Synthetic oligonucleotide primers and method for thereof for indicating immunodeficiency viruses and leukaemia of cats in clinical material by multiplex polymerase chain reaction
SUBSTANCE: invention relates to biotechnology and a set of synthetic oligonucleotide primers and method for use thereof. Proposed set comprises two pairs of primers having following structure - FIV F: 5′-AAGAGTCCCAAATATGCCATAGG-3′ and FIV R: 5′-TCCATCCAAATTGCTACTGTTC-3′; FeLV F: 5′-GAATAAACCTCTTGCTGTTTGC-3′ and FeLV R: 5′-AATCAGATCGAATGACAGAGACAC-3′. Presented primers do not contain palindromic repetitions of nucleotides, do not form expressed secondary structure and do not have extended G-C portions, and temperature of annealing is 55 °C for both pairs of oligonucleotides. Described method involves preparation of reaction mixture by mixing buffer of 10-fold for PCR - 2.5 mcl, a dNTP (25 mM) - 0.4 mcl, primers FIV F, FIV R, FeLV F and FeLV R (15 pmol/mcl) in 1 mcl, Taq polymerase (5 units/mcl) - 0.2 mcl MgCl2 (50 mM) - 1.5 mcl bidistilled water - 11.4 mcl and test DNA 5 mcl, amplification is carried out in following mode: total denaturation 95 °C - 3 minutes, cycle denaturation (95°C - 20 or 60 s) - annealing (55 °C - 20 or 60 s) - elongation (72 °C - 40 or 60 s), cycle of denaturation-annealing-elongation is repeated 35 times, final elongation 72 °C - 5 minutes, storing 4 °C - 10 minutes, and detection of obtained results is carried out by 1.5 % agarose gel. Presence or absence of amplified fragments of nucleotide sequences length 397 base pairs for FIV (feline immunodeficiency virus) and 221 base pairs for FeLV (feline leukemia virus) indicates presence or absence of viruses in organism cat.
EFFECT: invention can be used in scientific research for detecting a genetic material of immunodeficiency viruses - FIV and leukemia FeLV cats by multiplex polymerase chain reaction (PCR) in animal blood.
2 cl, 3 dwg, 3 tbl
SUBSTANCE: method includes treating a sample with a denaturing buffer solution, sorbing short RNA on a fibre glass sorbent in the presence of a chaotropic agent, followed by washing off non-bonded biopolymers and chemical reagents from the sorbent and eluting the end product. The biological fluid sample undergoes two-step denaturing, wherein at the first step two volumes of the denaturing buffer solution are added to the sample and at the second step two volumes of 96% ethanol and equal volume of chloroform are added to the obtained mixture, followed by stirring and separating the residue by centrifuging. Non-bonded biopolymers are washed off from the sorbent twice with the buffer solution and elution of the end product from the sorbent is carried out with the buffer solution.
EFFECT: simple method, short duration of the method and high output of short RNA.
3 cl, 3 tbl, 4 ex
SUBSTANCE: claimed invention relates to the field of biotechnology and deals with a method of detecting a provirus of cattle leukosis. The characterised method includes the detection of LTR (Long Terminal Repeat) fragment - a sequence of the leukosis provirus. Determination of the size of an amplified fragment of a nucleotide sequence by the electrophoretic separation in an agarous gel is performed. As primers applied are oligonucleotides: BL1.F 5-GAGTTAGCGGCACCAGAAGC-3 and BL1.R 5-ATAGAGCTCGCGGTGGTCTC-3. The product of synthesis constitutes 175 pairs of the nucleotides. A genomic DNA is extracted by a sorbent method with the following elution in TE buffer, amplification is carried out in the mode: 95°C for 3 minutes 1 time, 94°C for 20 seconds - denaturation, 66°C for 20 seconds - annealing of the primers, 74°C for 25 seconds - elongation, 45 times, 74°C for 3 minutes - chains completion. As a positive control of reaction proceeding applied is a preparation of a positive control of BLV antigen.
EFFECT: invention can be applied in the molecular-genetic diagnostics of animal diseases and scientific research in veterinary.
2 dwg, 1 tbl
SUBSTANCE: invention relates to biochemistry. Described is method of detecting presence or absence of several serotypes of human papillomavirus (HPV) in biological sample by means of multi-channel analysis system, where said analysis system detects larger quantity of serotypes than the number of existing channels of detection. Method peculiarity consists in the fact that first and second sets of primers and probes, which are degenerate with respect to each other, are used; as well as the fact that each degenerate probe has signal grouping, each of which studies signal, detected on one and the same channel. Third set of primers and probes is not degenerate with respect to two other sets of primers and probes and is distinctive for third serotype, which does not belong to serotypes, to which mainly annealing of degenerate sets of primers and probes takes place, and where third set of primers and probes has signal grouping, which emits signal with wavelength, which is the same or differs from wavelength, emitted by signal grouping of degenerate probes. Respective primers and probes are presented.
EFFECT: invention makes it possible to detect larger quantity of HPV types than the number of existing channels of detection in analysis system.
11 cl, 3 dwg, 6 tbl
SUBSTANCE: inventions relate to the field of DNA-genealogy and deal with a method of determining haplogroups of human Y-chromosomes, a test-system and oligonucleotide primers. The characterised method is realised in two stages. At the first stage genetic typing is carried out by basic haplogroups R,N,J,I,Q,C,E,D,G,O of an Y-chromosome tree by multiplex PCR with the application of specific oligonucleotide primers of the first and second sets of the test-system. At the second stage, if mutation is detected, additional multiplex PCR for typing by subhaplogroups is carried out. If at the first stage mutation is not detected, multiplex PCR for typing by rare haplogroups A,C3,F,H,K,L,O3 and T is carried out. The area of annealing primers outside mutation zones within the limits of species specificity corresponds to sequences from the first set of the test-system. The area of annealing of the oligonucleotide primers, directly adjoining the mutation zone within the limits of species specificity corresponds to sequences from the second set of the test-system.
EFFECT: inventions can be applied for the determination of haplogroups of the human Y-chromosome.
3 cl, 2 dwg, 7 tbl, 2 ex
SUBSTANCE: DNA is recovered from peripheral venous blood which is followed by a genetic typing of the APOE gene and detecting polymorphous alleles APOE*2, APOE*3, APOE*4; if the assays shows any genetic types containing alleles APOE*2, a high risk of endometrial cancer is predicted.
EFFECT: invention provides a highly specific criterion for predicting the risk of hyperproliferative diseases of the endometrium, including endometrioid adenocarcinoma in females with hyperplastic processes in the endometrium.
2 dwg, 10 tbl, 4 ex
SUBSTANCE: invention refers to medicine, namely to a method for the prediction of developing occupational hyperkeratosis. Substance of the method consists in recovering DNA from peripheral venous blood lymphocytes, genotyping TP53 gene rs1625895 polymorphism by polymerase chain reaction with restriction enzyme digest analysis. Finding G/A genotype and A allele ensured predicting a risk of developing occupational hyperkeratosis.
EFFECT: using the invention enables predicting developing occupational hyperkeratosis after the body has been exposed to occupational hazards.
2 tbl, 2 ex
SUBSTANCE: presented group of inventions refers to medicine, molecular biology and biotechnology. A method for determining lung cancer cell sensitivity to cisplatin involving determining MLH1, ERCC1, DDB2, AKR1B1, FTL genes expression patterns vs. cisplatin IC50 for known cell lines; constructing cisplatin IC50 for these cell lines vs. derived gene expression patterns calibration straight, and hybridising on a microchip. What is also presented is a set of oligonucleotide probes presented by SEQ ID NO: 9-13.
EFFECT: presented group of inventions provides effective aids and methods for determining lung cancer cell sensitivity to cisplatin.
2 cl, 1 dwg, 2 tbl, 3 ex
SUBSTANCE: invention relates to field of biochemistry, in particular to method of identifying changes in gene expression, characteristic of ageing, in selected tissue. Selected tissue represents cardiac, muscular, cerebral or adipose tissue. Method includes selection of one or several genes, differentially expressed in tissue of old subjects in comparison with young subjects, with application of two criteria. Said criteria are: change of expression in at least 50% of lines, breeds or ethnic groups of tested species at preliminarily determined significance level p<0.10, as well as at least partial reversion of changes in gene expression by restriction in caloric content.
EFFECT: invention provides obtaining reliable tissue-specific biomarkers of ageing.
7 cl, 11 tbl, 5 ex
SUBSTANCE: invention refers to molecular biology and can be used in diagnosing cardiomyopathies of various origin Presented is a set of synthetic oligonucleotides for identifying mutations of a coding part of desmin (DES) gene associated with cardiomyopathies. The mutations are identified by detecting a full sequence of the coding part of DES gene. The coding part of DES gene is amplified by means of 7 pairs of synthetic oligonucleotides at the same temperature and annealing time, and the prepared amplification products are sequenced by means of one pair of universal primers.
EFFECT: presented invention enables the sensitive and specific detection of DES gene mutations, with reducing the amplification reaction time, the number of manipulations, the time of agent application for the sequencing reaction, and reducing a probability of a reaction error.
3 cl, 1 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, more specifically to AXL signalling pathway inhibitors, and can be used in medicine. What is produced is a soluble AXL polypeptide version free from AXL transmembrane domain, which contains at least one amino acid modification in position No. n, wherein n is specified in 32, 72, 87, 92 or 127 or a combination thereof, wherein n+7 is described by numbering SEQ ID NO: 1 that are wild-type AXL sequences, wherein the above modification increases a AXL polypeptide binding affinity to protein 6 specifically inhibiting the growth (GAS6), which is twice as strong as the wild-type AXL polypeptide affinity. The polypeptide can be fused with Fc fragment and used in a method of treating, reducing or preventing tumour dissemination and invasion in a mammalian patient.
EFFECT: invention enables inhibiting the AXL/GAS6 signalling pathways effectively.
8 cl, 15 dwg, 6 tbl, 3 ex
SUBSTANCE: present invention relates to biotechnology, particularly to genetic engineering and can be used in the biomedical industry. Proposed is a recombinant plasmid pBMC-RT(A)-hum, meant for expressing reverse transcriptase of the human immunodeficiency virus, and is distinguished by that, it contains an artificial gene (RT(A)-hum) synthesised by an enzyme, which is characterised by a sequence of nucleotides, optimally adapted to expression in cells of mammals.
EFFECT: use of the recombinant plasmid provides for significant increase (6-8 times) in output of the target protein compared with the primary standard.
4 dwg, 1 tbl, 3 ex
FIELD: gene engineering.
SUBSTANCE: vector of present invention includes expression cassette containing transgene located under promoter control and central polypurine tract (cPPT) located upstream from cassette. Said vector provides delivery of desired transgenes into target cells and high level expression thereof in such cells. Also disclosed are host cell transduced with lentiviral vector, methods for transduction and uses thereof.
EFFECT: new agent for gene therapy.
26 dwg, 2 tbl, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to the field of biotechnology, namely, to obtaining inhibitors of adhesion and/or aggregation of platelets, and can be used in medicine. A polypeptide, used as a component of a pharmaceutical composition and in sets for screening of the inhibitors of platelet adhesion or aggregation, is obtained in a recombinant way with the application of a matrix of the salivary gland cDNA of Anopheles stephensi.
EFFECT: invention makes it possible to obtain the polypeptide, possessing inhibiting activity with respect to platelet aggregation and/or inhibiting activity with respect to platelet adhesion.
10 cl, 4 dwg, 5 ex
SUBSTANCE: expression vector is also provided, comprising the polynucleotide and a transformant obtained from S. cerevisiae, for production of the said protein, comprising the polynucleotide or vector.
EFFECT: efficient production of fatty acids with long-chain, having 18 carbon atoms.
9 cl, 3 dwg, 3 tbl
SUBSTANCE: invention relates to biotechnology and genetic engineering. Claimed is expression plasmid vector for monocistronic heterologous expression of recombinant proteins in cells of Chinese hamster ovary (CHO), possessing property of high-frequency integration of expression cassette in said cells, in the following sequence containing region of initiation of plasmid pUC replication with functional gene of ampicilline resistance; site of Epstein-Barr virus terminal repetition (EBVTR), functional promoter of elongation factor 1 alpha gene of Chinese hamster, flanked with 5' untranslated region of said gene; site for cloning open reading frame of recombinant protein (polylinker); functional terminator of elongation factor 1 alpha gene of Chinese hamster, flanked with 3' untranslated region of said gene, promoter, functioning in mammalian cells; gene of antibiotic resistance; and functional terminator of antibiotic resistance gene. Also claimed is line of cells - recombinant protein producers and method of obtaining recombinant protein with application of said cells.
EFFECT: invention makes it possible to increase stability and productivity of expression systems of recombinant proteins.
13 cl, 7 dwg, 6 tbl, 8 ex
SUBSTANCE: invention refers to biotechnology. What is presented is nucleic acid coding protein possessing acetyl-CoA-carboxylase activity making up the deficiency of acetyl-CoA-carboxylase in yeast, wherein a nucleotide sequence is specified in nucleic acid, which contains a nucleotide sequence: (a) coding protein consisting of the amino acid sequence SEQ ID NO:2; (b) hybridised in the hard conditions with nucleic acid complementary to SEQ ID NO:1; (c) SEQ ID NO:1; and (d) hybridised in the hard conditions with nucleic acid consisting of the complementary nucleic sequence coding protein SEQ ID NO:2; wherein SEQ ID NO:1 and 2 are disclosed in the description. There are also described: acetyl-CoA-carboxylase (SEQ ID NO:2) increasing the host-specific arachidonic acid content; a recombinant vector containing the above nucleic acid; and a cell transformed by the above vector for producing the fatty acid composition rich in arachidonic acid. What is presented is a method for producing the fatty acid composition involving culturing the above cell and collecting the fatty acid composition from the transformed cell culture.
EFFECT: invention enables producing the fatty acid composition rich in arachidonic acid in the host cell.
11 cl, 8 dwg, 5 tbl, 8 ex
SUBSTANCE: claimed inventions deal with a modified protein, nucleic acid, coding such protein, a vector, containing nucleic acid, and a carrier for biotin binding, which such protein is immobilised on. The characterised modified biotin-binding protein is obtained by the introduction of a mutation from one to several amino acid residues into a sequence, represented in SEQ ID NO:2, or an amino acid sequence, identical to the said sequence by 98% or more, and the presence of the biotin-binding activity, where at least one residue, selected from the group, consisting of residues from 1) to 4), presented below, is substituted with the residue of acidic amino acid or residue of neutral amino acid; 1) residue of arginine in position 104 SEQ ID NO: 2; 2) residue of lysine in position 141 SEQ ID NO: 2; 3) residue of lysine in position 26 SEQ ID NO: 2 and 4) residue of lysine in position 73 SEQ ID NO: 2.
EFFECT: claimed inventions make it possible to obtain the biotin-binding protein and can be applied for biotin binding.
14 cl, 6 dwg, 11 tbl, 3 ex
SUBSTANCE: invention relates to biotechnology and gene engineering. A method for selecting at least one transfected eukaryotic host cell expressing a target product, the eukaryotic host cells comprise at least an introduced polynucleotide encoding the target product, an introduced polynucleotide encoding a DHFR enzyme using at least one expression vector, providing a plurality of eukaryotic host cells, whose viability is dependent upon folate uptake, wherein the said host cells comprise at least a foreign polynucleotide encoding the target product, a foreign polynucleotide encoding a DHFR enzyme, culturing the said plurality of the eukaryotic host cells in a selective culture medium comprising folic acid in a concentration of 12.5-50 nM combined with a concentration of MTX of 2.3-500 nM, selecting at least one eukaryotic host cell expressing the target product.
EFFECT: described is a method of the target product and culture medium preparation.
11 cl, 2 tbl, 2 ex
SUBSTANCE: invention relates to field of medicine, genetic engineering and biotechnology. Claimed is method of providing tumour-free tissue-substituting therapy on the basis of obtained from adult somatic cells induced pluripotent stem cells (iPSC), where the latter are genetically modified by means of artificial chromosome (AC), carrying bicistronic cassette with suicide-gene and gene of sensitivity to antibiotic under control of regulatory element, specific for pluripotent stem cells, with modified iPSC being selected in presence of respective antibiotic, absence of AC integration into iPSC genome is confirmed, are introduced into recipient organism directly, without preliminarily differentiation in vitro, after 1-14 days patients are given a 5-10-day course of therapy with inductor of toxicity of suicide-gene product.
EFFECT: invention makes it possible to reduce to zero risk of cancer transformation of transplanted cells and development of teratomas, increase clinical efficiency of recovery of cell mass of injured organ or tissue, reduce waiting time for potential recipients.
6 cl, 1 tbl, 1 dwg
SUBSTANCE: synthetic DNA is proposed, encoding human erythropoietin, having the sequence Seq ID No. 1, comprising its expression vector, the method of production of erythropoietin producer strain, and a strain of a Chinese hamster ovary cells - producer of recombinant human erythropoietin, deposited under the number RKKK(P) 761 D.
EFFECT: invention enables to increase the expression level of recombinant human erythropoietin.
5 cl, 1 tbl, 8 dwg, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to molecular biology and medicine. There are presented ribonucleic acids of general formula (NuGlXmGnNv)a and derivatives thereof as an immunostimulating agent, compositions containing them.
EFFECT: treating cancer diseases, infectious diseases, allergic and autoimmune diseases.
21 cl, 2 dwg, 5 tbl