Method of obtaining recombinant antigen g2 of dobrava hantavirus in e.coli cells
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.
The technical field of the invention: the invention relates to the field of biotechnology, in particular, to obtain recombinant antigen G2 Hantaviruses with the capability to improve the reproducibility and sensitivity of enzyme immunoassay in the diagnosis of HFRS.
Level of technology: hemorrhagic fever with renal syndrome (HFRS) virus retransmissions zoonotic disease is one of Russia's leading position in the number of cases among the natural focal infections.
The most effective method of dealing with HFRS is specific prevention, i.e. vaccination of the population in endemic regions [1-3]. Currently, the commercial vaccine against hemorrhagic fever with renal syndrome produced only in China, but none of these vaccines cannot be applied in the European regions of Russia, because it does not provide protection from virus, Puumala and Dobrava - causative agents of HFRS in these regions [4-7].
For production and control of vaccines against the virus, Puumala and Dobrava necessary to solve the problem of developing an effective serological methods based on proteins of the outer shell - envelope of Hantaviruses. To date, however, the problem of obtaining a full-sized commercial preparations of proteins envelope G1 and G2 of Hantaviruses not solved anywhere in the world, due to their toxicity in relation to bacteria inim cells.
Was recently obtained recombinant antigen NT-Δ12, representing a fragment of a protein G2 Hantavirus Dobrava, preserving the immunogenicity of a full size product, but does not exhibit toxicity towards cells of the producer (U.S. Pat. application RU # 2010141819 from 13.10.2010). This protein was accumulated in E. coli cells as Taurus enable [8-10]. Consequently, when using antigen NT-Δ12 in immunological tests, it is necessary to pre-renaturation product, which in turn negatively affects the sensitivity and reproducibility of the immunological test.
The aim of our work is to develop ways of improving the quality characteristics of antigen NT-Δ12 by changing its structure.
Disclosure of the invention: the invention is a method of improving the quality characteristics of recombinant antigen G2 (HT-Δ12) for use in immunological tests in the diagnosis of HFRS due to the improved solubility of the target protein antigen G2 Hantavirus Dobrava by stabilizing its globular structure of beta-structural mini-domain foldon fibritin coliphage JS98C3. Thus, the present invention relates to a method for producing a protein-antigen G2 Hantavirus Dobrava with specified performance characteristics (protein GHF) to improve FOTS is izvedenosti and sensitivity of enzyme immunoassay in the diagnosis of HFRS. The method involves the following stages:
1) preparation of plasmid constructs pGHF carrying trifunctionally fused gene;
2) expression and analysis of protein yield GHF;
3) chromatography protein GHF on a column of Sepharose 6 FF;
4) conduct an immunochemical tests.
A brief description of graphics:
Figure 1. Plasmid construction pGHF concept.
Figure 2. The sequence trifunctional fused gene encoding a derivative of the peptide HT, in the structure of the pGHF.
The GFP gene sequence is underlined with a wavy line;
The gene sequence of the peptide HT of the composition of the cDNA of the virus Dobrava grayed out;
The gene sequence foldon fibritin highlighted by the dotted line;
Figure 3. Electrophoretic analysis of the products of expression of design pGHF soluble in (1) and insoluble (2) fraction of the cell lysate E. coli JM109, detransforming design pGHF. Proteins are separated in denaturing 15% SDS page. Figure (a) shows the total protein (gel stained Coomassie Blue R-250), figure (b) presents the glow of the protein in the gel under UV radiation.
Figure 4. Chromatography of protein GHF on a column of Sepharose 6 FF. The optical density at λ=280 nm are shown in the graph (1).
Dashed lines mark the range of fractions containing protein GHF (B1-B8).
Figure 5. The study of the antigenic activity of the protein GHF met the house enzyme-linked immunosorbent assay. Tablets activated protein-antigens GHF (a) and NK6 (b). Pools of blood sera of patients with hemorrhagic fever with renal syndrome (virus Dobrava, Puumala, Hantaan) and healthy donors were used in a dilution of from 50 to 3200 times in increments of 2 times. On the ordinate axis is specified As450each point minus background (control without the addition of human serum). The graph shows the reaction of protein antigens from the blood sera of patients with HFRS (1) and with sera of healthy blood donors (2).
The implementation of the invention:
1) obtaining design pGHF,
2) expression and analysis of protein yield GHF,
3) chromatography protein GHF,
4) conduct an immunochemical tests.
1) Obtaining design
Getting constructs was performed according to the following scheme:
In the previously described construction of pet-NT-Δ12 (U.S. Pat. application RU # 2010141819 from 13.10.2010)that carries the gene peptide NT cloned gene foldon fibritin of design pET-cd-32a . This vector construct pet-NT-Δ12 uncoupled sites SpeI and XhoI, and the insertion of the constructs pET-cd-32a removed the restriction sites XbaI and XhoI.
Later in the unique XbaI site of the resulting construction on the basis of the vector Rita have introduced a synthetic DNA duplex made up of pre-phosphorylated oligonucleotides:
5' CTAGCGGGGGCGCCGGAGGA 3',
5' CTAGTCCTCCGGCGCCCCCG 3'.
Next, the resulting structure-based rate COI is litovali as template for PCR with a unique primer
5' ggagatctGCTAGCGGGGGCGCCGGAGGAATTaattcgag 3'
and with a standard primer T7terminator.
The resulting PCR product length 600 BP were digested with restrictase BglII and XholI and cloned in the vector pGem3z, split across sites BamHI and SalI.
Insert "NT+ foldon fibritin from design pGem3z removed the restriction sites SacI and > PST and cloned in the expression vector pQE30-GFP, containing the gene for green fluorescent protein GFP-based vector pQE30 (Qiagen)  on the same site.
As part of the design (Fig 1 and 2) we can identify the following elements:
- expression vector pQE30 (Qiagen);
gene green fluorescent protein GFP (Evrogen);
- fragment of polylinker vector pGem-3z (Promega);
gene Ht from the construction of pet-HT-12;
- fragment of polylinker vector pGem-3z (Promega);
- gene foldon fibritin phage JS98C3 .
2) Expression and analysis of protein yield
Design pGHF was introduced into cells of E. coli strain JM109. Selection of colonies was carried out by two parameters: ampicillinampicillin and by the presence of green fluorescent staining of the colonies. The resulting product was cultured at 30°C in liquid medium (0.5% of yeast extract, 1% peptone and 0.5% NaCl) supplemented with 100 μg/ml ampicillin in Erlenmeyer flasks with a volume of 750 ml (30 ml medium per flask) for 14-18 hours. Seed material was a washed cell culture plates with agar medium (0.5% of yeast extract, 1% Pepto is, 1.5% agar, 0.5% of NaCl)obtained by seeding primary transformants. The age of transformants about 40 hours since the end of the transformation, the cultivation temperature of 30°C. the Dose of inoculation with 5×108cells per flask. Induction of the promoter in the cells of the producer spent IPTG.
Assessment of the accumulation of the target product in E. coli cells was performed using electrophoretic analysis of total proteins recombinant producer according to laemmli's method. This of coarse cell lysate of each culture was collected in 100 μl. The lysate was subjected to centrifugation at 14000 g for 15 min and separated soluble and insoluble cell fractions. Proteins were solubilizers in 30 ál of buffer laemmli's method and analyzed the composition of proteins by denaturing SDS-electrophoresis in 15% SDS page (figure 3). Fluorescence proteins soluble fraction in the gel was observed in the region of 47 kDa, which corresponds to the calculated mass of the protein GHF. Proteins insoluble fraction fluorescent glow had not.
3) Chromatography protein
Coarse cell lysate culture of JM109 (GHF) was subjected to centrifugation at 8000 g for 1 hour and separated soluble and insoluble cell fractions. Protein from the soluble fraction was subjected to initial purification by double vysalivaniya ammonium sulfate. The first precipitate, obtained by deposition of a 1.2% solution (NH)
The main purpose of the GC protein - derived peptide NT under denaturing conditions was the removal of impurities nucleic acids, forming colloid involving proteins and preventing the effective application of other methods of chromatography. With the removal of drug impurities cellular proteins E. coli was considered only as a side task.
The resulting protein solution GHF in the volume of 15 ml was applied on the column Sepharos 6 FF (GE Healthcare, USA, height 24 cm, volume of 48 ml), equilibrated with buffer A. the Elution was performed with a linear gradient of NaCl from 0 to 0.5 M in chromatography buffer (Tris-HCl, 100 mm) at a rate of 8 ml/min (figure 4).
Fractions for further work containing the target protein GHF, were selected by evaluating the presence of fluorescent green color in solution. Selected fractions of 10 ml volume each were combined and subjected to concentration and purification on HisLink Protein Purification Resin (Promega). Material protein fractions GHF obtained, was immunochemical pure and homogeneous electrophoretic that made it possible to use it to conduct and monogenically tests.
4) conduct an immunochemical tests
As the format of analysis was selected typhoid direct sorption of recombinant protein antigen GHF on the surface of immunological tablet. Drug for comparison served as a protein-antigen NK6. In the process of sorption of protein preparations GHF and NK6 was dissolved in 20-50 times carbonate-bicarbonate buffer (KGB), bringing the concentration of total protein in the solution to 10 μg/ml of the resulting solution was made in immunological tablets for 1 day, then spent the blocking of nonspecific binding to the substrate with 1% BSA in buffer KGB. In the next stage of analysis was conducted serial titration pools of blood sera of patients with HFRS and, as a comparison of healthy donors. When this pool of blood sera included HFRS patients, infected with the virus, Dobrava, Puumala, and Kuantan. Serum was taken in a dilution of from 50 to 3200 times in increments of 2 times. As a control sample used wells, in which, instead of diluted human serum contributed PBS buffer ("conjugate control).
In conclusion, all wells were treated individuum conjugates against human IgG labeled with peroxidase and TMB substrate in the presence of hydrogen peroxide. The signal was measured on a flatbed-spectrophotometer at λ=450 nm. Resulttemplate presented in figure 5.
The results demonstrate that increasing the solubility of the recombinant antigen by changing its structure leads to increased resolution immunoassay for the diagnosis of HFRS.
List of used sources
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2. Cho H.W., Howard C.R., Lee H.W. Review of an inactivated vaccine against hantaviruses. - Intervirology. - 2002 - V.45, no. 4-6, P.328-333.
3. Song G. Epidemiological progresses of hemorrhagic fever with renal syndrome in China. - Chin Med J (Engl). - 1999 - V.112, No. 5, P.472-477.
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5. Choi Y. Ahn C.J., Seong C.M., M.Y. Jung, B.Y. Ahn Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells. - Vaccine. - 2003 - V.21, No. 17-18, P.1867-1873.
6. Lee H.W., Chu Y. K., Woo Y.D. Immune responses after two or three doses of Hantavax vaccination against Hantaan virus // Proc. fifth international conference on hemorrhagic fever with renal syndrome, hantavirus pulmonary syndrome and hantaviruses. Lion, Franch - 2001. - P.234-242.
7. Ruan Y., Xu X., Liu W" Deng X, Weng S, Zhou W, Wang Q, Chen L, Fang L, Xu Z, Yan Q, Liu W, Dong G, Gu H, Yu Y, Xu Z. A study on immunogenicity and safety of left-hand drive vehicles inactivated vaccine against hemorrhagic fever with renal syndrome. - Zhonghua Yu Fang Yi Xue Za Zhi. - 1999 - V.33, No. 6, P.340-342.
8. Hooper J.W., Kamrud K.I., Elgh F., D. Custer, C.S. Schmaljohn DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against seoul virus infection. - Virology. - 1999 - V.255, No. 2, P.269-278.
9. Kallio-Kokko, H., Leveelahti R., Brummer-Korvenkontio, M., Lundkvist, A., Vaheri, A., Vapalahti O. Human immune response to Puumala virus glycoproteins and nucleocapsid protein expressed in mammalian cells. J Med Virol. - 2001 - V.65, No. 3, P.605-613
10. Huang H., Li X., Zehua Z. Genetic immunization with Hantavirus vacine combining expression of G2 glycoprotein and fused interleukin-2. - Genetic Vaccines and Therapy. - 2008 - V.6. - P.15-21.
11. Latypov O.R., Besides A.K., Litrov AV product Characteristics wac gene of bacteriophage JS98C3 closely related to phage JS98 // Bulletin of the KSU. 2007. T. S.112-122.
12. Shevelev A.B., Fomenkov VG, Kuznetsova T.V., Kovalev, L.I., Lagodna NV creating a producer of myostatin in the form of a fused protein with 6His-GFP-based E. coli and analysis of its immunogenicity. The collection of articles "biomedical technology to increase efficiency in the conditions of intense physical activity. The Ministry of education and science of the Russian Federation, Moscow, 2004, P.140-150.
A method of obtaining a recombinant antigen G2 Hantavirus Dobrava in E.coli cells, characterized by the fact that DNA constructs pGHF encoding a protein of three parts, where the N-terminal position of the green fluorescent protein GFP, Central - peptide length 73 S.A. with the amino acid sequence SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEWKDPDGMLKDHLNILVTKDIDFDT and C-terminal mini-domain foldon fibritin coliphage JS98C3 (figure 2), is introduced into cells of E. coli cultured transformed this design cells are lysed biomass, separating the insoluble fraction of the lysate by centrifugation, the expression product in the form of Taurus VK is Uchenie solubilizing using denatured alcohol, spend chromatography, and used the product for the detection of specific antibodies in the serum of patients with hemorrhagic fever with renal syndrome.
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
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
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
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
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
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
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
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
SUBSTANCE: invention describes nucleotide sequence (dwg.2), coding immunogenic polypeptide LcrV(G113), serving the base for construction of recombinant plasmid DNA pETV-I-3455, with the size 6538 bp, which codes immunogenic polypeptide LcrV(G113). Plasmid consists of plasmid pBR322 replicon, β-lactamase gene, determining resistance to ampicillin, T7-promoter, 1ac-operator, f1-replicon and DNA fragment, flanked by the sites for restrictases Ndel and Hindlll, coding synthesis of protein LcrV(G113), which starts from initiating codon ATG. Described is recombinant strain of bacteria E. coli BL21 (DE3)/pETV-I-3455 - producer of immunogenic polypeptide LcrV(G113) with amino acid sequence, represented on dwg.3, where tryptophan in position 113 (W113) is substituted with glycin. Described is method of obtaining said polypeptide by cultivation of strain E. coli BL21(DE3)/pETV-I-3455. Cells are destroyed in buffer solution by ultrasound and polypeptide is isolated successively by gel-permeation chromatography with application of carrier TSK HW-40, anion-exchanging and hydrophobic chromatography.
EFFECT: invention makes it possible to obtain product with high immunogenic and protective activity.
5 cl, 10 dwg, 2 tbl, 5 ex
SUBSTANCE: invention refers to recombinant plasmid DNA pER-Hir coding a hybrid protein capable to autocatalytic breakdown to form [Leul, Thr2]-63-desulphatohirudin, to Escherichia coli to an ER2566/pER-Hir strain - a producer of said protein and a method for producing genetically engineered [Leu 1, Thr2]-63-desulphatohirudin. The presented recombinant plasmid DNA consists of the SapI/BamHI fragment of DNA plasmid pTWIN-1 containing a promoter and a terminator of T7-RNA-polymerase transcription, an amplifier of phages T7 gene 10 translation, β-laktamase (Ap) gene, modified mini-intein Ssp DnaB gene, with an integrated sequence of a chitin-binding domain, and the SapI/BamHI-fragment of DNA containing a sequence of a gene of recombinant [Leul, Thr2]-63-desulphatohirudin-1 containing β-laktamase (Ap) gene as a genetic marker, and unique recognition sites of restriction endonucleases located at the following distance to the left from the site BamHI: Nrul - 186 base pairs, Ndel - 594 base pairs, Xbal - 882 base pairs, EcoRV - 2913 base pairs, Hpal - 2966 base pairs.
EFFECT: inventions allow producing said compound which is used as a drug applied to prevent blood hypercoagulation.
3 cl, 1 dwg, 4 ex
SUBSTANCE: invention refers to biotechnology and gene engineering and represents recombinant pHisTevTSIB0821 plasmid for expression in Escherichia coli cells of TSIB_0821 prolidase from Thermococcus sibiricus archean. The proposed plasmid includes NdeI/SalI-fragment of pET-22b(+) (Novagen) plasmid and a DNA fragment with the size of 1196 pairs of bases, which contains a fused gene consisting of the following structural elements: nucleotide sequence coding 6-histidine tag, nucleotide sequence coding the site of recognition/decomposition of TEV protease, and nucleotide sequence of TSIB_0821 gene, which are connected so that at their biosynthesis in E. coli cells a continuous reading frame can be maintained. E. coli Rosetta(DE3) strain is obtained, which is transformed with the above plasmid, - a producer of chimeric protein including amino acid sequence of TSIB_0821 prolidase, fused on N-end with the 6-histidine tag and the site of recognition/decomposition of TEV protease. A growth and induction method of a producer strain and a method for separation and cleaning from the obtained biomass of functionally active recombinant TSIB_0821 prolidase including the following technological process: two metal affine chromatographies, gel filtration, TEV protease treatment, dialysis, and concentration, have been developed.
EFFECT: invention allows obtaining recombinant prolidase that is similar as much as possible as to structure to its natural equivalent with high and stable yield, level of cleaning and functional activity.
3 cl, 3 ex
SUBSTANCE: recombinant plasmid DNA contains BamHI/Hindlll - fragment of pQE30 plasmid DNA; BamHI/Hindlll - fragment of gene Yp2 Galleria mellonella, coding fermentative-active part of lipase polypeptide, protein of pyralid bee digestive tract; as genetic marker - bla gene of B-lactamase; nucleotide sequence, coding polyhistidin tract within Yp2 gene reading frame.
EFFECT: invention may be used for creation of pollution decomposer preparation containing hard alkanes.
4 dwg, 2 tbl, 2 ex
SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use. Provided particle contains expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus, haemagglutinin gene of A/Perth/16/2009(H3N2) strain with pre-optimised for expression in human being cells nucleotide sequence presented in SEQ ID NO:2. The specified haemagglutinin gene of influenza virus of A/Perth/16/2009(H3N2) strain is cloned in expressing cassette containing polyadenylation signal SV40 under control of cytomegalovirus promoter. Presented invention may be used for induction of specific immunity to influenza virus A of H3N2 subtype during injection in efficient quantity.
EFFECT: providing intense expression of recombinant haemagglutinin of the specified influenza virus.
6 cl, 9 dwg, 1 tbl, 4 ex
SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use as a component for production of vaccine for influenza virus A of H1N1 subtype. Presented recombinant particle contains expressing cassette including SV40 polyadenylation signal and cytomegalovirus promoter with influenza virus haemagglutinin gene being included. As influenza virus haemagglutinin gene, haemagglutinin gene of strain A/California/07/2009(H1N1) is used with pre-optimised for expression in human being cells nucleotide sequence provided in SEQ NO:2. These inventions allow raising specific immunity to influenza virus A of H1N1 subtype by provision of overexpression of haemagglutinin gene of influenza virus A/California/07/2009(H1N1).
EFFECT: improvement of the method.
6 cl, 9 dwg, 1 tbl, 4 ex
SUBSTANCE: invention represents the method for obtaining 2S,4R-monatin or its salt providing the contact of 4R-IHOG with aminotransferase of L-amino acid in presence of L-amino acid and method for 2S,4R-monatin or its salt obtaining with provision of 2S,4R-monatin isomerisation. The present invention also reveals aminotransferases of L-amino acid for performing the methods of monatin obtaining and method for obtaining such L-aminotransferases providing the cultivation of bacterial cell, into which there input is an expression vector containing polynucleotide that codes the presented aminotransferase.
EFFECT: invention allows obtaining 2S,4R-monatin and 2R,4R-monatin with improved output due to the use of revealed aminotransferases of L-amino acid.
27 cl, 6 dwg, 23 tbl, 48 ex
SUBSTANCE: invention pertains to the versions of antibodies against IL-6 receptor, variable regions of light and heavy chains of which are modified by introduction of amino-acid replacement. There revealed is a pharmaceutical composition for treating the diseases associated with IL-6 and containing the said version of antibody.
EFFECT: invention allows efficient treatment of the diseases associated with IL-6.
8 cl, 48 dwg, 16 tbl, 20 ex
SUBSTANCE: method involves cultivation in the appropriate conditions of yeast Saccharomyces cerevisiae and release of target protein; besides, release is directed with leader polypeptide, which has amino acid sequence SEQ ID NO1 and representing a variant of a pro-area of leader polypeptide of protein PpPIRl Pichia pastoris.
EFFECT: invention enlarges the range of methods for obtaining target protein in yeast Saccharomyces cerevisiae and increases possibilities for effective synthesis of such proteins.
2 dwg, 4 ex
SUBSTANCE: invention describes two-stranded RNA modified with lipids and including antisense thread, the nucleotide sequence of which is complementary to target sequence in the target gene, and a sense thread, the nucleotide sequence of which is complementary to antisense thread. A two-stranded lipid is connected to the first nucleotide on the 5'-end of sense thread, either directly or through a linker. RNA has high stability to action of nuclease and is effectively absorbed with a cell, as well as generates excellent effect of RNA interference.
EFFECT: improvement of efficiency.
15 cl, 14 dwg, 3 ex
SUBSTANCE: invention also refers to an expression vector and a transformant containing polynucleotide, as well as a production method of lipid composition or composition based on fat acids using the transformant.
EFFECT: invention allows producing a new ferment with improved properties, which has glycerol-3-phosphatocyltransferase activity.
11 cl, 4 dwg, 14 tbl, 4 ex
SUBSTANCE: recombinant hybrid inhibitor of angiogenesis represents a protein shown in dwg. 1. This protein includes amino acid sequence of plasminogen of a human being from amino acid 82 to 341 and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, which are covalently connected to each other. An inhibitor production method involves expression of its gene in cells of E. coli producer strain, which are transfected with recombinant plasmid DNA pBSRK13 with a physical map presented in dwg. 2, which has the size of 4155 pairs of bases. This plasmid includes a gene coding the recombinant hybrid inhibitor of angiogenesis, as well as a gene of signal peptide OmpA, lac-operator, a gene of stability to kanamycin, replicative origin pUC ori and a gene coding the lac-operator under control of promoter T7. A target protein is extracted from periplasmatic area of bacterial cells by affine and gel-filtration chromatography. The invention can also be used in medicine for creation of new medicinal agents with antiangiogenic therapeutical effect.
EFFECT: invention allows producing a new protein having antiangiogenic activity and increased selectivity of action in relation of tumoral endothelium.
2 cl, 3 dwg, 1 tbl, 5 ex
FIELD: medicine, genetics, biochemistry.
SUBSTANCE: invention relates to new NOS-variants or mutants that comprise structural modifications in site Akt-dependent phosphorylation. Modified NOS-proteins or peptides, in particular, human proteins or eNOS-peptides having change of amino acid residue corresponding to S/T in motif of the consensus-sequence RXRXXS/T of NOS-polypeptide of wild type and nucleic acid molecules encoding thereof can be used in genetic therapy and proteins and NOS-peptides can be used in screening methods of agents modulating activity of NOS. The advantage of invention involves the creature of new NOS-variants or mutants that can be used in genetic therapy.
EFFECT: valuable medicinal properties of mutants.
25 cl, 1 tbl, 9 dwg, 3 ex