Method of production of recombinant antigen g2 of hantavirus dobrava in cells of e coli
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
The technical field of the invention: the invention relates to the field of biotechnology, in particular, to obtaining vaccines for the prevention of viral infections, namely infection, hemorrhagic fever with renal syndrome.
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 . Inactivated vaccine Hantavax  was developed for use in the Republic of Korea, dominated by species of Hantaviruses Canaan and Seoul. Currently, the vaccine under the brand name Hantavax is produced industrially and commercially available. It is approved for clinical use in the Republic of Korea.
Along with Hantavax in China and South Korea developed, licensed and actively used in the treatment of HFRS other variants of killed vaccine against hemorrhagic fever with renal syndrome:
1) In China is formalin inactivated vaccine based on virus Seoul, grown in primary cell cultures of Syrian hamster kidney; for inactivation of virus in the production of this vaccine is used β-propiolactone.
2) China is a vaccine on the basis of the virus Hantaan, grown in primary cultures of kidney cells Mongolian gerbils, inactivated by formalin.
3) Purified vaccine based on virus Hantaan obtained from the brain of experimentally infected rats suckling .
4) currently, China has also developed and is undergoing clinical trials of the bivalent vaccine based on virus Hantaan and Seoul obtained on transplantable cell cultures Vero-E6 (inactivation of β-propiolactone). According to the developers, this drug should replace the inactivated vaccine, since the latter is due to the low level of treatment cause serious side effects, not providing the maximum level of the synthesis of neutralizing antibodies.
However, 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 [4-10].
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 against bacterial the m cells.
Was previously obtained recombinant antigen G2 Hantaviruses NT-Δ12 . However, the output of this protein did not exceed 20 mg per liter of culture, which together with the losses during the purification of the drug prevents the use of this antigen in immunological tests.
The aim of our work is to develop ways of increasing the number of product accumulation in the producer strain due to changes in the structure of the target protein.
Disclosure of the invention: the invention is a method of increasing the yield of recombinant antigen G2 Hantaviruses in E.coli cells by optimizing the structure of the antigen to trifunctional derived peptide NT.
Based on the design of pet-NT-Δ12 that encodes the peptide HT in the isolated state, was designed and obtained design, where this peptide was part trifunctional fused protein. This N-terminal position occupied by the green fluorescent protein GFP, Central - peptide neurotensin and C-terminal light chain double-stranded protein inhibitor Konitza of potatoes (PKPI-B1). When assembling structures instead of vector RET based on the T7 promoter was used pQE30 containing less powerful T5 promoter. This decision was motivated by a desire to improve the conditions of the folding of the product by reducing the intensity of its accumulation. For the same purpose Gras on the Itza domains GFP, HT and PKPI-B1 design trifunctional fused protein were introduced artificial flexible peptide linkers, enriched in Gly, Ser and Ala encoded by synthetic oligonucleotides. In addition, when designing a new plasmid gene peptide NT was shortened by 40 BP from the 3'-end, which will not affect the immunochemical properties of the protein, but reduces to it the number of disulfide bonds and hydrophobic areas, which improves the solubility of the product and reduces its negative impact on cell viability producer.
A brief description of graphics:
1. Plasmid construction RNC, schematic diagram.
2. The sequence trifunctional fused gene encoding a derivative of the peptide HT, in the structure of RNC.
• The sequence of the GFP gene is underlined with a wavy line;
• The sequence of the peptide HT of the composition of the cDNA of the virus Dobrava
• light chain inhibitor Konitza of potatoes
• artificially introduced by linker sequences, providing the flexibility of articulation of the globular domains of different origin in italics.
3. Electrophoretic analysis of the products of expression of design RNC in soluble and insoluble fraction of the lysate is one E. coli TG1, detransforming: (1) the Vector pQE30, (2) Design RNC. Proteins are separated in denaturing 15% SDS page and stained for total protein with Coomassie Blue R-250. The arrow indicates the location of the protein NK6 (estimated weight 42 kDa).
Fig.4. Anion-exchange chromatography protein NK6 on a column of Sepharose-Q in the presence of 4M urea. Thin gray lines indicate the concentration of NaCl in the eluting buffer and the conductivity of the solution, and the black line is the optical density at λ=280 nm. Dashed lines indicate the range of fractions selected for electrophoretic analysis, black line - fractions selected for further work.
5. Electrophoretic analysis of protein fractions NK6 after ion-exchange chromatography under denaturing conditions. Proteins are separated in denaturing 15% SDS page and stained for total protein with Coomassie Blue R-250. The position of the target protein by mass of 42 kDa indicated by the dotted oval. For further work were selected fractions D6-F6.
Fig.6. The study of the antigenic activity of the protein NK6 by the method of enzyme-linked immunosorbent assay. Tablets activated protein-antigen NK6 and blocked with normal rabbit serum at a dilution of 100 times. Serum of patients with hemorrhagic fever with renal syndrome (virus Dobrava, Puumala) and healthy donors were practicability in a series of dilution from 20 to 1280 times in increments of 2 times. On the ordinate axis is specified As492each t is his minus background (control without the addition of human serum).
The implementation of the invention:
1) obtaining design
2) expression and analysis of protein yield
3) preparation of test samples for chromatography
4) ion-exchange chromatography.
5) conduct immunochemical tests.
1) Obtaining design
Getting constructs was performed according to the following scheme:
1. Using a matrix design pET-Ht-Δ12 and primers Li4 (GGAGATCTCTAGCGGGGGCGCCGGAGGAATT) and NT (GGACTAGTATCAAAGTCAAGTGCCTT) conducted PCR. The product is treated with the restriction enzyme BglII.
2. On the matrix plasmids pQE-GFP performed PCR using primers ECFP1 (GGGGATCCATGGTGAGCAAGGGCGAGGAand GFP10 (GGAGATCTCTTGTACAGCTCGTCCATGCC). The product is treated with the restriction enzyme BglII.
3. The products of stages 1 and 2 combined and subjected to legirovanie.
4. The matrix product of ligation stage 3 performed PCR with primers ECFP1 and NT. The PCR product purified by separation from the gel and subjected to cleavage by restrictase BamHI and SpeI.
5. The design of the pQ-Knl representing gene PKPI-B1 (Tuberosum cultivar Istra, NCBI access number AY692479) in the vector pQE30-treated restrictase SpeI and BamHI.
6. Products of reaction of the enzyme with stages 4 and 5 combined, subjected to legirovanie and introduced into cells of E. coli TG1 by transformation.
7. The obtained intermediate design 6-based vector pQE30 were digested with restriction enzyme SpeI and ligated with a synthetic is NC-duplex, obtained by mixing in advance phosphorylated oligonucleotides CTAGCGGGGGCGCCGGAGGAATT and CTAGTTCCGGCGCCCCCG to introduce the linker sequence on the border areas of NT and easy C-terminal chain of the protein inhibitor PKPI-B1. The obtained expression design on the basis of the vector pQE30 was designated RNC (Fig.1 and Fig.2).
2) Expression and analysis of protein yield
Design RNC was introduced into the cells of the strain E. coli TG1 by taking ampicillin-resistant 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% peptone, 1.5% agar, 0.5% of NaCl)obtained by seeding primary transformants. The age of the transformants is not more than 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 did not.
Evaluation of the yield of the target product 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 4000 G. within 15 min and was divided 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).
The analysis showed that trifunctionally protein-based peptide NT - design product RNC as free peptide NT, accumulated in the insoluble cell fraction, with a yield of about 40 mg/l of culture.
3) Preparation of test samples for chromatography
Coarse cell lysate culture TGl(pHK6) was subjected to centrifugation at 8000 g for 1 hour and separated soluble and insoluble cell fractions. The combined precipitate of insoluble cellular fractions washed three times in 25 ml of 4 M NaCl with addition of 1% Triton, 25 ml of saline with 1% Triton and 25 ml of water. Sediment suspended in 8 ml of buffer (Tris-HCl 10 mm, pH=8,6)containing 8 M urea and 0.1% β-mercaptoethanol. The solution was boiled on a water bath for 10 minutes and centrifuged. Received the clarified supernatant was applied to gelfiltration chromatographic column filled with Sephadex G-25 fine at a speed of 5 ml/min using a liquid chromatographic system low pressure "AKTA-Purifile" (GE Healthcare, USA). Separation was carried out in buffer (Tris-HCl 10 mm, pH=8,6)containing 4 M urea, in order to remove salts, excess recover the I and low-molecular cell impurities. The solubilized under denaturing conditions, the protein was subjected to purification using anion exchange chromatography.
Isolation and purification of target proteins from the resulting supernatant was performed using a liquid chromatography system "AKTA-Purifile" (GE Healthcare, USA).
4) Ion-exchange chromatography
The main purpose anionoobmennoi chromatography protein - derived peptide NT under denaturing conditions, was the removal of impurities nucleic acids, forming colloid involving proteins and preventing the effective use of gel filtration and other methods of chromatography. With the removal of drug impurities cellular proteins E. coli was considered only as a side task.
Obtained after gel filtration the solution of denatured protein NK6 in the volume of 15 ml was applied to the anion exchange column of Sepharose-Q (XL) (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 10 mm, pH=8,6, 4 M urea, 1 M NaCl) at a rate of 8 ml/min (Figure 4).
Collected 20 fractions of 10 ml each were analyzed using denaturing SDS-electrophoresis in 15% SDS page. For electrophoresis were selected by 50-100 ál from each fraction. Proteins were subjected to deposition of 1 ml of a mixture (500 ál of 100% acetone, 100 ál of 70% THU 1 ml). The obtained salt precipitation which was ilizarova under denaturing conditions in 15 μl of buffer laemmli's method (with the addition of urea) (Fig.5).
Material protein fractions NK6 obtained by anion-exchange chromatography under denaturing conditions, was immunochemical clean and electrophoretic homogeneous, allowing it to be directly used for renaturation and immunochemical tests (Fig.6).
5) Conduct immunochemical tests
As the format of analysis was selected typhoid direct sorption of recombinant protein antigen NK6 on the surface of immunological tablet. Were used in the experiment chromatographically purified protein NK6 in denatured form. In the process of sorption protein preparation is stored in the form of concentrated solutions in the presence of 4 M urea, 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. A necessary step in the analysis was blocking the normal binding of antibodies present in the serum of healthy donors, at the expense of competition. To this end, the tablets are subjected to blocking with 1% BSA, optionally processed, contributing to each well of normal rabbit serum, rasb is run by PBS buffer at a ratio of 1:100.
In the next stage of analysis was conducted serial titration test sera of patients with HFRS (or healthy donors in the comparison group). In this study the specificity of the reaction in the sample comparison included a) HFRS patients infected with the virus Dobrava, b) HFRS patients infected with the virus, Puumala, C) healthy donors. 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=492 nm. The results of the determinations are shown in figure 6.
The results show high reactivity of antibody positive sera against antigen NK6. The signals of positive sera of patients in 2-2,5 times higher than the signal sera of healthy donors in the same breeding that allows you to reliably diagnose the presence in the blood of people of specific antibodies against the virus Dobrava and Puumala.
List of used sources
1. Maes P., Clement J., Van Ranst M. Recent approaches in hantavirus vaccine development. Expert Rev Vaccines. - 2009 - V.8, No. 1, Page 67-76.
2. Cho H.W., Howard C.R., Lee H.W. Review of an inactivatedvaccine 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 (Engi). - 1999 - V.112, No. 5, P.472-477.
4. Oya A. Japanese encephalitis vaccine. - Acta Paediatr Jpn. - 1988 - V.30, №2, R-184.
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., Y.K. Chu, 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., Eigh 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 vaccine combining expression of G2 glycoprotein and fused interleukin-2. - Genetic Vaccines and Therapy. - 2008 - V.6. - P.15-21.
11. Abelev, Mosnarbank, Mvelopes, Eueope, Oageoojs, Tchudakorova, Eaatem. A method of obtaining a recombinant antigen G2 Hantaviruses in E. coli cells. The patent application. Entrance. No. 060029. Reg. No. 2010141819. Date 13.10.2010.
A method of obtaining a recombinant antigen G2 Hantavirus Dobrava in cells of E. coli, features resouses fact, the DNA constructs RNC shown in figure 1, 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 SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEWKD PDGMLKDHLNILVTKDIDFDT and C-terminal light chain double-stranded protein inhibitor type Konitza of potatoes (PKPI-BI), is introduced into E. coli cells, 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 include solubilizers using denaturant, spend chromatography under denaturing conditions and use the product for detection of specific antibodies in the serum of patients with hemorrhagic fever with renal syndrome.
SUBSTANCE: method is proposed to produce a polypeptide, including cell cultivation, which intensely expresses a bicarbonate carrier and has a transferred DNA, which codes the desired polypeptide.
EFFECT: invention makes it possible for the cell to produce the specified polypeptide and the appropriate cell.
12 cl, 15 dwg, 6 ex
SUBSTANCE: invention relates to chemical engineering and techniques for producing veterinary, medical and pharmaceutical preparations. The method of producing a novel antiviral substance based on 2,5-dihydroxybenzoic acid and gelatine includes oxidising 2,5-dihydroxybenzoic acid with laccase enzyme to intermediate phyenoxy radicals and semiquinones, which are then copolymerised with gelatine, and separating the obtained copolymer from low-molecular weight components by dialysis; optimum concentrations of components of the reaction mixture are as follows: 2,5-dihydroxybenzoic acid - 15-80 mM, gelatine - 1-13 mg/ml reaction mixture, laccase - 0.5-10 units of activity/ml reaction mixture.
EFFECT: obtained copolymer has antiviral activity on herpesvirus, particularly Aujeszky's disease virus.
2 tbl, 1 dwg, 3 ex
SUBSTANCE: method includes cultivation of previously prepared culture of the recombinant strain B. anthracis 55ΔTPA-1Spo-. The cell mass is separated using the filtration module with a membrane having a pore diameter of 0.2 mcm. Protein EA1 is extracted from the washed cell mass using a buffer with 1% sodium dodecyl sulfate, and purified by diafiltration using membrane filters and two-stage ion-exchange chromatography on hydroxyapatite. The protective antigen is isolated from the culture filtrate and purified by successive steps of concentration and diafiltration.
EFFECT: use of the invention enables to obtain in one processing chain the highly purified antigens of anthrax microbe - protective antigen and protein EA1 needed to create chemical vaccines.
3 dwg, 5 ex
SUBSTANCE: culture fluid of producer strain is concentrated on hollow fibers truncating macromolecules with a molecular weight higher than 15 kDa. The cell concentrate is added to dry NaCl in a final concentration of 0.5 M. Mixed on a shaker for 20 min. The suspension is centrifuged for 15 min at 10000 g and the supernatant is separated, the supernatant pH is made up to 3.0 by four-molar solution of HCl. The suspension is centrifuged. The residue is added to water in a volume of 0.1% of the initial volume of the culture fluid, the residue is suspended and alcohol is added in amount of 0.1% of the initial volume of the culture fluid. Ethanol or propanol or isopropanol is used as alcohol. Exposed for 30 minutes at 0°C and centrifuged. The alcohol is removed from the solution by evaporation. The peptide fraction is purified. Water is added to 0.1% of the initial volume of the culture fluid, active carbon is added in an amount of 0.5% w/v (v - water volume). Centrifuged removing impurities adsorbed on the carbon. The aqueous solution is passed through the membrane truncating macromolecules with a molecular weight more than 10 kDa.
EFFECT: invention enables to accelerate preparation of bacteriocin and to increase the degree of purification of the resulting product in a yield of 90% of the total activity in the culture fluid.
1 dwg, 1 tbl, 4 ex
SUBSTANCE: present invention refers to biotechnology and medicine. There are presented versions (aCt1 and aCt2) of one-domain antibodies specifically binding the Chlamydia trachomatis antigen. There are described versions of the method of inhibiting an infection caused by Chlamydia wherein the method involves the preparation of elementary bodies C.trachomatis by a therapeutically effective amount of the nanoantibody aCt1 or aCt2 before being attached to infected target cells.
EFFECT: use of the invention provides the antibodies to detect and block the infections Chlamydia trachomatis that can find application in medicine.
6 cl, 4 dwg, 5 ex
SUBSTANCE: invention relates to a method of preparing a pharmaceutical composition, CHO cell to obtain the desired protein, the CHO cell - DNA recipient encoding the desired polypeptide, the method of production of the desired polypeptide. The method of production of the desired polypeptide comprises cultivation of CHO cell which is transformed DNA encoding alanine aminotransferase and DNA encoding the desired polypeptide. In the particular case the CHO cell is cultured in α-ketoglutarat-containing medium. The method of preparing of the pharmaceutical composition comprises preparing of the desired polypeptide with the method described above. The polypeptide is mixed with pharmaceutically acceptable carriers or additives. The preparation is prepared. The CHO cell for preparing of the desired protein has DNA transferred into it, encoding alanine aminotransferase, and DNA transferred into it, encoding the desired polypeptide.
EFFECT: invention enables to prepare a desired polypeptide with a high yield.
10 cl, 22 dwg, 3 ex
SUBSTANCE: recombinant host cell Pichia pastoris includes a cascade of fucosylation reactions, where ferments make up the specified cascade of reactions. The specified host cell Pichia pastoris includes nucleic acids, which code GDP-mannose-dehydratase (GMD), GDP-ketodesoxymannose-epimerase/GDP-ketodesoxygalactose-reductase (FX), a carrier of GDP-fucose (GFTr) and fused protein, which contains a catalytic domain of α1,6-fucosyltransferase EC 2,4.1.68, fused with amino acids 1-36 Mnn2 S.cerevisiae. A hybrid vector includes regulatory elements of DNA, which are functional in the host cell and which are functionally connected with a coding DNA sequence, which codes the fused protein, including amino acids 1-36 Mnn2 S.cerevisiae, fused with the catalytic domain of α1,6-fucosyltransferase EC 220.127.116.11. The specified vector is used to transform the host cell Pichia pastoris.
EFFECT: invention makes it possible to create a host cell Pichia pastoris, which will produce glycoproteins with fucosylated N-glycans.
8 cl, 6 dwg, 3 ex
SUBSTANCE: protein of flu virus hemagglutinin is disclosed, as well as a molecule of nucleic acid, which codes such protein, a vaccine for treatment or prevention of infections, which are mediated by a flu virus, a set and also methods to produce protein and vaccines. The protein of hemagglutinin H5 of the flu virus is characterised by the fact that in the position 223 of the series it is substituted with asparagine, and the second lysine is built into the position 328.
EFFECT: modified protein has improved immunogenic properties.
14 cl, 1 tbl, 4 ex
SUBSTANCE: group of inventions relates to feed production, particularly a method for microbiological production of feed yeast from grain wastes. The method involves preparation of grain material by grinding to 120-160 mcm particles. Further, the ground grain material is used to prepare an aqueous suspension containing 15-25% dry substances. The obtained suspension is saccharified with α-amylase and glucoamylase to 6-10% glucose content in the suspension. Nitrogen and phosphorus sources are added to the obtained suspension such that 90 mg of nitrogen and 45 mg of phosphorus are required for synthesis of 1 g of biomass. A culture is added to the obtained nutrient medium, said culture being a producer of the Saccharomyces cerevisiae yeast strain VKPM U-3585, having amylase activity, which is deposited in the Russian National Collection of Industrial Microorganisms (VKPM) and can be used in producing feed protein. The Saccharomyces cerevisiae yeast strain VKPM U-3585 is continuously grown in a multiple-section apparatus while feeding the nutrient medium simultaneously into several sections, followed by concentration of the suspension by vacuum evaporation and drying. Moisture freed at the vacuum evaporation and drying steps is used to prepare the aqueous suspension of grain material.
EFFECT: invention increases output of crude protein and true protein.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention discloses a method for preparing an anthrax diagnostic serum by hyperimmunisation of ox producers with an antigen of the strain Bacillus anthracis M-71. Hyperimmunisation is conducted in increasing doses: first subcutaneous introduction of the antigen 100-120 mln microbial cells together with saponin 2.5-3 mcg; 12-14 days later, the antigen is introduced intracutaneously in a dose of 2.5-3.0 bln microbial cells together with saponin 2.5-3 mcg; 6-7 days later, every 3-4 days, the antigen is introduced intravenously 12-14 times in increasing doses 10.0 to 210 bln. microbial cells. It is followed by blood sampling, keeping at temperature 37-38°C for 2-3 hours, placing in a fridge at 2-8°C for 3-5 days, separating serum. The prepared serum is preserved in 5-7% carbolic acid in isotonic solution in ratio (9-10):1 respectively. The ready serum is sterile and has an AR titre min. 1:1000, a CFT titre min. 1:20. A diagnostic set for anthrax diagnosis comprises the antigen of the strain Bacillus anthracis M-71, the anthrax diagnostic serum and healthy bovine's native serum.
EFFECT: invention provides higher specific activity of the serum and diagnostic effectiveness for an anthrax agent.
5 cl, 1 tbl, 3 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: what is offered is a new method for producing human methionine-free interferon-alpha2b. The method starts with recombinant plasmid DNA containing a human interferon-alpha2b gene which is foregone by an enteropeptidase proteolysis site, and used to transform Escherichia coli cells. The cells are cultured, and inclusion bodies of the synthesised predecessor are isolated. It is followed by partial renaturation of the isolated predecessor in the presence of dithioerythrole preventing closure of disulphide bonds. The predecessor is hydrolysed by the enteropeptidase enzyme with producing human methionine-free interferon-alpha2b. After completion of the predecessor hydrolysis reaction, complete renaturation of human interferon-alpha2b is carried out in the presence of a pair of cystine and cysteine compounds promoting closure of disulphide bonds. The produced protein is purified by a chromatography in a KM-sepharose.
EFFECT: higher yield.
5 dwg, 1 ex
SUBSTANCE: invention can be used for producing recombinant human interleukin-11 (rIL-11). The recombinant plasmid DNA pET32M/mTrx-rhIL-11 is produced by a method which involves synthesis of four fragments of a recombinant human interleukin-11 gene from oligonucleotide primers, cloning of each fragment in a plasmid vector pGEM5z restricted by EcoRV endonuclease, transformation of each of 4 plasmids of E.coli cells of strain DH5a, selection of clones coding the related fragments of the recombinant human interleukin-11 gene, selection of plasmids with the recombinant human interleukin-11 gene without mutations, combination of all fragments of the recombinant human interleukin-11 gene in the plasmid vector pUC19, construction of the vector pET32M of the plasmid pET32b, recloning of the recombinant human interleukin-11 gene in an expression vector pET32M, coding thioredoxin 1 E.coli with substitution of Asn84/Gln. The produced DNA is used to transform the E.coli BL21 (DE3) strain cells to produce the E.coli BL21 (DE3)/pET32M/mTrx-rhIL-11 strain that is a producer of recombinant human interleukin-11 as an ingredient of soluble fused protein mTrx-rhlL-11.
EFFECT: effective production of recombinant interleukin-11 in Escherichia coli cells.
3 cl, 5 dwg, 1 tbl, 4 ex
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