Recombinant plasmid dna pg1-rm7, facilitating synthesis of hybrid protein g1-rm7, and hybrid protein binding tumour necrosis factor and having bioluminescence

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

 

Group of inventions relates to biotechnology, genetic and protein engineering and is used to obtain genetically fused bifunctional hybrid protein consisting of single-stranded human antibodies against tumor necrosis factor alpha man and a modified light-emitting protein coelenterazine-dependent luciferase Renilla muelleri.

The tumor necrosis factor alpha (TNF) is a major mediator of acute inflammatory response to gram-negative bacteria and other infectious agents. The main source of TNF are activated mononuclear phagocytes, although stimulated T-cells, natural killer cells and mast cells can also secrete this protein [1]. The main stimulator of the start of production of TNF by macrophages is bacterial lipopolysaccharide (LPS) or gram-negative bacteria, releasing LPS [1]. Interferon-gamma produced by T-cells and NK-cells, increased production of this cytokine LPS-activated macrophages [2].

TNF is responsible for many systemic complications arising from severe infectious processes, while TNF produced in large quantities, can cause systemic clinical and pathological manifestations. By acting on the hypothalamus, it induces fever and is therefore called natural endogenous feast of the genome. As a result of fever increases the synthesis of prostaglandins cytokine-stimulated cells of the hypothalamus [3]. In addition, TNF has an effect on the liver, increasing, in particular, the synthesis of hepatocytes, serum amyloid protein a and fibrinogen - serum inflammatory proteins. Prolonged production of TNF causes and metabolic damage, such as cachexia: it inhibits the synthesis of lipoprotein lipase, the enzyme required to release fatty acids from circulating of lipoprotein complexes. When the concentration of TNF in the serum reaches high values, exceeding 10-7M, it causes a decrease in vascular tone and contractility, which, ultimately, leads to a sharp drop in blood pressure and shock [4]. In addition, TNF and make a significant contribution to the development of local inflammatory responses, particularly in autoimmune diseases [5]. Therefore, the high-sensitive quantitative determination of the level of TNF in the body is necessary for timely diagnosis of a number of pathological conditions.

Luciferase soft coral Renilla muelleri is a relatively small, 36 kDa, single-chain polypeptide. The enzyme catalyzes the oxidation of substrate - coelenterazine, molecular oxygen with the formation of CO2and molecules to coelenteron is Yes in the excited state. The transition of this compound in the ground state accompanied by the release of light in the visible region of the spectrum (λmax=482 nm). The gene encoding this protein was cloned obtained and studied recombinant protein wild type, method of site-directed mutagenesis derived thermostable variant of this protein Rm7 [6, 7].

Known gene and recombinant coelenterazine-dependent luciferase coral Renilla reniformis, and improved derivatives of this protein. The literature describes bifunctional hybrid proteins on the basis of this luciferase fused with other proteins such as green fluorescent protein (GFP) [8, 9], receptor [10], etc. that are used for in vivo imaging, research-based phenomena BRET and others

The closest analogue is the prototype of the claimed group of inventions is recombinant plasmid DNA, obtained on the basis of bacterial expressing plasmids pKKtac the insert, gene T anti-CEA encoding the antibody to carcinoembryonic antigen and gene modified luciferase Renilla reniformis, United by the oligonucleotide coding for the 18-membered peptide-linker. The plasmid encodes a hybrid protein synthesis anti-CEA diabody-Renilla luciferase (Db-18-RLuc8)having affinity to carcinoembryonic antigen and bioluminescent luciferase activity, which can be used televisualization tumors in vivo [11].

Design, synthesis and use for immunoassay of tumor necrosis factor hybrid proteins using luciferase Renilla reniformis or its analogs as reporters are not described in literature. Also not described any hybrid protein with luciferase soft coral Renilla muelleri or genetically modified variants.

The technical result of the group of inventions is to obtain bifunctional hybrid protein having the ability to bind TNF person and at the same time bioluminescent activity as potentially suitable highly sensitive reporter for the detection of tumor necrosis factor method bioluminescent immunoassay.

This result is achieved by constructing recombinant plasmid DNA pG1-Rm7, containing a unique gene single-stranded human antibodies directed against TNF, and gene modified luciferase Renilla muelleri; the expression of target hybrid protein in transformed mentioned plasmid DNA cells of Escherichia coli HB2151, followed by separation of periplasmatic fraction and purification of metal-chelate chromatography hybrid protein G1-Rm7, which has the ability to bind TNF person and at the same time bioluminescent activity. The essence of invention is to track the future.

Genetic engineering methods receive plasmid pG1-Rm7, bearing a unique gene single-stranded human antibodies directed against TNF, and gene modified luciferase Renilla muelleri. Cells of E. coli HB2151, transformed constructed a plasmid, capable of producing a hybrid protein G1-Rm7, consisting of single-stranded human antibodies directed against TNF, and modified luciferase Renilla muelleri, catalyzing the oxidation reaction coelenterazine with emission of light in the visible region of the spectrum.

The source of genetic material for constructing the recombinant plasmid pG1-Rm7 are:

a) the modified plasmid vector pHEN2-g1 [12], containing the gene for the variable domains of the heavy and light chains of the monoclonal antibodies gl against tumor necrosis factor person representing two fragment of 360 BP and 333 BP, respectively, between which is the connector size 57 BP, encoding the linker peptide (Gly4Ser)2(AlaProGlySer)(Gly4Ser), hydrolyzed by NotI and ApaI sites and contains the site of initiation of replication of phage M13, the lacZ promoter and a unique restriction sites HindIII (235), SfiI (328), NcoI (332), AvaI (2939), PST (986), NotI (1076), BamHI (1737), ClaI (2045);

b) a DNA sequence encoding a peptide GGSGGS and a modified luciferase Renilla muelleri [6, 7], built on the sites of the restriction endonucleases NotI The ApaI.

The resulting plasmid pG1-Rm7 characterized by the following features:

- has a molecular 3.69 MDA and the size of the BP 6161;

- contains a NotI-ApaI fragment size 954 BP sequence encoding the peptide GGSGGS and modified coelenterazine-dependent luciferase from the soft coral Renilla muelleri;

- contains genetic markers: bla gene, the ampicillin resistance gene (3-lactamase), which defines the resistance to ampicillin in transformation of Escherichia coli.

The expression of a hybrid protein is carried out in bacterial cells Escherichia coli HB2151, transformed DNA plasmid pG1-Rm7, providing induced isopropylthioxanthone (IPTG) synthesis of a hybrid protein G1-Rm7.

The display expression is carried out using gel electrophoresis under denaturing conditions (SDS-PAGE). The level of expression was determined by densitometry polyacrylamide gel, colored Kumasi-K using software Image Lab Ver. 3.0 provided with the device GelDocXR+(Bio-Rad). The expression level is about 2% of the total cellular protein.

The target protein is recovered from periplasmic fractions and purified using metal chelate chromatography. The degree of cleaning is determined by scanning the gel on the densitometer GelDocXR+(Bio-Rad). The concentration of purified hybrid protein G1-Rm7 define spec is reformations with a set of DC Protein Assay (Bio-Rad), no Protocol of the manufacturer.

The resulting hybrid protein has bioluminescent activity, which is determined using a luminometer cuvette (model BLM 8802, SKB Science, Krasnoyarsk) in the buffer of the following composition 50 mm Tris-HCl pH 7.0, 25 mm NaCl, 1 mm EDTA immediately after making coelenterazine (10-5M solution in methanol). When conducting bioluminescent immunoassay measurements were carried out using tablet luminometer Mithras LB 940 (Berthold, Germany). Bioluminescence initiated by the introduction of a freshly prepared solution coelenterazine in 50 mm Tris-HCl pH 7.0, 25 mm NaCl, 1 mm EDTA.

The resulting hybrid protein has the ability to bind TNF man, which was shown by direct bioluminescent solid-phase immunoassay. While TNF is found in the solution with a concentration of 1.6 ng/ml (9,4 10-11M).

Thus, for the first time obtained plasmid DNA pG1-Rm7, containing in the same reading frame gene single-stranded human antibodies capable of binding a tumor necrosis factor, a sequence encoding a peptide GGSGGS, and gene modified luciferase Renilla muelleri; the resulting hybrid protein - single-stranded antibody-luciferase (G1-Rm7)that binds the tumor necrosis factor and with bioluminescent activity, which provides detection of tumor necrosis factor biolum nascentium by immunoassay.

The invention is illustrated by the following figures:

Figure 1. The General scheme of the structural organization of the plasmid pG1-Rm7. Legend: g1 - gene that encodes a single-chain antibody able to bind tumor necrosis factor; Rm7 gene encoding a modified luciferase Renilla muelleri; Linker sequence encoding a peptide GGSGGS; His6 sequence encoding a peptide NNNNNN; bla - gene resistance to ampicillin; are some restriction enzymes cut sites.

Figure 2. The nucleotide and encoded by its amino acid sequence of the hybrid protein G1-Rm7.

Figure 3. 12% SDS-gel electrophoresis of fractions when selecting a hybrid protein G1-Rm7. Tracks: 1, 2 - a lysate of cells NV/pG1-Rm7 before and after induction IPTG, respectively; 3 - cytoplasmic fraction, 4, 5 - periplasmatic fraction before and after cleaning, the metal-chelate chromatography, 6 - fraction Taurus inclusion, 7 - markers molecular masses. The arrow shows the band corresponding to the target hybrid protein.

Figure 4 (A-B). Solid-phase immunoassay TNF using a hybrid protein G1-Rm7.

For a better understanding of the essence of the present invention, they are illustrated by the following examples of implementation.

Example 1. Construction of plasmid pG1-Rm7.

Pre-teach the construction of the gene encoding a recombinant luciferase from the soft coral Remll muelleri.

Amplification of the gene encoding a luciferase, carried out by PCR using Pfu DNA polymerase (SibEnzyme Novosibirsk, Russia). For the synthesis of a DNA fragment encoding a luciferase, the reaction mixture was added Prime time? 92Rm7No5 5'-CAGGCTGCGGCCGCAGGTGGGTCAGGTGGCTCTACGTCAAAAG encoding a flexible peptide linker (Gly2Sei)2and containing the restriction site for NotI. In use as a reverse primer 115Rm7Ap3 5'-GTTAGCAGCGGGCCCTCAGTGGTG containing the restriction site for ApaI. The product of amplification encoding the peptide (Gly2Ser)2and recombinant luciferase Remlla muelleri, purified using QIAquick PCR Purification Kit (Qiagen, USA) and split restrictase NotI and ApaI in the reaction mixture, containing 10 mM TpcHCl, pH 7.6; 10 mM MgCl2; 50 TM NaCl, 1 mM DTT and 20% activity of the respective enzymes. The reaction leading to 2 hours at 37°C. After that, the fragment length of 990 BP purified by electrophoresis in agarose gel, followed by purification using the QIAquick Gel Extraction Kit (Qiagen, USA).

Plasmid DNA pHEN2-g1, containing the gene for single-chain antibody g1 with a leader peptide pelB, treated with restriction endonucleases NotI and Apal in standard buffer at 37°C for 2 hours. Then spend the dephosphorylation within 1 hour with 20 units of CIP in the same buffer. Enzymes inactivate by heating at 65°C for 20 minutes After that, the DNA plasmids purified by electrophoresis in 1% agarose gel with the consequences is the fact that purification using QIAquick Gel Extraction Kit (Qiagen, USA.

The linearized plasmid are ligated with a DNA fragment coding for the peptide (Gly2Ser)2and recombinant luciferase Renilla muelleri treated with the same restriction endonucleases. Ligation is carried out in standard buffer. Received ligase mixture transform cells of E. coli XL1Blue. Using restriction analysis of selected clones containing plasmid DNA with an insert of the correct size. Thus obtained target plasmid is designated as pG1-Rm7. Scheme of plasmid DNA pG1-Rm7 presented in figure 1.

The correct construction confirmed by sequencing. The result is shown in figure 2.

Example 2. Getting a hybrid protein G1-Rm7.

Cells of E. coli HB2151 transforming DNA plasmid pG1-Rm7, incubated in YTx2 environment with the addition of 0.1% glucose and 100 mm of ampicillin at 37°C to an optical density OD600=0.4 to 0.5. Then the cell suspension is cooled and the expression of a hybrid protein G1-Rm7 induce the addition of 0.5 mm IPTG. The cells are incubated at 28°C overnight and precipitated by centrifugation. A hybrid protein G1-Rm7 separated from periplasmic fractions and purified using metal chelate chromatography sorbent Talon (Clontech) according to the manufacturer's Protocol. Results isolation and purification are presented in figure 3. The figure 3 shows (track 1, 2)that after induction in the cells of a new protein, molecule the full weight of 64.5 kDa close to the calculated mass of the hybrid protein G1-Rm7 (arrow). Protein detected in the cytoplasmic and periplasmic fractions (lanes 3, 4), as well as in inclusion bodies (lane 6). After cleaning periplasmatic fraction of the metal-chelate chromatography the resulting preparation in which a hybrid protein is 15.5% (lane 5). On track 7 shows the molecular markers of the masses.

Example 3. The use of a hybrid protein G1-Rm7 for bioluminescent solid-phase immunoassay TNF.

In the wells of an opaque immunological tablet (Costar, USA) add 100 ál of a solution of recombinant tumor necrosis factor [13] in different concentrations (1000, 200, 40, 8, 1.6, 0 ng/ml) in 50 mm K-Na phosphate buffer pH 7.0 with 0.15 M NaCl (PBS) and incubated at 37°C for one hour. After washing (PBS containing 0.1% Tween-20 and 5 mm EDTA) in the wells contribute 100 ál of 2% solution of skim milk powder in PBS and incubated at 37°C, 1 hour. After washing (PBS containing 0.1% Tween-20 and 5 mm EDTA) in the wells contribute to the solution of the resulting hybrid protein, incubated at 23°C 1 hour under stirring. After washing (PBS containing 0.1% Tween-20 and 5 mm EDTA) add to the wells, 100 μl of a solution coelenterazine (2×10-6M in 50 mm Tris-HCl pH 7.0, 25 mm NaCl, 1 mm EDTA) with simultaneous measurement of bioluminescent signal of bound peroxidase hybrid protein using tablet luminometer Mithras LB 940 (Berthold, Germany). Signal integrate within 30-40 C. Obtained the results shown in Figure 4(A-B). On Figa shown bioluminescent signals of the hybrid protein from holes on the surface which barbirolli different amounts of TNF. On Figb shows the dependence of the integrated for 40 seconds bioluminescent signal (average value from two repetitions, with less of the averaged signal from the control wells with zero content TNF) concentration solutions TNF taken for sorption on the surface of the hole (logarithmic coordinates). The dependence of the bioluminescent signal from the concentration of tumor necrosis factor in the range from 1.6 ng/ml to 1 mg/ml is close to linear (R2=0,988). This means the possibility of constructing a calibration curve for quantitative immunoassay of tumor necrosis factor using the obtained hybrid protein as a reporter.

Thus, for the first time obtained plasmid DNA pG1-Rm7, containing in the same reading frame gene single-stranded human antibodies that can bind tumor necrosis factor and gene modified luciferase Renilla muelleri. The obtained recombinant hybrid protein - single-stranded antibody-luciferase (G1-Rm7)that binds the tumor necrosis factor and with bioluminescent activity that makes possible its use as a highly sensitive reporter for the quantitative immunoassay necrosis factor tumor is.

SOURCES of INFORMATION

1. Abbas, A.K., Lightman, A.H., Pober, J.S. Cellular and Molecular Immunology. Fourth Edition, 553. - 2000. P.240-242, 244-246.

2. Konur, A., Schuiz U., Eissner g, Andreesen R, Holler E. Interferon (IFN)-gamma is a main mediator ofkeratinocyte (HaCaT) apoptosis and contributes to autocrine IFN-gamma and tumour necrosis factor-alpha production. Br. J. Dennatol. - 2005. - Vol.152(6). - P.1134-42.

3. Zailae MZ. Decreased proinflammatory cytokine production by peripheral blood mononuclear cells from vitiligo patients following aspirin treatment. Saudi Med J. - 2005. - Vol.26(5). - P.799-805.

4. Carlson DL., Willis MS., White DJ., et al., Tumor necrosis factor-alpha-induced caspase activation mediates endotoxin-related cardiac dysfunction. Crit Care Med. - 2005. - May; 33(5). - P.1021-8.

5. Feldmann m and Maini R.N. Lasker Clinical Medical Research Award. TNF defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases. Nat. Med. - 2003 - Vol.9. - P.1245-1250.

6. M.S.Titushin, S.V.Markova, L.A.Frank, N.P.Malikova, G.A.Stepanyuk, J.Lee, E.S.Vysotski. Coelenterazine-binding protein ofRenilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase. // Photochem. Photobiol. Sci. - 2008. - Vol.. - P.189-196.

7. G.A.Stepanyuk, J.Unch, N.P.Malikova, S.V.Markova, J.Lee, E.S.Vysotski. Coelenterazine-v ligated to the Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase. Anal. Bioanal. Chem. - 2010 - Vol.398 - P.1809-1817

8. Wang Y., Yu Y.A., Shabahang S., Wang G., A.A. Szalay Renilla luciferase-Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals. Mol. Genet. Genomics. - 2002 - Vol.268. - P.160-168

9. J.Liu, Y.Wang, A.A.Szalay, A.Escher. Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein. Luminescence - 2000 - Vol.15. - P.45-49

10. Zeng F.-Y., A.J. Mclean, G. Milligan, M. Lemer, D.T. Chalmers, Behan D.P. Ligand specific up-regulation of a Renilla reniformis luciferase-tagged, structurally unstable muscarinic M3chimeric G protein-coupled receptor. Mol. Pharmacol. - 2003. -Vol.64. - P.1474-1484

11. Venisnik C.M., T. Olafsen, A.M. Loening, M. Iyer, Gambhir SS, Wu A.M. Bifunctional antibody-Renilla luciferase fusion protein for in vivo optical detection of tumors. Prot. Eng. Des. Sel. - 2006. - Vol.19. - P.453-460.

12. Vihrova M.A., Batanova T.A., Lebedev LR, Shingareva L.N., Frank L.A., Kirpichnikov M.P., N.V. Tikunova single-Stranded human antibodies directed to tumor necrosis factor. Bioorg. chemistry. - 2011. - V.37. - S-343.

13. Shingareva L.N., Sagaidak, L.N., Turkish R.L., Nedospasov S.A., Esipov, D.S., Korobko VG Mutants of the tumor necrosis factor human: production and some properties. - 1996. - T. - S-251.

1. Recombinant plasmid DNA pG1-Rm7 for synthesis of a hybrid protein G1-Rm7 in Escherichia coli cells, which binds the tumor necrosis factor person and has a bioluminescent luciferase activity Renilla muelleri, where indicated plasmid DNA physical map presented in figure 1, has a size of BP 6161, molecular weight 3.69 Md, a unique restriction sites HindIII (235), SfiI (328), NcoI (332), AvaI (2939), PST (986), NotI (1076), BamHI (1737), ClaI (2045) and includes nucleotide sequence of SEQ ID NO: 1 encoding a hybrid protein with the amino acid sequence SEQ ID NO: 2 consisting of single-chain antibodies against tumor necrosis factor, peptide GGSGGS and modified luciferase Renilla muelleri.

2. A hybrid protein G1-Rm7, binding tumor necrosis factor person and possess bioluminescent AK is ewnetu, derived from Escherichia coli cells, transformed with recombinant plasmid DNA pG1-Rm7, having a molecular weight of 65.4 kDa, consisting of single-chain antibodies against tumor necrosis factor, peptide GGSGGS and modified luciferase Renilla muelleri, having amino acid sequence SEQ ID NO: 2 represented in figure 2.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns preparing a genetic construct providing a synthesis of p35d recombinant protein in Escherichia coli cells. There are presented: recombinant plasmid DNA pQE-p35d providing the synthesis of p35d recombinant protein of cowpox virus and containing in accordance with physical and genetic map presented on Fig. 2: pQE30 plasmid vector, a fragment coding MRGSHHHHHHG oligopeptice and a fragment of 17 base pairs, coding a fragment of p35 protein of cowpox virus within 1 to 239 amino acid residues (Fig.1a); Escherichia coli XL1Blue/pQE-p35d B-1252 bacterial strain that is a producer of p35d recombinant protein of cowpox virus, containing recombinant plasmid DNA pQE-p35d deposited in the Collection of Bacteria, Bacteriophages and Fungi of FBUN GNTs VB Vector, registration No. B-1252, and p35d recombinant protein of cowpox virus.

EFFECT: solutions may be used to engineer the test systems and to prepare orthopoxvirus split vaccines.

3 cl, 7 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of obtaining a recombinant antigen G2 of hantaviruses. The disclosed method is characterised by that the DNA structure pGHF, which encodes a fused protein of three parts, where N-terminal position is occupied by a green fluorescent protein GFP, central position is occupied by a peptide of 73 amino acid residues with the amino acid sequence SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and the C-terminal position is occupied by a mini-domain Foldon of coliphage fibritin JS98C3 (dwg 2), is introduced into E. coli cells; cells transformed by this structure are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifuging, the product of expression in the form of inclusion bodies is solubilised with methylated spirit, chromatography is carried out and the obtained product is used to detect specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.

EFFECT: disclosed solution improves repeatability and sensitivity of immunoenzymatic assay when diagnosing hemorrhagic fever with renal syndrome.

7 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to microbiology and molecular genetics and pertains to a recombinant polypeptide A2, DNA coding said polypeptide, a strain which produces polypeptide A2 and methods of using such a recombinant polypeptide. The disclosed recombinant polypeptide A2 is characterised by an amino acid sequence of 346 amino acids in which the first 13 amino acids are coded by the plasmid sequence pQE 32 and are covalently bonded with the next 333 amino acids which are coded by a sequence of the HSA-binding fragment of chromosomal DNA of the strain DG 13 of streptococci of the group G-CFG.

EFFECT: group of inventions can be used in diagnosis, eg, when making a test system for determining microalbuminuria based on a laboratory criterion of the preclinical phase of diabetic nephropathy, as well as a reagent for separating human serum albumin by affinity chromatography and for freeing serum from HAS, which enables to determine other proteins present in the serum in lower concentrations.

9 cl, 11 dwg, 1 tbl, 9 ex

FIELD: biotechnology.

SUBSTANCE: method is characterised in that the DNA of the structure RNAb indicated on Figure 1, which encodes the fused protein of three parts, where N-terminal position is green fluorescent protein GFP, central - peptide of 73 amino acid residues with the amino acid sequence of SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and C-terminal - light chain of double-stranded protein Kunitz-type inhibitor from potato tubers (PKPI-BI), are introduced into cells of E. coli. The cells transformed by this construction are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifugation. The product of expression in the form of inclusion bodies is solubilised with the denaturant. Chromatography is carried out under denaturing conditions. The resulting product is used for detection of specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.

EFFECT: invention enables to obtain the recombinant antigen G2 of Hantavirus Dobrava with increased yield.

6 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology. The invention discloses a composition for coexpression in an eubacterial host cell orthogonal tRNA (O-tRNA) and orthogonal aminoacyl-tRNA synthetase (O-RS), which preferably aminoacylates said O-tRNA with an unnatural amino acid. The disclosed composition consists of two nucleic acid constructs: the first construct contains promoter and terminator nucleotide sequences derived from an E.coli proline tRNA gene and which is derived from the archaea of the expressed sequence, which encodes one O-tRNA or is a polycistronic operon, and the second construct contains a modified E.coli glnS promoter and the expressed nucleotide sequence which encodes the corresponding O-RS. Described is a translation system which includes the disclosed vector constructs, and a method of obtaining the polypeptide of interest which contains an unnatural amino acid in a genetically defined position.

EFFECT: obtaining properties with new properties, which are defined by inclusion of unnatural amino acids into a predetermined position.

54 cl, 8 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: disclosed is an E.coli strain which produces a recombinant protein p30 of the African swine fever virus. The strain is homogeneous, stable during passage and culturing in liquid and solid culture media and is resistant to chloramphenicol.

EFFECT: invention can be used to produce a recombinant protein p30 of African swine fever virus for diagnostic purposes.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: recombinant plasmid DNA pTB323 under the invention coding the hybrid polypeptide glutathione-8-transferase (GST) and a shorter version of the protein MPT64 (rΔMPT64), has an average molecular weight 3.6 MDa, size 5574 base pairs, consists of: a) EcoRI-BamHI-fragment of the vector plasmid pGEX-2T of size 4938 base pairs containing the β-lactamase gene inducing tac-promotor, the internal gene Iaclg coding the lactose operone repressor protein, a glutathione-5-transferase gene fragment from S. japonicum with a multiple sites of gene cloning (MSC) in 3'-terminal part of this gene and a nucleotide sequence coding a thrombine proteolysis site and found in front of the MSC; b) EcoRI-BamHI-fragment of 636 base pairs containing a truncated gene MPT64 flanked by EcoRI and BamHI restriction endonuclease sites and prepared by amplification of the gene-related fragment with genome DNA M. tuberculosis; c) a genetic marker - β-lactamase gene determining resistance of pTB323 plasmid transformed cells E. coli to the antibiotic ampicillin; d) unique restriction sites: BamHI - 930/934, EcoRI ~ 1566/1570. The recombinant bacterial strain Escherichia coli BL21/pTB323 - producer of hybrid polypeptide GST-ΔMPT64 with the properties of the mycobacterial antigen ΔMPT64 is deposited in the Collection of Microorganisms of Federal State Research Institution State Science Centre Vector, No. B-1028. The recombinant polypeptide GST-ΔMPT64 produced by the recombinant strain under the invention contains as a carrier protein the N-terminal polypeptide fragment glutathione-S-transferase S.j. (226 amino acid residues, 26.31 kDa) and has a complete amino acid sequence (431 amino acid residues, 48.76 kDa) presented in the description.

EFFECT: using the invention enables developing the high-purity polypeptide in the preparation amounts with the preserved immunogenic properties and provided separation of the target protein from the amino acid sequence of the carrier protein for studying of the immunogenic properties of the target protein.

3 cl, 4 dwg, 4 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: polyepitope proteins consisting of two or more protein fragments of the foot-and-mouth disease virus, particularly serotype O/Taiwan/99, connected by linker amino acid sequences, are constructed. The vaccine polyepitope protein can be characterised by general formula VP4(X1 -X2)-GRL-VP1(X3-X4)-GRL-VP1 (X5-X6)GRL-2C(X7-X8)-GRL-3D(X9-X10)-GRL-3D(X11-X12), where GRL is a glycine-rich linker, Xn-Xm are integers denoting the number of amino acid residues of the corresponding foot-and-mouth disease virus protein, VP4, VP1, 2C, 3D are names of foot-and-mouth disease proteins. The invention discloses a nucleotide sequence (NS) which codes peptides included in the polyepitope protein, a recombinant plasmid which facilitates synthesis of a hybrid polyepitope protein in procaryote (E.coli) and eucaryote (plant) cells, and a solvent composition for a foot-and-mouth disease vaccine based on the polyepitope protein.

EFFECT: polyepitope protein has high immunising power and can be considered a potential recombinant vaccine against foot-and-mouth disease.

15 cl, 7 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: engineered recombinant protein molecule for preparing a recombinant vaccine for an infection caused by swine influenza virus (HlNlv-2009). The molecule consists of a residue of methionine, a sequence of extracellular M2 protein domain of 2 to 24 amino acids and a sequence of a nuclear hepatitis B virus antigen of 4 to 149 amino acids. The molecule is able to form virus-like particles. There are also disclosed a recombinant nucleic acid coding such molecule, a vector for its expression, virus-like particles formed by such molecules and a vaccine based on the prepared virus-like particles.

EFFECT: prepared vaccine can be considered as a candidate recombinant swine influenza virus vaccine.

6 cl, 8 dwg, 3 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: what is offered is an expression construct for expression of single- or multipass transmembrane polypeptides in a bacterial host cell. Said construct contains a protein-coding polynucleotide, a strictly sensitive promoter of lower basal activity in the host-cell, and a leader sequence comprising a translation initiation enhancer. The strictly sensitive promoter comprises at least one positive control element and at least one negative control element. One or more positive and negative control elements represent a heterologous control element. Besides, what is offered is a method for producing the expressed transmembrane polypeptide and a method of recovering it form the host cell.

EFFECT: methods provide producing the high-yield transmembrane polypeptides either of native conformation, or in a soluble form.

53 cl, 28 dwg, 5 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering, specifically to creation of fibroblast growth factor receptor (FGFR) muteins, and can be used in medicine. The polypeptide of the FGFR4 receptor extracellular domain (ECD) acidic region mutein is an FGFR4 ECD chimera or a FGFR4 long acid box version and has more acid residues in the D1-D2 linker region than the wild-type FGFR4 ECD. The muteins may include a point mutation that inhibits glycosylation. The mutein is used to treat a disease associated with one or more FGFR ligands, e.g., proliferative diseases, including various types of cancer, angiogenic disorders and macular degeneration.

EFFECT: invention enables to obtain an FGFR4 ECD acidic region mutein, having low capacity to bind with tissue, by increasing the number of amino acid residues within the D1-D2 linker region.

32 cl, 22 dwg, 11 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically PTH receptor agonists, and can be used in medicine. A polypeptide of formula PTH(1-X)/PTHrP(Y-36) is constructed, where denotes an integer between 11 and 18, and Y=X+1, where PTH(1-X) is an amino acid from 1 to X of the human PTH sequence (SEQ ID NO:5) and PTHrP(Y-36) is an amino acid from Y to 36 of the human PTHrP sequence (SEQ ID NO:6). The polypeptide contains one or more of the following mutations in the PTH(1-X) sequence: Ala in position 1, Ala or Aib in position 3, Gin in position 10, Arg or homoarginine in position 11, Ala in position 12, and Trp in position 14. The obtained polypeptide is used to repair fractures, treat hypoparathyroidism, hyperphosphatemia, tumoral calcinosis, osteoporosis, osteomalacia, arthritis, thrombocytopenia, as well as increase stem cell mobilisation in a subject.

EFFECT: invention enables to obtain a polypeptide having prolonged activity on the PTH receptor.

18 cl, 37 dwg, 8 tbl, 11 ex

FIELD: biotechnologies.

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

FIELD: biotechnologies.

SUBSTANCE: inventions refer to polynucleotide sequence coding the structured pertactin protein (Prn); a vector including such a sequence and compositions containing protein or vector. The characterised polynucleotide sequence codes 300 first amino acids that are the closest to N-end of this type of natural mature Prn (PrnX300), and amino-acid sequence including 620 last amino acids that are the closest to C-end of this type of natural mature Prn (PrnY620) so that structured pertactin PrnX300-PrnY620 of Bordetella class is obtained. Structured molecules Prn include polymorphisms of different B. Pertussis strains and cause immune responses with increased protective ability and opsonophagocytic activity, which exceed the corresponding properties of the preceding vaccines.

EFFECT: protein obtained as per the presented invention can be used in medicine and veterinary as a component of antibacterial vaccines against Bordetella pertusis.

12 cl, 3 dwg, 2 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.

EFFECT: proposed inventions allow performing simultaneous monitoring of product of hydrogen peroxide and phosphatidyl inositol-3,4,5-triphosphate in a living cell.

4 cl, 4 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: microorganism-producent is cultivated in a suitable nutrient medium with subsequent extraction and treatment of target protein. At the same time the producent is yeast strain Saccharomyces cerevisiae, transformed with the expression vector, which contains an area of initiation of replication of endogenic 2-mcm plasmid of yeast Saccharomyces cerevisiae, and also a yeast promotor GAL1, which controls gene expression, including a DNA sequence SEQ ID NO: 1, coding a fused protein, the composite parts of which are aminoacid sequences of the protein E7-HSP70 and the protein of ubiquitin of yeast Saccharomyces cerevisiae, occupying within the fused protein an N-end position ending with a processing site, which separates it from the sequence of the protein E7-HSP70 and recognised natural ubiquitin-specific yeast proteinases. The yeast strain Saccharomyces cerevisiae VKPM Y-3853 - producent of the protein E7-HSP70 - is produced by transformation with the expression vector pPDX3U-E7-HSP70 of the yeast strain Saccharomyces cerevisiae D702.

EFFECT: group of inventions provides for high level of biosynthesis and yield of treated protein E7-HSP70, production of a water-soluble correctly processed protein E7-HSP70, principal absence of toxic and pyrogenic bacterial LPS in preparations of the protein E7-HSP70.

2 cl, 4 dwg, 12 ex

FIELD: biotechnologies.

SUBSTANCE: fusion peptide is presented for neutralisation and destruction of organophosphorous compounds, comprising a signal peptide TAT3 with amino acid sequence SEQ ID NO: 5, presented in the description, functionally connected to the sequence of organophosphate hydrolase SEQ ID NO: 18, classified as protein EC 3.1.8. The following components are described: extracted polynucleotide, which codes the specified fusion protein; a vector containing the specified polynucleotide; and a procaryotic host cell containing the specified vector and expressing the specified fusion protein. The method is proposed to produce a ferment, which destroys organophosphorous compounds, including expression of the specified polynucleotide in the procaryotic host cell and production of the specified ferment.

EFFECT: invention makes it possible to increase expression of organophosphate hydrolase in a host cell.

13 cl, 1 tbl, 9 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.

9 cl, 5 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions refers to biotechnology and concern hypoallergic fused proteins. A presented hypoallergic fused protein consists of one hypoallergic molecule originated from an allergen, fused with a second non-allergic protein originated from a pathogen, or a fragment thereof with the hypoallergic molecule originated from the allergen showing 50% decreased ability to bind IgE, 30% decreased T-cell responsiveness as compared with a wild-type allergen. A nucleic acid molecule coding the fused protein, an expression vector, a host cell expressing the same protein, and a vaccine composition containing the protein are also presented.

EFFECT: presented invention enables preparing therapeutic and prophylactic drugs for an allergy or diseases caused by pathogens with using no toxic adjuvants, showing no side effects.

14 cl, 23 dwg, 17 tbl, 27 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically preparing a tumour necrosis factor receptor and may be used in medicine. Genetic engineering technique is used to produce mutant TNFRp75 bound to a tumour necrosis factor and lymphotoxins substantially consisting of N-terminal 257 amino acid residues TNFRp75 wherein the N-terminal residue Glu92 is substituted by Asn, His, Ser or Ala and wherein the N-terminal residue Trp89 is optionally substituted by Tyr or Phe. The produced mutant is used to construct a fused protein with an additional amino acid fragment specified in a constant area of human immunoglobulin and one of five functional areas of albumin found on C-terminal of the soluble mutant TNFRp75. The produced mutant TNFRp75 and the fused protein is used as a part of a pharmaceutical composition for treating the diseases associated with TNFα overexpression which involve rheumatoid arthritis, psoriasis, sclerodermatitis, Sjogren syndrome, Strumpell-Marie disease, lupus erythematosus, acute disseminated myositis and syndrome similar to systemic lupus erythematosus.

EFFECT: invention enables producing soluble TNFRp75 mutant able to be bound to tumour necrosis factor and lymphotoxins at a high affinity degree.

9 cl, 12 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

Up!