Recombinant plasmid dna pci-ub-polyepi containing epitopes of tumour-associated antigens for colorectal cancer and method of its application for stimulation of specific antitumour immune response against colorectal cancer cells
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
The invention relates to biotechnology, protein engineering and medicine, specific to the creation of recombinant plasmids providing expression polyepitopic tumor-associated antigens in dendritic cells, which are able to stimulate specific T-helper and cytotoxic T-lymphocytes.
The tumor may be a wide range of antigens. Some antigens are presented in all tumors of a particular type, and some antigens are unique and can only be detected in a given patient.
There are many ways to include antigens in the antigenic composition of the vaccine.
Proteins or protein fragments of tumor cells directly injected into the body as a vaccine.
- The body is introduced genetic material encoding these proteins (DNA and RNA vaccines).
- As a "means of delivery" of the antigen in the patient's body can be used virus. The viruses used in this way, is called "viral vectors" and do not have any infectious properties. These viruses in the laboratory to infect cells of the human body and become carriers on the surface of tumor antigens. The virus is able to infect only a small number of cells is sufficient to generate an immune response, but insufficient for stabilizati disease.
Using genetic engineering techniques can also be used viruses to produce cytokines or embed the proteins in the surface of the virus, which contributes to the activation of immunocompetent cells. Thus, the modified viruses can be entered into the patient alone or in combination with a vaccine to enhance the generation of an immune response.
Sometimes as antigens in the vaccine used antibodies. The patient is given antibodies to tumor antigens, then B-lymphocytes produce antibodies to these antibodies, which recognize tumor cells. This so-called "antiidiotypic vaccine that is different from passive antibody therapy.
The described method of generating antigen-specific antitumor cytotoxic cells, taken as a prototype, in which JC was obtained from mononuclear cells (MNCs) in the peripheral blood of patients with cancer of the gastrointestinal tract by cultivation OLS enriched fraction of monocytes for 7 days in the presence of IL-4 (1000 u/ml) and GM-CSF (1000 u/ml). Then in culture derived dendritic cells (DC) were added CEA (peptide) at a concentration of 40 μg/ml and 3 μg/ml β2-macroglobulin, were incubated for 4 h at 20°C. Next peptidylglycine DC were irradiated (55 Gy) and were carried in a ratio of 1:20 with autologous cytotoxic lymphocytes. Qi is amoxicot estimated standard method for the release of 51Cr-tag from the dead cell lines of colorectal cancer. The death of tumor cell lines HT29, WiDr, KM12LM) did not exceed 16% (Matsuda K., Tsunoda T., Tanaka N., Umano Y., Tanimura, N., Nukaya I., K. Takesako, Yamaue H. Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignancies following vaccination with CEA peptide-pulsed dendritic cells. // Cancer Immunol. Immunther. - 2004. No. 53. - P.609-616.).
The disadvantage of this approach is the long term cultivation of dendritic cells (7 days), which requires the use of large amounts of recombinant cytokines (GM-CSF, IL-4), and the use of additional proteins (β2-macroglobulin), which leads to the considerable increase of technology. Used allogeneic tumor cells are alien antigens that may shape the response, however, remains not known whether the thus activated cells to exhibit cytotoxic activity against tumor antigens and cells, respectively. Uses a tumor-associated protein CEA, which can to varying degrees be expressed on tumor cells. In addition, the efficiency of cytotoxicity of cells achieved in the prototype does not exceed 16%.
The present invention is the creation of a design polyepitopic of immunogens, including epitopes of tumor-associated antigens for colorectal cancer, and the procedure of its use to stimulate specific antitumor immune response against colorectal cancer cells.
The problem is solved by constructing recombinant plasmid DNA pCI-UB-POLYEPI carrying the gene of artificial polyepitope protein under the control of the CMV promoter, and transfection of it in autologous dendritic cells to ensure the expression polyepitopic tumor-associated antigens and stimulation of specific cytotoxic cells.
The essence of the invention consists in the following. The obtained recombinant plasmid DNA pCI-UB-POLYEPI containing 11 epitopes of tumor-associated antigens in colorectal cancer. This polyepitopes design transferout Mature autologous dendritic cells. Then pCI-UB-POLYEPI-transfected DC were cultured with autologous mononuclear cells of peripheral blood of colorectal cancer patients. Mature DC are obtained by adding to immature DC proinflammatory cytokine TNF-α.
The technical result of the invention is to provide polyepitopes construction, transfection of which DK is achieved efficient stimulation of specific cytotoxic lymphocytes against colorectal cancer cells.
The principal difference of the developed method from the prototype is used for activation of the DC polyepitopes design, including epitopes of tumor-associated antigens for colorectalcancer, and application protocols activation and maturation of DC, which also decreases the stage of getting a Mature antigen-transfected DC to 6 nights.
The present invention improves the efficiency of generation of cytotoxic cells, and also allows you to shorten cocultivation DK and MNCs, which ultimately leads to cost reduction method.
The invention is carried out as follows.
For a better understanding of the essence of the present invention the following are examples of its implementation.
Example 1. The preparation of recombinant plasmids pCI-UB.
Plasmid pCI-UB contains ubiquitin, a small protein conservative affecting the cellular localization of the attached protein. Ubiquitin guides attached to protein in the proteasome. The DNA fragment encoding ubiquitin, obtained by PCR of genomic human DNA. Used oligonucleotides containing the recognition sites endonucleases Xhol and EcoRI. The obtained amplication DNA fragment cloned into the vector pCI-neo. In the clone obtained plasmid pCI-UB, the nucleotide sequence of the obtained constructs were checked by sequencing.
Example 2. Design and obtaining tumor polyepitope.
To construct polyepitope protein analyses of amino acid sequences selected proteins (that is L.1) used computational prediction and analysis of existing experimental data.
For prediction of MHC-I epitopes have been using the program (SYFPEITHI; http://www.syfpeithi.de; ProPredl; ; NetMHC-3.0 ). Selected epitopes having the maximum rating for the alleles HLA-A*02, and have the testimony of experimental validation in the literature (if any). While priority was given to the experimental data. For predicting MHC-II epitopes used the program (EpiTOP; http://www.pharmfac.net/EpiTOP/). Selected epitopes, with a total maximum rating for the alleles DRB1-01, 04, 07, 13, 15.
The design was also introduced helper T-cell epitope of diphtheria-tetanus toxoid (QYIKANSKFIGITEL (TT), 830-844 amino acids tetanus toxoid), not specific amplifying the response of CD8+T-lymphocytes. Early work showed that ARY N-terminal amino acids flanking each epitope significantly increase the affinity to TAP the conveyor, and which encourages a more pronounced response of CD8+T-lymphocytes. These sequences are also used to separate epitopes in polyepitopes design. Each epitope requires proteolysis in the specific website above its N-Terminus for efficient translocation, the excess amino acids can be deleted by aminopeptidase. Thus, in the absence of rational design neighboring epitopes can be broken. Design con which e.g. enables separation of epitopes AAY spacer elements sequence, which is the target for proteasome cleavage. DYKDDDDK introduced in polyepitope design for ease of monitoring the expression of polyepitope protein. This peptide is not masked in different positions with the introduction of recombinant proteins and can be effectively detected by using monoclonal antibodies. In polyepitope design was also introduced CTL-peptide from ovalbumin (SIINFEKL) for monitoring immunization. Design and amino acid sequence developed polyepitope protein is shown in figures 1 and 2, respectively. For artificial gene encoding polyepitopic protein, its amino acid sequence was subjected to reverse-transcribed with regard to the frequency of occurrence of codons in humans, for cloning 5'-end was added to the EcoRI site at the 3'-end was added XbaI site, containing a stop codon in frame. The gene was synthesized by PCR with overlapping oligonucleotides (length of 70-80 N.O.) and cloned in plasmid pMTL22 sites EcoRI and XbaI. The nucleotide sequence of the obtained clone was verified by sequencing Tengeru. As a result, the selected clones with the desired nucleotide sequence. The result obtained plasmid DNA pMTL22-POLYEPI.
MUC4-1-EpCAM-1-EpCAM-2-EpCAM-3-CEA-1-CEA-2-CEA-3-MUC1-TT-OVA-MUC4-2 - EpCAM-4-CEA-4
1. Schematic structure polyepitope the CSOs protein.
2. Amino acid sequence polyepitope protein. Bold tumor-specific epitopes, underlining auxiliary motives for processing.
Example 3. The preparation of recombinant plasmids pCI-UB-POLYEPI Plasmid DNA pCI-UB and pMTL22-POLYEPI was subjected to hydrolysis with restriction endonucleases EcoRI and XbaI in series with intermediate presidenial DNA with isopropanol. Restrictive DNA fragments obtained by the hydrolysis of pMTL22-POLYEPI, were separated in agarose 1% gel using gel electrophoresis. Cut a strip of the gel containing the DNA fragment encoding polyepitope. Was suirable DNA from the gel by adsorption of DNA on the soft-feel. Gidralizovanny DNA pCI-UB was extracted with phenol-chloroform mixture and subsequent presidenial isopropanol. Next was performed ligation of the resulting DNA fragments with subsequent transformation of E. coli cells strain XLBlue ligase mixture. Selected clones containing polyepitope. The nucleotide sequence of the obtained clone was verified by sequencing Tengeru.
Example 4. Obtaining dendritic cells, transfected with the plasmid pCI-UB-POLYEPI
Isolated from the peripheral blood of patients colorectal the cancer adherent fraction of mononuclear cells cultivated at a concentration of 1 million/ml in complete medium containing 10% fetal calf serum, 2 mm L-glutamine, 10 mm HEPES, 5×10-5mm 2-mercaptoethanol, 80 μg/ml gentamicin, 100 μg/ml ampicillin in an atmosphere of 5% CO2at 37°C with the addition of rcgm-CSF (50 ng/ml) and rcil-4 (100 ng/ml). After 96 hours of cultivation to derived immature DC is added to maturation paid rcfn-α at a dose of 25 ng/ml After 24 hours of cultivation was performed procedure magnetic transfection of Mature dendritic cells using reagents firm "Homoki", the research Protocol was based on the proposed manufacturer of the scheme. After 24 hours was carried out by the joint cultivation of transfected dendritic cells and unattached fraction of mononuclear cells at a ratio of 1:10/ 96 hours test conducted on the cytotoxicity of culture of mononuclear cells against autologous tumor cells.
To determine the modulation of the cytotoxic activity of T-lymphocytes using the derived DC were evaluated by the death of tumor cells in the cytotoxic test. For this to mononuclear cells of the patient, who had previously acultural transfected with plasmids DK, were added to autologous tumor cells. Cytotoxicity was assessed by the increase in the content of the intracellular enzyme lactate dehydrogenase in conditioned medium resulting from the loss of tumor cells. Shown significant improvement values cytot is sicnosti, that is an indicator of activation of antitumor cytotoxic cells, necessary for the destruction of tumor cells (figure 1).
Joint cultivation of MNCs with DC transfected with a recombinant polyepitope the plasmid pCI-UB-POLYEPI, is an effective method of generating specific cytotoxic cells mononuclear origin in patients with colorectal cancer that is manifested in the strengthening of their cytotoxic anti-tumour response. Thus, as a result of the research has developed a method of stimulation of antitumor cytotoxic immune response using autologous dendritic cells transfected polyepitopes recombinant plasmid providing the expression polyepitopic tumor-associated antigens for colorectal cancer in dendritic cells and stimulation of specific cytotoxic cells.
1. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumor-associated antigens, colorectal cancer, has a size of 6 355 BP and expresses the following amino acid sequence:
recombinant plasmid pCI-UB-POLYEPI contains the CMV enhancer (5251-5910 P.N.), the CMV promoter (5919-6001),T7 promoter (6317-6336 P.N.), ubiquitin (1-229), also contains a unique restriction sites EcoRI (233), XbaI(891), NotI (908), BamHI (3164), NdeI (5638), SacI (5980), XhoI (6342).
2. The method of generating antigen-specific antitumor cytotoxic cells, which consists in co-cultivation of mononuclear cells (MNCs) in peripheral blood of colorectal cancer patients with dendritic cells (DC), activated by tumor antigens, characterized in that cultivation of use Mature DK, obtained by adding to immature DC proinflammatory cytokine TNF-α, activated by transfection in them recombinant plasmid DNA pCI-UB-POLYEPI claimed in claim 1.
SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.
EFFECT: invention can be used in therapy and diagnosis of diseases associated with AXL.
23 cl, 20 dwg, 24 ex, 3 tbl
SUBSTANCE: invention proposes an antibody that specifically binds heparin-binding EGF-like growth factor (HB-EGF) and its antigen-binding fragment. Invention describes a nucleic acid molecule, an expressing vector, a host cell and a method for obtaining an antibody or its antigen-binding fragment, as well as use of antibody or its antigen-binding fragment for obtaining pharmaceutical composition for diagnostics, prevention or treatment of hyperproliferation disease, methods and sets for diagnostics and prevention or treatment of the state associated with HB-EGF expression. This invention can be further found in therapy of diseases determined with or related to HB-EGF expression.
EFFECT: improving efficiency of composition and treatment method.
34 cl, 43 dwg, 28 ex, 12 tbl
SUBSTANCE: invention describes polynucleotide, expression vector, host cell and production method of humanised antibody together with their use, as well as medical preparation against rheumatoid arthritis, prophylaxis or treatment method of rheumatoid arthritis and use of humanised antibody at production of pharmaceutical preparation for prophylaxis or treatment of rheumatoid arthritis. This invention can be used in therapy of human diseases associated with α9 integrin.
EFFECT: improved activity and thermal stability.
14 cl, 6 dwg, 6 tbl, 11 ex
SUBSTANCE: invention refers to eucariotic vector for expression of target recombinant product in a mammal cell and to its use, to a mammal cell for production of target recombinant product and to a method for its production, a method of a mammal cell selection and a method for obtaining a target recombinant product. Vector includes the first polynucleotide coding a functional folate receptor bound to a membrane as a selective marker and the second polynucleotide coding the target product that is expressed in a recombinant manner. Target product represents a pharmaceutically active, therapeutically active or diagnostic polypeptide. Functional folate receptor bound to the membrane and target product are expressed from the above expression vector. Sampling system is based on introduction of a gene of exogenic functional folate receptor bound to the membrane to a mammal cell.
EFFECT: invention allows effective selection of transformed cells and high yield of target product.
26 cl, 3 tbl, 2 ex
SUBSTANCE: expression vector includes: (a) replication origin OriP obtained from Epstein-Barr virus (EBV), where replication origin contains: 1) symmetry element of the second order (DS); and 2) duplication section (FR) that contains fixation point EBNA; (b) replication origin SV40; (c) insertion section for inserting a gene of concern; (d) promoter EF-1b functionally bound to the insertion section; (e) poly-A signal; (f) bacterial replication origin; (g) selected marker; and unnecessarily containing (h) sequence of nucleic acid, which codes constant area of heavy or light chain of antibody, which is functionally bound to the insertion section. With that, replication origin OriP is bound to an initiation factor of replication EBNA 1, which acts from outside and is not coded with an expression vector.
EFFECT: use of an expression vector in an extracted host cell, a set and a method for obtaining recombinant protein provides production of abundant protein expression.
26 cl, 25 dwg, 3 tbl, 4 ex
SUBSTANCE: invention can be used for obtaining recombinant human blood coagulability factor VIII with deletion of B-domain (hFVIII-BDD). Recombinant plasmid DNA pAP227 coding polypeptide with sequence hFVIII-BDD also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 2H5 producing recombinant hFVIII-BDD with highly stable yield at the level of about 20 IU/ml/24 h. Cultivation of cells-producers is performed in medium DME/F12 containing 2-4% of Fetal Bovine Serum, 1% of dimethylsulphoxide and 50 IU/l of insulin.
EFFECT: improvement of the method.
4 cl, 5 dwg, 9 ex
SUBSTANCE: recombinant plasmid DNA pBK415 coding polypeptide with sequence of tissular activator of human plasminogen, also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 1F8 producing recombinant protein of tissular activator of plasminogen with highly stable yield at the level of up to 190 mg/l. Cultivation of cells-producers is performed under perfusion conditions in presence of a mixture consisting of additive CHO Bioreactor supplement and sodium butyrate or dimethylsulphoxide with further separation of a target product.
EFFECT: improvement of the method.
5 cl, 5 dwg, 3 tbl, 8 ex
SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.
EFFECT: improvement of the method.
4 cl, 5 dwg, 7 ex, 3 tbl
SUBSTANCE: invention proposes an antibody that specifically connects segment M1' IgE and that induces apoptosis in IgE-expressing B-cells and its antigen-binding fragment. Besides, compositions and curing methods of IgE-mediated abnormalitiy, an item, a specific elimination method of IgE-producing B-cells, methods for prophylaxis and reduction of IgE products induced with an allergen, as well as isolated nucleic acid, an expression vector, a host cell and a method for obtaining an antibody as per the invention together with their use are considered.
EFFECT: invention can be further used in therapy of diseases associated with IgE.
46 cl, 19 dwg, 5 tbl, 13 ex
SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.
EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.
20 cl, 7 dwg, 9 ex
SUBSTANCE: strain of diploid cells of lamb synovial membrane is obtained by culture method of growing tissular explants in DMEM nutritional medium with 10% of fetal bovine serum (FBS). Strain of lamb synovial membrane cells is sensitive to lentiviruses of small-size ruminants (goat arthritis-encephalitis virus and visna-maedi virus). Strain of lamb synovial membrane cells is stored in cell culture collection of All-Russian Research Institute of Veterinary Virology and Microbiology under number 64 and is deposited in Specialised collection of transferred somatic cell cultures of agricultural and field animals in All-Russian Research Institute of experimental veterinary named after Ya. R. Kovalenko under number 82.
EFFECT: obtaining strain of diploid cells of lamb synovial membrane Ovis aries, stable by biological characteristics, acceptable for scientific research and diagnostic work, characterised by permissiveness to lentiviruses of small-size ruminants, acceptable for scientific research and development of new antigenic preparations.
SUBSTANCE: method for obtaining hair microfollicle of mammal includes the following stages: de novo papilla and another cell population chosen from the group including fibroblasts, keratinocytes and/or melanocytes are provided, and de novo papilla is incubated together with the other cell population under conditions of unattached culture. Obtaining de novo papillae includes the following: dermal papilla from mammal hair follicle is provided; fibroblasts of dermal hair papilla are extracted from dermal papilla by mechanical fixing of the dermal papilla on the surface of vessel for cell culture, at that basal plate is punched to provide extraction of dermal hair papillae; extracted dermal hair papillae are thickened in monolayer culture without collagen-coated surface, where the dermal hair papillae are subcultivated; thickened dermal hair papillae are concentrated in cell families, which indicate size and form of physiologic dermal papillae, where the dermal hair papillae are differentiated in vessels without culture adhesion under concentration of cells on the vessel surface within range of 1000-100000 of dermal hair papillae per cm2, and de novo papillae are covered with matrix proteins.
EFFECT: inventions allow formation of hair microfollicle capable of beard hair formation.
16 cl, 4 dwg, 5 tbl, 6 ex
SUBSTANCE: invention relates to biotechnology, in particular to obtaining a population of central memory T-lymphocytes, and can be used in medicine. A population of cells is obtained, having a central memory T-lymphocyte (Tcm) phenotype and which do not induce anti third party GVHD, wherein at least 50% of the cells have the CD8+, CD62L+, CD45RA-, CD45 RO+ signature.
EFFECT: invention enables to obtain a population of tolerance inducing cells that are capable of homing to lymph nodes after transplantation.
15 cl, 13 dwg, 9 ex
SUBSTANCE: invention relates to virology, particularly to culturing cells and tissue for producing and studying viruses. The strain of cells of the synovial membrane of a young pig is obtained by cultivating growing tissue explants of the synovial membrane of a young pig in a DMEM culture medium with 10% fetal bovine serum (FBS). The cell culture of the synovial membrane of a young pig is sensitive to viruses of classical swine fever, African swine fever, Aujeszky's disease and can be used to study viruses as well as in diagnostic studies. The strain of the synovial membrane of a young pig is stored in the collection of cell cultures of the National Research Institute for Veterinary Virology and Microbiology of Russia under No.65 and is deposited in the Special Collection of Grafted Somatic Cell Cultures of Agricultural and Industrial Animals RKKK(P) (SKHZH RASKHN) of the Y.R. Kovalenko National Research Institute for Experimental Veterinary (VIEV) under No.81.
EFFECT: obtaining strain of cells of the synovial membrane of a young pig having stable biological properties, which is suitable for virology and diagnostic studies, which is sensitive to causal viruses of swine diseases such as African and classical swine fever and Aujeszky's disease.
SUBSTANCE: invention relates to cellular engineering, biotechnology and food industry. Disclosed is a method of culturing myoblasts in vitro to obtain myoblast biomass, where the cultured cells used are immotalised animal myoblasts; cells are grown using a culture medium with non-animal proteins, and also containing haemoglobin, wherein the cells are kept in a proliferative state using fibroblast and/or hepatocyte growth factors to form biomas, and differentiation of myoblasts into myocytes is then induced by removing fibroblast and/or hepatocyte growth factors from the medium, and in the case of cells with normal myostatin expression, a myostatin inhibitor is added to the medium.
EFFECT: method can be used in food industry to produce safe meat food products in large amounts over a short period of time.
6 cl, 3 tbl, 9 ex
SUBSTANCE: there described is a method for obtaining resident stem cells of mammal heart expressing surface markers c-kit or sca-I and/or MDR1, in the course of which the samples of myocard tissues are separated, ground, treated by collagenase and trypsin, cultivated at cultural bowl with coating from fibronectin by method of explant culture of ground samples with the following immunoselection.
EFFECT: invention allows obtaining enriched culture of resident stem cells of heart for the purposes of medicine and transplantology.
8 cl, 3 dwg, 4 ex
SUBSTANCE: method for preparing a corneal biotransplant involving the application on a matrix representing a polymer film of non-modified nanostructured hyaluronic acid, corneal cells represented by an autogenous mixed cell culture containing epithelial corneal progenitors and corneal fibroblasts taken in specific proportions and culture on a medium containing the cultural concentration of the antibiotics.
EFFECT: using the method described above leads to reducing the length of culture up to 9-12 days and maintaining the additional prolonged maintenance of biotransplant cell migration and proliferation.
2 dwg, 1 tbl
SUBSTANCE: what is presented is medium and method for the vital immobilisation of human and animal sperm cells, wherein the sperm cells of native or thawed sperm prepared by the ejaculate centrifugation in a density gradient, or sampled by the flotation; the sperm cells are transferred into an immobilisation medium with low osmotic pressure 100-170 mOsm/kg for 1 min to 2 hours with the gas mixture composition containing 0.1 to 8% of carbon dioxide, and a medium temperature of 0 to 39°C.
EFFECT: invention may be used for diagnostic and therapeutic purposes for animal reproduction and fertilisation.
7 cl, 1 tbl
SUBSTANCE: invention provides a method for producing a heterogenous skin cell population implying the sampling of a skin biopsy material at a depth of 2 mm, tissue homogenisation in 0.9% aqueous sodium chloride at temperature +23 … +25°C, recovery of homogenate, filtration of the homogenate through an inert filter cloth with the pore diameter of 20 mcm, centrifugation of the homogenate at 400 g for 5 minutes at temperature +23 … +25 C.
EFFECT: skin cell separation and viability preservation.
2 cl, 1 dwg
SUBSTANCE: group of inventions refers to biotechnology and deals with new nucleotide sequences of Torque teno virus (TTV) and vectors containing such sequences. Extracted polynucleotide molecule contains polynucleotide sequence chosen from the group consisting of SEQ ID NO:4, sequence complementary to SEQ ID NO:4 and polynucleotide that is at least 95% identical to SEQ ID NO:4.
EFFECT: inventions can be use for production of vaccines to prevent diseases of pigs and other animals, which are caused by Torque teno virus.
4 cl, 7 dwg, 3 tbl, 10 ex
SUBSTANCE: created is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype containing expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain haemagglutinin gene with pre-optimised for expression in human being cells nucleotide sequence was used providing overexpression of haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain. Haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain with optimised nucleotide sequence was cloned in expressing cassette under promoter control and contains polyadenylation signal. Promoter is cytomegalovirus promoter, and polyadenylation signal is SV40. Expressing cassette is located in zone of E1 deletion of human being adenovirus genome of the 5-th serotype. Also method of use of recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype for induction of specific immunity to influenza virus B.
EFFECT: possibility of use in pharmaceutical industry for production of vaccine preparations.
6 cl, 9 dwg, 1 tbl, 4 ex