Recombinant plasmid pet-15b_t3_rl dna providing synthesis of recombinant fusion protein consisting of tumor-specific peptides and antitumor peptide rl2, and recombinant fusion protein possessing anti-tumor activity against human breast cancer

FIELD: biotechnology.

SUBSTANCE: pET-15b_T3_RL plasmid DNA is constructed which provides synthesis of recombinant fusion protein consisting of a tumor-specific peptide and an antitumor peptide RL2. The recombinant fusion protein isolated from Escherichia coli cells transformed with pET-15b_T3_RL plasmid, has a molecular weight of 15.9 kDa, consists of methionine residue, tumor specific peptide YTYDPWLIFPAN, glycine GGGS spacer, and a recombinant lactaptin RL2 analogue.

EFFECT: production of a protein providing cytotoxic activity against cancer cells in culture and targeted properties for tumor tissue.

2 cl, 6 dwg, 5 ex

 



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.

EFFECT: invention makes it possible to increase efficiency of delivery of molecules of nucleic acids into mammalian cell due to peptide, capable of internalisation into mammalian cell with efficiency, constituting at least 200% of efficiency of internalisation of peptide TAT, which has amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

8 cl, 16 dwg, 1 tbl, 8 ex

FIELD: biotechnology.

SUBSTANCE: method comprises immunoaffinity chromatography using mini-antibodies a-hLF-1 and a-hLF-4, which amino acid sequences are presented as SEQ ID NO:1 and SEQ ID NO:2. The invention may be used to obtain highly purified fraction of human lactoferrin.

EFFECT: invention enables to carry out with high efficiency the separation of proteins of lactoferrin of human and goat consisting in milk, using the single-domain mini-antibodies.

6 dwg, 2 ex

FIELD: chemistry.

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

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for recombinant production of human tissue factor (hTF). Constructed is a plasmid pHYP-10ETFCS6 having a length of 5,912 b.p. with a physical map presented on Fig. 2, for expression in a bacterium of the genus Escherichia, which is a precursor of the mutant [C245S] hTF containing an inseparable N-terminal leader peptide containing a deca-histidine cluster and an enterokinase identification sequence fused in a frame with a sequence coding the above mutein fused in the frame with the sequence coding the additional inseparable C-terminal peptide containing the deca-histidine cluster. A method for producing the precursor of the mutein[C245S]hTF contains culturing the producing bacterium in a nutrient medium, recovering inclusion bodies, solubilising the precursor protein, performing a metal chelator chromatography in the denaturation environment, re-folding and diafiltration of the protein solution. A method for producing the mature mutein[C245S] hTF involves detecting the N-terminal leader peptide from the above mutein precursor with using enterokinase and recovering the target protein.

EFFECT: invention enables increasing the level of biosynthesis and yield of pro-coagulation active hTF.

9 cl, 5 dwg, 1 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to versions of a method for microbiological synthesis of hybrid protein E7-HSP70. Synthesis of protein E7(6)-HSP70, E7(11)-HSP70, E7(16)-HSP70 or E7(18)-HSP70 is carried out by cultivating the respective yeast strain Saccharomyces cerevisiae VKPM Y-3919, yeast strain Saccharomyces cerevisiae VKPM Y-3853, yeast strain Saccharomyces cerevisiae VKPM Y-4057 or yeast strain Saccharomyces cerevisiae VKPM Y-4058 in suitable conditions in a medium containing saccharose as a carbon source. The cultivation process is carried out in two phases. The first phase is carried out at 25-26°C and initial saccharose concentration of 25-30 g/l. Upon achieving saccharose concentration in the medium of 15 mg/l, the second phase of the cultivation process is carried out, where temperature is lowered and kept not higher than 23°C, pH is kept at 5.7-5.9, and saccharose concentration is maintained via exponential replenishment at 15 mg/l.

EFFECT: group of inventions enables to obtain the end product in amount of not less than 550 mg/l and enables to carry out the cultivation process for not more than 65 hours.

4 cl, 12 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a constructed plasmid for expression in a cell of a Chinese hamster, in the following sequence, which mainly contains the following elements: pUC plasmid replication beginning region; an open reading frame (ORF) of beta-lactamase providing immunity to ampicillin; procaryotic gene promoter bla; a section of terminal repetition of Epstein-Barr virus of a human being; a functional gene promoter of elongation factor 1 alpha of the Chinese hamster, 5' non-translated region of this gene and a non-transcribed region flanking this gene, coding Kozak sequence for cap-dependent initiation of translation; ORF of the gene of subunit 1 of complex of 2,3-epoxyreductase of vitamin K (VKORC1) of the Chinese hamster with stop codon; a functional terminator and a signal of polyadenilation of the gene of elongation factor 1 alpha of the Chinese hamster, 3' non-translated region of this gene and a non-transcribed region flanking this gene; a promoter of early genes of virus SV40; gene of immunity to a selective agent; and a polyadenilation signal and terminator of virus SV40. Cells of the Chinese hamster - producer of protein with Gla-domain are transformed by the obtained plasmid, which are used in a method of recombinant obtainment of proteins with Gla-domain.

EFFECT: invention allows increasing productivity of the above cells owing to increasing activity of native VKORC1.

12 cl, 12 dwg, 2 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology, namely to novel analogues of insulin and can be used in medicine. The said insulin analogue is characterised by one of the following structures: Arg(A0), His(A8), Gly(A21), Arg(B31), Arg(B32)-NH2-insulin; His(A8), Gly(A21), Arg(B31), Arg(B32)-NH2-insulin; Arg(A0), Glu(A15), His(A8), Gly(A21), Arg(B31), Arg(B32)-NH2-insulin. The invention also relates to a method of obtaining the said analogue, a pharmaceutical composition and a medication, including its application for treatment of diabetes mellitus.

EFFECT: invention makes it possible to obtain the insulin analogue, characterized by the delayed release and steady and longer action, for instance, in comparison with human insulin or glargine insulin.

18 cl, 2 dwg, 24 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, namely to leukolectins, and can be used in medicine. What is prepared is the polypeptide leukolectin characterised by SEQ ID NO:1-8. The recombinant preparation is ensured by using a nucleic acid coding it and integrated into an expression vector which is used to transform a host cell. Testing absence-presence or determining an amount of the polypeptide leukolectin are ensured by using an antibody or an antigen-binding fragment of a variable region of the above antibody which is specifically bound to the polypeptide leukolectin. The polypeptide leukolectin or the nucleic acid coding it are used as ingredients of a pharmaceutical composition in therapy of pathological disorders of skin and mucous membranes.

EFFECT: invention enables treating or preventing autoimmune disorders of skin, inflammatory diseases of skin or mucous membrane, or injured skin in an animal effectively.

16 cl, 19 dwg, 3 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to modified von Willebrand factor (VWF), and can be used in medicine. A recombinant method is used to preparing modified VWF fused in C-terminal of its primary translation product with N-terminal of albumin by the linker SSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS. The prepared modified VWF is used as a part of the pharmaceutical composition for treating or preventing coagulation failure.

EFFECT: invention enables preparing the modified VWF which maintains its ability to N-terminal dimerisation and C-terminal multimerisation with a prolonged half-period of functional blood plasma occurrence as compared to the half-period of functional VWF occurrence.

17 cl, 5 dwg, 4 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biochemistry. Disclosed is L-fucose α1→6 specific lectin, which is extracted from a basidiomycete or an ascomycete or an ascomycete, characterised by peak molecular weight of about 4500 m/z, determined via MALDI-TOF mass spectrometry analysis. The novel L-fucose α1→6 specific lectin has high affinity for a L-fucose α1→6 sugar chain, represented by an association constant of 1.0×104 M-1 or higher (at 25°C), and has an association constant of 1.0×103 M-1 or lower (at 25°C) with high-mannose sugar chains and/or glucolipids which do not contain an L-fucose α1→6 sugar chain. In one version, the disclosed L-fucose α1→6 specific lectin is a protein or a peptide which consists of an amino acid sequence selected from SEQ ID NO:2-6. The L-fucose α1→6 specific lectin is used for specific detection of a L-fucose α1→6 sugar chain and effective purification of the L-fucose α1→6 sugar chain or a sugar chain which does not contain L-fucose α1→6.

EFFECT: obtaining L-fucose α1→6 specific lectin.

16 cl, 38 dwg, 8 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology and can be applied for the selection of affine molecules. Constructed is a plasmid DNA pGST/APT/X 6193 bp long, with the weight of 4.09 MDa, containing as a genetic marker the gene β-lactase and unique sites of the recognition of restriction endonucleases, located at the following distance to the right from the site NdeI: BgIII 665bp, BamHI 779 bp., XhoI 801 bp. The plasmid pGST/APT/X consists of a NdeI-XhoI DNA fragment of the commercial plasmid pET22b(+) and nucleotide sequence SEQ ID NO:1, inserted by sites NdeI-XhoI and combining in its composition the sequences, which code a fragment of a gene of glutathione-S-transferase, a peptide substrate of a lethal factor of anthrax RRKKVYPYPME, peptide GGLNDIFEAQKIEWHED, biotinylated in vivo under the influence of E.coli biotin-ligase, and a linker sequence, substituted by genetic-engineering manipulations by sites of restriction endonucleases BamHI and XhoI for a sequence, coding a target protein B-subunit of shiga-toxin I.

EFFECT: invention makes it possible to carry out the selection of highly specific aptamers to the said target protein.

2 cl, 4 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, the modified polypeptide having homoserine-O-acetyltransferase activity having the amino acid sequence of SEQ ID NO:17 or at least 95% homologous thereto, in which the amino acid at position 111 from the start amino acid methionine, of the sequence is substituted with glutamic acid. The invention also relates to a method for producing O-acetyl homoserine, comprising culturing the microorganism belonging to the genus Escherichia, transformed with polynucleotide, encoding the said polypeptide.

EFFECT: high production yield of O-acetyl homoserine.

18 cl, 5 dwg, 3 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions concerns recombinant DNA pA3, recombinant plasmid DNA pQE 30-pA3, strain E.coli M 15-A3, recombinant polypeptide A3, test systems for the qualitative detection and quantitative determination of microalbuminuria. The characterised recombinant DNA pA3 is produced by a polymerase chain reaction with the use of chromosomal DNA of the strain DG 13 of group B streptococci and structured primers. An amplification fragment has been cloned by a system of expression vectors pQE-pQE 30 in E.coli M 15 to produce recombinant DNA pQE 30-pA3, which codes an amino acid sequence of the recombinant polypeptide A3 having an ability to bind human serum albumin (HSA) selectively.

EFFECT: presented inventions can be used in the medical practice and industry for producing the test systems for the detection and quantitative determination of microalbuminuria - one of the earliest signs of renal irritation in the patients suffering from diabetes mellitus and essential arterial hypertension.

6 cl, 9 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for recombinant production of human tissue factor (hTF). Constructed is a plasmid pHYP-10ETFCS6 having a length of 5,912 b.p. with a physical map presented on Fig. 2, for expression in a bacterium of the genus Escherichia, which is a precursor of the mutant [C245S] hTF containing an inseparable N-terminal leader peptide containing a deca-histidine cluster and an enterokinase identification sequence fused in a frame with a sequence coding the above mutein fused in the frame with the sequence coding the additional inseparable C-terminal peptide containing the deca-histidine cluster. A method for producing the precursor of the mutein[C245S]hTF contains culturing the producing bacterium in a nutrient medium, recovering inclusion bodies, solubilising the precursor protein, performing a metal chelator chromatography in the denaturation environment, re-folding and diafiltration of the protein solution. A method for producing the mature mutein[C245S] hTF involves detecting the N-terminal leader peptide from the above mutein precursor with using enterokinase and recovering the target protein.

EFFECT: invention enables increasing the level of biosynthesis and yield of pro-coagulation active hTF.

9 cl, 5 dwg, 1 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: claimed inventions deal with an isolated polynucleotide, coding a polypeptide, involved in biosynthesis of pyripyropene A, a vector and a host cell, including such a polypeptide, and methods of obtaining pyripyropene A precursors, including the host cell cultivation. The claimed polynucleotide codes the polypeptide, possessing any one or more of the activities - polyketide synthase, prenyltransferase, hydroxylase, acetyltransferase or adenylate synthase.

EFFECT: claimed inventions make it possible to synthesise pyripyropene A, which is an insecticidal agent, and can be used in the formation of plant resistance to pest insects.

16 cl, 11 dwg, 1 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, in particular to obtaining recombinant enzyme-labelled antigen G2 of hantavirus Dobrava. Essence of invention consists in the following: claimed method of obtaining antigen G2 of hantavirus Dobrava consists in expression of antigen in cells of E.coli in form of enzyme-labelled antigen of G2 hantaviruses based on HT protein antigen. Beta-galactosidase, which is highly active stable enzyme, serves enzyme label for protein antigen. Invention can be used to increase specificity and reproducibility of immunosorbent assay in HFRS diagnostics.

EFFECT: presence of commercially available chromogenic substrate of beta-galactosidase (X-gal) makes it possible to quickly estimate result of immunosorbent reaction visually or by change of optic density of solution in visible area.

6 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry and represents a new bifunctional PSH protein containing human peroxiredoxin Prx6 and manganese superoxide dismutase MnSOD possessing the antioxidant activity of superoxide dismutase and peroxidase. What is also described is a chimeric nucleic acid coding the presented protein. A method for preparing the presented protein by culturing cells of the strain E.coli BL21(PSH) transformed by constructed recombinant expression vector based on pET22b(+) plasmid is disclosed.

EFFECT: invention enables producing high-yield protein PSH possessing the high antioxidant activity.

4 cl, 6 dwg, 3 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to recombinant obtaining G-CSF, and can be used for production of G-CSF in cells of E.coli. For effective production of protein in cells of E.coli G-CSF-coding DNA sequence is optimised. On the basis of obtained optimised DNA sequence plasmid pAS017, also including NdeI/BamHI-fragment of DNA of pETM-50 vector and having physical map, presented on the drawing, is constructed.

EFFECT: invention provides effective production of protein in cells of Ecoli.

2 cl, 1 dwg, 1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to genetically engineered production of human proteins, and may be used for preparing human epidermal growth factor (hEGF) in bacterial cells in the form of glutathione-3-transferase fusion protein. What is constructed is the recombinant DNA coding GST-hEGF fusion protein which consists of an amino acid sequence of glutathione-S-transferase and an amino acid sequence of human epidermal growth factor divided by a cleavage site by enterokinase, and characterised by the nucleotide sequence SEQ ID NO:1. The KpnI/XhoI fragment of the vector pET41 and the above recombinant DNA are used to create the recombinant plasmid pAS007 for expression of GST-hEGF fusion protein in E.coli cells.

EFFECT: invention enables reaching high GST-hEGF expression levels in Ecoli cells.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.

EFFECT: invention enables to obtain a highly sensitive reporter for detecting a tumour necrosis factor via bioluminescent analysis.

2 cl, 4 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.

EFFECT: invention makes it possible to increase efficiency of delivery of molecules of nucleic acids into mammalian cell due to peptide, capable of internalisation into mammalian cell with efficiency, constituting at least 200% of efficiency of internalisation of peptide TAT, which has amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

8 cl, 16 dwg, 1 tbl, 8 ex

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