Recombinant plasmid dna pfk2 providing synthesis of recombinant peptide being human kappa casein fragment analogue, method for preparing recombinant peptide, and recombinant peptide, human kappa casein fragment analogue exhibiting apoptotic activity in relation to malignant cells
SUBSTANCE: invention can be used in medical and biologic industry for preparing antineoplastic drugs. Plasmid DNA pFK2 providing synthesis of recombinant analogue of human kappa casein fragment, in Escherichia coli cells is designed; and a method for preparing a recombinant product with using it is described. The recombinant analogue of human kappa-casein fragment recovered from Escherichia coli cells transformed by recombinant plasmid DNA pFK2 has molecular weight of approximately 16 kDa; consists of residual methionine, human kappa-casein fragment with 24 on 134 amino acid residue and C-terminal histidine path and exhibits apoptotic activity in relation to malignant cells.
EFFECT: higher anticancer activity of the compounds.
3 cl, 6 dwg, 4 ex
The invention relates to biotechnology, genetic and protein engineering, specifically to obtain genetic design for synthesis in Escherichia coli cells recombinant peptide which is an analog of a fragment of Kappa-casein man from 24 to 134 amino acid residue C-terminal his-tag tract and having apoptotic activity against cancer cells.
Cancer is one of the main causes of adult mortality in Russia. The lower mortality of people from malignant tumors is one of the tasks of modern medicine. One of the directions in solving this task is the development and introduction of modern anticancer agents. Special attention is given to the creation of drugs that induce apoptosis of cancer cells. Currently, several of these protein drugs are already used in clinics for the treatment of certain cancers. Basically these drugs on the basis of tumor necrosis factor (TNF-α) and its analogues (TRIAL, Fast). However, the preparations on the basis of anti-inflammatory cytokines cause serious side effects, which limits their application. Therefore, the search for new proteins that can inhibit the growth and induce apoptotic death of cancer cells is an important task.
Previously biobarrier and studied natural peptide, named lactation with cytotoxic effects on cancer cells . This peptide, which is a fragment of Kappa-casein from human milk, has a molecular mass of about 8.6 kDa and contains a defined sequence of 74 amino acid residues , which is a proteolytic fragment of Kappa-casein with 63 123 amino acid residues . Known peptide has apoptotic activity against cultures of cells of mammary adenocarcinoma (MCF-7 .
The main disadvantage of the drug on the basis of natural Kappa-casein person is expensive and limited source of raw materials for its production. The same reason makes it impossible to manufacture such drug in sufficient quantities for clinical use, and therefore it is necessary to develop a recombinant analogue of the Kappa-casein person. In the Russian Federation and abroad such analogues are missing.
An object of the invention is the creation of recombinant plasmids for synthesis of recombinant peptide which is an analog of a fragment of Kappa-casein person and obtaining recombinant peptide analogue of a fragment of Kappa-casein person having apoptotic activity against cancer cells.
The goal of the project is achieved, the design is rirovanie plasmids pFK2 by insertion in the plasmid vector pGSDI  the DNA fragment, encoding a methionine residue, a fragment of Kappa-casein man from 24 to 134 amino acid residue and a C-terminal his-tag tract. The transformation obtained by plasmid of Escherichia coli cells provides a synthesis of the recombinant peptide, analogue, fragment of Kappa-casein person having apoptotic activity against cancer cells.
The invention consists in the following.
Genetic engineering methods receive plasmid pFK2, carrying the gene encoding the recombinant peptide analogue of a fragment of Kappa-casein man, created on the basis of the DNA sequence that encodes a fragment of Kappa-casein man from 24 to 134 amino acid residues his-tag and Oligopeptide.
Cells of E. coli XL-blu carrying gene RNA polymerase of phage T7 under the inducible lacUV5 promoter transform the constructed plasmid pFK2 and grown over night. Night culture (1/50) seeded in fresh medium YTx2 with ampicillin (50 μg/ml). The synthesis of RNA polymerase induce the addition isopropylthioxanthone to a concentration of 0.5 mm at the time when the culture reaches srednetehnicheskoy phase of growth. Induced cells grow 6 hours at 30°C, and then harvested by centrifugation at 5000 g. Induced cells of E. coli XL-blu/pFK2 used for purification of recombinant fragment of Kappa-casein person using affin the th chromatography. The result is a recombinant peptide analogue of a fragment of Kappa-casein person having a molecular weight of about 16 kDa, consisting of a methionine residue, a fragment of Kappa-casein man from 24 to 134 amino acid residue and a C-terminal his-tag tract with apoptotic activity against cancer cells, including amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 (figure 1).
The source of genetic material for constructing recombinant plasmids pFK2 are:
(a) plasmid vector pGSDI , providing the insert DNA fragment encoding a fragment of Kappa-casein man, his expression under control of the late promoter T7 DNA polymerase;
b) a DNA fragment encoding a fragment of Kappa-casein, which is produced in a polymerase chain reaction using as a template genomic DNA extracted from human placenta, and oligonucleotide primers: CDCex3U55 5'GCCCAGATCTATGAA-CCAGAAACAACCAGCATGCCATGAG-AATG (underlined entered the start codon) and IPTI376L67 5'GCCCGTCGA-CTTAGTGATGGTGATGGTGATGTGATCCGCCGATGGTAGGGA-TGAT-TATTTTATCCTG (underlined introduced a stop codon), the respective 5'- and 3'-end of DNA encoding the fragment of Kappa-casein from 24 to 134 amino acid residue, and ensuring the presence of amplication fragment sites plays the guitar and sings the AI SalI and BglII, respectively.
The resulting plasmid pFK2 (figure 2) is characterized by the following features:
- has a molecular weight of 2.09 MDA and size 3164 P.O.;
- encodes the recombinant peptide analogue of a fragment of Kappa-casein rights containing a methionine residue, a fragment of Kappa-casein from 24 to 134 amino acid residue peptide with the amino acid sequence GGSHHHHHH;
- consists of the following elements:
a) DNA fragment size 373 BP, encoding a methionine residue, a fragment of Kappa-casein from 24 to 134 amino acid residue peptide with the amino acid sequence GGSHHHHHH,
b) plasmid vector pGSDI , providing efficient transcription of the obtained gene encoding a fragment of Kappa-casein man, and his expression
a) the site of initiation of replication of plasmids pBR322;
b) the promoter of bacteriophage T7;
C) genetic markers: AMPr gene, the ampicillin resistance gene, β-lactamase), which defines the resistance to ampicillin in transformation of Escherichia coli;
d) artificial gene that encodes a start codon, a fragment of Kappa-casein man from 24 to 134 amino acid residue, an Oligopeptide sequence GGSHHHHHH and stop codon;
d) a unique recognition sites of restriction endonucleases, with the following coordinates: EcoRI (1595), BglII (1613), SalI (1985), PST (1995), HindIII (2000), ClaI (2053).
Thus the om, first obtained plasmid DNA, providing products in Escherichia coli cells recombinant peptide analogue of Kappa-casein person, consisting of a fragment of Kappa-casein man from 24 to 134 amino acid residue and a C-terminal his-tag tract and having apoptotic activity against cancer cells.
The invention is illustrated by the following drawings:
Figure 1. The nucleotide sequence and encoded by its amino acid sequence SalI/BglII fragment of plasmid pFK2 encoding a fragment of Kappa-casein man from 24 to 134 amino acid residue C-terminal his-tag channel.
Figure 2. The General scheme of the structural organization of the plasmid pFK2. FK2 - gene that encodes a recombinant analogue of the Kappa-casein person, SD website landing ribosomes, T7 - promoter of phage T7, AMPr-gene resistance to ampicillin; are some restriction enzymes cut sites.
Figure 3. Electrophoregram in 12% SDS-page of cell lysates of E. coli XL-blu/pFK2, track 1 and E. coli XL-blu/pGSDI (track 2). Track 3 - markers of molecular weights.
Figure 4. Electrophoregram in 12% SDS-page of protein fractions and purified recombinant fragment of Kappa-casein person. Tracks: 1 - the lysate of E. coli cells containing plasmid pFK2, 2 - fractions, not interacting with chromatographic sorbent, 3 - faction, allereie 50 mm imidazole, 4 - fractions, allereie 250 mm imida the I, 5 - fractions, allereie 6M guanidine-HCl, 6 markers molecular weights.
Figure 5. Cell viability adenocarcinoma human breast lines MCF-7 and control of mesenchymal stem cells MSC, incubated for 3 days in complete culture medium in the presence of different concentrations of recombinant analogue of Kappa-casein person. 100% accepted the viability of cells incubated in the absence of recombinant analogue of Kappa-casein person.
6. Histograms of the distribution of cells F-7 intensity of staining with FITC-labeled annexin V (X scale) and propedy iodide (Y). Cells were incubated with recombinant analogue of Kappa-casein person (24, 48, 72 h) and without analogue (72 h, control), after which the adherent (monolayer) and detached cells were stained with FITC-annexin V/PI and analyzed by flow cytometry. G1-G3 - subpopulations of cells whose contribution % indicated after the designation of a subpopulation. Border subpopulations are indicated by lines.
For a better understanding of the essence of the present invention the following are examples of its implementation.
Example 1. Construction of plasmids pFK2.
As the source of the gene encoding the fragment of Kappa-casein person, using genomic DNA extracted from human placenta. Amplification of the gene encoding the fragment of Kappa-casein person, about who W ill result by PCR using DNA polymerase "rTth DNA Polymerase XL (Applied Biosystems", USA), which has the editing activity and allows to synthesize products with the frequency of mutation is about 1×10-6errors / nucleotide, which is one to two orders of magnitude less than the frequency of mutations when using Tag DNA polymerase. For the synthesis of a DNA fragment encoding a fragment of Kappa-casein of human rights, the reaction mixture was added primers CDCex3U55 5'GCCCAGAT-CTATGAACCAGAAACAACCAGCATGCCATGAGAATG (underlined entered the start codon) and IPTI376L67 5'GCCCGTCGACTTAGTGATGGTGATGGTGATGTGATCCGCCGATGGT A-GGGATGATTATTTTATCCTG (underlined introduced a stop codon), the respective 5'- and 3'-end of DNA encoding the fragment of Kappa-casein from 24 to 134 amino acid residue, and providing amplication fragment, the presence of the restriction sites SalI and BglII, respectively. The reaction is performed using hot start to the amplifier "GeneAmp PCR System 9700" ("Applied Biosystems", USA). The PCR conditions: pre-denaturation of 2 minutes at 94°C; 10 cycles of 30 seconds at 94°C, 40 seconds at 60°C (with subsequent lowering of the temperature by 1°C per cycle), 30 seconds at 68°C; 20 cycles of 30 seconds at 94°C, 40 seconds at 50°C, 30 seconds at 68°C; and a final stage for 20 minutes at 72°C.
Product aplicatii encoding a fragment of Kappa-casein person, split by restrictase SalI and BglII in the reaction mixture containing 50 mM l, pH 7.5; 10 mM MgCl2; 50 mM NaCl and 50 units of the asset the spine of the respective enzymes. Similarly handle restrictable DNA plasmids pGSDI. The reaction are 1 hour at 37°C. Similarly handle restrictable DNA vector plasmid pGSDI. After this PCR fragment and the linearized vector purified by electrophoresis in a 1.5% agarose gel with subsequent DNA isolation using the kit QIAquick Gel Extraction ("Qiagen, USA) according to the manufacturer's recommendations.
Ligation of a DNA fragment encoding a fragment of Kappa-casein person, and vector DNA are in standard conditions .
Example 2. Obtaining strain-producer of a fragment of Kappa-casein person - product plasmids pFK2.
Cells of E. coli XL-blu carrying gene RNA polymerase of phage T7 under the inducible lacUV5 promoter transform the constructed plasmid pFK2. Cells of E. coli XL-blu transformed by the plasmid pFK2, grown during the night. Night culture (1/50) seeded in fresh medium YTx2 with ampicillin (50 μg/ml). The synthesis of RNA polymerase induce the addition isopropylthioxanthone to a concentration of 0.5 mm at the time when the culture reaches srednetehnicheskoy phase of growth. Induced cells grow 6 hours at 30°C, and then harvested by centrifugation at 5000 g and analyzed by electrophoresis according to laemmli's method  in 12% SDS-polyacrylamide gel (SDS page). The results of this analysis are presented in figure 3, show the presence of indutsirovanno the cell culture of E. coli XL-blu/pFK2 additional protein with a molecular weight of about 16 kDa (figure 3, track 1), which is absent in the control cell lysate of E. coli XL-blu/pGSDI (figure 3, lane 2).
Example 3. The purification of the recombinant peptide, analogue, fragment of Kappa-casein person from cells of E. coli XL-blu/pFK2.
Induced cells of E. coli XL-blu/pFK2 used for purification of recombinant fragment of Kappa-casein person using affinity chromatography on Ni-NTA agarose (Sigma, USA), according to manufacturer's instructions. On a chromatographic column Packed with 5 ml Ni-NTA agarose (Sigma, USA) and equilibrated with buffer a containing 50 mm Na-phosphate buffer pH 8.0, 300 mm NaCl, 10 mm imidazole, 1 mm phenylmethylsulfonyl fluoride, 10 mm 2-mercaptoethanol, put a lysate of induced cells with a flow rate of 1 ml/min To remove non-specific adsorbed proteins of E.coli conduct preliminary elution with 20 ml of buffer a containing 50 mm imidazole. Recombinant peptide elute with 10 ml buffer a containing 250 mm imidazole, and then spend an additional elution under denaturing conditions 10 ml of buffer containing 50 mm Tris-HCl pH 8.0, 6 M guanidine-HCl. The obtained protein fractions cialiswhat against 150 mm NaCl, Tris-HCl pH 7.5 (two shifts for 18 h at 5°C) and analyzed by electrophoresis in SDS page with SDS according to laemmli's method , preparing samples for application to the gel in the presence of 2-mercaptoethanol. An example of such purification is shown in figure 4. Fractions containing recombinant peptide,analogue fragment of Kappa-casein person, concentrate ion exchange chromatography on DEAE-cellulose.
Determining the concentration of protein in preparations of recombinant analogues lactating carried out by the method of Bradford . To construct a calibration curve using bovine serum albumin (Serva, USA). Protein concentration shows that the total output is 150 mcg from 500 ml culture of Escherichia coli cells.
Example 4. The study apoptotic activity of recombinant peptide analogue of a fragment of Kappa-casein person in relation to cancerous cells.
To analyze the effect of recombinant peptide analogue of a fragment of Kappa-casein person, use cell adenocarcinoma human breast line MCF-7 from the Russian cell culture collection of vertebrates, ISC RAS, St.-Petersburg, which is cultivated in IMDM medium with 10 mm L-glutamine and 40 mg/ml gentamicin in the presence of 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2in the culture flasks with square bottom surface 25 cm2. Counting the number of cells is carried out in the camera Goryaeva. 2×106the MCF-7 cells were cultured in 96-well tablet for 2 days (exponential growth phase, 50% of the monolayer). To the culture medium add a recombinant analogue of the Kappa-casein person and incubated for 3 days, after which assessed the t of morphological changes by light microscopy and analyze cell viability using the MTT-test . For this, cells MCF-7 incubated in the presence of 1 mg/ml MTT for 1 h, 37°C, the medium with MTT removed. MTT-formazan dissolved in isopropyl alcohol, and determine the optical density of the solution at a wavelength of 570 nm with control at a wavelength of 630 nm on the device "Multiskan RC", ThermoLabs (USA).
The results of the experiment are presented in figure 5. The experimental results show that incubation of MCF-7 in the presence of the generated recombinant analogue of Kappa-casein person leads to a significant reduction in their viability (62%, 3 days incubation). Decreased viability is accompanied by morphological changes characteristic of apoptosis of cells in culture: detaching from the plastic substrate, condensation of cytoplasm and nuclei and the appearance of secondary necrotic cells, painted Trifanova blue. On the third day of incubation, the number of secondary necrotic cells reaches 20-25%.
In addition, conduct quantitative determination of apoptotic cells in culture using flow cytometry. For this, cells MCF-7 were cultured in 24-hole tablet 2 days (2×105cells/well, the exponential growth phase, 50% of the monolayer). To the culture medium add a recombinant analogue of the Kappa-casein person according to the method described in . Cells incubated for 24, 48 or 72 hours.
After incubation, the cells ukrepivshis from p is Daiki, harvested from the culture medium, centrifuged 15 min at 2000 g, after which the medium was removed and the cells are suspended in buffer for color OPTIONS (30 mm HEPES-NaOH pH 7.5, 3 mm CaCl2, 140 mm NaCl, 100 µg/ml PI and 1 ál/(ml cell suspension) of a solution of FITC-labeled annexation V (BD Biosciences, USA). The cells in the composition of the monolayer was washed with TBS (140 mm NaCl, Tris-HCl pH 7.5), incubated with 100 μg/ml of trypsin in phosphate-buffered saline. Then inactivate the trypsin by adding IMDM medium with 10% fetal bovine serum, the cells are precipitated by centrifugation for 15 min at 2000 g, the medium removed and the cells are suspended in buffer for color OPTIONS. All cells analyzed by flow cytometer BD FACSAria using system filters 585/42 BP (PI) and BP 530/30 (FITC).
The results of the experiments presented in Fig.6. Experimental results confirm that the generated recombinant analogue of the Kappa-casein person induces apoptotic death of these cells: incubation of the cells in the presence of recombinant analogue of Kappa-casein person leads to the destruction of the monolayer and detaching the cells from the plastic substrate. The increase in the number of necrotic cells is due to loss of apoptotic cells. Therefore, necrotic cells are secondary necrotic, i.e. the result of apoptotic death.
Thus, the first floor is Jena plasmid DNA pFK2, containing the gene encoding the recombinant peptide analogue of a fragment of Kappa-casein man from 24 to 134 amino acid residue and a C-terminal his-tag tract that provides products in Escherichia coli cells recombinant peptide analogue of a fragment of Kappa-casein person having apoptotic activity against cancer cells.
SOURCES of INFORMATION
1. Vlasov V.V., Richter, V.A., Semenov D.V., Nakapila CENTURIES, Kuligina E.V., Potapenko MO Peptide having apoptotic activity against cancer cells. // Patent RU 2317304 C1. 2006.
2. Nakapila V.V., Semenov D.V., Potapenko MO, Kuligina E.V., Keith UA, Romanova I.V., Richter, VA Lactatin - protein of human milk induces apoptosis in adenocarcinoma cells MCF-7. // Dokl. An. - 2008. - T. - S-271.
3. Belavin P.A., Netesova N.A., S. Reshetnikov, Ivanisenko V.A., Eroshkin A.M., Protopopov E.V., Loktev V.B. have been, Malygin EG the expression of the gene fragments F of Japanese encephalitis virus in Escherichia coli cells. // Biotechnology. - 1997. - V.3. - P.3-9.
4. Maniatis T., Fritsch E., Sambrook D. Molecular cloning. M.: Mir, 1984.
5. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. // Nature. - 1970. - V.227. - P.680-685.
6. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. // Anal. Biochem. - 1976. - V.72. - P.248-254.
7. Mosmann T.J. Rapid colorimetric assay for cellular growth andsurvival: Application to proliferation and cytotoxic assays. // J. Immunol. Meths. - 1983. - V.65. - P.55-63.
1. Recombinant plasmid DNA pFK2 for synthesis of recombinant peptide which is an analog of a fragment of Kappa-casein person, in Escherichia coli cells, with a molecular mass of 2.1 MDa and size 3164 BP, containing:
plasmid vector pGSDI, hydrolyzed by SalI and BglII sites, and contains the site of initiation of replication of plasmids pBR322, the promoter of phage T7 and unique restriction sites EcoRI (1595), BglII (1613), SalI (1985), PST (1995), HindIII (2000), ClaI (2053);
BglII-SalI fragment size 372 BP, including the start codon, a segment that encodes a fragment of Kappa-casein man from 24 to 134 amino acid residue and the Oligopeptide sequence GGSHHHHHH, stop codon;
genetic markers: Amrg gene ampicillin resistance gene, β-lactamase), which defines the resistance to ampicillin in transformation of Escherichia coli.
2. A method of obtaining a recombinant peptide which is an analog of a fragment of Kappa-casein person providing cells transformation of E. coli XL-blu carrying the gene for RNA polymerase of phage T-7 under the inducible lacUV5 promoter, constructed the plasmid pFK2 according to claim 1; growing the transformed cells in a medium with ampicillin to srednetehnicheskoy stage of growth induced with 0.5 mm by isopropylthioxanthone and subsequent cultivation for 5-7 h at 28-30°C; separation of cells zentrifugenbau the m with subsequent purification of the recombinant fragment of Kappa-casein person using affinity chromatography on Ni-NTA agarose.
3. Recombinant peptide analogue of a fragment of Kappa-casein person having a molecular weight of about 16 kDa, consisting of a methionine residue, a fragment of Kappa-casein man from 24 to 134 amino acid residue and a C-terminal his-tag tract with apoptotic activity against cancer cells, including amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: I.
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to genetic engineering and can be used to optimise expression of the antigen protein of the human epidermal growth factor-2 (HER2/neu). To obtain the HER2/neu protein, a nucleic acid synthetic molecule is used, which is codon-optimised for high level of expression of the said protein in a human cell.
EFFECT: invention increases production of the recombinant HER2/neu protein during expression in human cells.
8 cl, 10 dwg, 14 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.
EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.
3 dwg, 5 ex
SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.
EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.
17 cl, 1 dwg, 7 tbl, 7 ex
SUBSTANCE: in dissolvent, which contains from 55% to 70% of water (wt/wt), precursor of insulin or precursor of insulin derivative is exposed to fermentative splitting at alkaline values of pH. In process of fermentative splitting, they use tripsin or lysil-specific protease, preferably Achromobacter lyticus protease I. Then without separation of intermediate product from reaction mixture, mentioned intermediate product is fermentatively complemented with nucleophilic compound, which represents aminoacid ether, aminoacid amide, peptide, peptide ether or peptide amide in reaction mixture, having water content in the range from 10% to 50% of water (wt/wt), at acidic values of pH, close to neutral pH value. If required, protective group (s) is/are removed.
EFFECT: preparation of insulin compound from its precursor by efficient improved method.
24 cl, 5 ex
SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.
EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.
16 cl, 3 tbl, 7 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to a method of producing recombinant protein human albumin-interleukin-2 or recombinant protein human albumin-alpha 16-interferon, modified by attachment of human albumin. The method involves technology of culturing yeast strain Pichia pastoris PS106/pPIC9HAbIL-2 or yeast strain Pichia pastoris PS106/pPIC9HAbIFNa-16 in modified culture medium BMGY, after which induction synthesis of target proteins is carried out at low temperature. Further, cells are removed and the medium is concentrated. Target proteins are then precipitated using ammonium sulphate or polyethyleneglycol 3350. Target proteins are then separated by gel filtration on Sephacryl HR 200 or BioRad P-300 sorbents. Finally, affinity chromatography is then done on Cibacron F3GA sorbent.
EFFECT: invention simplifies and increases efficiency of the technology of purifying target proteins, and also allows for obtaining biologically active hybrid proteins, suitable for making medicinal agents.
3 cl, 1 tbl, 5 ex
SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.
EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.
29 cl, 5 dwg, 6 tbl, 7 ex
FIELD: medicine; microbiology.
SUBSTANCE: way is intended for reception of functionally active LF form, the basic toxic protein defining cellular disturbances, leading to death of an organism at infection with a malignant anthrax bacterium. For realisation of the way a recombinant plasmid pETHIS-LF (7816 items) is designed, containing a full-size gene of the lethal factor (LF) of malignant anthrax under the control of the promotor of bacteriophage T7 and to a determinant of ampicillin tolerance. The plasmide provides effective synthesis of LF protein of malignant anthrax merged with sequence of six Histidinums for clearing with the metal-chelate chromatography. The strain Escherichia coli BL-HISLF is designed using transformation of the specified plasmid DNA in the strain E.coli BL21 (DE3), synthesizing active LF protein. The target product is separated with the way including clearing on a metal-chelate sorbent with the subsequent additional clearing of the LF protein by gel-filtration.
EFFECT: reception of active recombinant protein LF on the simplified technology and with a high output of synthesised protein of the lethal factor.
3 cl, 3 dwg, 3 ex
FIELD: genetic engineering.
SUBSTANCE: invention refers to genetic engineering and can be used in medical and biologic industry for making recombinant heterocarpine that is an antagonist of human release factor of growth hormone (GHRH). There is disclosed complete nucleotide sequence coding polypeptide heterocarpine; there are disclosed the related primer sequences to be used in heterocarpine gene cloning, as well as genetic make-ups including specified sequence, particularly hybrid gene coding fused protein containing polypeptide heterocarpine, as well as expression vectors for said hybrid gene. There is described method for making recombinant heterocarpine as His-tag fused protein, providing application of the host cells transformed or transfected with the disclosed genetic make-ups.
EFFECT: recombinant heterocarpine according to the invention can be used in making a medicinal agent for cancer treatment.
9 cl, 6 ex
SUBSTANCE: peptide is obtained from phage peptide library including set of static peptides with length of 12 aminoacid residues, by affine selection of phage clones containing peptide capable of specific linking to antibodies of benzo[α]pyrene and benzo[α]anthracene. Peptide displays specific interaction effect on antibodies of benzo[α]pyrene and benzo[α]anthracene and features molecular weight of 1.3 kDa and registered aminoacid sequence LHLPHHDGVGWG encoded by nucleotide sequence SEQ ID NO:1.
EFFECT: application in medicine as a base for peptide medicine development for immunologic prevention of malignant tumours of humans.
4 dwg, 3 ex
SUBSTANCE: invention represents plasmid DNA pD4spGBD enabling the expression of a recombinant antigen - protein LigA L. interrogans domain 4 in chimeric protein D4-GBD and 1,3-β-glucan-binding domain (GBD), and a method for making a based subunit engineered leptospirosis vaccine.
EFFECT: invention allows ensuring the development of an intensive immune response and high level of antibodies synthesis with no additional adjuvants.
6 cl, 1 dwg, 6 ex
SUBSTANCE: invention represents plasmid DNA pD5spGBD enabling the expression of a recombinant antigen - protein LigA L. interrogans domain 5 in chimeric protein D5-GBD and 1,3-β-glucan-binding domain (GBD), and a method for making a based subunit engineered leptospirosis vaccine.
EFFECT: invention allows ensuring the development of an intensive immune response and high level of antibodies synthesis with no additional adjuvants.
6 cl, 1 dwg, 6 ex
SUBSTANCE: recombinant plasmid DNA pTrcIFdL coding polypeptide with human gamma interferon activity of molar mass 3.06 Md (4.642 "т.п.о."), and physical map presented on dwg 1. is engineered. Said plasmid DNA pTrcIFdL is used to prepare BL21 (DE3)/pTrcIFdL strain, a producer of polypeptide with human gamma interferon activity.
EFFECT: invention allows preparing polypeptide with biological activity of human gamma interferon and improved thermal stability, and increasing its biosynthesis level.
2 cl, 2 dwg, 5 ex
SUBSTANCE: recombinant DNA is produced, which codes functionally active hybrid protein (BrdGl7ACA-cbd), consisting of amino-acid sequence of acylase glutaryl-7- aminocephalosporanic acid of strain Brevundimonas diminuta All-Russian collection of industrial microorganisms B-1297 and chitin-binding domain of chitinase Al Bacillus circulans. Recombinant plasmid pSVH0108 is constructed for expression of BrdGl7ACA-cbd in cells E.coli, containing sequence of recombinant DNA that codes hybrid protein under control of promotor and terminator of RNA-polymerase of phage T7. As a result of E.coli strain tranformation with this recombinant plasmid and selection of transformed clones, a new strain E.coli BL21(DE3)/pSVH0108 cbd is produced - producer of hybrid protein BrdGl7ACA-cbd.
EFFECT: high yield of recombinant ferment.
4 dwg, 2 tbl, 7 ex
SUBSTANCE: invention can be used in producing vaccines against Streptococcus agalactiae - a representative of streptococci group B (SGB) in diagnostics of the diseases - for creation of a detection system of immunoglobulin A level in biological fluids, in immunochemistry as accessible immunochemical reagents (affine recovered IgA fragments). Offered unique recombinant DNA are produced by polymerase chain reaction (PCR) with using chromosomal DNA of strain 219/4849 Ibc of serotype SGB and unique primers. One recombinant DNA contains three nucleotide substitutes in comparison with an initial site of chromosomal DNA. The following cloning of amplified fragments is carried out in a linear vector pGEM-T Easy, and at the final stage by the system of express ionic vectors pQE30/31/32 in E coli JM 109. The produced recombinant DNA code amino acid sequences of recombinant polypeptides exhibiting ability to connect selectively various molecular forms of IgA and designated as P6, P7, P8. Polypeptide P6 causes synthesis of long circulating high-affine anti-Rb antibodies possessing protective properties against SGB.
EFFECT: application of the invention provides production of recombinant polypeptides based N-terminal conservative part of surface Bac SGB of Ibc serotype and containing a first IgA-connecting site A with changed or native sequence MLKKIE, polypeptide exhibits immunologically relevant and protective properties, and they also high selectively connect IgA.
12 cl, 20 dwg, 4 tbl, 19 ex
SUBSTANCE: invention concerns the method for making an immunogenic reagent which causes immune response on infection Bacillus anthracis, including one to several polypeptides which together represent three domains of a full-size protective antigen (PA) from B anthracis or their versions, and at least, one of specified domains contains domain 1 or domain 4 of the PA, or its version. Said polypeptides of specified immunogenic reagent, and the full-size PA are produced as a result of expression in a recombinant cell E.coli. The invention also discloses an expression vector and nucleic acid with percent of residual guanidine and cytosine more than 35%, coding immunigenic polypeptide which is said protective antigen (PA).
EFFECT: high-yield immunogenic polypeptide.
13 cl, 5 dwg, 3 tbl, 6 ex
SUBSTANCE: there is designed recombinant plasmid DNA pYfi-gfp enabling production of chimeric fluorescent protein GFPaav with five modified amino acids, corresponding to the first five amino acids of protein YfiA with controlling a promotor of gene YfiA in presence of toxic agents. Plasmid has size 3730 base pairs. Recombinant strain Escherichia coli JM109-pYfi contains a genetic design under given invention and produces fluorescent protein GFPaav in presence of toxic agents damaging a cell.
EFFECT: invention allows registering the presence of toxic agents in a medium with increasing the fluorescence level of cells of produced strain.
2 cl, 4 dwg, 4 ex
SUBSTANCE: there is designed recombinant plasmid DNA pSC13D6 containing a gene of one-chained antibody to tick-borne encephalitis virus sc13D6. Plasmid consists of 3782 basepairs and contains: a plasmid vector pGEMl, phage T7 promotor and unique restriction sites. There is also discovered strain of bacteria Escherichia coli BL21 (DE3)/pSC13D6 - a producer of a virus neutralising one-chained antibody sc13D6 to tick-borne encephalitis virus.
EFFECT: invention allows producing one-chained antibodies sc13D6 to tick-borne encephalitis virus able to inhibit it effectively.
2 cl, 5 dwg, 5 ex
FIELD: chemistry, medicine.
SUBSTANCE: novel antibodies and fragments of human antibodies are bound with GDF-8 in a specific way and inhibit its activity in vitro and/or in vivo. On the basis of said invention pharmaceutical composition is created, which can be used for diagnostics, prevention or treatment of degenerative dysfunctions of muscle or bone or disorders of insulin metabolism.
EFFECT: extending range of arsenal of technical means used in treatment of diseases related to muscular, bone tissue or insulin metabolism.
SUBSTANCE: there are offered recombinant protein molecule M2E-HBC, and virus-like particles formed of such molecules. The recombinant virus-like particle based on nuclear antigen of hepatitis B virus represents surface polypeptides of outer domain M2 of avian influenza virus protein. The produced virus-like particles are characterised with high immunogenicity. There is also disclosed vaccine for the infection caused by avian influenza virus, including such virus-like particles as an active agent.
EFFECT: preparations are active to various strains of avian influenza virus and can be considered as a candidate for universal avian influenza virus type A vaccine.
7 cl, 8 dwg, 4 tbl, 8 ex
SUBSTANCE: thymus-specific protein T101 consisting of 84 amino acids is recovered from human thymus. The recovered full-length peptide T101 includes the signal peptide consisting of 33 amino acids and the peptide sequence T101 consisting of 51 amino acids and exhibiting immunomodulating activity. The full-length peptide T101, and also peptide fragments and derivatives are used as a part of a pharmaceutical composition for treating autoimmune and inflammatory diseases.
EFFECT: invention allows preparing polypeptide capable to stimulate lymphocyte proliferation of human peripheral blood, to inhibit the tumour growth and to modulate the immune system.
10 cl, 15 dwg, 1 tbl, 11 ex