment.
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SUBSTANCE: propanediol-oxydoreductase coding gene is introduced into an E.coli cell, which makes it possible to produce high levels of 1,2-propanediol, in fact, without 1,3-propanediol, with growing on glycerol as the only carbon source. Genes of glycerol dehydrogenase (gldA), dihydroxyacetone kinase (dhaK) and/or methylglyoxal synthase (mgsA) can be additionally inserted to express the said enzymes together with propanediol-oxydoreductase (fucO), as well as a gene of glycerol dehydratase or aldo-keto reductase in order to express the said glycerol dehydratase or aldo-keto reductase together with propanediol-oxydoreductase. The E.coli cell, transformed by the propanediol-oxydoreductase coding gene, can be defective in arabinose, methylglyoxal and/or dihydroxyacetonephosphate metabolism.
EFFECT: invention relates to the field of genetic engineering and can be used for recombinant production of 1,2-propanediol (1,2-PD).
9 cl, 4 dwg, 9 tbl, 3 ex
SUBSTANCE: bacterial strain Rhodococcus aetherivorans ofRussian classification of microorganisms BKM Ac-2610D is proposed. The bacterial strain is different in growth on the common organic-mineral medium at high nitrile hydrase activity, reaching 332 U/mg at 20°C or 521 U/mg at 25°C. The nitrile hydrase of the bacterial strain Rhodococcus aetherivorans of Russian classification of microorganisms BKM Ac-2610D is thermostable. Also the method of culturing this strain is provided. The cells of the strain are inoculated on the slant meat-and-peptone agar and grown for 24-48 hours. Then the biomass is washed with sterile saline solution with pH 7.0-7.4. The resulting suspension is inoculated to the first container with the nutrient medium. The process is carried out for 24-48 hours at 28-30°C, with the circumferential stirring at a speed of 140-160 rev/min until obtaining the optical density of the suspension of 2-16 units at 540 nm and the magnitude of the optical layer of 5 mm. The resulting suspension is inoculated to the second container, which volume is 10-100 times greater than that of the first container. The value of optical density in the second container is adjusted to 0.1-0.3. The strain is cultured for 48-120 hours at 26-31°C, the aeration of 0.5-1.0 the air volume/medium volume per minute until the value of optical density reaches 36-40 and pH 7.5-7.8. The resulting biomass is separated. Also the method of production of acrylamide is provided. Hydration of acrylonitrile is carried out with the acrylonitrile concentration not exceeding 0.5%. The hydration is carried out using the biomass of bacterial strain Rhodococcus aetherivorans of Russian classification of microorganisms BKM Ac-2610D based on the order of 400-500 g of dry weight of strain per 1 ton of the final product - acrylamide with the concentration of 45-49%. The inventions enable to obtain cells of Rhodococcus aetherivorans of Russianclassification of microorganisms BKM Ac-2610D of 10-18 g/l with the nitrile hydrase enzyme activity of 250-332 U/mg.
EFFECT: improvement of strain quality.
5 cl, 6 ex, 1 tbl
FIELD: medicine; pharmaceutics.
SUBSTANCE: invention relates to virology and biotechnology. The human immunodeficiency virus strain type 1 IV741 is isolated on the territory of the Russian Federation from a patient who has not received ARV therapy. The strain is deposited in the State Collection of Viruses of the D. I. Ivanovsky Research Institute of Virology of the Ministry of Health and Social Development of Russia under number N1186. The strain belongs to HIV subtype AE. The strain has stable reproductive activity. The infectious titre is 5-6 lg TCD50.
EFFECT: obtained strain is a suitable natural model for studying antiviral (specific) activity of new-generation chemotherapeutic agents and for making vaccines.
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SUBSTANCE: bacteria strain Salmonella enteritidis var. Issatschenko 32/3 deposited in the Departmental Collection of Beneficial Microorganisms for Agricultural Purpose of All-Russia Research Institute for Agricultural Microbiology (GNU VNIISHM Rosselkhozakademii) with registration No. RCAM 00149 has expressed pathogenic properties against mouse-like rodents. Efficiency of bioattractant obtained based on Salmonella enteritidis var. Issatschenko 32/3 bacteria strain and grain in grain and vegetable warehouses and green-houses was more than 90% in relation to sewer rat and house mouse.
EFFECT: improving efficiency.
SUBSTANCE: invention proposes Aeromonas bestiarum bacteria strain - producer of alkaline ribonuclease having antiviral activity and deposited in a collection of bacteria, bacteriophages and fungi of the Federal Budgetary Scientific Institution "The State Scientific Centre of Virology and Biotechnology "Vector" with registration No. B-1270. Strain has high production rate of alkaline ribonuclease - 921.8 U/ml and activity against A/H5N1 bird and A/Aichi/2/68 (H3N2) human being flu viruses.
EFFECT: improving strain properties.
2 dwg, 7 tbl, 8 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.
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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: 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: carrier is proposed for targeted delivery of nucleic acids to cells expressing the receptor CXCR4, which consists of a sequence-ligand to the receptor CXCR4 with the amino acid sequence KPVSLSYRSPSRFFESH, the linker part of two molecules of ε-aminohexanoic acid linking the sequence-ligand to the sequence for compaction of nucleic acids, the sequence providing compaction of nucleic acids and the complex output from endosomes CHRRRRRRHC.
EFFECT: invention can be used for targeted delivery of genetic structures into cells with the receptor CXCR4 on the surfaces, such as malignant tumour and stem cells, in order to correct genetic defects, influence on processes of implementation of the genetic information and prevention of diseases.
3 cl, 7 dwg, 4 ex